1

SECTION 3. GENETICS.

CHAPTER 1. GENETICS AS A SCIENCE STUDYING HEREDITY AND

VARIABILITY.
Genetics (G. genesis = descent) is a scientific study of the mechanism of inher-
itance and causes of variability in living organisms related by descent.
The subject-matter of genetics is the main properties of living-matter - heredi-
ty and variability of prokaryotes, animal and plant organisms, including a human be-
ing, there development and abnormity.
Heredity is the property of living matter, providing the transmission of the pa-
rental qualities and characters for the continuity of morphological, physiological and
biochemical structure of living nature from one generation to the next. It provides the
transmission of the typical type of individual development of organism.
Variability is the property of living matter to take or to lose the qualities or
characters. It is the differences that permit us to distinguish between the offspring of
the same parents.

1.1. The main goals of genetics.

1. To study the laws, determining the inheritance of genes.
2. To establish the hereditary basis of variability.
3. To understand the origin of species and distinction between them.
4. To study the structure of genes and their function.
5. To study the distribution of genes within the population.
6. To discovery the factors, regulating gene activity during embryogenesis in the
normal and pathological conditions.

1.2. The modern fundamental tendencies of genetic study.

1. To solve the problems of molecular nature of gene mutations.
2. To study the mechanisms and processes of regulation of gene activity.
3. To solve the problems of artificial synthesis of amino acids, proteins.
4. To decode the human genome.

1.3. The main methods of genetics.

Genetic Analysis - the complex of methods for studying the hereditary charac-
teristics of organisms. Genetic analysis includes:
1. Hybridological method. It studies the laws of heredity and also the structure
and behavior of genetic structures by means of special forms of crossbreeding.
2. Cytogenetic method. Study the chromosomes and their implications in ge-
netics and study the cellular structures and mechanisms associated with genetics.
3 Genealogical method. Method of drawing and analysis of pedigree.
4 Twin method. It studies the role of environmental and genetic causes on the
manifestation of any character.
5. Biochemical method. Biochemistry is carried out at the cellular or subcellular
level, generally on cell extracts. Biochemical methods are applied to the main chemi-
 2
cal compounds of genetics - notably DNA, RNA, and protein. Biochemical tech-
niques are used to determine the activities of genes within cells and to analyze sub-
strates and products of gene-controlled reactions. In one approach, cells are ground
up and the substituent chemicals are fractionated for further analysis. Special tech-
niques (e.g., chromatography and electrophoresis) are used to separate the compo-
nents of proteins so that inherited differences in their structures can be revealed. For
example, more than 100 different kinds of human hemoglobin molecules have been
identified. Radioactively tagged compounds are valuable in studying the biochemis-
try of whole cells. For example, thymine is a compound found only in DNA; if radio-
active thymine is placed in a tissue-culture medium in which cells are growing, genes
use it to duplicate themselves. When cells containing radioactive thymine are ana-
lyzed, the results show that, during duplication, the DNA molecule splits in half, and
each half synthesizes its missing components.
Chemical tests are used to distinguish certain inherited conditions of humans;
e.g., urinalysis and blood analysis reveal the presence of certain inherited abnormali-
ties - phenylketonuria (PKU), cystinuria, alkaptonuria, gout, and galactosemia. Ge-
nomics has provided a battery of diagnostic tests that can be carried out on an indi-
vidual’s DNA. Some of these tests can be applied to fetuses in utero.
6. Population method. It studies the allele frequencies and their distribution
within the population. The population genetics is based largely on mathematical log-
ic—for example, the Hardy-Weinberg equilibrium and its derivatives.
7. Mathematic method. Because much of genetics is based on quantitative data,
mathematical techniques are used extensively in genetics. The laws of probability are
applicable to crossbreeding and are used to predict frequencies of specific genetic
constitutions in offspring. Geneticists also use statistical methods to determine the
significance of deviations from expected results in experimental analyses.
Bioinformatics uses computer-centred statistical techniques to handle and ana-
lyze the vast amounts of information accumulating from genome sequencing projects.
The computer program scans the DNA looking for genes, determining their probable
function based on other similar genes, and comparing different DNA molecules for
evolutionary analysis. Bioinformatics has made possible the discipline of systems bi-
ology, treating and analyzing the genes and gene products of cells as a complete and
integrated system.
8. Molecular-genetic method. Although overlapping with biochemical tech-
niques, molecular genetics techniques are deeply involved with the direct study of
DNA. This field has been revolutionized by the invention of recombinant DNA
technology. The DNA of any gene of interest from a donor organism (such as a hu-
man) can be cut out of a chromosome and inserted into a vector to make recombinant
DNA, which can then be amplified and manipulated, studied, or used to modify the
genomes of other organisms by transgenesis. A fundamental step in recombinant
DNA technology is amplification. This is carried out by inserting the recombinant
DNA molecule into a bacterial cell, which replicates and produces many copies of the
bacterial genome and the recombinant DNA molecule (constituting a DNA clone). A
collection of large numbers of clones of recombinant donor DNA molecules is called
 3
a genomic library. Such libraries are the starting point for sequencing entire genomes
such as the human genome. Today genomes can be scanned for small molecular vari-
ants called single nucleotide polymorphisms, or SNPs (“snips”), which act as chro-
mosomal tags to associated specific regions of DNA that have a property of interest
and may be involved in a human disease or disorder.

1.4. Historical overview on the development of Genetics.

The history of genetics goes back to the earliest days of agriculture. Archeologi-
cal evidences are available that suggest that in the Middle East, at least 10000 years
ago sheep were domesticated and deliberate breeding was done according to the
commonsense applied genetics.
Genetics began as the study of heredity. If is an ancient
discipline at least 4000 years ago in Egypt, and then in other
parts of the world, formers recognized that they could im-
prove their crops and their animals by selective breeding.
Their knowledge was primitive, but they recognized that
many features of plants and animals were passed from gen-
eration to generation. There more, they discovered that de-
sirable traits such as size, seed and weight of animals should
sometimes be combined by controlled mating. Nothing was
known of genes or any of the rules of heredity. Modern ge- Fig. 1.1.Gregor Mendel
netics began in the middle of 19th century with Gregor Men-
del's careful analysis of inheritance in peas, which will be shown later (fig.1.1). Men-
del's paper remained unnoticed for 34 years until it was rediscovered in 1900 by De
Vries, Correns and Tshermak and just this year is considered the birth of genetics
as a science. At the beginning of 20th century when biochemistry and cytology be-
came developed sciences genetics began to work more
intensely than ever.
Drosophila melanogaster, the common fruit fly, was
discovered in the early eighteenth century as an experi-
mental animal in which genetic changes could we identi-
fied with changes in the physical characteristics. This an-
imal is easy and cheap to keep and breeds easily in the
laboratory. Thanks to this fruit fly many openings were
done:
1) Morgan (fig.1.2.) formulated the main principles
of the chromosomal theory of heredity on the basis
Fig. 1.2. Thomas Morgan of experiences on Drosophila;
2) Drosophila was the first representative of animals
whose genes were mapped.
Not long before the beginning of the second World War it was acknowledged that
DNA was the main heredity material, bearing biological information. In 1953
J.Watson and F.Crick (fig. 1.3.) presented the structure of DNA and showed how it
 4
stored the genetic information. For this discovery they were awarded the Nobel
Prize. Since then onwards, molecular genetics became firmly establish.
1970 became the year of the most revolutionary development of the recombinant
DNA technology. The collection of these techniques enabled geneticists to model
genes that are to transfer them from one organ-
ism to another. It was a great impact in genetic
research, particularly in our ability to understand
gene expression and its regulation in plants and
animals, a formerly difficult problem. Currently
it is providing new tools of great economic im-
portance and of value medical practices. This
branch of genetics is known as genetic engineer-
ing. According to Schulty, an American geneti-
cist, the history of genetics since 1900 is like a
Fig. 1.3. J. Watson and F.Crick play in which the first act begins with the dis-
covery of a buried treasure (Mendel's paper
which laid the foundation of the classical genetics), the second with the discovery of
the theory of the gene, and the third act with the discovery of DNA as the genetic ma-
terial leading to the checking of the genetic code.
We cannot but mention the Soviet priority in studying of Genetics. Many dis-
coveries especially in Medical Genetics were done in our country in the 20-s. They
are connected with the names of Vavilov, Chetverikov, Davidenkov, Koltsov and

S.N. Davidenkov N.I. Vavilov S.S. Tchetverikov A.A. Prokofyeva-

Belgovskaya

N.K. Koltsov N.P. Botchkov Y.K. Ginter

Fig. 1.4. Russian geneticists.
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many others who were unfortunately repressived and genetics was clammed as bour-
geoisie science till the 60-s (fig. 1.4.). I want you to remember some Soviet geneti-
cists: Prokofyeva-Belgovskaya, Zakharov, Botchkov, Ginter, Kozlova and others,
who are known all over the world due to their discoveries and monographs (fig. 1.4.).
There are three stages in the history of genetics. The first one is: studying of he-
redity at the level of organism; the second - at the cellular level and the third - at the
molecular level.

1.5. The main terms used in Modern Genetics.

Gene - is a structural and functional elementary unit of hereditary material, is a
segment of DNA molecule, which is carried in chromosomes and controls the expres-
sion of a character in an organism on cooperation with its allele, other genes and en-
vironment. Chemically, a gene is a segment of DNA molecule, coding the primary
structure of polypeptide or m-RNA, or t-RNA.
The genes determining the genetic traits are represented by symbols for describ-
ing breeding experiments. By the earlier convention (introduced by Mendel) a capital
letter is used to denote a gene for the dominant form of a trait, and a corresponding
small letter is used for the gene of a recessive form of that trait.
Alleles (Allelomorphs, Allelic genes). There are two genes for every character,
one controlling each of its two contrasting expressions. A pair of genes that control
the two alternative expressions of the same character and have the same loci (sites) in
the homologous chromosomes were originally called the alleles or allelomorphs or
allelic genes.
The term allele is used for any genes of a single character.
Example: The genes A and a for tallness and dwarfness are alleles for height in
garden pea, the genes B and b for brown and blue colour are alleles for colour of eyes
according to originally definition.
Locus - is a specific position occupied by a gene or of its alleles on a chromo-
some (plural, loci).
Dominant and recessive genes. When two allelic genes representing the alter-
natives of a character come together in an individual, usually one gene is expressed
fully (or in part) and the other is not expressed at all. The gene which always express-
es itself even in the present of its contrasting gene is known as the dominant gene.
The phenomenon in which one member of a pair of allelic genes expresses in
whole (complete dominance) or in part (incomplete dominance) over the other mem-
ber is called dominance.
On the other hand, the gene which fails to express itself in the present of its con-
trasting dominant gene is called the recessive gene.
The phenomenon, in which one member of a pair of allelic genes exhibits the
appearance in whole or in part is called recessiveness.
Example: A human being with genes Bb for colour of eyes is brown because the
gene B for brown colour of eyes, being dominant, expresses itself and the gene b for
blue colour of eyes, being recessive, does not expresses itself.
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Homozygous (Pure) and Heterozygous (hybrid) organisms.
In an individual the two allelic genes of a character may be similar, in which
case both of them strand for the same alternative character, or dissimilar, in which
case they represent both the alternatives of a character. The individuals with these
conditions are referred to by different terms.
Homozygous (Pure) organism.
An organism on which both the allelic genes of a character are identical is called
homozygous or genetically pure for that character.
Example: The human being with BB and bb gene pairs is homozygous for colour
of eyes. The combination of BB genes is homozygous dominant, and the combination
of bb genes, homozygous recessive.
Heterozygous (Hybrid) organism.
An organism in which the two allelic genes of a character are unlike is said to be
heterozygous or hybrid for that character (tab. 1.1.).
Example: the human being with Bb gene pairs is heterozygous for colour of
eyes. In heterozygote (heterozygous individual), one allele codes for dominant trait
and the other for recessive trait, and usually the dominant allele is expressed and the
recessive allele remains latent.
An organism may be homozygous for the certain traits or gene pairs and hetero-
zygous for the other traits or gene pairs.
Table 1.1.
The differences between homozygous and heterozygous individuals.
Homozygous individuals Heterozygous individuals
1. Have similar alleles for a specific 1. Have dissimilar alleles for a spe-
trait (BB or bb); cific trait (Bb);
2. Produce only one kind of gametes 2. Produce two kinds of gametes
(B, B or b,b); (B, b);
3. Breed true for a specific trait, i.e. 3. Do not breed true for a specific
produce offspring with similar gen- trait, i.e. produce offspring with
otype and phenotype on inbreeding; three genotypes and two or tree
phenotypes on inbreeding;

Homozygousity is known as a property of the organism, developed from a zy-

gote formed by the merging of two gametes similar according to their hereditary fea-
tures.
Heterozygousity - is a property of the organism formed by the merging of two
gametes different according to their hereditary features.
Hemizygousity -is a condition of the organism connected with the existence of
one or two unpaired genes, i.e. these genes have no allelic partners.
Hemizygousity. This is a condition of the organism connected with the exist-
ence of one or two unpaired genes, i.e. these genes have no allelic partners.
Genotype and phenotype. The concepts of genotype and phenotype were for-
mulated by Johansson in 1909 to distinguish the genetic traits from the expressed
 7
traits.
Genotype of an individual refers to the sum total of genes inherited from both
the parents irrespective of whether they are expressed or not (tab. 1.2.).
Example: BB, bb, Bb are genotypes for the colour of eyes.
Phenotype of an individual refers to the expressed or observable structural and
functional traits produced by the interaction of genes and environment (tab. 1.2.).
Example: brown colour of eyes in human being, tallness in garden pea plant.
Individuals of the same genotype have the same phenotype and breed alike. In-
dividuals of different genotypes may have the same phenotype, e.g. the genotypes BB
and Bb dive rise to brown colour of eyes; may have the different phenotypes, e.g. the
genotypes BB and bb give rise to brown and blue colour of eyes respectively.
Individuals of the same phenotypes may or may not breed alike. Human being
with the phenotype brown colour of eyes may have genotype BB or Bb, and will
breed accordingly.
Table 1.2.
The differences between the genotype and phenotype.
Phenotype Genotype
1. It is the expression of a trait. 1. It is the gene equipment that deter-
mines a trait, e.g. BB or Bb for a
brown colour of eyes.
2. It can be noted by direct observa- 2. It can be ascertained from the an-
tion of an individual. cestry or progeny of an individual.

3. In individuals having similar 3. In individuals having similar geno-

phenotype may or may not have type develop similar phenotype in the
similar genotype. same environment.

4. Individuals having different phe- 4. Individuals having different geno-

notypes have different genotypes. types may have the same phenotype,
e.g. genotypes BB and Bb produce
brown colour of eyes.

Genome. A complete set of genes (DNA instructions) contained in the haploid

dose of chromosomes and inherited as a unit from one parent is known as a genome.
A haploid cell contains a single genome. A diploid cell contains two genomes,
one parental, other maternal. A cell may have more than two genomes also, such cells
are called polyploid.
Gene pool. All the genotypes of all the individuals in a population make up the
gene pool.
Inheritance. This is the process of transmission of distinctive features from
generation to generation. There are autosomic, autosomo-dominant, autosomo-
recessive (dominant, recessive), cytoplasmic inheritance, inheritance related to sex
and others.
Hereditability. This is the degree of phenotypic variability depending on ge-
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netic distinctions between individuals.

CHAPTER 2. MENDELIAN GENETICS.

In 1866 Gregor Mendel published the results of experiments in his paper «Ex-
periments in plants hybridization», in which he had investigated inheritance in garden
peas. From these findings he discovered the existence of discrete hereditary elements
and the principles and analytic rules determining their transmission from parents to
offspring. The principles and rules of heredity that Mendel recognized ultimately be-
came major factor in the development of Modern biology.

2.1. Gregor Mendel.

Gregor Johann Mendel (Austrian, 1822-1884) was born in a peasant family. He
received school education mainly by his awn effort. Due to poverty, he becomes a
monk (religious man). He was later made an abbot (head) of Augustinian monastery
(religious place) at Brunn, Austria (now Brno in Czechia). Here he also worked as a
teacher of physics, mathematics and natural history in a local school. However, the
study of natural phenomenon, especially inheritance, was his main interest. From
1856 to 1865, Mendel conducted breeding experiments on garden peas in the garden
of his monastery. He was not the first to experiment in the field of inheritance, but
was luckily the first to formulate clear-cut laws of heredity. The principal difference
between Mendel’s approach and that of others scientists who were interested inher-
itance is that he thought in quantitative terms. He preceded by starting simple ques-
tions to be answered, by experiments, and then looking for statistical regulations that
might identify general rules.
Finally he published his findings in a Biological Journal in which he explained:
1) the unit characters, as the factors of inheritance,
2) existence of them in alternate forms called alleles,
3) their transmission through gametes as pure forms,
4) their segregation in equal ratios,
5) their dominance' over recessive traits,
6) independent but random assortment of factors,
7) their inheritance in a regular pattern in successive generation in set laws.
But later lie drifted towards other experimental materials like Hieralcium (a
composite member) and others. Unfortunately the materials were unsuitable and the
results were quite contrary to the previous findings. He could not get any where. He
started to doubt about his own findings and he was a distraught man. He was over-
burdened; he over worked; in this kind of stress and strains he passed away as any
other mortal.
His laws form the basis of the science of genetics even today, and he is remem-
bered as the father of genetics. Unfortunately, the great significance of Mendel’s dis-
covery was not appreciated by contemporary biologists, because:
1) his work was ahead of his time;
2) he published it in an obscure (unknown imperceptible) journal;
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3) he was himself not sure of his findings;
4) mind of the biologists was pre-occupied with the Darwin’s theory of evolu-
tion.
Mendel died in 1884 without knowing that he had the foundation of modern ge-
netics. In 1900, however, his results were independently rediscovered by three biolo-
gists, namely De Vries of Netherlands, Carl Correns of Germany and Erichvon
Tshermak of Austria in 1900. These scientists deserve all the credits for naming them
after Mendel, and for brining them to the notice of the biologists. Mendel’s work was
got republished by De Vries in Flora in 1901. A few years later, W. Bateson and oth-
ers confirmed Mendel’s work and found that the same laws applied to animals also.
Experiments with garden pea plants have made Mendel to lay down some princi-
ples of inheritance, which are now called as Mendel’s Laws of inheritance:
1. Law of unit characters.
2. Law of Purity of gametes.
3. Law of dominance.
4. Law of Segregation.
5. Law of Independent Assortment.

2.2. Mendel’s experimental plant.

Just as geneticists do today, Mendel chooses the organism for his experiments
very carefully. The garden pea, Pisum Sativum, had a number of advantages. These
are given below:
1). Pea plants are easy to grow and to cultivate.
2). Pea plant has a short life-cycle so that results can be had within a year.
3). Controlled pollinations.
Cross-pollination (hybridization) can be easily achieved by removing the sta-
mens (emasculation) before the pollen grains mature and dusting the pistil of this
flower (female parent) with the pollen from a desired plant (male parent).
4). Garden peas were found to differ in certain definitive and easily detectable
traits. Mendel noted which always appear in two opposing conditions, one dominant
and other recessive are called the contrasting traits. Pea plants pure for each of the
seven characters he selected was available.
5). The traits of each kind of pea plant were preserved in generation after gener-
ation because the plants had bisexual flowers to self-pollination. Complete enclosure
of reproductive parts by petal ensures self-pollination.
6). It produces many seeds in one generation. This helps in drawing correct con-
clusions.
7). It can be raised, maintained and handled.

2.3. Mendel's experiments and his principles of inheritance.

The characteristics Mendel studied were constant in inheritance and at the be-
ginning of his experiments he established true-breeding lines. He studied seven char-
acters of pea plant. Firstly, Mendel made the monohybrid cross with each pair of the
characters.
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Monohybrid cross – is the cross between the organisms which differed by one
character.
For example. One pair of characteristics that he studied was yellow versus green
seeds. When pollen from plants of a line with green seeds was used to cross-pollinate
plants from a yellow-seeds line, all of the hybrid seeds produced were yellow (abbre-
viated as F1, for first filial generation).
The F1 hybrid plants were then allowed to self-pollinate. The progeny plants are
called the F2 (second filial generation). Seeds of these plants were like both of the
original parental types - that is, yellow and green. Mendel counted 5474 F2 seeds that
were yellow and 1850 that were green and noted that this ratio was approximately
3:1.

Parental lines: yellow seeds x green seeds

F1: all yellow

Parental lines: yellow seeds x yellow seeds

F2: 3 yellow : 1 green

He used the symbols for the explanation of his results. The dominant character he
indicated by the capital latter (A), the recessive character – be the small letter (a).
The symbol of female genotype is ♀, the symbol of male genotype is ♂, the
symbol of cross – x. Parental lines – capital letter P, the gametes – G, the first filial
generation – F1, the second filial generation – F2.

P (Parents): ♂ AA x ♀ aa
mark of male mark of female
individual individual
MEIOSIS
G (Reproductive A a
Cells):

F1 (Zygotes): Aa

P1: ♂ Aa x ♀ Aa

G: a A A a

F2: AA : Aa : Aa : aa
homo- hetero- homozygous

Similar results were obtained when Mendel crossed plants of six other pairs of al-
ternative characteristics. Mendel concluded that the traits from the parental lines were
transmitted as two different elements of a particulate nature that retained their purity
in the hybrids. We called the element associated with the trait seen in the hybrids
 11
(yellow seeds, used in the example) dominant, and the other element, associated with
the trait not seen in the hybrids but seen in their progeny (green seeds), recessive.
Mendel's conclusions were reinforced by the results of experiments in which F 3
progeny produced by the self-pollination of individual F2 plants were observed. In the
experiment with yellow versus green, for example, F2 plants grown from green seeds
produced only green F3 seeds. When 565 F2 plants were grown from yellow seeds,
193 of them produced yellow seeds but the other 372 plants produced both yellow
and green seeds in a proportion very close to 3 to 1 the results can be diagrammed
as follows:
F 2 yellow plants (565 were tasted):
1/3 (193) - gave plants producing only yellow F3 seeds
2/3 (or 372) - gave plants producing both yellow and green seeds;
- 3/4 were with yellow seeds;
- 1/4 were with green seeds;
Phenotypic ratio F3 seeds is 3:1

F 2 green plants:
All plants with - all gave plants producing only green F 3 seeds
green seeds

The ratio 1:2:1 was observed each time in progeny from F 42 plants. Mendel
formulated the following simple explanation of the 1:2:1 ratio in F2:
1. A pea plant has two hereditary determinants for each observed trait.
2. Each reproductive cell (gamete) of a plant has only one of the two determi-
nants. The two determinants of the pair occur with equal frequencies in the reproduc-
tive cells.
3. The union of male and female reproductive cells in the formation of new zy-
gotes is a random process. If is shown in the figure. We use the symbols A and a to
represent the dominant and recessive determinants.
Using the results of the experiments with pea plants, differed by one pair of al-
ternative characters, Gregor Mendel postulated the principles (laws) of inheritance.

1. Law of unit characters.

Every organism has certain distinct morphological as well as physiological char-
acters. These characters are controlled by specific units of heredity. These were
termed as factors, now they are called as Genes. Genes exist in two alternate forms
called alleles. Now it is known that the genes are located in chromosomes. For exam-
ple, the color of the flower: pea plants produce yellow seeds and some green seeds.
So the genes responsible for producing yellow color and green color must be differ-
ent. As both the genes control the same character namely the color of the seeds, they
are called alleles or allelomorphs.

2. Law of Purity of gametes: During the formation of gametes the paired char-
 12
acteristics (genes) separate and segregate at random so that each gamete re-
cessive one or other characteristics (alleles).
The unit characters or hereditary units are transmitted to their off springs
through gametes. During reproduction parents produce gametes which contain all the
genes for all the characters of an organism. However, these genes are present in sin-
gle doses. For example-pea plant may have hundreds of morphological and physio-
logical characters. Among them, if we choose one character, then it is controlled by a
particular pair of allelic genes. During reproduction only one gene enters into one
gamete, thus gametes contain only one gene for this character. Thus the gamete is
considered as pure and uncontaminated with respect to the said character. The same
principle holds good for other characters also.
3. The Principle of Uniformity (Principle of Dominance): The hybrids of
cross between the pure line organisms are uniformity in genotype and phenotype.
4. The Principle of Segregation: The segregation of characters of F2 hybrids
in the complete dominant inheritance is occurred in the definite quantitative propor-
tions.
Cytological basis of the Principle of Uniformity.
Characters:
A – yellow colour of seeds;
a - green color of seeds;

P: ♂ AA x ♀ aa
Yellow Green

G: A a

F1: Aa
100% yellow

Cytological basis of the Principle of Segregation.

Characters:
A – yellow colour of seeds;
a - green color of seeds;

P1: ♂ Aa x ♀ Aa
yellow yellow

G: a A A a

F2: AA : Aa : Aa : aa
Genotypic ratio: 1 2 1
Phenotypic ratio: 3 yellow : 1 green
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Dihybrid cross – is the cross between the organisms which differed by two char-
acters.
After the monohybrid cross, Mendel crossed the parental plants that differed in
two or three pairs of alleles with different characteristics. For example, plants from a
true-breeding line having round seeds which were yellow were crossed with plants
with wrinkled green seeds. The F1 seeds from this cross were round and yellow, so
they were hybrids for either characters, or dihybrid. This phenotype was expected
from the results of the individual monohybrid crosses. Round seed shape and yellow
color were dominant. The F2 seeds had four possible combinations of phenotypic
characteristics:
9/16 round, yellow 3/16 wrinkled, yellow
3/16 round, green 1/16 wrinkled, green

Mendel also noticed that four phenotypes in the F2 from the dihybrid cross oc-
curred approximately in the proportions 9/16 round and yellow; 3/16 wrinkled and
yellow; 3/16 round and green; 1/16 wrinkled and green. Similar 9:3:3:1 ratio of F2
phenotypes were found in the progeny of other dihybrid crosses.
So, Mendel formulated The Principle of Independent Assortment: Inher-
itance of pairs of characters located in the different chromosomes is independently
from each other.
Cytological basis of the Principle of Independent Assortment .

round, yellow wrinkled, green

Parents: ♀ AABB x ♂ aabb

Gametes: AB ab

F1 progeny: AaBb
round, yellow

Parents: ♀ AaBb x ♂ AaBb

AB Ab aB ab AB Ab aB ab
F2:

♂ AB Ab aB ab
♀
AB AABB AABb AaBB AaBb
Ab AABb Aabb AaBa Aabb
aB AaBB AaBb aaBB aaBb
ab AaBb Aabb aaBb aabb
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F2 progeny: Genotypes Phenotypes
1/16 AABB + 2/16 AABb +
2/16 AaBB + 4/16 AaBb = 9/16 round, yellow

1/16 aaBB + 2/16 aaBb = 3/16 wrinkled, yellow

1/16 AAbb + 2/16 Aabb = 3/16 round, green
1/16 aabb = 1/16 wrinkled, green
The format used to show which combinations of F1 female and male gam-
etes produce F2 genotypes is called a Punnet square.

2.4. Test Cross and Back Cross.

Test cross is a simple method devised by Mendel to verify the genotype of the F 1
hybrid.
When the F1 hybrid with a dominant character is crossed with the homozygous
recessive parent, it is called a test cross. The characters of a test cross are:
1. It involves crossing the individual to a true-breeding recessive (homozygous
recessive).
2. If the unknown is heterozygous, approximately half the progeny will have the
dominant trait and half the recessive trait.
3. If the unknown is homozygous dominant, all the progeny will have the domi-
nant trait
Test cross is also used for checking the correctness of Mendel’s law of segrega-
tion (using a monohybrid test cross) and the law of independent assortment of charac-
ters (using a dihybrid test cross). For example,
(1) Monohybrid test cross: In a monohybrid cross of Tall x Dwarf, the F1 are all
tall (Fig 2.1). Let us see what happens when this F1tall is test crossed with the homo-
zygous recessive parent i.e. Dwarf with (tt) genotype.
We aim to check and verify two things:
a) determine the genotype of F1 Tall and;
b) check the correctness of the law of segregation.
Thus, F1 tall is back crossed (test
crossed) with the homozygous recessive
dwarf parent and the progeny of the
cross examined. We know that the re-
cessive dwarf with (tt) genotype will
produce only one type of gametes (i.e.,
all with (t) only). However, as regards
the F1 tall, there can be two possibili-
Fig. 2.1. Test Cross in monohybrid cross.
ties:
If the F1 tall is homozygous with (TT) genotype, it shall produce only one type of
gametes (i.e. all with (T) only). As a result, the progeny of the cross should be all tall.
(T) x (t) = (Tt) Tall.
If, on the other hand, the F1 tall is heterozygous with (Tt) genotype and, if Men-
del's law of segregation of characters is correct, then the F1 should produce two types
 15
of gametes, (T) and (t) in equal proportion. The recessive dwarf parent produces only
(T) type of gametes. The resulting test cross progeny should be 50% Tall and 50%
Dwarf or in 1:1 ratio as follows: The results obtained in the actual test cross experi-
ments completely agree with the theoretical expectations. Thus it is proved that F 1
Tall is a heterozygous dominant (monohybrid) with (Tt) genotype and that the alleles
segregate during gamete formation.
(2) Dihybrid test cross: In the dihybrid cross of yellow round X green wrinkled
the F1, dihybrid is a double heterozygous yellow round with (YyRr) genotype. It is
test crossed with the double homozygous recessive parent, (i.e. green round with
(yyrr) genotype). We aim to test the correct-
ness of Mendel's law of independent assort-
ment of characters (fig. 2.2).
If the principle of independent assortment
is correct, then as Mendel explained, the
(YyRr) dihybrid should produce four types of
gametes: YR, Yr, yR and yr in equal propor-
tions. The recessive parent (yyrr) shall pro-
duce only one type of gametes i.e. yr type. It is
expected that the maximum possible chance Fig. 2.2. Test Cross in dihybrid cross.
combinations between these gametes should
produce four kinds of phenotypes in the ratio of 1:1:1:1 as follows. The actual num-
bers of these four types of offspring obtained in one of Mendel's test crosses were:
Yellow round = 55, Yellow Wrinkled = 49, Green Round = 51 and Green Wrinkled =
52. This gives an approximate 1:1:1:1 ratio as per the theoretical expectations and
thus, confirms the law of independent assortment of characters.
In such a cross both the genotypes and their relative frequencies in the gametes
produced by heterozygote are known directly from examination of the phenotypes of
the progeny, because one parent contributes only recessive allele.
Another type of cross is back cross, a cross between a hybrid and an individual
with the same genotype as one or the other of its parents. Backcrosses are commonly
used by geneticists and by plant and animal breeders.
Significance of back cross in plant breeding:
1. It is a rapid method of improving a variety of crop.
2. It is an easier and quicker method of obtaining a desirable trait in a pure ho-
mozygous condition (true breeding trait).
3. Back cross is also use frequently in hybridization experiments because of its
simple ratio. Moreover, this needs the analysis of fewer progeny.

2.5. Mendelian characters in a human being.

A mendelian trait is one that is controlled by a single gene (monogenetic) how
simple Mendelian inheritance pattern. In such cases, a mutation in a single gene can
cause a disease that is inherited according to Mendel's laws. Examples include sickle-
cell anemia, Tay-Sachs disease, cystic fibrosis and xeroderma pigmentosa. A disease
controlled by a single gene contrasts with a multifactorial disease, like arthritis,
 16
which is affected by several loci (and the environment) as well as those diseases
inherited in a non-Mendelian fashion. The mendelian inheritance in man database is a
catalog of, among other things, genes in which Mendelian traits cause disease, some
of them in the table 2.1.
Table 2.1
Mendelian traits in a human being
№ Signs Dominant Recessive
1 Eyes large small
2 Eye colour brown blue
3 Shape of eyes straight squint
4 Type of eyes Mongolian European
5 Sight short-sighted Normal
6 Eyelid epicanthus normal
7 Ears wide narrow
8 Chin long short
9 Gap between incisors there is there is not
10 Hair curly simple
11 Grey hair at the age of 25 years after the age of 40 years
12 Balding In men In women
13 Growth of hair in middle line there is there is not
of forehead
14 Shaggy eyebrows there is there is not
15 Lower lip thick and hangig normal
down
16 Ability to turn the tong back there is there is not
17 Ability to roll the tong up there is there is not
18 Tooth in newborns there are there are not
19 Skin thick thin
20 Skin colour dark white
21 Freckles there are there are not
22 Hand with 6 or 7 fingers with 5 fingers
23 Prevaling hand right left
24 Antigens of AB0 system A, B 0
25 Voice (in women) soprano alto
26 Voice (in men) bass tenor
27 Absolutely ear for music there there is not
28 Hereditary deafness there is not there is
 17
2.6. Statistics in Genetics (mono-, di - and polyhybrid crosses).
Many testable questions can be posed with respect to inheritance of mono-, di-
and polyhybrid crosses. To answer this type of question (and others), geneticists use
the scientific method. And major tools used in the scientific method are the common
laws of Probability and Statistics (tab. 2.2).
Table 2.2
The Statistics in mono-, di- and polyhibrid crosses.
TYPE OF CROSS
CHARACTERS monohybrid dihybrid polyhybrid
2
Number of kinds of gametes formed by F1 2 2 2n
Number of genotypes in F2 3 32 3n
Number of phenotypes in F2 2 22 2n
2
Segregation in phenotype in F2 3+1 (3+1) (3+1)n
Segregation in genotypes in F2 1+2+1 (1+2+1)2 (1+2+1)n
*n – number of allelic pairs
 18
CHAPTER 3. THE MAIN TYPES OF INHERITANCE.
There are monogenic and polygenic inheritances. Monogenic inheritance is di-
vided into autosomic and six-linked inheritances which are subdivided into dominant
and recessive.
These types correspond to the rules of inheritance of separate Mendelian charac-
ters (fig. 3.1).

Fig. 3.1. The types of inheritance.

3.1. Autosomal dominant type of inheritance.

Autosomal dominant type of inheritance is conditioned by transmission of dom-
inant gene allele in generation, becoming localized in autosomes. It has some traits,
showing itself in pedigrees (fig. 3.2):
1) Characteristic is found in every generation.
2) It is inherited by approximately one half of children;
3) Both males and females can be affected equally and the condition can be in-
herited from either parent (fig. 3.3).
4) Both parents transfer to children
equally.
5) There is no such thing as "skipping"
people (or generations).
6) Multiple generations may be affected
in a family.
7) Some people are the first one to be af-
fected in their family, as a result of a new ("de
novo"), sporadic mutation in the germ cells
(egg and sperm cells).

Risks to children:
1. When only one parent is affected, Fig. 3.2. Autosomal dominant type of
every child he/she has a 50% chance to be af- inheritance.
fected and a 50% chance to be unaffected.
 19
2. When both parents are affected, every child they have has a 75% chance to
be affected, and a 25% chance to not be affected. Of note, in some conditions having
two damaged copies of the gene is too severe and may not be compatible with life.
Examples of disorders with autosomo-dominant type of inheritance.
- Antithrombin III deficiency;
- Familial
hypercholesterolaemia;
- Huntington's disease;
- Myotonic dystrophy;
- Neurofibromatosis Type I
(von Recklinghausen's disease)
- Osteogenesis imperfecta;
- Achondroplasia;
- Brachydactyly;
- Hypertrichosis; Fig. 3.3. Pedigree, analyzing the Autosomal
- Skin aplasia; dominant type of inheritance.
- Marfan syndrome.

3.2. Autosomal recessive type of inheritance.

Autosomal recessive type of inheritance is conditioned by transmission of reces-
sive gene allele in gene ratios, becoming localized in autosomes (fig. 3.4, fig. 3.5).
The typical traits of this type are:
1) The characteristic may be absent in genera-
tion of children, but appear in generation of grand-
children;
2) The characteristic may develop in children in
its absence in both parents; among children of those
familiar is found in 25% of cases.
3) The characteristic is inherited by all children
if it is found in both parents;
4) It is developed in 50% in children if it is
found in one parent;
5) Characteristic is inherited by male and fe-
male progeny equally often.
Fig. 3.4. Autosomal recessive type.
Risks to children:
1. When both parents are carriers, every child
they have has a 25% chance of being affected, a 50% chance to be a carrier, and a
25% to neither be affected nor a carrier.
2. When one parent is a carrier and the other is not a carrier or affected, every
child they have has a 50% chance to be a carrier and a 50% chance to neither be a
carrier nor affected. No child will be affected.
3. When one parent is affected, and the other parent is a carrier, every child
they have has a 50% chance to be affected and a 50% chance to be a carrier.
 20
4. When one parent is affected and the other is not a carrier or affected, every
child they have will be a carrier. No child will be affected.

Examples of disorders with autosomo-recessive type of inheritance.

- á1-antitrypsin deficiency;
- Congenital adrenal hyperplasia due to 21-hydroxylasedeficiency;
- Cystic fibrosis;
- Growth hormone deficiency (rare
familial form);
- Phenylketonuria;
- Tay-Sachs disease;
- Albinism;
- Anophthalmia;
- Holoprosencephaly; Fig. 3.5. Pedigree, analyzing Autosomal reces-
- Xeroderma pigmentosum. sive type of inheritance.

The specialties of sex-linked inheritance are connected with localization of

genes in sex chromosomes: X and Y. Y-linked inheritance is also called holandric
inheritance, which is differed from other types by direct transmission of character-
istic from father to all sons. It is found in every generation in progeny of males.

3.3. Y-linked type of inheritance.

Y-linked or holandric inheritance (fig. 3.6, fig. 3.7) implies that only males are
affected and that an affected male transmits the trait to all his sons but to none of his
daughters. Y-linked conditions typically include the following characteristics:
1. No females are ever affected.
2. There is no such thing as "skipping" generations.
3. All males with a damaged copy of the gene
are affected, as there is no second copy.

Fig. 3.7. Y-linked type of inheritance.

Fig. 3.6. Pedigree, analyzing Y-linked type of inheritance.
In the past it has
 21
been suggested that such bizarre-sounding conditions as porcupine skin, hairy ears
and webbed toes are Y-linked traits. With the possible exception of hairy ears, these
claims of holandric inheritance have not stood up to more careful study. Recent evi-
dence clearly indicates, however, that the H-Y histocompatibility antigen (it seems to
play little part in transplantation in humans. It seems to be important for testicular dif-
ferentiation and function, its expression does not, however, necessarily correlate with
the presence or absence of testicular tissue) and genes involved in spermatogenesis
are carried on the Y chromosome and therefore manifest holandric inheritance. The
family surname also shows this pattern of inheritance!
Risks to children:
When a male is affected, every male child he has will be affected. No female
child will ever be affected.

3.4. X-linked type of inheritance.

And now some specialties of X-linked inheritance which is depended on
hemizygousity of locus in man and on func-
tional and genetic inaction of one of X-
chromosomes in woman. Genes carried on the
X chromosome are referred to as being X-
linked.

3.4.1. X-linked recessive type of inheritance.

Females have two X chromosomes, while
males have one X and one Y. X-linked reces-
sive conditions are caused by genes that are lo-
cated on the X chromosome. In a male, the
condition is expressed in everyone with one
damaged copy of the gene, as males do not
have a second gene to compensate. In females,
the condition is expressed in everyone with two Fig. 3.8. X-linked recessive type of
damaged copies of the gene, while everyone inheritance.
with one damaged copy is a carrier. X-linked
recessive conditions typically include the following characteristics:
1. As it is rare for females to have two damaged copies, far more males are af-
fected than females.
2. Carrier females usually do not have any symptoms of the condition, but there
are some conditions in which a carrier female may present with a milder expression
(or degree) of symptoms.
3. Affected males often born to unaffected, carrier mothers.
4. In some cases, a male is affected as result of a new ("de novo"), sporadic mu-
tation.
 22
Risks to children (fig. 3.8, 3.9):
1. When a female is a carrier and the male is unaffected, every male child they
have has a 50% chance of being affected and a
50% chance to not be affected; while every fe-
male childe they have has a 50% chance to be a
carrier and a 50% to neither be affected nor a car-
rier.
2. When a male is affected and the female is
not a carrier, every male child they have will not
be affected, while every female child born will be
a carrier. These conditions are never passed down
from father to son.
3. When a male is affected and the female is
a carrier, every male child they have has a 50%
chance to be affected and a have a 50% chance to
not be affected; while every female they have has Fig. 3.9. X-linked recessive type of
a 50% chance to be affected and a 50% chance to inheritance.
be a carrier.

Examples of disorders with X-linked recessive type of inheritance.

- Haemophilia A and haemophilia B;
- Color blindness;
- Aarskog syndrome;
- Agammaglobulinemia X-linked infantile;
- Hydrocephaly;
- Granulomatous disease X-related, chronic;
- Diabetes insipidus nephrogenic.

Fig. 3.10. X-linked recessive type of

inheritance.

3.4.2. X-linked dominant type of inheritance.

Females have two X chromosomes, while males have one X and one Y. X-linked
dominant conditions are caused by genes that are located on the X chromosome. X-
linked dominant conditions typically include the following characteristics (fig. 3.11):
1. Both males and females can be affected.
2. There is no such thing as "skipping" people (or generations).
3. Multiple generations may be affected in a family.
4. Male often has more severe (sometimes lethal) symptoms, as they do not have
a second X to compensate.
5. In some cases, a person is affected as result of a new ("de novo") sporadic mu-
tation.
6. Women inherit it more often than men;
 23
7. If, a woman have it, all children or one half of then inherit it;
8. If, a man has it, it inherited by all children of female sex, but none of his sons.

Fig. 3.11. X-linked dominant type of inheritance.

Risks to children (fig. 3.12):

1. When a female is affected and the man is not affected, every child (both male
and female) they have has a 50% chance of being affected and a 50% chance to be
unaffected.
2. When a man is affected and the woman is not affected, every male child they
have will be unaffected, while every female child they have will be affected. These
conditions are never passed down from father to son.
3. When a females is affected and the man is affected, every male child they
have has a 50% chance to be affected, while every female child they have will be af-
fected.

Examples of disorders with X-linked

dominant type of inheritance.
- Hypophosphatemia (vitamin D-
resistant rachitis);
- Focal dermal hypoplasia;
- Coffin-Lowry syndrome.

Fig. 3.12. X-linked recessive type of inheritance.

 24
3.5. The types of correlative inheritance.
Independent inheritance differs by accident combination of parent’s signs in
offsprings. It is characteristic for signs which are controlled by genes of
nonhomologous chromosomes and genes which are localized in one chromosome in
the distance more than 50 morganids. For example, in human beings: ability of taste
feeling of phenylketonuria and secretion of erythrocyte antigens of blood group sys-
tem AB0 into saliva are inherited independently.
In complete-linked inheritance corresponding signs of parents are developed in
offsprings always as one block. Signs controlling by genes of one chromosome,
which are located so closely that this exclude crossing over, are inherited in such
way. For example, in human beings: synthesis of polypeptides of á- and â-
hemoglobin.
In incomplete inheritance corresponding signs of parents are reproduced in part
of offsprings jointly and in other part - independently. Such inheritance may be ob-
served in the genes of one linkage group, distance between which in the chromosome
assumes regular crossing over. For example, in human being: genes controlling secre-
tion of antigens of blood group system AB0 and system Lutheran are located in
one chromosome in the distance of 15 morganids. These signs are inherited as partly
linked type of inheritance.

Fig. 3.13. The types of correlative inheritance.

A - INDEPENDENT, B - COMPLETE, C - INCOMPLETE LINKED INHER-
ITANCE OF SIGNS A AND B;
1 - genes of this signs are localized in nonhomologous (a) chromosomes or in the different
distance of one chromosome (b, c); 2 - frequency of gametes formation with different compo-
sition of alleles (in per cents); 3 - testcross with specimen, forming one type of gametes; 4 -
correlation of off-springs with different genotypes, being born in the result of testcross.
 25

CHAPTER 4. THE CHROMOSOMAL INHERITANCE.

4.1. The development of ideas about the Chromosome Theory.

Scientists were able to identify chromosomes under the microscope as early as
the 19th century. The second half of the nineteenth century was a time of remarkable
advances in genetic research. As early as the 1860s, Gregor Mendel and Charles
Darwin began to explore possible mechanisms of heredity. Then, over the next few
decades, Walther Flemming, Theodor Boveri, and Walter Sutton made a series of
significant discoveries involving chromosomes, including what these structures
looked like, how they moved during mitosis, and what role they likely played in the
transmission of genetic characteristics. However, it wasn't until the work of Thomas
Hunt Morgan in the early twentieth century that researchers were finally able to di-
rectly link the inheritance of genetic traits to the behavior of chromosomes, thereby
providing concrete evidence for what became known as the chromosome theory of
heredity.

The year 1865 was marked by two profound biological breakthroughs: Mendel's
publication of his experiments in plant hybridization, and Darwin's provisional hy-
pothesis of pangenesis. Although neither man had any direct evidence for the theo-
ries he proposed, Mendel speculated that cells contained some type of factor that car-
ried traits from one generation to the next, while Darwin proposed that traits could be
passed down via units he termed "gemmules," which he believed traveled from every
body part to the sexual organs, where they were stored (Benson, 2001). Because these
first attempts to explain the mechanisms of heredity lacked any scientific support,
their profound importance went unrecognized by the scientific community for dec-
ades. Nonetheless, Mendel and Darwin's work laid the foundation for formulating a
testable, research-based theory of heredity.

4.1.1. Flemming, Boveri, and Sutton connect Chromosomes to Heredity.

Meanwhile, several independent advances-namely, discovery of chromosomes
and their behavior during mitosis as described by, and discovery of the link between
chromosomes and heredity as described by Theodor Boveri-began to shed light on
the events that take place in the nucleus during cell division and their implications.
Then, some thirty-five years later, the significance of Mendel's work was emphasized
by Walter Sutton, whose observations of chromosome behavior during cell division
and gamete formation were consistent with Mendel's findings. Thus, the basis for
chromosome theory, and the field of cytogenetics, was created.
Walther Flemming Describes Chromosomes. By the middle of the nineteenth
century, scientists understood that cells derived from other cells and that the heredi-
tary information was located in the nucleus, but the physical nature of the hereditary
material remained unknown. The microscopes of the time provided very poor resolu-
tion of living cellular structures, making it necessary for investigators to treat fixed
cells with various stains to enhance the contrast of their contents. Thus, using innova-
 26
tive microscopy techniques and painstaking precision, German anatomist Walther
Flemming recognized and explored the fibrous network within the nucleus, which he
termed chromatin, or "stainable material." (Flemming had actually discovered the
chromosome, although the term would be coined a few years later by Heinrich
Waldeyer.) Flemming noted that during cell division, the chromatin formed thread-
like bodies, which he termed mitosen, from the Greek word for thread. Based on
many observations of cells in various stages of division, Flemming correctly deduced
the sequence of chromosome movements during mitosis, which would be confirmed
decades later by microscopy of live dividing cells. With his characteristic attention to
detail, Flemming also made the important observation that chromosomes split along
their length during mitosis, and he correctly hypothesized that the split chromosomes
were partitioned into different daughter cells at the end of mitosis.
Thus, Flemming recognized that chromosomal movement during mitosis offered
a mechanism for the precise distribution of nuclear material during cell division. His
work provided an invaluable description of the initial mechanisms underlying the
process of cell division, and it helped paved the way for the discovery of hereditary
mechanisms.

Theodor Boveri Links Chromosomes and Heredity. The end of the nineteenth
century was marked by advancements in cytological techniques and microscopy.
During this period, German embryologist Theodor Boveri took Flemming's findings
to the next level by providing the first evidence that chromosomes of germ cell line-
age provide continuity between generations. Boveri found evidence for this hypothe-
sis through his research of early development in the roundworm species Ascaris
megalocephala (now known as Parascaris equorum). Ascaris embryos provided an
excellent experimental model for Boveri's observations; this is because the large,
clear cells of the Ascaris embryo have only two pairs of chromosomes, and the em-
bryos develop distinct somatic cell and germ cell lineages during the first few cleav-
age divisions. Boveri was thus able to trace the fate of the chromosomes in individual
cell lineages with great precision, and he made the surprising observation that the full
complement of chromosomes was retained only in the Ascaris germ cell lineage, the
source of future gametes. By contrast, the chromosomes in somatic cells, the source
of all other adult tissues, underwent a curious process of fragmentation and elimina-
tion known as chromosome diminution (chromosome diminution does not occur in
mammals).
Boveri also recognized that chromosome number was reduced in the gametes. In
Ascaris does not occur until fertilization is complete eggs, meiosis, so Boveri was
able to observe the behavior of egg and sperm chromosomes following fertilization.
He noted that Ascaris eggs retained only two chromosomes after the polar body
formed, and that the normal number of four chromosomes was restored following fu-
sion of the sperm and egg pronuclei. Boveri's work provided one of the first descrip-
tions of meiosis.
 27
Walter Sutton Finds Evidence for Mendel's Principles. Working independently
near the dawn of the twentieth century, American graduate student Walter Sutton
confirmed and expanded upon Boveri's observations using a superior cytological
model. Sutton, a Kansas farm boy, had made the serendipitous discovery that it was
possible to distinguish individual chromosomes in cells undergoing meiosis in the
testes of the lubber grasshopper, Brachystola magna. Thus, in his classic paper pub-
lished in 1902, Sutton described the configurations of individual chromosomes in
cells at various stages of meiosis. Sutton was able to identify 11 pairs of chromo-
somes that could be distinguished by their sizes, as well as an accessory singleton
that he correctly presumed to be a sex chromosome. Due to its apparent lack of a
partner, this chromosome sorted into only half of the sperm cells, while the remaining
half never received a copy. Though the accessory chromosome was unpaired, it still
replicated and entered stages of mitosis in the same manner as all other chromo-
somes, which prompted Sutton to declare it a "true chromosome" and not simply an
accessory.
Sutton postulated that all chromosomes have a stable structure, or "individuali-
ty," that is maintained between generations, and he used this property to follow the
behavior of individual chromosomes through the various stages of meiosis, including
synapsis. Most notably, Sutton recognized that his observations were consistent with
the, whose findings had only recently been rediscovered. In fact, Sutton closed his
1902 paper with the statement, "I may finally call attention to the probability that the
association of paternal and maternal chromosomes in pairs and their subsequent sepa-
ration during the reducing division as indicated above may constitute the physical ba-
sis of the Mendelian law of heredity." With these words, Sutton articulated the chro-
mosomal theory of inheritance.
Sutton subsequently went on to explain the basis for the ongoing variation among
heritable traits. He noted that the position of each chromosome at the midline during
metaphase was random, and that there was never a consistent maternal or paternal
side of the cell division. Each chromosome was, therefore, independent of the others.
When they separated into gametes, the set of chromosomes in each daughter cell
could contain a mixture of the parental traits, but not necessarily the same mixture as
that of other daughter cells. The newly discovered chromosomal independence during
meiosis meant that the number of possible chromosomal combinations for each gam-
ete could be calculated based on the number of chromosomes in the organism: specif-
ically, there are 2n possible combinations of chromosomes in gametes, with "n" repre-
senting the number of chromosomes in the gamete. Furthermore, considering all the
possible pairings of one gamete with another, the variation in zygotes is (2n)2, which
results in some fairly large numbers. Indeed, Sutton provided examples of the poten-
tial variation among hypothetical organisms with gamete chromosome numbers rang-
ing from 1 to 19.He also correctly assumed that there was more than one trait present
on each chromosome, so the actual total variation was even higher than any of those
included in the table.
This observation of potential variation added strength to Sutton's assertion that he
had discovered the physical basis for Mendel's principle of independent assortment.
 28
Still lacking, however, was definite proof. Scientists thus needed an experimental
system in which the inheritance of genetic traits could be linked directly to the behav-
ior of chromosomes. Such an opportunity presented itself soon thereafter, with a dis-
tinct mutation in the fruit fly Drosophila melanogaster.

4.1.2. Thomas Hunt Morgan experimentally demonstrates chromosome theory.

During the early years of the twentieth century, Thomas Hunt Morgan and his
colleagues at Columbia University identified hundreds of Drosophila genes and made
many pivotal discoveries about ge-
netic transmission. Cytological ex-
amination showed that Drosophila
possesses four pairs of chromo-
somes, including a pair of sex
chromosomes. Female flies normal-
ly have two identical X chromo-
somes, whereas males have a single
Fig. 4.1. Karyotype of Drosophila melanogaster.
X chromosome and a smaller, gene-
poor Y chromosome (fig. 4.1). Unlike humans, however, sex in fruit flies is deter-
mined by the number of X chromosomes, rather than by the presence of the Y chro-
mosome.

The role of chromosomes in transmission of hereditary information was proved

owing to:
a) discovery of genetic determination of sex;
b) establishment of groups of linkage of signs;
c) construction of genetic and then cytological maps of chromosomes.

4.2. The main States of Chromosome Theory.

1. Genes are located in chromosomes; different chromosomes keep different
number of genes. The set of genes of every non-homologous chromosome is unique.
2. Allele genes occupy appointed and identical loci of homologous chromo-
somes.
3. Genes are localized in the chromosome in appointed sequence in linear order.
4. Genes of one chromosome form group of linkage owing to which linked in-
heritance of some signs takes place. The force of linkage is in the inversely propor-
tion to the distance between genes.
5. Every biological species is characterized by specific chromosome set - karyo-
type.

4.3. Sex-linked inheritance.

4.3.1. Morgan’s Experiments with Drosophila melanogaster.
In fact it is described as genetically inert or genetically empty since it carries so
few genes. In Drosophila it is thought that the genes determining male characteristics
 29
are carried on the autosomes and their phenotypic effects are masked by the presence
of a pair of X chromosomes. Male characteristics, on
the other hand, appear in the presence of a single X
chromosome. This is an example of sex-limited inher-
itance, as opposed to sex-linked inheritance, and in hu-
mans is thought to cause suppression of the genes for
growth of beard in females.
Morgan and his co-workers noticed that inheritance
of eye colour in Drosophila was related to the sex of the
parent flies. Red eye is dominant over white eye. A red-
eyed female crossed with a white-eyed male produced
the F1, all are red, and equal number of red-eyed fe-
males and males. Self-cross between red-eyed male and
female of F1 produced three red-eyed and one white-
eyed part of fruit-flies, but, among the red-eyed there Fig. 4.2. Sex-linked inheritance.
are both female and male and white-eyed were only
males, no white-eyed females. The fact that male flies showed the recessive charac-
teristic more frequently than female flies suggested that the white eye recessive allele
was present on the X chromosome and that the Y chromosome lacked the eye color
gene (Fig. 4.2).

Parental lines: ♀ Red x ♂ White

F1 generation: all Red (♀ ♂)

P1: ♀ Red x ♂ Red

F2 in sex: 2 ♀ : 1♂ : 1♂
F2 phenotype: 3 Red : 1 White

In the reciprocal cross, a white-eyed female crossed with a red-eyed male pro-
duced of F1 red-eyed females and white-eyed males. Inbreeding these F1 flies pro-
duced equal numbers red-eyed females, red-eyed males and white-eyed males, white-
eyed females (fig. 4.3).

Parental lines: ♀ White x ♂ Red

F1 generation: 1 ♀ Red : 1 ♂ White

P1: ♀ Red x ♂ White

F2 in sex: 1 ♀ : 1♂ : 1♀: 1♂

F2 phenotype: Red : White

Fig. 4.3. Sex-linked inheritance
(reciprocal cross).
 30

From this Morgan rightly concluded that only the X chromosome carries the
gene for eye color. There is no gene locus for eye color on the Y chromosome. This
phenomenon is known as sex linkage.

4.3.2. The cytological basis of Sex-linked inheritance.

XW – Red
Xw – White

Parental lines: ♀ XW XW x ♂ Xw Y
W w
G: X X Y

F1 in sex : ♀ XW Xw ♂ XW Y

all are Red

P1: X W Xw x ♂ XW Y
W w W
G: X X X Y

F2 in sex: 1 ♀ XW XW : 1♀ XW Xw : 1 ♂ XW Y : 1 ♂Xw Y

F2 phenotype: 3 Red : 1 White

Reciprocal cross:

Parental lines: ♀ X w Xw x ♂ XW Y
w W
G: X X Y

F1 generation: 1 ♀ XW X w : 1 ♂ Xw Y
Red White

P1: ♀ XW Xw x ♂ Xw Y
W w w Y
G: X X X

F2 in sex: 1 ♀ XW Xw : 1♂ XW Y : 1 ♀ X w Xw : 1 ♂ X w Y

F2 phenotype: Red : White

 31

4.4. The sex determination and differentiation.

4.4.1. Types of sex determination.
A sex-determination system is a biological system that determines the
development of sexual characteristics in an organism. Most sexual organisms have
two sexes. In many cases, sex determination is genetic: males and females have
different alleles or even different genes that specify their sexual morphology. In
animals, this is often accompanied by chromosomal differences. In other cases, sex is
determined by environmental variables (such as temperature) or social variables (the
size of an organism relative to other members of its population). The details of some
sex-determination systems are not yet fully understood.
1. Progamic - determined from oocyte size in Dinophilus apatris.
2. Epigamic - determined by the stochastic choice of the zygote implantation
place in Bonellia viridis.
3. Syngamic (chromosomal) - determined by a stochastic choice of the spermat-
ocyte which enters the oocyte in Homo sapiens.
4. Eusyngamic – with fertilization and without fertilization.

4.4.2. The role of chromosomes in sex determination.

The technique of relating phenotypic characteristics of organisms to the structure
of their chromosomes is seen most clearly in the determination of sex. In Drosophila
the observed phenotypic differences between the two sexes appear to be related to the
differences in the size of their chromosomes.
Examination of the chromosome structure of a range of animals revealed that
males and females showed certain chromosomal differences. Pairs of chromosomes
(homologous chromosomes) are found in all cells, but one pair of chromosomes al-
ways shows differences between the sexes. These are the sex chromosomes or
heterosomes.
All other chromosomes are known as autosomal chromosomes or autosomes. As
can be seen in Drosophila has four pairs of chromosomes. Three pairs appear identi-
cal in both sexes (numbers II, III and IV), but the other pair, whilst appearing identi-
cal in the female, differ in the male. The chromosomes are known as X and Y chro-
mosomes, and the genotype of the female is XX and that of the male is XY. These
characteristic sex genotypes are found in most animals, including humans; but in the
case of birds (including poultry), moths and butterflies the sex genotypes are re-
versed: the females are XY and the males are XX. In some insects, such as the grass-
hopper the Y chromosome may be absent entirely and so the male has the genotype
XO.
In the production of gametes the sex chromosomes segregate in typical
Mendelian fashion. For example, in mammals each ovum contains an X chromo-
some; in males one half of the sperms contain a Y chromosome. The sex having the
XX genotype is described as homogametic as it produces gamete cells containing on-
ly X chromosomes. Organisms with the XY genotype are described as heterogamet-
 32
ic since half their gametes contain the X chromosome and half the Y chromosome.
In humans, the genotypic sex of an individual is determined by examining non-
dividing cells. One X chromosome always appears in the active state, which has the
normal appearance. If another is present, it is seen in a resting state as a tightly coiled
dark-staining body called the Barr body or sex chromatin.
Barr body. Barr (1944) observed a body heavily stained by nucleic acid stains in
the nerve cells of female cats. Later on it was found that almost all somatic cells of
mammalian females show the occurrence of this body which was named as sex
chromatin or Barr body. This was found to be missing in normal males. Ohno (1959)
observed that one of the two X-chromosomes in the nuclei of liver cells of female rat
was darkly stained at mitotic prophase while the X chromosome of males was stained
normally. This suggested the involvement of heterochromatic female X chromosome
in the formation of sex chromatin or Barr body.
The Barr body lies against the nuclear mem-
brane and appears as a rounded disc. Davidson and
Smith (1954) showed that it takes a special form in
the polymorphonuclear leucocytes and is known as
the 'drum stick' because it can be seen as an appen-
dix to the nucleus.
The number of Barr bodies is always one less
than the number of X chromosomes present, that is Fig. 4.4. The female somatic cell
male (XY) = 0, female (XX) = 1. The function of with inactivated X-chromosome.
the Y chromosome appears to vary according to species.
In humans the presence of a Y chromosome controls the differentiation of the
testis which subsequently influences the development of the genital organs and male
characteristics. In some organisms, however, the Y chromosome does not carry genes
concerned with sex.
These organisms are divided into two types: unisexual and bisexual. Bisexual
organisms have both female and male reproductive organs in one organism (her-
maphrodites) (plants, some animals -snails).
In human, most of animals and plants, the sex determination and differentiation
is by the sex chromosomes.

4.4.3. The chromosomal type of sex determination.

1. ♀ XX; ♂XY (mammalian, most insects);
In the XY sex-determination system, females have two of the same kind of sex
chromosome (XX), while males have two distinct sex chromosomes (XY). Some
species (including humans) have a gene SRY on the Y chromosome that determines
maleness; others (such as the fruit fly) use the presence of two X chromosomes to
determine femaleness. The XY sex chromosomes are different in shape and size from
each other unlike the autosomes, and are termed allosomes.
2. ♀ XX; ♂XO (insects such as cockroach and some roundworms);
In this variant of the XY system, females have two copies of the sex chromosome
(XX) but males have only one (X0). The 0 denotes the absence of a second sex
 33
chromosome. This system is observed in a number of insects, including the
grasshoppers and crickets of order Orthoptera and in cockroaches (order Blattodea).
The nematode C. elegans is male with one sex chromosome (X0); with a pair of
chromosomes (XX) it is a hermaphrodite.
3. ♀ ZW; ♂ZZ (many vertebrates (fish, reptile, birds), some insects (but-
terflies);
The ZW sex-determination system is found in birds and some insects and other
organisms. The ZW sex-determination system is reversed compared to the XY
system: females have two different kinds of chromosomes (ZW), and males have two
of the same kind of chromosomes (ZZ). In the chicken, this was found to be
dependent on the expression of DMRT1.
4. ♀ ZO; ♂ZZ (some butterflies).
In this type, the females have only one sex chromosome and hence represented as
ZO. Females are heterogametic. Males carry two identical sex chromosomes desig-
nated ZZ. Males are homogametic. On fertilization by a Z-carrying sperm the Z-
carrying ovum would develop into a male (ZZ) and on fertilization, the Z-lacking
ovum would develop into a female.

4.5. The Lyon Hypothesis.

Muller (1932) described the equal expression of genes of X chromosomes in
male and female Drosophila although the X chromosomes in females are twice in
number than in males. This was termed 'dosage compensation by him. Human fe-
males also have two X chromosomes in their diploid cells whereas the males possess
only one X chromosome in their somatic cells.
The inactive X usually lies along the edge of the interphase nucleus in a highly
condensed state. It is always the last to replicate. In 1948, before the discoveries of
Lyon, Barr and Bertram found that in the interphase nucleus of female cat neurons
there were a significant number of cells that had one "darkly staining body" lying
along the edge of the nucleus, but they never found a "darkly staining body" in the
neurons of male cats. Similar "darkly staining bodies" are found in buccal epithelial
cells of human females, although they can usually be found in only 30% to 40% of
the cells. Normal males never express these "Barr bodies." In all cases, the number of
Barr bodies is one less than the number of X chromosomes in an individual. One Barr
body means the individual has two X chromosomes, two Barr bodies means the indi-
vidual has three X chromosomes, etc. We now know that the "darkly staining" Barr
body is the condensed, inactive X chromosome.
The mechanism of dosage compensation does exist in humans too and although
this was formulated by several workers (Russell, 1961; Beutler, 1962) its clear expla-
nation was given by Lyon in 1961 and 1966.

The significant features of Lyon's hypothesis are as follows:

1. One of the two X chromosomes of a diploid cell of a normal mammalian fe-
male is genetically active, the other one is inactive.
2. The inactive X chromosome can be maternal or paternal.
 34
3. Different cells of the same animal can have inactivation of different X chro-
mosomes, i.e. one cell can have inactivation of maternal and the other cell can have
inactivation of paternal X chromosome.
4. The inactivation occurs early in the embryonic development. Once it is done in
a cell, the progeny of that cell has the same inactive X chromosome, i.e., it remains
fixed throughout the further development of each cell line. This means that the fe-
male is a genetically mosaic, i.e., paternal X is inactive in some cells whereas mater-
nal X is inactive in other cells. Such a mechanism may have some evolutionary sig-
nificance though limiting the genetic differences between the sexes while at the same
time, taking advantage of XX/XY sex determination. If the genes are present in het-
erozygous condition on two X-chromosomes, one of the allele will be expressed in
some cells, the other in other cells. This explains the mosaicism of the coat color in
mice.

4.6. The sex related genetic effects.

Sex-limited genes are ones that are inherited by both men and women but are
normally only expressed in the phenotype of one of them. The heavy male beard is an
example. While women have facial hair it is most often very fine and comparatively
sparse.
Sex-controlled genes are expressed in both sexes but differently. An example of
this is gout , a disease that causes painfully inflamed joints. If the gene is present,
men are nearly eight times more likely than women to have severe symptoms.
Genome imprinting or genetic imprinting is a phenomenon in which, some
genes are known to have a different effect depending on the gender of the parent from
whom they are inherited. This phenomenon is referred to as Apparently, diabetes ,
psoriasis , and some rare genetically inherited diseases, such as a form of mental re-
tardation known as Angelman syndrome, can follow this inheritance pattern.
 35

CHAPTER 5. LINKED INHERITANCE.

Mendel's law of independent assortment is not universal. It holds good only for
the characters whose genes are located on the different (nonhomologous) chromo-
somes. But there are always many more inheritable characters in an individual than
there are chromosomes. Every individual organism bears several heritable characters.
Which are represented by the innumerable genes present on the chromosomes. Dur-
ing meiosis, the chromosomes move into the gametes as units, all the genes present
on any given chromosome will segregate as a group and move together from genera-
tion to generation. This tendency of the genes located on the same chromosome, to
stay together in hereditary transmission, is known as linkage. The genes located on
the same chromosome are called linked genes.
The principle of linkage was discovered by Bateson and Punnet in 1906 in the
sweat pea, plant, Lathyrus odoratus. However, linkage, as a concept was put forth by
Thomas Hunt Morgan in 1910 based on his experiment on Drosophila melano-
gaster.

5.1. Morgan’s experiments with Drosophila melanogaster.

Experimental research of phenomenon of linkage groups was carried out by
Thomas Morgan and his workers in 1910 - 1916 and was assumed as a basis (based
on) of "chromosome theory of heredity".
Experiments with Drosophila melanogaster have shown that genes are divided
into 4 groups according to sign of combined transmissions. Number of such "linkage
groups" is equal to number of chromosomes in haploid set so development of signs
which are inherited linking together is controlled by the genes of one chromosome.
This conclusion is based on the data of following observations.
A cross of grey fruit-fly (B) with normal wings (W) and black fruit-fly (b) with
rudimentary wings (w) gives in F1 grey hybrids with normal wings BbWw.
CYTOLOGICAL BASIS:
B - grey body color
b - black body color
V - Long wing
v - vestigial wing
Parental lines: B B b b
♂ x ♀
V V v v

Grey Long Black Vestigial

F1 generation: B b
V v

all Grey Long

 36
(♂ ♀)
Crossing of male-hybrid of F1 BbWw with the black female with rudimentary
wings bbww (test cross I) individuals of two kinds similar to parents forms are born
in equal quality.
TEST CROSS I:
P1: ♂ B b x ♀ b b

V v v v
Grey Long Black Vestigial

F1: B b : b b

V v v v
Grey Long Black Vestigial
50% 50%
The results of two crosses convince that development of analyzing signs is con-
trolled by different genes localized in one chromosome.
Cross of female-hybrid of F1 with black male with rudimentary wings (test cross
II) gives 4 variants of individuals.
They have combinations of signs which are formed in independent inheritance,
but quantitative correlation of hybrids is another.
TEST CROSS II:
P1: ♀ B b x ♂ b b

V v v v
Grey Long Black Vestigial

F1: B b b b B b b b

V v v v v v V v

Grey Long Black Vestigial Grey Vestigial Black Long

41,5% 41,5% 8,5% 8,5%

Parental combinations Recombinant combinations

This result shows the partial linkage of hereditary signs and breach of linkage is
explained by crossing over.
The comparison of results of the second (female-hybrid in the cross) and the first
(male-hybrid in the cross) experiments testifies to possibility of incomplete and com-
plete linked inheritance of one pair of signs.
This opposition is explained by peculiarity of biology of fruit-fly which is in-
cluded in absentee of crossing over in gametogenesis of males.
 37
In own example quantity of offspring with changed correlation of sings (such in-
dividuals are called crossovers) and therefore controlling alleles form 17%. This fig-
ure indicates the number of crossing-over gametes. Other combinations of genes of
fruit-fly differ from by other frequency of crossing over. In the constant conditions
(temperature, age of female and other) this value is constant for every pair of genes of
one chromosome. For example, the number of crossing-over gametes for genes of
white eyes and yellow color of body is 1,5%; for genes of yellow body and rudimen-
tary wings is 47%. Power of gene linkage is inversely proportional to the distance be-
tween them in chromosome (this is the rule, or the law of Th. Morgan).
Constancy of percentage of crossing-over between the genes is used as index of
relative distance between them and order of inter disposition during composition of
chromosome maps. The unit of distance between the genes is called morganid. It
corresponds to the distance by which crossing over takes place in 1% of gametes.
At the distance of 50 morganids and more over signs are inherited independently
in spite of localization of genes in one chromosome.

5.2. The kinds of linkage.

Complete linkage. The genes closely located in the chromosome, show complete
linkage as they have no chance of separating by crossing-over and are always trans-
mitted together to the same gamete and the same offspring. Thus, the parental combi-
nation of traits is inherited as such by the young one in case of complete linkage.
Complete linkage is very rarely case and is known at heterogametic sex of Drosophila
and silkworm.
Incomplete linkage. The genes distantly located in the chromosome show in-
complete linkage because they have a chance of separation by crossing-over and of
going into different gametes and offspring.

5.3. The linkage groups.

All the genes in a particular chromosome form a linkage group. Since the genes
in a particular chromosome have their alleles in the corresponding homologous
chromosome, a pair of homologous chromosomes are said to form a single linkage
group. Thus, the genes occur in as many linkage groups as there are pairs of homolo-
gous chromosomes. In other words, the number of linkage groups in a species corre-
sponds to its haploid number of chromosomes. This principle is known as the limita-
tion of linkage groups. Size of the linkage group depends upon the size of the chro-
mosome. Therefore, small chromosomes have small linkage groups and large chro-
mosomes have large linkage groups. The human X chromosome carries over 102
genes whereas the Y chromosome bears only over 10 genes.

5.4. The factors affecting linkage.

Besides the distance between the genes, the following physiological and envi-
ronmental factors affect the linkage of genes:
1. Age. With advancing age, the chances of crossing over lessen, increasing the
strength of linkage.
 38
2. Temperature. Rise in temperature increases the chances of chiasma formation,
decreasing the strength of linkage.
3. X-Rays. Exposure to X-rays reduces the strength of linkage.

5.5. The significance of linkage.

The phenomenon of linkage is important in many ways:
1. It greatly reduces the chances of the formation of new combinations of genes
in the gametes. It, thus, helps in keeping the parental, racial and specific traits togeth-
er.
2. It helps in maintaining the valuable traits of a newly developed variety.
3. Linkage disallows the plant and animal breeders to combine all the desirable
traits in a single variety.
4. The fact that there are as many linkage groups as the chromosome pairs is an
important evidence for the location of genes in the chromosomes.

5.6. The linkage analysis.

According to Tomas Morgan, the genes which are situated on the chromosome,
are linearly arranged. The distance between the two genes on the chromosome is cor-
related with the amount of crossing over indicated by the two alleles. The direct pro-
portionality exists between the distance of alleles and the number of crossing over.
According to Jacob, Lwoff, Monod and Wolfinan (1960), it is possible to determine
the exact locus (location in the chromosome) of any gene and the order of loci of all
genes on chromosomal thread. The representation of such figure is known chromo-
some map or linkage map or genetic map. The chromosome map is sometime known
as crossing over map. Sturtevant (1911) was the first who constructed chromosome
map followed by Bridges and others.
Gene mapping. The major significance of calculating crossover frequencies is
that it enables geneticists to produce maps showing the relative positions of genes on
chromosomes. Chromosome maps are constructed by directly converting the crosso-
ver frequency or value between genes into hypothetical distances along the chromo-
some. A crossover frequency or value (COV) of 4% between genes A and B means
that those genes are situated 4 units apart on the same chromosome. A COV of 9%
for a pair of genes A and C would indicate that they were 9 units apart, but it would
not indicate the linear sequence of the genes.

C? A B C?

4
9 9

In practice it is usual to determine crossover values for at least three genes at

once, as this triangulation process enables the sequence of the genes to be determined
as well as the distances between them. Consider the following crossover values as
determined by a series of breeding experiments involving four genes P, Q, R and S.
 39
P - Q = 24%
R - P = 14%
R - S = 8%
S - P = 6%
To calculate the sequence and distances apart of the genes, a line is drawn repre-
senting the chromosome and the following procedure carried out.
1) Insert the positions of the genes with the least COV in the middle of the chro-
mosome, that is S - P = 6%.
S P

2) Examine the next largest COV, that is R - S = 8%, and insert both possible po-
sitions of R on the chromosome, relative to S.

R? S P R?

8 6
8

3) Repeat the procedure for the next largest COV, that is R -P = 14%. This indi-
cates that the right-hand position of R is incorrect.
R S P

8 6

14

4) Repeat the procedure for the COV for P - Q = 24%. The position of Q cannot
be ascertained without additional information.

Q? R S P Q?

24 24

If, for example, the COV for Q - R = 10% this would confirm the left-hand posi-
tion for gene Q.
A problem which arises in preparing chromosome maps is that of double crosso-
ver, particularly when considering genes which are widely separated, since the num-
ber of apparent crossovers will be less than the actual number. For example, if cross-
overs occur between alleles A and B and B and C, A and C will still appear linked,
but the chromosome will not carry the recessive allele b.
 40
Chromosome mapping involves techniques used to assign a gene or DNA se-
quence to a specific chromosome or a particular region of a chromosome.
 41
CHAPTER 6. EXTRANUCLEAR INHERITANCE.
Extranuclear inheritance is the transmission of genes that occur outside the
nucleus. It is found in most eukaryotes and is commonly known to occur in
cytoplasmic organelles such as mitochondria and chloroplasts or from cellular
parasites like viruses or bacteria. It’s also called cytoplasmic inheritance (tab. 6.1).

Table 6.1.
The cytoplasmic inheritance.
Prokaryotes Eukaryotes
Characters

Nucleus Cytoplasm Nucleus Cytoplasm

DNA Nucleoid Plasmids Nucleotype Cytotype

Obligatory Nucleoid genes Plasmids, Genes DNA

genetic Episomes Of Mitochondria
elements and Chloroplasts
Facultative 1. Inseration 1. Symbiotic 1. Mobile gene 1. Spiroplasm,
genetic 2. Transposons bacteria, (MDG), 2. Viruses,
elements 3. Bacteriophagy 2. Symbiotic algae 2. Viruses, 3. Extra-
4. Bacteria 3. Tox+transposon 3. B-chromosomes, chromosomal el-
plasmids 4. Amplificatory ements
copies of DNA

6.1. Extranuclear inheritance of eukaryotic organelles.

Mitochondria are organelles which function to produce energy as a result of
cellular respiration. Chloroplasts are organelles which function to produce sugars
via photosynthesis in plants and algae. The genes located in mitochondria and
chloroplasts are very important for proper cellular function, yet the genomes replicate
independently of the DNA located in the nucleus, which is typically arranged in
chromosomes that only replicate one time preceding cellular division. The
extranuclear genomes of mitochondria and chloroplasts however replicate
independently of cell division. They replicate in response to a cells increasing energy
needs which adjust during that cells lifespan. Since they replicate independently,
genomic recombination of these genomes is rarely found in offspring contrary to
nuclear genomes, in which recombination is common. Mitochondrial disease are
received from the mother, sperm does not contribute for it.

6.2. Extranuclear inheritance of facultative genetic elements.

Extranuclear transmission of viral genomes and symbiotic bacteria is also
possible. An example of viral genome transmission is perinatal transmission. It
occurs from mother to fetus during the perinatal period, which begins before birth
and ends about 1 month after birth. During this time viral material may be passed
from mother to child in the bloodstream or breastmilk. This is of particular concern
with mothers carrying HIV or Hepatitis C viruses. Examples of cytoplasmic
 42
symbiotic bacteria have also been found to be inherited in organisms such as insects
and protists.

6.2.1. Plasmids and episomes.

A plasmid is a DNA molecule that is separate from, and can replicate
independently of, the chromosomal DNA. They are double stranded and, in many
cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found
in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).
Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp). The number of
identical plasmids within a single cell can range anywhere from one to even
thousands under some circumstances. Plasmids can be considered to be part of the
mobilome, since they are often associated with conjugation, a mechanism of
horizontal gene transfer.
An episome is a portion of genetic material that can exist independent of the
main body of genetic material (called the chromosome) at some times, while at other
times is able to integrate into the chromosome.
Examples of episomes include insertion sequences and transposons. Viruses are
another example of an episome. Viruses that integrate their genetic material into the
host chromosome enable the viral nucleic acid to be produced along with the host
genetic material in a nondestructive manner. As an autonomous unit (i.e., existing
outside of the chromosome) however, the viral episome destroys the host cell as it
commandeers the host's replication apparatuses to make new copies of itself.
Another example of an episome is called the F factor. The F factor determines
whether genetic material in the chromosome of one organism is transferred into
another organism. The F factor can exist in three states that are designated as
FPLUS, Hfr, and F prime.
FPLUS refers to the F factor that exists independently of the chromosome. Hfr
stands for high frequency of recombination, and refers to a factor that has integrated
into the host chromosome. The F prime factor exists outside the chromosome, but
has a portion of chromosomal DNA attached to it.
An episome is distinguished from other pieces of DNA that are independent of
the chromosome (i.e.,plasmids) by their large size.
Plasmids are different from episomes, as plasmid DNA cannot link up with
chromosomal DNA. The plasmid carries all the information necessary for its
independent replication. While not necessary for bacterial survival, plasmids can be
advantageous to a bacterium. For example, plasmids can carry genes that confer
resistance to antibiotics or toxic metals, genes that allow the bacterium to degrade
compounds that it otherwise could not use as food, and even genes that allow the
bacterium to infect an animal or plant cell. Such traits can be passed on to another
bacterium.
Transposons and insertion sequences are episomes. These are also known as
mobile genetic elements. They are capable of existing outside of the chromosome.
They are also designed to integrate into the chromosome following their movement
from one cell to another. Like plasmids, transposons can carry other genetic material
 43
with them, and so pass on resistance to the cells they enter. Class 1 transposons, for
example, contain drug resistance genes. Insertion sequences do not carry extra
genetic material. They code for only the functions involved in their insertion into
chromosomal DNA.
Transposons and insertion sequences are useful tools to generate changes in the
DNA sequence of host cells. These genetic changes that result from the integration
and the exit of the mobile elements from DNA, are generically referred to as
mutations. Analysis of the mobile element can determine what host DNA is present,
and the analysis of the mutated host cell can determine whether the extra or missing
DNA is important for the functioning of the cell.

CHAPTER 7. BIOTECHNOLOGY AND GENE ENGINEERING.

7.1. Gene engineering.
Genetic engineering can be defined as the science dealing with the correction of
nature's mistakes at their source, the DNA molecule, comprising the gene. Through
it's techniques, genetic engineering carries out the deliberate modification of specific
genes or implants desired new genes into chromosomes. The genetic or biochemical
techniques by which genetic information contained in two or more different DNA
molecules could be combined in one unit fall within the score of genetic engineering.
It can also be defined as the science of adding, removing or replacing genetic
units in order to achieve permanent and heritable changes in plants or animals for the
benefit of mankind. Terms such as “gene surgery”, “gene therapy”, “gene manipula-
tion”, “gene transplantation”, “gene intervention” and “algeny” have been synony-
mously used with genetic engineering. A detailed knowledge of the molecular nature
of the gene and the ability to manipulate cells of higher organisms as well as bac-
teria, may eventually allow the possibility of genetic engineering, i.e., the artificial
production of new varieties of animals or plants by altering the genotype of existing
varieties without the slow hit-and-miss of selective breeding. For humans it is only a
distant future possibility and is based on the work of similar nature on lower organ-
isms especially microorganisms. Artificially induced mutations by man in the strict
sense are also covered under genetic engineering.

7.2. Biotechnology.
Biotechnology is technology based on biology - biotechnology harnesses cellu-
lar and biomolecular processes to develop technologies and products that help im-
prove our lives and the health of our planet. We have used the biological processes of
microorganisms for more than 6,000 years to make useful food products, such as
bread and cheese, and to preserve dairy products.
Modern biotechnology provides breakthrough products and technologies to
combat debilitating and rare diseases, reduce our environmental footprint, feed the
hungry, use less and cleaner energy, and have safer, cleaner and more efficient indus-
trial manufacturing processes.
Currently, there are more than 250 biotechnology health care products and vac-
cines available to patients, many for previously untreatable diseases. More than 13.3
 44
million farmers around the world use agricultural biotechnology to increase yields,
prevent damage from insects and pests and reduce farming's impact on the environ-
ment. And more than 50 biorefineries are being built across North America to test
and refine technologies to produce biofuels and chemicals from renewable biomass,
which can help reduce greenhouse gas emissions.
Recent advances in biotechnology are helping us prepare for and meet society’s
most pressing challenges.
Biotech is helping to heal the world by harnessing nature's own toolbox and us-
ing our own genetic makeup to heal and guide lines of research by:
1) Reducing rates of infectious disease;
2) Saving millions of children's lives;
3) Changing the odds of serious, life-threatening conditions affecting millions
around the world;
4) Tailoring treatments to individuals to minimize health risks and side effects;
5) Creating more precise tools for disease detection; and
6) Combating serious illnesses and everyday threats confronting the developing
world.
Biotech uses biological processes such as fermentation and harnesses biocata-
lysts such as enzymes, yeast, and other microbes to become microscopic manufactur-
ing plants. Biotech is helping to fuel the world by:
1. Streamlining the steps in chemical manufacturing processes by 80% or more;
2. Lowering the temperature for cleaning clothes and potentially saving $4.1
billion annually;
3. Improving manufacturing process efficiency to save 50% or more on operat-
ing costs;
4. Reducing use of and reliance on petrochemicals;
5. Using biofuels to cut greenhouse gas emissions by 52% or more;
6. Decreasing water usage and waste generation; and
7. Tapping into the full potential of traditional biomass waste products.
Biotech improves crop insect resistance, enhances crop herbicide tolerance and
facilitates the use of more environmentally sustainable farming practices. Biotech is
helping to feed the world by:
1. Generating higher crop yields with fewer inputs;
2. Lowering volumes of agricultural chemicals required by crops-limiting the
run-off of these products into the environment;
3. Using biotech crops that need fewer applications of pesticides and that allow
farmers to reduce tilling farmland;
4. Developing crops with enhanced nutrition profiles that solve vitamin and nu-
trient deficiencies;
5. Producing foods free of allergens and toxins such as mycotoxin; and
6. Improving food and crop oil content to help improve cardiovascular health.
 45
CHPATER 8. GENOTYPE AS INTEGRATIVE GENETIC SYSTEM OF
AN ORGANISM.
Soon after the rediscovery of Mendel's laws in 1900 there were reports of an in-
heritance pattern that differed from those established by Mendel. The variations were:
1) The degree of dominance differed and incomplete dominance was observed
in certain cases.
2) Various genes were found to possess more than two alleles, or multiple allelic
inheritance was observed.
3) It was found that a trait is governed by the interaction of the interaction of
two or more genes.
This result in F2 phenotypic ratios deviating from the typical Mendelian ratios.
Such variations indicated that Mendel's laws do not apply universally to all sexually
reproducing organisms. There were attempts to explain such a phenomenon by many
researches including Bateson and Punnet. They described genes not as separate ele-
ments producing distinct phenotypes as postulated by Mendel but as producing prod-
ucts which could interact in a predictable way to produce new phenotypic expres-
sions. Depending upon the form of interaction Mendelian phenotypic ratios is modi-
fied in various ways.
For example, if gene A produces a product A resulting in A phenotype and gene
B produces another product B, an organism with genotype AB might show a third
phenotype, different from the one produced by alleles A and B separately.
Today we'll make out some of the important cases of gene interaction resulting
in variations in inheritance patterns.

8.1. The types of allelic gene interaction.

8.1.1. Incomplete dominance.
Absence of dominance of one member of a pair
of alleles over the other is quite common in most or-
ganisms. For examples, in crosses between Snap-
dragon plants from a red-flowered variety and a va-
riety with white-coloured flowers, the F1 plants pro-
duce only pink flowers intermediate in colour be-
tween those of the parental varieties. In the experi-
ment, the F2 obtained by self-pollination of the F1
hybrids consisted of 22 plants with red flowers, 52
with pink flowers and 23 with white flowers. The
numbers agree with the Mendelian monohybrid ratio
of 1 dominant homozygote: 2 heterozygotes : 1 re-
cessive homozygote.
Fig. 8.1. Incomplete dominance.
In agreement with the predictions from this in-
terpretation, the red-flowered F2 plants produced
 46
only red-flowered progeny, the white-flowered plants also were true-breeding, and
the pink-flowered plants again produced progeny of all three phenotypes in the pro-
portions 1 red: 2 pink: 1 white.
In this example of flower-colour inheritance the absence of dominance is ex-
plained by the mechanism of formation of the red pigment. The pigment is formed by
a complex sequence of enzymatic reactions.
A critical enzyme is determined by the dominant allele, and a defective enzyme
is determined by recessive allele. In heterozygotes concentration of the enzyme re-
quired for synthesis of the red pigment are reduced.
Cytological basis of Incomplete Dominance:
Characters:
AA – red colour of flower;
aa - white colour of flower;

P: ♂ AA x ♀ aa
Red White

G: A a

F1: Aa
100% pink

P1: ♂ Aa x ♀ Aa
Pink Pink

G: a A A a

F2: AA : Aa : Aa : aa
Genotypic ratio: 1 2 1
Phenotypic ratio: 1 red 2 pink : 1 white

8.1.1. Overdominance.
The condition of a heterozygote having a phenotype that is more pronounced or
better adapted than that of either homozygote. The overdominance is the main cause
for the fitness advantages of heterosis, and then there should be an over-expression of
certain genes in the heterozygous offspring compared to the homozygous parents.

8.1.2. Codominance and Multiple Allelism.

Another exception from simple dominance occurs when the two different alleles
in a heterozygote are both fully expressed, resulting in a phenotype that is qualita-
tively different from those of the homozygotes. This is called codominance. One of
the best examples is the effect of the genes that determine human blood groups of
 47
which the ABO group is the best known. The blood group genes determine the syn-
thesis of polysaccharides (polymers of sugar) on the surface of red blood cells. Two
distinct polysaccharides, A and B, are made and these are determined by the alleles IA
and IB, respectively. The third allele I0, which is a recessive, is defective in that it
does not determine synthesis of either A or B polysaccharides (fig. 8.2).
The results are that a homozygous recessive I0I0 individual produces red blood
cells lacking both polysaccharides, and such an individual has type 0 blood. An IAIA
individual or IAI0 heterozygote has type A blood (because the A polysaccharide is
present) and IBIB individual or an IBI0 heterozygote has type B blood. The IAIB indi-
vidual illustrates codominance, for his or her blood cells possess both A and B poly-
saccharides and hence has type AB blood.
Blood groups are important in medicine because of the ABO system in that
most human blood contains antibody to either the A or B polysaccharides. An anti-
body is a protein made by the immune system, capable of binding to a foreign mole-
cule (called an antigen) and inactivating it. Antibodies are specific such as only a sin-
gle foreign molecule is recognized. Inactivation is usually accomplished by formation
of a precipitate or at least a large molecular aggregate. Thus, type A blood contains
red cells with the A surface antigens and only anti-B antibodies and type B contains
only anti-A antibodies. Type 0 blood which lacks both A and B antigens contains
both anti-A and anti-B antibodies; and type AB bloods lacks both antibodies since
both A and B antigens are present.
Mind that the AB0 system illustrates both codominance (both A and B antigens
are produced by an IAIB individ-
ual) and multiple allelism (three
alleles - IA; IB; I0 - comprise the
system and any allelic pair is a
possible genotype). Many ex-
amples of multiple allelism are
known; they do not always ex-
hibit codominance. Also, ab- Fig. 8.2. Genotypes of ABO system of human blood
sence of dominance, as seen groups.
with Snapdragon colour, pro-
duces a blending effect in heterozygotes, but in codominance gene products of both
alleles is present.
Cytological basis of Co-Dominance:
Characters:
A
I _ - I blood group;
IB_ - II blood group;
I0I0 - III blood group;
IAIB - IV blood group
 48
P: ♂ IAI0 x ♀ IBI0

G: IA I0 IB I0

F1: IAIB IAI0 IBI0 I0I0

Genotypic ratio: 1 : 1 : 1 : 1
Phenotypic ratio: 1 : 1 : 1 : 1

8.3. The types of non-allelic gene interaction.

8.3.1. Epistasis.
Since all organisms are complex biochemical systems and all traits are deter-
mined ultimately by a series of chemical reactions. It should not be unexpected that
different genes and their particular alleles often influence one another. Several types
of mutual influences are possible; for example, the product of one allele might either
inhibit or enhance the activity of another gene, or a single trait might require the ac-
tivity of two or more genes.
So some genes may be inhibitor, some complementary and some additive.
One of phenomena, which cause departures from Mendelian ratios, is called epistasis.
It differs from dominance, which always refers to the modification of the expression
of one member of a pair of alleles by the other (inhibitory action of one of alleles in
pair).
If dominant allele has epistatic action they say about dominant epistasis. When
recessive allele in homozygous condition has epistatic action we have recessive epis-
tasis.
Dominant epistasis:
B - black coat of horse;
b - chest-nut coat of horse;
C - epistatic gene (grey coat - as a result of epistasis);
c - allele which not inhibit the manifestation of activity of gene B (b).
grey coat horse chest-nut coat horse
P: CCBB x ccbb

G: CB cb

F1: CcBb - grey horses – uniformity of hybrids.

P2: CcBb x CcBb

G: CB Cb cB cb CB Cb cB cb
 49

F2: 9 B_C_ : 3 bbC_ : 3 B_cc : 1 bbcc

12 grey 3 black 1 chest-nut horses.
So, disintegration in horse coat is: 12 : 3 : 1. The grey coat in horses develops
from a result of inhibitory activity of dominant allele C on gene B in any allelic
condition. The absence of dominant allele C in genotype ensures the development of
black coat (B) or chest-nut coat of horse (b).

Fig. 8.3. Dominant epistasis.

Recessive epistasis.
A - dominant allele of zonal distribution of pigment in the hair length in mice
(accumulation of pigment in base or end of hair) – grey colour;
a - recessive allele which is not determine the zonal distribution of pigment –
agouti colour;
C - the allele of synthesis of pigment;
c - the absence of pigment (or albinism); gene c doesn't give the possibility to
show itself gene A (a).
agouti mouse white mouse
P: aaCC x AAcc

G: aC Ac

F1: AaCc - grey mice - uniformity of hybrids.

P2: AaCc x AaCc

G: AC Ac aC ac AC Ac aC ac
 50

F2: 9 A_C_ : 3aaC_ : 3 A_cc : 1 aacc

9 grey 3 agouti 4 white
So distribution of hair colour in mice is 9 : 3 : 4. The grey hair in mice develops
from a result of activity of dominant alleles A and C; the black hair develops from a
result of activity of dominant C allele and recessive a, and a white colour of hair de-
velops in the case of interaction of both recessive alleles a and c and dominant A al-
lele with recessive c, which inhibit the activity of dominant A.
The example in man is: the development of albinism in people having homozy-
gous recessive allele a, which inhibit the manifestation of gene activity P1; P2; P3;
P4, being responsible for synthesis of pigment melanin.

Fig. 8.4. Recessive epistasis.

8.3.2. Complementary.
The next example of departure from Mendelian ratios is complementation (the
complementary interaction of dominant alleles of genes).
The first example of complementation
(Complementary interaction of dominant non-allelic genes).
A - allele being responsible for synthesis of propigment in sweet pea plant;
a - absence of propigment;
 51
B - allele being responsible for synthesis of enzyme which catalyzes the
transmutation of propigment to pigment.
b - absence of this enzyme.
A_bb - white flowers;
aaB_ - white flowers;
aabb - white flowers
A_B_ - purple flowers.

white white
P: AAbb x aaBB

G: Ab aB

F1: AaBb - all purple.

P2: AaBb x AaBb

purple purple

G: AB Ab aB ab AB Ab aB ab

F2: genotypes: 9 A_B_ : 3 A_bb : 3 aaB_ : 1 aabb

purple white
So disintegration in flower colour in sweet pea-plants in phenotypes is: 9 pur-
ple : 7 white.
The purple colour of flowers of sweet-pea-plant in hybrid of F1 and 9/16 of F2
is a result of complementary interac-
tion of dominant alleles of genes A
and B. One of dominant alleles
doesn’t determine the development of
colour in absence of the other domi-
nant allele of another gene.

Fig. 8.5. Complementary interaction of domi-

nant non-allelic genes.
 52
The second example of complementation.
There are two genes being responsible for the form of fruit of pumpkin:
A - globe-shaped form of fruit of pumpkin;
B - globe-shaped form of fruit of pumpkin;
ab - long form of fruit;
AB -disk-like form of fruit.
globe-shaped globe-shaped
P: AAbb x aaBB

G: Ab aB
b b
F1: AaBb - disk-like form

P2: AaBb x AaBb

G: AB Ab aB ab AB Ab aB ab
b b b b b b b b
F2: 9 A_B_ : 3 A_bb : 3 aaB_ : 1 aabb
disk-like globe-shaped long

Fig. 8.6. Complementary interaction of dominant and recessive non-allelic genes.

 53
So distribution in signs of form of fruit of pumpkin is: 9 : 6 : 1.
The appearance of F1 hybrids and 9/16 of F2 progeny with disk-like form of fruit
is the result of complementary interaction of dominant alleles of genes A and B.

The third example of complementation:

A - blue colour of plumage in wavy parrots;
B - yellow colour of plumage;
AB - green colour of plumage;
ab - white colour of plumage.

blue x yellow
P: AAbb x aaBB

G: Ab aB
b b
F1: AaBb - all green.

P2: AaBb x AaBb

AB Ab aB ab Ab Ab Ab Ab
G: b b b b b b b

F2: 9 A_B_ : 3 A_Bb : 3 aaB_ : 1 aabb

green blue yellow white.

So distribution in signs of plumage colour in parrots is: 9 : 3 : 3 : 1.

The examples of complementary interaction in man: the appearance of special
hair glitter (shining or glossy) in certain combinations of alleles of two genes being
responsible separately for synthesis of black and red pigments.

8.3.3. Polymery.
And at last some words about additive interaction of genes (phenomenon of
polymery or polymeric genes). It consists in two or more genes of dominant alleles
controlling the development of one sign in the same degree. These genes are called
polymeric or polygenes and marked the same letter in Latin with figure indexes.
The first example in polymery.
A1 A2 - red colour of wheat grain;
a1 a2 - absence of colour.
 54
P: red x white
A1 A1 A 2 A2 x a1 a1 a2 a2
G: A 1 A2 a1 a2
F1: A1a1A2a2 - all brown.
P2: A1 a1 A2 a2 x A1 a1 A2 a2
F2: 9 A1_ A2_ : 3 A1_a2 a2 : 3 a1 a1 A2_ : 1 a1 a1 a2 a2
15 coloured 1 non-coloured.

The distribution (disintegration) in presence or absence of wheat grain colour is

15: 1.
The activity of dominant alleles of polymeric genes A1 and A2 in genotype is
summed.
There are some examples of polymeric inheritance in animals and plants: laying
of eggs in hen; productivity of dairy cattle; weight and height in animals; contents of
saccharides in root crops, length of corn-cob.

The second example in polymery.

P1 P2 P3 P4 - high level of synthesis of melanine in skin cells in man.
p1 p2 p3 p4 - low level of synthesis of melanine in skin cells in man.

P: Negroes x white-skin woman

P1 P1 P2 P2 P3 P3 P4 P4 x p1 p1 p2 p2 p3 p3 p4 p4

G P1P2P3P4
p1p2p3p4

F1: P1 p1 P2 p2 P3 p3 P4 p4
mulatto
The distribution in intensivity of skin pigmentation in progeny of mulatto - tet-
raheterozygotes are very large. The intensivity of skin pigmentation in man depends
on number of dominant genes in genotype.
There are inherited with polymeric genes: height, weight, body-building and
mental abilities in man.

8.4. Pleiotropy.
During his study of inheritance in pea plants, Gregor Mendel made several inter-
esting observations regarding the color of various plant components. Specifically,
Mendel noticed that plants with colored seed coats always had colored flowers and
colored leaf axils. (Axils are the parts of the plant that attach leaves to stems.) Mendel
also observed that pea plants with colorless seed coats always had white flowers and
 55
no pigmentation on their axils. In other words, in Mendel's pea plants, seed coat color
was always associated with specific flower and axil colors.
Today, we know that Mendel's observations were the result of pleiotropy, or the
phenomenon in which a single gene contributes to multiple phenotypic traits. In this
case, the seed coat color gene, denoted a, was not only responsible for seed coat col-
or, but also for flower and axil pigmentation (Fairbanks & Rytting, 2001).
The term pleiotropy is derived from the Greek words pleio, which means
"many," and tropic, which means "affecting." Genes that affect multiple, apparently
unrelated, phenotypes are thus called pleiotropic genes (Figure 1). Pleiotropy should
not be confused with polygenic traits, in which multiple genes converge to result in a
single phenotype.
Examples of Pleiotropy. In some instances of pleiotropy, the influence of the
single gene may be direct. For example, if a mouse is born blind due to any number
of single-gene traits (Chang et al., 2002), it is not surprising that this mouse would
also do poorly in visual learning tasks. In other instances, however, a single gene
might be involved in multiple pathways. For instance, consider the amino acid tyro-
sine. This substance is needed for general protein synthesis, and it is also a precursor
for several neurotransmitters (e.g., dopamine, norepinephrine), the hormone
thyroxine, and the pigment melanin. Thus, mutations in any one of the genes that af-
fect tyrosine synthesis or metabolism may affect multiple body systems. These and
other instances in which a single gene affects multiple systems and therefore has
widespread phenotypic effects are referred to as indirect or secondary pleiotropy
(Grüneberg, 1938; Hodgkin, 1998).

Pleiotropy in Humans. As touched upon earlier in this article, there are many
examples of pleiotropic genes in humans, some of which are associated with disease.
For instance, Marfan syndrome is a disorder in humans in which one gene is respon-
sible for a constellation of symptoms, including thinness, joint hypermobility, limb
elongation, lens dislocation, and increased susceptibility to heart disease. Similarly,
mutations in the gene that codes for transcription factor TBX5 cause the cardiac and
limb defects of Holt-Oram syndrome, while mutation of the gene that codes for DNA
damage repair protein NBS1 leads to microcephaly, immunodeficiency, and cancer
predisposition in Nijmegen breakage syndrome.
One of the most widely cited examples of pleiotropy in humans is phenylketonu-
ria (PKU). This disorder is caused by a deficiency of the enzyme phenylalanine hy-
droxylase, which is necessary to convert the essential amino acid phenylalanine to ty-
rosine. A defect in the single gene that codes for this enzyme therefore results in the
multiple phenotypes associated with PKU, including mental retardation, eczema, and
pigment defects that make affected individuals lighter skinned (Paul, 2000).
The phenotypic effects that single genes may impose in multiple systems often
give us insight into the biological function of specific genes. Pleiotropic genes can
also provide us valuable information regarding the evolution of different genes and
gene families, as genes are "co-opted" for new purposes beyond what is believed to
be their original function (Hodgkin, 1998). Quite simply, pleiotropy reflects the fact
 56
that most proteins have multiple roles in distinct cell types; thus, any genetic change
that alters gene expression or function can potentially have wide-ranging effects in a
variety of tissues.

CHPATER 9. VARIABILITY.

9.1. Historical overview on the development of variability ideas.

The paleontologist Wilhelm Waagen applied the term «mutation» to morpholog-
ical discontinuities in a temporal series of fossil ammonites. Hugo de Vries in Hol-
land and William Bateson in England described mutations as discontinuous heredi-
tary variations causing major, easily recognizable changes. De Vries argued that the
building blocks of evolutionary change are sudden mutational changes rather than the
gradual «individual variability..... [that] cannot lead to a real overstepping of the spe-
cies limits even with the most intense steady selection». De Vries based his muta-
tion theory of evolution on the results of segregation in progenies of crosses of the
evening primrose, Oenothera.
Actually, the «mutants» described by de Vries were due to a variety of heredi-
tary changes, particularly chromosomal aberrations. Richard Goldschmidt and others
argued that there are two kinds of mutations: some produce variation among individ-
uals, modifying their adaptation to the environments, while others produce new spe-
cies, genera, families, etc.
The mutations allegedly responsible for drastic changes were called «systemic
mutations», «macromutations», and the like. The modern concept of mutation is due
to Thomas Hunt Morgan and his associates who worked with the fruit fly, Drosophi-
la melanogaster. Mutations are defined as changes in single genes with effects rang-
ing from barely detectable to very drastic. Some mutations cause morphological vari-
ations, but others result in behavioral changes or modify the viability, fertility, or rate
of development of their carriers.
Mutations are said to be «spontaneous», i.e., due to unknown naturally occurring
agencies. H.J. Muller showed that mutations arise with definite regularity, at the
rates that can be measured. He also discovered that the frequency of mutation is in-
creased in the progenies of flies treated with X-rays. Other high-energy radiations
were later shown to be also mutagenic. Charlotte Auerbach was the first to demon-
strate unambiguously that a chemical, namely mustard gas, had mutagenic properties.
It was later shown that exposure to a variety of chemical agents, or to higher tempera-
tures, increases the frequency of mutations.
Since the 1940-s, mutations have been extensively studied in the mold Neu-
rospora, in yeast and other unicellular organisms, in bacteria such as Escherichia
coli, and in viruses. Morphological mutations modifying the appearance of colonies
were discovered in these organisms, but major contributions to genetic knowledge
and to the understanding of the mutation processes were made through the study of
so-called auxotroph mutations. In contrast to prototrophic (wild-type) organisms,
 57
auxotrophic organisms require special nutritional supplements for growth and repro-
duction. Other types of mutations commonly studied in haploid organisms are those
causing resistance or sensitivity to certain drugs, such as penicillin or streptomycin,
or to infection by specific viruses. In the viruses themselves, mutations were discov-
ered that modify the viruses’ ability to infect their bacterial hosts or to multiply in
them.
The discovery of the double-helical structure of DNA, and later of the genetic
code, opened the way to an understanding of the process of mutation and its causes in
physicochemical terms.

9.2. Classification of variability.

Phenotypic variability is the changes of condition of signs which take place un-
der development or factors of environment. Hereditary material is not involved in
such changes. Phenotypic variability is divided into modificational and accidental
variability.
How to prove that some signs are Variability
not inherited?
For example, parents from the Genotypic Phenotypic
northern country decide to have a rest on
the south. Certainly, they get sunburn,
Combinative Mutational
their skin become brown. At the same
time they have a conception of a child. Modificational Accidental
What color of skin will this child have?
Of course, white. So sunburn is not he-
reditary sign. The levels of
modificational variability depend on Fig. 9.1. Type of variability.
«norm of reaction». There are wide and
limited norms of reaction (fig. 2). The examples neither of wide nor of reaction in
human being are: height, weight, color of skin; and limited norm of reaction are: eye
color; number of tooth, hands, legs; blood groups.

WINTER SUMMER

Fig. 9.2. Modificational variability.

 58

Norm of reaction

limits of variation of the character determining

by genetically

Fig. 9.3. Norm of reaction.

Accidental variability appears in some individuals in the result of unfortunate
events (catastrophe, accident disaster). Modificational variability explain phe-
nomenon of phenocopies.
For example, let's discuss hypospadias. The reason of hypospadias may be
changes in genetic material or deviation during embryogenesis. In the last case
change presents itself phenocopy. Tactics of physician is equal in the both cases -
surgery correction of defect. But for solution of the problem about the probability of
the birth of the child with hypospadias in the family of this patient differentiation of
cases of genotypical and phenotypical nature is necessary.

Genotypical variability is divided into combinative and mutational variability.

Combinative variability is variability which depends on recombination of al-
leles in genotypes of offspring in accordance with genotypes of parents.
There are three mechanisms of combinative variability. Change of genetic mate-
rial between homologous chromosomes during crossing over allele composition of
sex cells chromosomes of offspring changes in comparison with parental gametes. In
connection with unknown type of divergence of paternal and maternal chromosomes
of each homologous pair in anaphase of reductional division in chromosomal sets of
off-spring, chromosomes, inherited from two parents are represented at the same time
 59
in the different combinations. The third mechanism of combinative variability is con-
nected with fertilization. Owing to accidental combination of gametes, taking place
in fertilization, variety of combinations of chromosome and allelic composition of
zygotes is created.
So, briefly, 3 mechanisms of combinative variability:
1) Independent divergence of chromosomes during meiosis;
2) Crossing over;
3) Accidental combination of genes during fertilization.
Combinative variability has great importance in natural and artificial selections.

Mutational variability. Heredity is a conservative process, although not per-

fectly so. The information encoded in the nucleotide sequence of the DNA is repro-
duced during replication so that each replication results in two DNA molecules iden-
tical to each other and to the parental one. Occasionally, «mistakes» in the process of
replication lead to different nucleotide sequences in parental and daughter DNA mol-
ecules. Changes in the hereditary materials are known as mutations. Mutations are
spasmodic changes of hereditary signs. Mutations arise suddenly, without transi-
tional conditions in comparison with initial form of characteristic.
Without the occurrence of hereditary changes, life could not have evolved or
diversified. The term «mutation» is used to designate both the processes by which he-
reditary changes arise and the outcomes of such processes («mutants»). It is usually
clear from the context whether the term refers to the process of change; to the
changed hereditary materials, or to both.

9.3. Darvin's continuous variation and De Vries' discontinuous variations

Darvin's continuous variation. Darvin’s theory of evolution was based upon the
slow variations shown by individuals of the same race. According to Darwin the he-
reditary variability which is advantageous, i.e. better adapted to the environment is
selected by nature. These variations are produced in gradual fashion and have addi-
tive effect. Small variations added up in various generations give rise to new varie-
ties. The characters produced by adding up these small variations are heritable.
De Vries' discontinuous variations. The hereditable variations according to De
Vries are not slow or gradual or continuous but sudden and discontinuous. The ap-
pearance of a short-legged lamb in the flock of a Norwegian farmer or the hornless
individuals in the breeds of horned cattle is examples of these types of mutations.
This sudden change in the genetic material (gene) giving a phenotype different from
the normal was called mutation by De Vries.
This was because they are caused by changes in a gene. Gene mutations can
either occur spontaneously in nature, as the short-legged lamb mutant mentioned
above, or can be induced artificially by certain chemicals or with radiation effects.
The spontaneous mutations also may be caused by mutagenic agents present in the
environment.
 60
Phenotypic changes and mutations. Each biochemical reaction in an organism
is catalyzed by an enzyme. Enzymes are controlled by one or more genes. If the
gene mutates, it produces a different enzyme, the result being some block in the bio-
chemical pathway or an altered pathway. This results in a different phenotype. All
mutations cannot be immediately detected. When a dominant gene mutates to its re-
cessive form, it can be detected only if it becomes homozygous in an individual.

9.4. Classification of mutations.

I. According to the level of hereditary material:
a) genomic mutations;
b) chromosomal mutations;
c) gene mutations;
II. According to the cell type:
a) somatic;
b) germinal;
III. According to the origin:
a) spontaneous mutations;
b) induced mutations.

9.4.1. Gene mutations.

A gene or point mutation occurs when the DNA sequence of a gene is altered
and the new nucleotide sequence is passed to the offspring. The change may be due
to the substitution of one or a few nucleotides for others, or to the addition or deletion
of one or a few nucleotides. Nucleotide substitution can be either transitions or
transversions.
Transitions are replacements of a purine by another purine (A by G, or vice
versa), and of a pyrimidine by another pyrimidine (C by T, or vice versa).
Transvertions are replacements of a purine by a pyrimidine, and vice versa (C
or T by either G or A, and vice versa).
Substitutions in the nucleotide sequence of a structural gene may result in
changes in the amino acid sequence of the polypeptide encoded by the gene, although
this is not always the case owing to the degeneracy of the genetic code. Consider the
triplet AAT in DNA (corresponding to UUA in messenger RNA), which codes for
the amino acid leucine. If the first A is replaced by G the triplet will still code for
leucine but if it is replaced by C it will code for valine instead. A nucleotide substitu-
tion in the DNA that results in an amino acid substitution in the corresponding poly-
peptide may or may not severely affect the biological function of the protein. Nucleo-
tide substitutions that change a triplet coding for an amino acid into a terminating tri-
plet are likely to have severe effects. If the second A in the AAT triplet is replaced
by T, the resulting triplet will code in messenger RNA for UAA, which is a ter-
 61
minator codon; the following triplets in the DNA sequence will not be translated into
amino acids. Point mutations
that result in the replacement
of one amino acid for a dif-
ferent one are called mis-
sense mutations (fig. 3);
when a triplet coding for an
amino acid changes to a ter-
minating codon, the muta-
tions are called nonsense mu-
tations (fig. 9.4).

Fig. 9.4. Missense mutation.

Fig. 9.5. Nonsense mutation.

Additions or deletions (fig. 9.5) of nucleotide pairs in the DNA sequence of a

structural gene often result in a much altered sequence of amino acids in the coded
polypeptide. The addition or deletion of one or two nucleotide pairs shifts the «read-
ing frame» of the nucleotide sequence from the point of the insertion or deletion to
the end of the molecule (fig. 9.6).
 62

Fig. 9.6. Deletion.

Fig. 9.7. Frameshift mutation.

For example, assume that a DNA segment is read as .......CAT-CAT-CAT-

CAT-CAT....... If a nucleotide base, say T, were inserted in the second position of
this segment, it would then be read as .......CTA-TCA-TCA-TCA-TCA....... The pol-
ypeptide segment corresponding to the original DNA sequence consists of five valine
amino acids; the segment corresponding to the altered sequence consists of one aspar-
tic acid and four serines. From the point of the insertion onwards the sequence of
amino acids becomes altered. However, if a total of three nucleotide pairs are either
added or deleted, the original reading frame is restored in the rest of the sequence.
Additions or deletions of nucleotide pairs in numbers other than three or multiples of
three are called frameshift mutations.
 63
Considerable progress has been made in recent years toward understanding the
causal processes of mutations. Transitions due to so called tautomeric shifts (changes
from one to another isomer) in the DNA nitrogen bases may occur. (Isomers are
chemical compounds that contain the same numbers of atoms of the same elements
but differ in structural arrangement and properties). The accuracy of base-pairing
during replication depends on the fact that the purines and pyrimidines of DNA usu-
ally exist in particular isomeric states. Occasionally, however, certain hydrogen at-
oms in the bases migrate to different unstable positions, modifying the pairing prop-
erties of the bases: C with the pairing properties of T, and vice versa; and A with the
pairing properties of G, and vice versa.
Insertion - an insertion changes the number of DNA bases in a gene by adding a
piece 9.8).

Fig. 9.8. Insertion mutation.

9.4.2. Somatic and germinal mutations.

The extent of phenotypic effect produced by a mutation depends upon various
factors like stage of the life cycle of organism at which the mutation takes place, the
type of cell in which it occurs, or whether the mutant gene is a recessive or a domi-
nant one. If the mutation occurs in a body cell of an individual, the progeny pro-
duced by that particular cell or the descendants of that cell only will inherit the
mutation and it cannot pass on to the germ cells. Such mutant cells die with the indi-
vidual. Cancer is caused by a mutation in a somatic cell. The gene regulating the divi-
sion of the cell mutates and the cell starts dividing when it should not. It replicates
repeatedly and produces many cancerous cells. The cancerous tissue thus dies with
the individual and is not inherited by the children.
 64
If a germ cell mutates, all the gametes produced by this cell will carry the mutat-
ed gene. Its expression in the next generation depends upon whether it is of dominant
or recessive form. If the mutation occurs in a gamete, only one member of the prog-
eny produced from that gamete receives the mutant gene. The example of a dominant
germinal mutation is the short-legged lamb observed by a Norwegian farmer in his
flock of sheep. This mutation has given rise to the Ancon breed of sheep by careful
breeding experiments.

9.4.3. Mutation rate. Mutable genes. Mutator genes.

The frequency of mutation is different in different genes. Genes with rapid mu-
tation rates are called mutable or unstable. Genes which do not mutate so easily are
called stable genes. In man genes associated with muscular dystrophy are estimated
to mutate once in 10 5 people. Bacterial cells mutate in the order of 1 in 10 8 and par-
ticular locus in Drosophila mutates in the order of 1 in 10 5 to 20 5 flies.
Emerson found that the R gene in maize (controls anthocyanin production) mu-
tates much more frequently than others, at the rate of 50 per 10 5 gametes. In some
organisms some genes mutate so frequently that the individuals are simply mosaics of
mutated and unmutated genes. Such mutable genes are more common in plants than
in animals and are more frequent in somatic tissues than in germ tissues.
Certain genes influence the mutation rates of other genes. Such genes are called
mutator genes. Mutator genes which control or reduce the frequency of mutation of
certain genes are known as antimutators.

9.4.5. Reverse mutations and directed mutations.

If the mutant allele changes back to the original wild type gene it is called the
reverse mutation. White-eye mutants of Drosophila can mutate back to wild type.
The frequency of forward mutation is generally higher than the frequency of reverse
mutation. Reverse mutations usually help to distinguish point mutations from large
mutational effects like deletions. A reverse mutation restores the original function of
the wild type of gene which is not possible in the case of a deletion effect.

Direct mutations. Study of mutations is easier in microorganisms, because

the spontaneous mutation rate is low in higher organisms. Bacteria are sensitive to
various antibiotics like streptomycin. If a large number of bacteria are plated on a pe-
tri-plate containing a medium that contains streptomycin, the bacteria are expected to
be killed, but a few bacteria survive, i.e. they are resistant to the antibiotic. These
bacteria give rise to colonies that are resistant to streptomycin. Now, these are two
explanations to the existence of these resistant colonies.
1) The resistant bacteria are able to adapt their physiology to the presence of
streptomycin, a case of adaptation.
2) The resistant cells are the mutants.
 65
If adaptation is considered as a case then there is one objection, why does the
adaptation persist so long? For it can be shown that the resistant cells maintain their
resistance for many generations, even in the absence of streptomycin. If adaptation is
rejected as an explanation to the appearance of resistant cells then mutation is the on-
ly alternative. If it is a case of mutation, then there are two possibilities:
1) It is a directed mutation, caused by the influence of the external agent,
streptomycin.
2) It is a spontaneous, pre-existing mutation.

9.4.6. Induced mutations. Effects of radiation (ionizing radiations, ultravio-

let rays) and chemicals.
The spontaneous mutation rate is very low at all the loci in all the organisms es-
pecially higher plants and animals. In 1927, Muller's experiments decided that the
mutation rate is much higher in the progeny of Drosophila treated with X-rays. Later
on, Muller's observations were confirmed by Stadler in barley and other workers con-
firmed it in microorganism. Soon after this, radiations were used at the most effective
tool in getting high frequency of mutations in plants, animals and microorganisms.
Radiations, X-rays, -, - and  - rays belong to the class of radiations called
ionising radiations. These can induce both gene and chromosomal mutations if
passed through biological materials and are powerful mutagens. X-rays were discov-
ered by Roentgen and have high penetrating power with a wavelength range between
10 A and 1 A. When X-rays penetrate biological material, they collide with its atoms
and release electrons. These electrons, since they are in continuous motion, collide
with other atoms, further releasing more electrons. Thus the stable atoms are trans-
formed into reactive ions. A large number of ions produced this way initiate a number
of chemical reactions. Since X-rays, -, - and  - rays all produce ions, they are
known as ionizing radiations. Ionizing radiations are produced from radioactive mate-
rials like radium, cobalt-60, etc.
Ionizing radiations are measured in terms of roentgen units (r). One roentgen
unit produces one electrostatic unit of charge in 1 cubic centimeter of air. «Rad» is
another unit by which irradiation is measured in terms of energy absorbed by the ma-
terial. Exposure to one rad produces 100 ergs of energy. Non-ionizing radiations
such as ultraviolet rays do not produce ions as they are less penetrating than ionising
radiations.

9.4.7. Effects of ionizing radiation.

1. They cause gene as well as chromosomal mutations.
2. The rate of mutations is directly proportional to the dosage.
3. The mutagenic effect is cumulative, i.e. the rate of mutation is a function of
the total amount of radiation which the organism has received. The effect of an acute
 66
X-ray dose of 6000 r is equal to the cumulative effect of three X-rays doses of 2000 r
each, spaced eight hours apart.
The entire human population is exposed to a natural radiation of the earth and
cosmic rays and the fallout radiation from the explosion of nuclear weapons. The
cumulative effect of these radiations poses a serious potential hazard for future hu-
man generations, since a newly arisen mutation has only a small probability of pro-
ducing favorable phenotype under normal environmental conditions. Therefore, any
increase in the amount of radiation to which we are exposed may produce new muta-
tions which may be deleterious to and inherited by the following generations.
Ultraviolet radiations may induce mutations by causing the formation of dimers
between identical pyrimidines adjacent in the same DNA chain. The dimerized bases
become displaced and are unable to form hydrogen bonds with the opposing purines
in the complementary chain. During DNA replication, gaps occur in the complemen-
tary chain and these gaps may then be filled with the wrong bases. Transversions as
well as transitions can result from this process.
Tautomeric shifts are transitory conditions of the DNA nitrogen bases. Stable
changes may also occur in the bases which then pair with the «wrong» bases during
replication.
Chemicals known to cause permanent alterations of the nitrogen bases are hy-
droxylamine, nitrous acid, and a variety of alkylating agents, such as mustard gas and
the epoxides (dimethyl and diethyl sulfonate, methyl and ethyl methane sulfonate,
and nitro-guanidine). Nitrous acid deaminates cytosine into uracil, which pairs with
adenine, and deaminates adenine yielding hypoxanthine which has the pairing proper-
ties of guanine. Hydroxylamine causes transitions from GC pairs to AT pairs. Alkyl-
ating agents alter the properties of the nitrogen bases, often leading to mispairing.
Base analogs are nitrogen bases resembling those normally occurring in DNA.
Instead of thymine, the analog 5-bromouracil may be incorporated, but it undergoes
tautomeric shifts and may pair with guanine upon replication. The base analog 2-
aminopurine is a purine that may pair with either cytosine or thymine. Some aro-
matic compounds, known as acridines, cause additions and deletions of one or to
more than 20 nitrogen bases in the DNA.
Acridines become intercalated between the stacked base pairs of the double he-
lix, but the process by which they result in insertions and deletions is not well known.
One model suggests that the acridines stabilize DNA «buckles» formed by dimers of
adjacent bases in the same chain. Insertions or deletions may occur during DNA rep-
lication whenever buckles exist in one of the chains.

9.5. DNA Repair.

DNA of all organisms is constantly under the tension of the internal and external
environment and is always at the risk of being damaged. The factors likely to dam-
age the genetic material include the nicking enzymes or the nucleases. Breaks may
 67
occur while the relaxed chromatin threads are being packed into metaphase chromo-
somes or breaks may occur in phage genetic material when it is being packed into the
phage head. Cellular chemicals, heat, UV light, sunlight, atmospheric pollutants
which may include various harmful gases can affect the integrity and survival of the
genetic material. The presences of all these damaging factors necessitate the exist-
ence of a DNA repair mechanism in the living organisms.
The DNA repair mechanism may operate at two stages, i.e. pre-replication and
post-replication. The pre-replication mechanism includes photoreactivation and exci-
sion repair.
Photoreactivation (fig. 8). Kelner discovered that the UV effect on genetic ma-
terial can be reversed before the genetic material is permanently affected. This can be
done by exposing the cells after UV treatment to visible light. This repair phenome-
non is called photoreactivation. This effect is observed is lower organisms like bacte-
ria, protozoans, algae, etc.
According to Setlow and Setlow, there are specific enzymes which split the di-
mers of pyrimidine bases produced by UV, thus restoring the normal DNA helix.
The enzyme responsible for converting the thymine dimer into two thymine mono-
mers is known as the photoreactivating enzyme. The enzyme absorbs a photon of vis-
ible light before breaking the dimer and hence is so named, although it can bring
about the conversion of a dimer into monomers in the dark. The enzyme is present in
all organisms including man.
People suffering from a hereditary disease called xeroderma pigmentosum, a
skin sensitivity to sunlight, are deficient in this enzyme.

Fig. 9.9. Photoreactivation.

 68
Excision repair (fig. 9.9). In this repair mechanism, the entire dimer and some
adjacent sequences are removed from the DNA strand and the gap is accurately filled
by the action of DNA polymerase followed by sealing by the enzyme ligase. An en-
donuclease, recognizing the distortion of the DNA strand due to the presence of a
thymine dimer introduces a nick in the dimer-containing area. The exonuclease then
digests that portion of the strand. DNA polymerase then synthesizes a piece of DNA
to be inserted in the gap produced by exonuclease. The synthesis is dictated by the
DNA template. The two broken ends are then joined by the enzyme ligase.

Fig. 9.10. Excision repair.

Post-replication repair. The post-replication repair mechanism has been studied

in Escherichia coli, and is equally applicable in higher organisms. Photoreactivation
and excision repair mechanisms, although efficient, still leave some thymine dimers
in the DNA strand. This demands the occurrence of a post-replication repair mecha-
nism. The replication of DNA strand with dimer skips this portion and recommences
on the other side, thus producing gaps in the daughter strand. These gaps are filled in
by DNA synthesis directed by the polymerase. The filled gaps are thus joined to the
main DNA strand on either side by the action of the enzyme - ligase. It is noted that
the gaps are filled in by DNA - synthesizing enzyme that does not require template
DNA for its activity. This post-replication system is error prone and may produce
mistakes that are frequently named as mutations. These mutations are of the transition
and transversion types. This repair mechanism may also cause deletions and additions
of bases which in turn cause mutations.
 69

The repair mechanisms are the result of biological adaptation during the evolu-
tionary process. If repair mechanism did not exist, there would not have been any life
on the earth which is being constantly exposed to sunlight and gaseous pollutants.
Pho-toreactivation seems to be the first developed repair mechanism followed by the
excision and the post-replication repair mechanisms. More than one repair system en-
sures the neutralization on harmful effects of UV and other mutagens. In case one
mechanism fails, the other can take over.

CHAPTER 10. Human Being as an Object of Genetic Analysis.

10.1. The deficiencies and advantages of human genetics.
The recognition of the role of genetic factors in the causation of human disease
has made clinical genetics one of the most rapidly developing fields in medicine. Im-
portant genetic contributions to the etiology of major diseases such as coronary artery
disease, diabetes mellitus, hypertension, and the major psychoses have been identi-
fied. At the same time, there has been a veritable explosion of knowledge in basic ge-
netics. Much of this progress has been propelled by recent advances in the area of
molecular genetics, which in turn have been applied directly to a better understanding
of the pathogenesis of disease and to improved diagnosis and management of pa-
tients. Appropriately, a major contribution of these new developments in genetics has
been in the area of prevention and/or avoidance of disease, the aspect of medicine
that must become the focus of modern medicine. Genetic screening programs to de-
tect individuals at risk, genetic counseling, and prenatal diagnosis are some of these
current applications of new genetic knowledge to medical practice.
The main deficiencies of human genetics study are following:
1) the great number of groups of linkage (karyotype);
2) impossibility of crossing;
3) slow change of generations;
4) slight number of progeny (offspring) in every family.
The advantages of human genetics are:
1) high level of studying of human biology, biochemistry, physiology etc.;
2) the large size of population in the earth (more than 5 milliards) in all the
territorial zones with the varieties of gene pool (normal and unnormal signs).

10.2. Genealogical method.

The classical method of performing a genetic test is by constructing a pedigree
in which the phenotypes of a line of ancestors are recorded.
Genealogical method allows geneticists to:
1. Determine the mode of transmission of a hereditary trait (dominant or reces-
sive, autosomal or sex-linked);
 70
2. Establish accurate or probable genotypes of the individuals in a family tree;
3. Estimate the probability for a couple to have a child with a certain hereditary
trait.
This is typically done with a family tree that shows the phenotype of each indi-
vidual; such a diagram is called a pedigree. An important application of probability
in genetics is its use in pedigree analysis.

10.2.1. The pedigree symbols.

Females and males are by convention represented by circles and squares, re-
spectively, and a diamond is used if the sex of an individual is unknown. Individuals,
having the phenotype of interest are indicated by shaded symbols.
The symbols of parents are joined by a horizontal line, which is connected verti-
cally to a second horizontal line that extends above the symbols for their offspring.
The offspring of two parents
are called siblings, or sibs, regard-
less of sex, and are represented
from left to right in order of their
birth.
Successive generations in a
pedigree are designated by Roman
numbers and the individuals in a
generation by Arabic numbers.
Twins are indicated by diagonal
lines, which converge at the hori-
zontal line above the sibship in the
case of non-identical (dizygotic)
twins or at the end of a short ver-
tical line from the sibship line for
10.1. The pedigree symbols. identical (enzygotic) twins (fig.
10.1).

Example: Pedigree of a human family showing the inheritance of albinism.

Next figure shows a simple pedigree that is easily analyzed.

Two affected individuals are present among the offspring of unaffected parents.
In such a situation a reasonable interpretation is that both parents carry the determin-
 71
ing allele (they are both heterozygous) and that the trait of interest is determined by
the homozygousity of the recessive allele.
A pedigree is not always interpretable, for example, the one in panel.

Since one parent carries the trait, there are two possibilities: 1) the affected fa-
ther is homozygous recessive, the mother is heterozygous, and the trait is inherited as
a homozygous recessive, or 2) the affected father is heterozygous, the mother is ho-
mozygous recessive, and the trait is inherited as a dominant.
Pedigree analysis in humans is frequently concerned with traits, such as albi-
nism, that are quite rare in the population. Thus it is quit unlikely that both parents
would carry the determining allele. The recessive inheritance of a rare condition is
indicated by several features of a pedigree. Specifically, the trait usually does not ap-
pear in every generation, it may reappear in a family after being absent in several
previous generations.
Children with the trait usually will have phenotypically normal parents, who
must therefore be heterozygous. Such a parent is often termed a carrier. As expected
from the pedigree 1 albinism is inherited as a recessive. In the infrequent case in
which both parents are affected, all of their offspring will be affected.
If a rare trait is inherited as a dominant, most affected individuals will be hetero-
zygous. Most matting of these individuals will be with homozygous recessive par-
ents and result in about equal numbers of affected and normal offspring. Thus, the
trait will appear in every generation, though some lines of descent may lack the trait
if by chance only normal offspring occur. In the case of dominant inheritance, the
trait will never be seen among the offspring of normal parents.

10.3. Population method.

Application of genetic principles to entire populations of organisms constitutes
the subject of population genetics.
The term population refers to a group of organisms of the same species living
within a prescribed geographical area, crossing to each other and produce the off-
spring. Such a group of interbreeding individuals is called a population.
Within a population a complete set of genetic information carried by the indi-
viduals is called as a gene pool. This pool includes not only the genes but all alleles
present in a population.
 72
10.3.1. The Hardy-Weinberg equilibrium.
For the medical geneticist, the most important concept in population genetics is
the Hardy-Weinberg equilibrium independently described in 1908 by the English
mathematician Y.H. Hardy and the German physician W.Weinberg. It was derived
to explain why dominant traits do not automatically replace recessive traits in the
population. Its utility for medical genetics, however, is in explaining why, in a
large population with random mating, allele frequencies do not change from gener-
ation to generation and how, for any genetic locus, the genotype frequencies
are determined by the relative frequencies of the alleles at that locus.
Consider a single autosome locus with two alleles (A and a), whose population
frequencies in both sperm and eggs are p = frequency of allele A, and q = fre-
quency of allele a. Because there are only two alleles, p + q = 1. Random, mating
without regard to the genotype is mathematically equivalent to random, mixing and
union of the sperms and eggs.
The genotype frequencies in the progeny are thus:
genotypes: AA, Aa, aa
frequency: p, 2pq, q

In the next generation, each of the three paternal genotypes can mate with each
of the three maternal genotypes. Thus the frequency of each genotype (AA, Aa and
aa), is stable over successive generation and the population is said to be in Hardy-
Weinberg equilibrium.
Of course, this principle depends upon certain assumptions of ideal population:
1. Mating in random.
2. Allele frequencies are the same in males and females.
3. The genotypes are all equal in viability and fertility (that is selection does not
occur).
4. Mutation does not occur.
5. Migration into population does not occur.
6. The population is sufficiently large that the frequencies of alleles will not
change from generation because of chance.
The most important medical application of the Hardy- Weinberg equilibrium
is the determination of allele frequency and heterozygote carrier frequency in a
population for which the frequency of a trait is known.
For example, cystic fibrosis occurs in approximately 1/2000 whites of Northern
and central European origin. Thus q2, or the frequency of homozygous affected indi-
viduals, is 1/2000, and q/ the frequency of the mutant allele is 2000 = 1/45 or 0.022.
The frequency of the normal allele is p = (1-q) = 44/45 or 0,978. The heterozygote
carries frequency is 2pq = 2x 44/45 x 1/45 = 1/23 or 0,044. Thus, more than 4% of
whites are heterozygous for the cystic fibrosis allele, and fact of considerable im-
portance in genetic counseling of families with cystic fibrosis.
 73

10.4. Biochemical method.

Biochemical method is used for indicating the hereditary diseases of metabo-
lism. Sir Archibald Garrod's pioneering studies on alkaptonuria established the field
of biochemical genetics and introduced the concept of the “inborn of metabolism”.
There are many biochemical methods for investigation of errors of metabolism
of amino acids, monosaccharide, lipids, proteins, etc.:
- chromatography;
- electrophoresis;
- mass-spectrography;
- hel-filtration, etc.

10.5. Cytogenetic method.

Cytogenetic method is used for indicating of the chromosomal diseases, which
are connected with genomic and chromosomal mutations. Metaphase chromosomes
are examined under microscope after the process of cultivating of peripheral human
blood lymphocytes and using of special staining. Chromosomes become unlike each
other and every (even micro-) aberrations become discovered.

10.6. Twins method.

Twins method is based on the signs of monozygotic and dizygotic twins.
Concordance - similarity of twins in analyzing sign (hair color, nose form, eye-
brow form, external ear form). It is typical for monozygotic twins.
Discordance is typical for dizygotic twins. Both characteristics are used in
studying of hereditability. Coefficient of heredity is calculated by the formula:
%Ec - %Dc
Ha = -----------------
100% - %Dc
Ec – concordance of the monozygotic twins
Dc – concordance of the dizygotic twins
If Ha approximately equal to 1, it means the most contribution of genotype; if Ha
is less than 0,5 till 0, it means the environmental contribution. This method is im-
portant for changing of possible additional pathological environmental signs in some
syndromes.

10.7. Molecular-genetic methods.

Molecular-genetic method is used for indicating of the hereditary diseases in
DNA level (identification of gene mutations).
 74

CHAPTER 11. INTRODUCTION TO MEDICAL GENETICS.

Human genetics is a science of biological variation, one can say that is the sci-
ence of biological variation in humans, and medical genetics is the science of human
biologic variation as it relates to health and disease. Clinical genetics is the part of
medical genetics concerned with the health of individual humans and their families.
It can be argued that medical genetics is a more extensive discipline than human ge-
netics because it is impossible to think of any aspect of human genetics that is not
relevant to medical genetics, but the converse is not true.
Indeed, medical genetics might be defined as “all of medicine that is genetic and
all of genetics that is medical”. Another definition of clinical genetics is, “the science
and practice ('art') of the diagnosis, prevention, and treatment of genetic disease”.

11.1. The human hereditary diseases and their classification.

Genetically determined diseases are often classified into:
a) gene diseases are due to single mutant genes with a large effect on the pa-
tient's health;
b) chromosomal diseases can result from either a variation in the chromosome
number or from structural changes;
c) multifactorial diseases are caused by a combination of environmental fac-
tors and mutations in multiple genes.

11.2. Human gene diseases.

Single gene disorders account for approximately 5-10% of pediatric hospital
admissions and childhood mortality. As might be expected, single gene disorders are
inherited in a simple Mendelian fashion and are also referred to as Mendelian diseas-
es. Some 3000 distinct disorders are now known or suspected to be single gene dis-
eases inherited in autosomal dominant, autosomal recessive or X-linked (dominant or
recessive) fashion.
Although many single gene disorders are rare, others are common and pose ma-
jor health problems. Familial hyperholesterolemia with its attendant high risk of
premature coronary artery disease occurs in 1 in 500 individuals.

11.2.1. Autosomal recessive gene disorders.

Sickle cell anemia. Sickle cell anemia was the first genetic disease to be defined
at the molecular level, and its study serves as a model for the application of modern
molecular genetic analysis to clinical disease. Single gene disorders have thus far
proven to be the area in which advances in molecular genetics have made the major
contribution to understanding and managing disease.
 75
Sickle-cell disease or sickle-cell anemia is a blood disorder characterized by
red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the
cells' flexibility and results in their restricted movement through blood vessels, de-
priving downstream tissues of oxygen. The disease is chronic and lifelong: individu-
als are most often well, but their lives are punctuated by periodic painful attacks and
a risk of various other complications. The disease occurs in individuals who are ho-
mozygous for a mutant hemoglobin gene.

Scheme of normal RBC Microphoto of normal RBC

and sickle cells and sickle cells

Fig. Human blood cells in norm and with sickle-cell disease.

Symptoms include chronic anemia,

shortness of breath, fever, and episodic
"crises" (severe pain in the abdomen,
bones, or muscles, in the joints, leg ulcers,
and jaundice). Life expectancy is short-
ened, with older studies reporting an aver-
age life expectancy of 42 and 48 years for
males and females, respectively. Sickle-
cell disease may lead to various acute and
chronic complications, several of which
are potentially lethal.
Frequency. It occurs in 1 in every
500 African Americans; the prevalence is
close to 1 in 2,000 for Hispanics whose
families came from the Caribbean, Central America, or South America. Even though
the sickle cell mutation began in India and Africa, it is now present throughout the
world. The disease is most frequently found in sub-Saharan Africa. It is also found in
some parts of Sicily, Greece, southern Turkey, and India, all of which have areas in
which malaria is endemic.
Type of inheritance – autosomal-recessive.
 76

Normal Red Blood Cells and Sickle Cells

Figure A shows normal red blood cells flowing freely in a

blood vessel. The inset image shows a cross-section of a normal red
blood cell with normal hemoglobin. Figure B shows abnormal, sick-
led red blood cells clumping and blocking blood flow in a blood ves-
sel. (Other cells also may play a role in this clumping process.) The
inset image shows a cross-section of a sickle cell with abnormal he-
moglobin forming abnormal strands.

Neurofibromatosis is characterized by
variable expression, pleiotropy and heterogeneity
which are both allelic and non-allelic. The dis-
covery of the NF1 gene has shed light on tumour
formation both in NF1 and in general.
The neurofibromatoses are genetic disor-
ders that cause tumors to grow in the nervous
system. Frequency is 1 in every 3,000–4,000
live births; type of inheritance is autosome-
dominant.

Multiple skin neurofibromas Typical café au lait spot

LLisch nodules

The tumors begin in the supporting cells that make up the nerves and the mye-
lin sheath - the thin membrane that envelops and protects the nerves. These disorders
cause tumors to grow on nerves and produce other abnormalities such as skin changes
and bone deformities. Although many affected persons inherit the disorder, between
 77
30 and 50 percent of new cases arise spontaneously through mutation in an individu-
al's genes. Once this change has taken place, the mutant gene can be passed on to
succeeding generations. Scientists have classified the disorders as neurofibromatosis
type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis, a type that was
once considered to be a variation of NF2. NF1 is the more common type of the
neurofibromatoses. In diagnosing NF1, a physician looks for changes in skin appear-
ance, tumors, or bone abnormalities, and/or a parent, sibling, or child with NF1.
Symptoms of NF1, which may be evident at birth and nearly always by the
time the child is 10 years old, may include light brown spots on the ski ("cafe-au-lait"
spots), two or more growths on the iris of the eye, a tumor on the optic nerve, a larger
than normal head circumference, and abnormal development of the spine, a skull
bone, or the tibia.
NF2 is less common and is characterized by slow-growing tumors on the
eighth cranial nerves. The tumors cause pressure damage to neighboring nerves. To
determine whether an individual has NF2, a physician looks for eighth nerve tumors,
cataracts at an early age or changes in the retina that may affect vision, other nervous
system tumors and similar signs and symptoms in a parent, sibling, or child.
The distinctive feature of schwannomatosis is the development of multiple
schwannomas (tumors made up of certain cells) everywhere in the body except on the
vestibular branch of the 8th cranial nerve. The dominant symptom is pain, which de-
velops as a schwannoma enlarges or compresses nerves or adjacent tissue. Some
people may develop numbness, tingling, or weakness in the fingers and toes.

Cystic fibrosis (also known as CF, mucovoidosis, or mucoviscidosis) is char-

acterized by striking molecular heterogeneity, with the most common DF508 mutation
showing linkage disequilibrium, an observation consistent with a single original mu-
tation and heterozygote advantage.
Frequency. Cystic fibrosis affects 1 in 2500 whites.
Type of inheritance is autosome-recessive.
Cystic fibrosis is a hereditary disease affecting the exocrine (mucus) glands of
the lungs, liver, pancreas, and intestines, causing progressive disability due to multi-
system failure.
Thick mucus production results in frequent lung infections. Diminished secre-
tion of pancreatic enzymes is the main cause of poor growth, greasy stools, and defi-
ciency in fat-soluble vitamins. Males can be infertile due to the condition congenital
bilateral absence of the vas deferens. Often, symptoms of CF appear in infancy and
childhood. Meconium ileus is a typical finding in newborn babies with CF.
Individuals with cystic fibrosis can be diagnosed prior to birth by prenatal ge-
netic testing. Newborn screening tests are increasingly common and effective (alt-
hough false positives may occur, and children need to be brought in for a sweat test to
distinguish disease vs carrier status). The diagnosis of CF may be confirmed if high
levels of salt are found during a sweat test, although some false positives may occur.
There is no cure for CF, and most individuals with cystic fibrosis die young:
many in their 20s and 30s from lung failure. However, with the continuous introduc-
 78
tion of many new treatments, the life expectancy of a person with CF is increasing to
ages as high as 40 or 50. Lung transplantation is often necessary as CF worsens.

Manifestations of cystic fibrosis

Clubbing of the fingersin a person with cystic fibrosis The scheme of mucus blockage

We'll discuss these disorders on the example of phenylketonuria. Children with

phenylketonuria (PKU), if untreated, are severely mentally retarded, frequently
have convulsions, and in the past were often placed in institutional care. In these
children, the particular enzyme necessary for the conversion of phenylalanine hy-
droxylase (PAH) is deficient; that is, there is a ‘genetic block' in the metabolic path-
way.
 79

Patients with phenylketonuria

This was, in fact, the first genetic disorder in humans shown to be due to a specific
enzyme defect by Jervis in 1953. As a result of the enzyme defect, phenylalanine ac-
cumulates and some is converted into phenylpyruvic acid and other metabolites
which are excreted in the urine. The enzyme block leads to a deficiency of tyrosine
with a consequent reduction in melanin formation. Affected children therefore often
have blond hair and blue eyes and areas of the brain which are usually pigmented,
e.g., the substantia nigra, can also lack pigment.

Biochemical effects of phenylketonuria.

Treatment. Bickel suggested that phenylalanine could be removed from the diet,
which has proved to be an effective treatment. If PKU is detected early enough in
childhood, mental retardation can be prevented by giving a diet containing a re-
stricted amount of phenylalanine. Phenylalanine is, however, an essential amino acid
and therefore cannot be entirely removed from the diet. By monitoring the level of
 80
phenylalanine in the blood, it is possible to supply sufficient amounts to meet normal
requirements and avoid levels which result in mental retardation.
Diagnosis. (Screening programs). PKU can be screened for by tests which de-
tect the presence of the
metabolite of phenylal-
anine, phenylpyruvic
acid, in the urine by its
reaction with ferric
chloride or through ele-
vated levels of phenyl-
alanine in the blood in
the Guthrie test. The
latter test involves tak-
ing blood samples from
children in the first
week of life and com-
paring the amount of
growth induced by the
sample with standards
in a strain of the bacte-
ria Bacillus subtilis
which requires phenylalanine for growth. This technique has been replaced in most
centers by use of direct fluorescent or immunological assays of phenylalanine levels.
Galactosemia is a rare disorder that affects the body's ability to break down a
food sugar called galactose (found in milk and other dairy products). Galactosemia
was first discovered in 1908 by the physician Von Ruess.
Normally, the body breaks down lactose into galactose and then into glucose (a
sugar used for energy). People with galactosemia are missing an enzyme called
GATL (galactose-1-phosphate uridyl transferase), which normally converts galactose
into glucose. Without this enzyme, harmful amounts of galactose build up in the
blood.
The most common form of the disorder, classic galactosemia, is passed down
in an autosomal recessive pattern. Classical galactosemia affects 1 in every 55,000
newborns.

Cataract A child with galactosemia

 81

To get the disorder, a child must inherit one defective gene from each parent.
Inheriting one normal gene and one mutated gene makes a person a carrier. A carrier
produces less of the GALT enzyme than normal, but is still able to break down glu-
cose and avoid having symptoms of galactosemia. However, carriers can still pass on
the mutated gene to their children.
The build-up of galactose in the body can cause several severe symptoms: kid-
ney failure, an enlarged liver, cataracts (clouding of the eye lens), poor growth, and
mental retardation.
People can inherit a milder form of the disorder when a different gene, also in-
volved in galactose metabolism, is mutated. These patients often suffer from cata-
racts, but not the other symptoms associated with classical galactosemia.
In most countries, babies are tested for galactosemia at birth. Using a tiny
blood sample taken from the baby's heel, the test checks for low levels of the GALT
enzyme. This allows for prompt treatment, which can substantially prevent the seri-
ous symptoms of this disorder.
For those families with a history of the disorder, a doctor can determine during
a woman's pregnancy whether her baby has galactosemia 1) by taking a sample of
fluid from around the fetus (amniocentesis), or 2) by taking a sample of fetal cells
from the placenta (chorionic villus sampling or CVS).
Newborn infants with galactosaemia present with vomiting, lethargy, failure
to thrive and jaundice in the second week of life. If untreated, they go on to develop
complications which include severe mental retardation, cataracts and cirrhosis of the
liver.

Clinical manifestations of galactosemia

 82
Galactosaemia can be screened for by the presence of reducing substances in
the urine which can be confirmed on further testing to be galactose. A definitive di-
agnosis of galactosaemia is achieved by demonstration of a deficiency of the enzyme
galactose-1-phosphate uridil transferase, necessary for the metabolism of the sugar,
galactose. The only way to treat galactosemia is through dietary restrictions. People
with the disorder must stay away from foods and drinks containing galactose, includ-
ing milk, cheese, and legumes (dried beans). The complications of galactosaemia can
be prevented by feeding affected infants commercially available milk substitutes
which do not contain galactose or lactose, the sugar found in milk which is broken
down into galactose. Early diagnosis and treatment are essential if the severe compli-
cations are to be prevented.

11.2.2. Autosomal dominant gene disorders.

Only one mutated copy of the gene will be necessary for a person to be affected
by an autosomal dominant disorder. Each affected person usually has one affected
parent. There is a 50% chance that a child will inherit the mutated gene. Conditions
that are autosomal dominant often have low penetrance, which means that although
only one mutated copy is needed, a relatively small proportion of those who inherit
that mutation go on to develop the disease. Examples of this type of disorder
are Huntington's disease, Neurofibromatosis 1, Marfan Syndrome, Hereditary
nonpolyposis colorectal cancer, and Hereditary multiple exostoses, which is a highly
penetrant autosomal dominant disorder. Birth defects are also called congenital
anomalies.
Huntington's disease, chorea, or disorder (HD), is an incurable neurodegenera-
tive genetic disorder that affects muscle coordination and some cognitive functions,
typically becoming noticeable in middle age. It is the most common genetic cause of
abnormal involuntary writhing movements called chorea. It is much more common in
people of Western Europe descent than in those from Asia or Africa. The disease is
caused by a dominant mutation on either of the two copies of a specific gene, located
on chromosome 4. Any child of an affected parent has a 50% chance of inheriting the
disease. In rare situations where both parents have an affected gene, or either parent
has two affected copies, this chance is greatly increased. Physical symptoms of Hun-
tington's disease can begin at any age from infancy to old age, but usually begin be-
tween 35 and 44 years of age. On rare occasions, when symptoms begin before about
20 years of age, they progress faster and vary slightly, and the disease is classified
as juvenile, akinetic-rigid or Westphal variant HD.
The Huntingtin gene normally provides the genetic code for a protein that is also
called "huntingtin". The mutation of the Huntingtin gene codes for a different form of
the protein, whose presence results in gradual damage to specific areas of the brain.
The exact way this happens is not fully understood. Genetic testing, which has been
possible since the discovery of the mutation, can be performed before the onset of
symptoms in the relatives of an affected individual, as an antenatal test, and also on
test-tube embryos, raising ethical debates. Genetic counseling has developed to in-
 83
form and aid individuals considering genetic testing and has become a model for oth-
er genetically dominant diseases.
The exact way HD affects an individual varies and can differ even between
members of the same family, but the symptoms progress predictably for most indi-
viduals. The earliest symptoms are a general lack of coordination and an unsteady
gait. As the disease advances, uncoordinated, jerky body movements become more
apparent, along with a decline in mental abilities and behavioral and psychiatric prob-
lems. Physical abilities are gradually impeded until coordinated movement becomes
very difficult, and mental abilities generally decline into dementia. Although the dis-
order itself is not fatal, complications such as pneumonia, heart disease, and physical
injury from falls reducelife expectancy to around twenty years after symptoms begin.
There is no cure for HD, and full-time care is often required in the later stages of the
disease, but there are emerging treatments to relieve some of its symptoms.
Self-help support organizations, first founded in the 1960s and increasing in
number, have been working to increase public awareness, to provide support for indi-
viduals and their families, and to promote research. These organizations were instru-
mental in finding the gene in 1993. Since that time there have been important discov-
eries every few years and understanding of the disease is improving. Current research
directions include determining the exact mechanism of the disease, improving animal
models to expedite research, clinical trials of pharmaceuticals to treat symptoms or
slow the progression of the disease, and studying procedures such as stem
cell therapy with the goal of repairing damage caused by the disease.

11.2.3. X-linked recessive gene disorders.

Duchenne and Becker muscular dystrophy emphasise the importance and
difficulties of carrier detection and demonstrate how disturbance or maintenance of
the reading frame provides an explanation for the relationship between genotype and
phenotype. Muscular dystrophies are a group of genetic conditions characterized by
progressive muscle weakness and wasting (atrophy). The Duchenne and Becker types
of muscular dystrophy primarily affect the skeletal muscles, which are used for
movement, and the muscles of the heart.
Frequency. These conditions occur much more frequently in males than in fe-
males. Duchenne and Becker muscular dystrophies together affect 1 in 3,500 to
5,000 newborn males.
Type of inheritance is X-linked recessive pattern.
Duchenne and Becker muscular dystrophies have similar signs and symptoms
and are caused by different mutations in the same gene. The two conditions differ in
their severity, age of onset, and rate of progression. In people with Duchenne muscu-
lar dystrophy, muscle weakness tends to appear in early childhood and progress rap-
idly. Affected children may have delayed motor skills, such as sitting, standing, and
walking. They are usually wheelchair-dependent by adolescence. The signs and
symptoms of Becker muscular dystrophy are usually milder and exhibit a large range
 84
of variation. In most cases, muscle weakness becomes apparent later in childhood or
adolescence and progresses at a much slower rate.

A patient with muscular dystrophy

Muscular dystrophy inheritance

Both the Duchenne and Becker forms of muscular dystrophy are associated
with a heart condition called dilated cardiomyopathy. This form of heart disease en-
larges and weakens the heart (cardiac) muscle, preventing it from pumping blood ef-
ficiently. Dilated cardiomyopathy progresses rapidly and is life-threatening in many
cases. In people with Duchenne muscular dystrophy, the signs and symptoms of car-
diomyopathy typically appear in adolescence. The onset of cardiomyopathy in people
with Becker muscular dystrophy is later, usually in early to mid-adulthood.

11.2.4. X-linked dominant gene disorders.

X-linked dominant disorders are caused by mutations in genes on the X chromo-
some. Only a few disorders have this inheritance pattern, with a prime example being
X-linked hypophosphatemic rickets. Males and females are both affected in these
disorders, with males typically being more severely affected than females. Some X-
linked dominant conditions such as Rett syndrome, Incontinentia Pigmenti type 2
and Aicardi Syndromeare usually fatal in males either in utero or shortly after birth,
and are therefore predominantly seen in females. Exceptions to this finding are ex-
tremely rare cases in which boys with Klinefelter Syndrome (47,XXY) also inherit an
X-linked dominant condition and exhibit symptoms more similar to those of a female
in terms of disease severity. The chance of passing on an X-linked dominant disorder
differs between men and women. The sons of a man with an X-linked dominant dis-
order will all be unaffected (since they receive their father's Y chromosome), and his
daughters will all inherit the condition. A woman with an X-linked dominant disorder
has a 50% chance of having an affected fetus with each pregnancy, although it should
be noted that in cases such as Incontinentia Pigmenti only female offspring are gener-
ally viable. In addition, although these conditions do not alter fertility per se, individ-
uals with Rett syndrome or Aicardi syndrome rarely reproduce.
 85
11.2.5. Y-linked gene disorders.

Neurofibromatosis, cystic fibrosis and Duchenne muscular dystrophy are all

serious conditions with implications for the extended family. In each of these disor-
ders there has been rapid progress over the last decade. The cloning of the prevalent
genes, identification of their mutational basis and isolation of their protein products
serve to illustrate important genetic principles and represent major scientific
achievements.
The genes and their protein products have been identified for all three of these
disorders, thereby opening up new opportunities and strategies for treatment includ-
ing the possibility of effective gene therapy and prevention with the help of prenatal
diagnosis. Positional cloning techniques have been successful in mapping and cloning
the NF1, CFTR and DMD genes for many other important single gene disorders. The
isolation of these genes has enhanced the prospect of successful gene therapy and
prevention of the birth of children with such disorders.

Hereditary diseases of metabolism

Inborn errors of metabolism comprise a large class of genetic diseases involving
disorders of metabolism. The majority are due to defects of single genes that code for
enzymes that facilitate conversion of various substances (substrates) into others
(products). In most of the disorders, problems arise due to accumulation of substanc-
es which are toxic or interfere with normal function, or to the effects of reduced abil-
ity to synthesize essential compounds. Inborn errors of metabolism are now often re-
ferred to as congenital metabolic diseases or inherited metabolic diseases.
Most inborn errors of metabolism are inherited in an autosomal recessive or X-
linked manner with only a few being inherited in an autosomal dominant manner.
This is because the defective protein in most inborn errors is an enzyme which is dif-
fusible and there is sufficient residual activity in the heterozygous state for it to func-
tion as normal in most situations. If, however, the reaction catalyzed by an enzyme is
rate-limiting or the gene product acts as an inhibitor of other reactions, the disorder
can manifest in the heterozygous state, i.e., be dominantly inherited.
Traditionally the inherited metabolic diseases were categorized as disorders of
carbohydrate metabolism, amino acid metabolism, organic acid metabolism, or
lysosomal storage diseases. In recent decades, hundreds of new inherited disorders of
metabolism have been discovered and the categories have proliferated.
Thus, principles of genetic counseling of hereditary disorders of metabolism
are directed firstly prevention of the development of disease after the birth of babies
with such anomalies; that's why only earlier diagnosis and treatment (diet) may help
to prevent hereditary diseases of metabolism.

11.3. Chromosomal diseases.

These diseases are the result of the addition or deletion of entire chromosomes
or parts of chromosomes.
 86
Aneuploidy is the gain or loss of individual chromosomes from the normal dip-
loid set of forty-six chromosomes. As in structural anomalies, the error may be pre-
sent in all cells of a person or in a percentage of cells. Changes in chromosome num-
ber generally have an even greater effect upon survival than changes in chromosome
structure. Considered the most common type of clinically significant chromosome
abnormality, it is always associated with physical and/or mental developmental prob-
lems. Most aneuploid patients have a trisomy of a particular chromosome.
Monosomy, or the loss of a chromosome, is rarely seen in live births. The vast major-
ity of monosomic embryos and fetuses are probably lost to spontaneous abortion dur-
ing the very early stages of pregnancy. An exception is the loss of an X chromosome,
which produces Turner's syndrome. Trisomy may exist for any chromosome, but is
rarely compatible with life.
Aneuploidy is believed to arise from a process called nondisjunction. Nondis-
junction occurs when chromosomes do not separate correctly during meiosis. The di-
rect result is that one gamete will have an extra chromosome and the other will be
lacking a chromosome. When these gametes are fertilized by a normal gamete, they
have either an extra chromosome (trisomy) or are missing a chromosome
(monosomy).
Four types of structural changes may occur in chromosomes: duplications, dele-
tions, translocations, and inversions. All may result when there is breakage of the
chromosomes and a rejoining or loss of chromosome fragments. If the same broken
ends rejoin, the chromosome becomes intact once again. The resulting effects of such
events depend on how large they are and where they occur on the chromosome. Rear-
rangements may occur in many forms and are less common than abnormalities of
chromosome number.
The most common type of rearrangement is called a balanced translocation be-
cause the amount of genetic information within that cell is normal even though it is
repositioned. Therefore the individual with a balanced translocation may appear nor-
mal. However, there will be a risk to the children of a carrier of a balanced transloca-
tion since that person is likely to produce unbalanced gametes (bearing too little or
too much genetic information), and therefore the risk of having abnormal offspring is
increased. Rearrangements such as aneuploidy may be found in all cells of an indi-
vidual, or they may occur only in a percentage of an individual's cells. This latter
condition is known as mosaicism. In general, mosaic individuals show a less severe
expression of their syndrome than those with chromosome abnormalities in all their
cells.
Most major chromosome disorders are characterized by growth retardation,
mental retardation, and a variety of somatic abnormalities. The loss of gain of whole
chromosomes other than the sex chromosomes is often incompatible with survival,
and such abnormalities are a major cause of spontaneous abortions or miscarriages.
Clinically significant chromosome abnormalities occurring nearly 1% of liveborn ba-
bies and account for about 1% of pediatric hospital admissions and 2.5% of child-
hood deaths. Major chromosomal anomalies are found in almost half of spontaneous
abortuses. Since approximately 15% of recognized pregnancies end in a miscarriage
 87
and it is estimated that 50% of conceptions may suffer from major chromosome prob-
lems.

Classification of chromosomal diseases.

Chromosomal diseases may be classified as follows:
1) Sex chromosome abnormalities in man;
2) Autosomal abnormalities in man;
3) Syndroms involving chromosome mutations.

Sex chromosome abnormalities in man.

Klinefelter syndrome was the first of all described by Klinefelter (1942). Barr
(1956) showed that the patients with this syndrome are chromatin positive and Jacobs
and Strong (1959) demonstrated that these people have 47,XXY chromosome num-
ber in their somatic cells, i.e., they possess two X-chromosomes and a Y-
chromosome. It occurs 1 per 1000 male live births. Most of their characters are due to
the intermediacy of the sex. These are enlargement of the breasts (gynecomastia),
feminine fat deposits and female distribution of abdominal and facial hair. In a popu-
lation the persons of this syndrome can be detected by their mental subnormality or
infertility.

Ap-
pearance of
patient
with
Klinefelter syndrome Gynecomastia

Karyotype of patient
with Klinefelter syn-
drome:
47,XXY.
(Archives of Kursk re-
gional hospital Medico-
genetical consultation,
2007)

Clinical manifestations
of Klinefelter syndrome
 88
Turner syndrome was first described by Turner (1938) in individuals who were
chromatin negative and phenotypically females with 45 (45,X) somatic number of
chromosomes. The frequency is about 1/5000 in liveborn girls.

Appearance of patient Low hairline

with Turner syndrome

Clinical manifestations
of Turner syndrome

Turner syndrome karyogram: 45,X

This is the only total monosomic condition normally compatible with life. Ovar-
ian dysgenesis, primary amenorrhea and fertility are the constant characteristics asso-
ciated with Turner's syndrome. The germ cells and endocrine tissue are completely
absent. Secondary sexual characteristics are also totally absent. The females show
other physical abnormalities like short stature, a short neck with webbing of the skin,
swelling of the hands and feet in infancy, increased carrying angle at the elbow, car-
diovascular and renal abnormalities, high arched palate. The chest is broad with
widely spaced nipples. In spite of these physical abnormalities of various organs, the
nervous system is not affected and the individuals with Turner's syndrome seem to
have normal brain development. They do not have tendencies towards mental retar-
dation.
 89
Autosomal abnormalities in man.
A duplication or deficiency of a part or whole of an autosomal chromosome re-
sults in a change in phenotype and is termed as autosomal abnormality. The somatic
chromosomes exist in homologous pairs carrying alleles of a gene at each locus. The
absence of one allele out of the two, when one of the chromosomes is missing, leads
to monosomic condition for that autosomal pair and this condition is lethal in most of
the cases. Similarly, an extra chromosome, a condition called trisomy, also leads to
serious abnormalities. The following conditions are of common occurence and are
social economically important.
Down syndrome. The individuals with Down's syndrome symptoms have 47
chromosomes (47, XX (XY),+21) instead of 46 in their somatic cells. This condition
was first described by Down (1866) and was called “mongolian idiocy” by him. It is
commonly known as “mongolism” because the suffers have a specific phenotypic re-
semblance to the individuals of oriental race, the Mongols.

Single palmer crease Appearance of baby with Down syndrome

Down syndrome is due to the presence of an extra chromosome number 21. This
was first demonstrated by Lejeune et al. (1959). Chromosome number 21 is the
smallest autosome in humans. It is present in the form of a complete extra chromo-
some or a major part of it, either free or translocated to another autosome of D or G
groups.
 90

Down syndrome karyogram

Clinical manifestations of Down syndrome

The two obvious characteristics of Down’s syndrome are that the inflicted per-
sons are mentally retarded and have characteristic facial resembling mongols. These
persons can be easily detected and diagnosed at birth. The condition of mongolism
occurs in all races and is presumed that the chance of a woman producing a mongol-
oid child than her younger counterparts. The frequency of the syndrome is 1 to 700 –
800 births.
A small and rounded head region with flat back is the most characteristic feature
of this syndrome. If carefully examined, almost every organ is defective in develop-
ment. The nose with a flattened bridge looks underdeveloped. “Epicanthic folds” of
skin and a protruding tongue which is coarsely fissured on the dorsum is another
prominent feature of the syndrome. The size of the external ear varies. It is smaller
or larger than the normal size. The hair on the head is always strait irrespective of the
racial characteristics. The skin is dry and coarse. The suffers are more susceptible to
infections due to low lymphocyte count in the blood. Endocrine glands are
hypoplastic and muscles are hypotonic in most suffers.
Patau syndrome. This syndrome was described by Patau et al.(1960) and is
associated with the trisomy of chromosome number 13 (D-group; 47,
XX(XY),+13).
 91

Cleft palate/lip

Appearances of deadborn with Patau syndrome

Polydactyly

Clinical manifestations of Patau syndrome

Individuals with this abnormality have a small and abnormally shaped head
(microcephaly) with small eyes. Hare lip and cleft palate is very common among the
suffers. Epicanthic folds on the face, a large flat nose, small mandibles and low set
malformed ears are other prominent features of this syndrome. Polydactyly is most
conspicuous. Internal organs show abnormalities. These are cardiovascular
anomalies, polycystic kidney, reduplicated ureters and excess of normal tissue in
liver, kidney, lung, pancreas, etc. The biochemical abnormality is the presence of
fatal haemoglobin at birth. This may be due to delayed development. The life ex-
pectancy is very poor, not more than a year or so.
 92
Edward's syndrome. This syndrome was first described by Edward et al.
(1960) and is associated with the trisomy of chromosome number 18 (E-group;
47,XX(XY),+18). Like Patau's syndrome this is also characterised by congenital
anomalies affecting almost all tissues and systems with short life span.

Appearance of newborn with Edward syndrome

Clinical manifestations of Edward syndrome

Syndroms involving structural chromosome aberrations.

Cri-du-chat or Cat-cry Syndrome. This syndrome was first described by
Lejeune et al. (1963) and is due to a deletion in a short arm of the chromosome num-
ber 5. The most prominent symptom is a curious, faint mewing cry. The babies have
small average birth weight and suffer from considerable mental defect. Cardiovascu-
lar anomalies are common. Facial features include a small cranium, low set ear,
rounded moon face, and epicanthic folds. The condition is quite compatible with sur-
survival. A deletion in the short arm of
chromosome number 4 is described by
Sidburg (1967) resulting in a syndrome
with defects more pronounced than in
cat-cry syndrome except that there is
no cat-cry.

A newborn with cri-du-chat syndrome

 93

Appearance of patients with cri-du-chat syndrome

Cri-du-chat syndrome karyogram

Principles of genetic counseling of chromosomal disorders direct firstly pre-

vention of the birth of babies with such anomalies; that’s why different types of pre-
natal diagnosis of foetus and embryo are used.
c) Multifactorial Diseases. This type is caused by a combination of environ-
mental factors and mutations in multiple genes.
Most phenotypic traits are determined by many genes collaborating at different
loci (polygenic) rather than by single gene effects. Parents and offspring, and usually
siblings, have 50 percent of their genes in common. Second-degree relatives share, on
average, one-fourth of all genes, and third-degree relatives (cousins) share one-
eighth. As the degree of relation becomes more distant, the probability of inheriting
the same combination of genes is reduced, and the degree of resemblance is likely to
be less.
Many common chronic diseases (e.g., essential hypertension, coronary artery
disease, and schizophrenia) and the common birth defects of children (e.g., cleft pal-
ate, cleft lip, and neural tube defects) that tend to run in families fit best into the cate-
gory of multifactorial genetic diseases. Multifactorial genetic diseases have both a
polygenic component and an environmental component of causative factors. Suscep-
tibility, or risk, genes are present in low frequency in the population at large. Howev-
er, if any one individual has a particularly large number of such genes, the disease
may manifest. When an individual is unfortunate enough to have inherited just the
right (or wrong) combination of risk genes, he or she passes beyond a "risk threshold"
at which environmental factors may determine the expression and severity of disease.
In order for another family member to develop the same disease, that individual
 94
would have to inherit the same, or a very similar, combination of genes. The likeli-
hood of such an occurrence is clearly greater in first-degree than in more distant rela-
tives. The chances of another relative inheriting the right combination of risk genes
decreases as the number of genes required to express a given trait increases. For ex-
ample, the recurrence risk for siblings in neural tube defects is almost 4 percent, or
ten times greater than the risk in the population as a whole.

11.4. Mitochondrial diseases

Mitochondrial medicine is a new and rapidly developing medical subspecialty.
Many specialists are involved in researching mitochondrial diseases, including doc-
tors specializing in metabolic diseases, cell biologists, molecular geneticists, neurolo-
gists, biochemists, pathologists, immunologists, and embryologists. Much of what we
know about these diseases has been discovered since 1940. In 1959, the first patient
was diagnosed with a mitochondrial disorder. In 1963, researchers discovered that
mitochondria have their own DNA or "blueprint" (mtDNA), which is different than
the nuclear DNA (nDNA) found in the cells' nucleus.
Mitochondrial and metabolic medical conditions are now referred to as mito-
chondrial cytopathies. Mitochondrial cytopathies actually include more than 40 dif-
ferent identified diseases that have different genetic features. The common factor
among these diseases is that the mitochondria are unable to completely burn food and
oxygen in order to generate energy.
The process of converting food and oxygen (fuel) into energy requires hundreds
of chemical reactions, and each chemical reaction must run almost perfectly in order
to have a continuous supply of energy. When one or more components of these chem-
ical reactions do not run perfectly, there is an energy crisis, and the cells cannot func-
tion normally. As a result, the incompletely burned food might accumulate as poison
inside the body.
This poison can stop other chemical reactions that are important for the cells to
survive, making the energy crisis even worse. In addition, these poisons can act as
free radicals (reactive substances that readily form harmful compounds with other
molecules) that can damage the mitochondria over time, causing damage that cannot
be reversed. Unlike nuclear DNA, mitochondrial DNA has very limited repair abili-
ties and almost no protective capacity to shield the mitochondria from free radical
damage.
With their central place in cell metabolism, damage — and subsequent dys-
function — in mitochondria is an important factor in a wide range of human diseases.
Mitochondrial disorders often present as neurological disorders, but can manifest as
myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifes-
tations. Diseases caused by mutation in the mtDNA include Kearns-Sayre syndrome,
MELAS syndrome and Leber's hereditary optic neuropathy. In the vast majority of
cases, these diseases are transmitted by a female to her children, as the zygote derives
its mitochondria and hence its mtDNA from the ovum. Diseases such as Kearns-
Sayre syndrome, Pearson's syndrome, and progressive external ophthalmoplegia are
thought to be due to large-scale mtDNA rearrangements, whereas other diseases such
 95
as MELAS syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with
ragged red fibers (MERRF), and others are due to point mutations in mtDNA.
In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial
proteins. This is the case in Friedreich's ataxia, hereditary spastic paraplegia, and
Wilson's disease. These diseases are inherited in a dominance relationship, as applies
to most other genetic diseases. A variety of disorders can be caused by nuclear muta-
tions of oxidative phosphorylation enzymes, such as coenzyme Q10 deficiency and
Barth syndrome. Environmental influences may interact with hereditary predisposi-
tions and cause mitochondrial disease. For example, there may be a link between pes-
ticide exposure and the later onset of Parkinson's disease.
Other pathologies with etiology involving mitochondrial dysfunction include
schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease,
epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus. A
common thread thought to link these seemingly-unrelated conditions is cellular dam-
age causing oxidative stress. How exactly mitochondrial dysfunction fits into the eti-
ology of these pathologies is yet to be elucidated.
Symptoms of mitochondrial diseases.
The types of mitochondrial diseases are categorized according to the organ sys-
tems affected and symptoms present. Mitochondrial diseases might affect the cells of
the brain, nerves (including the nerves to the stomach and intestines), muscles, kid-
neys, heart, liver, eyes, ears, or pancreas. In some patients, only one organ is affected,
while in other patients all the organs are involved. Depending on how severe the mi-
tochondrial disorder is, the illness can range in severity from mild to fatal.
Depending on which cells of the body are affected, symptoms might include:
poor growth, loss of muscle coordination, muscle weakness, visual and/or hearing
problems, developmental delays, learning disabilities, mental retardation, heart, liver,
or kidney disease, gastrointestinal disorders, severe constipation, respiratory disor-
ders, diabetes, increased risk of infection, neurological problems, seizures, thyroid
dysfunction, dementia (mental disorder characterized by confusion, disorientation,
and memory loss).
Frequency of mitochondrial diseases.
Researchers have identified more than 200 pathogenic mutations in mtDNA and
over 2000 in nDNA, and the combined incidence of mitochondrial diseases is cur-
rently estimated to range between 1 and 5 in 10,000.
In adults, many diseases of aging have been found to have defects of mito-
chondrial function. These include, but are not limited to, type 2 diabetes, Parkinson's
disease, atherosclerotic heart disease, stroke, Alzheimer's disease, and cancer. In ad-
dition, many medicines can injure the mitochondria.
Causes mitochondrial disease.
For many patients, mitochondrial disease is an inherited condition that runs in
families (genetic). An uncertain percentage of patients acquire symptoms due to other
factors, including mitochondrial toxins.
It is important to determine which type of mitochondrial disease inheritance is
present, in order to predict the risk of recurrence for future children.
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The types of mitochondrial disease inheritance include:
nDNA (DNA contained in the nucleus of the cell) inheritance. Also called au-
tosomal inheritance.
If this gene trait is recessive (one gene from each parent), often no other family
members appear to be affected. There is a 25 percent chance of the trait occurring in
other siblings.
If this gene trait is dominant (a gene from either parent), the disease often oc-
curs in other family members. There is a 50 percent chance of the trait occurring in
other siblings.
mtDNA (DNA contained in the mitochondria) inheritance.
There is a 100 percent chance of the trait occurring in other siblings, since all
mitochondria are inherited from the mother, although symptoms might be either more
or less severe.
Combination of mtDNA and nDNA defects.
Relationship between nDNA and mtDNA and their correlation in mitochondri-
al formation is unknown.
Random occurrences.
Diseases specifically from deletions of large parts of the mitochondrial DNA
molecule are usually sporadic without affecting other family member.
Medicines or other toxic substances can trigger mitochondrial disease.
Mitochondrial diseases diagnosis.
Diagnosis of mitochondrial disease can be invasive, expensive, time-
consuming, and labor-intensive. Therefore, evaluation is not taken lightly. Doctors
experienced in diagnosing and treating these diseases will take either a step-wise ap-
proach to diagnosis or, in some centers, the evaluation takes place over a few days.
The evaluation includes a combination of clinical observations and laboratory tests.
Under ideal circumstances, the evaluation will produce an answer. However,
even after a complete evaluation, the doctor might not be able to confirm a specific
diagnosis or put a name to the disorder. In many cases, however, the physician will
be able to identify which patients do and don't have metabolic diseases.
Mitochondrial disease is diagnosed by: evaluating the patient's family history;
performing a complete physical examination; performing a neurological examination;
performing a metabolic examination that includes blood, urine, and optional cerebral
spinal fluid tests; performing other tests, depending on the patient's specific condition
and needs. These tests might include: magnetic resonance imaging (MRI) or scan
(MRS) if neurological symptoms; retinal exam or electroretinogram if vision symp-
toms; electrocardiogram (EKG) or echocardiogram if heart disease symptoms; audio-
gram or BAEP if hearing symptoms; blood test to detect thyroid dysfunction if thy-
roid problems; blood test to perform genetic DNA testing.
More invasive tests, such as a skin or muscle biopsy, might be performed as
needed and recommended by the doctor.
Mitochondrial diseases treatment.
There are no cures for mitochondrial diseases, but treatment can help reduce
symptoms, or delay or prevent the progression of the disease.
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Treatment is individualized for each patient, as doctors specializing in metabol-
ic diseases have found that every child and adult is "biochemically different." That
means that no two people will respond to a particular treatment in a specific way,
even if they have the same disease.
Certain vitamin and enzyme therapies, along with occupational and physical
therapy, might be helpful for some patients.
Vitamins and supplements prescribed might include:
Coenzyme Q10; B complex vitamins: thiamine (B 1), riboflavin (B 2), niacin (B
3), B 6, folate, B 12, biotin, pantothenic acid; Vitamin E, lipoic acid, selenium, and
other antioxidants; L-carnitine (Carnitor ®); intercurrent illness supplement: vitamin
C, biotin.
Other treatments that might be prescribed include:
-diet therapy, as prescribed by your doctor along with a registered dietitian,
might be recommended;
-antioxidant treatments as protective substances are currently being investigat-
ed as another potential treatment method.
Important: Specific treatments should always be guided by a metabolic special-
ist. Patients should not take any of these supplements or try any of the treatments un-
less prescribed by a doctor. Taking inappropriate supplements or treatments might
lead to delays or failure in establishing an accurate diagnosis.

11.5. Prenatal diagnosis of genetic disease.

The objective of prenatal diagnosis is to offer prospective parents the assurance
of having unaffected children when the risk of having an affected child is unaccepta-
bly high. This is NOT equivalent to the assurance of having normal children. Prenatal
diagnosis allows one to convert a probability statement about the risk of a specific
disease to a certainty. It does not address all possible birth defects or genetic diseases.
Prenatal diagnosis represents a paradigm of the application of basic scientific
techniques to clinical problems. The development of these diagnostic procedures in
the 1960s resulted from advances in obstetrical techniques, in the ability to culture
human cells, and in cytogenetics and molecular genetics.
Prenatal diagnostics reveals over 500 specific dis-
orders and a large percentage of structural birth defects.
Current prenatal diagnostic techniques include tests in
which fetal cells are obtained: amniocentesis, chorionic
villus sampling (CVS), and fetal blood sampling. The
fetal cells are always analyzed for their chromosome set.
When family history or carrier testing has revealed a
risk, fetal cells can be analyzed for specific metabolic
diseases, DNA patterns, or other identification markers.
In addition to obtaining fetal cells, prenatal diagnostics
using ultrasound can visualize fetal structure and identi-
 98
fy a number of significant birth defects and medical conditions. Ultrasound testing is
Ultrasound image of a 12,5-week-old fetus.
noninvasive, but doesn’t show the cause of fetal defects.
A blood test called maternal serum alpha-fetoprotein (MSAFP) screening is
currently offered as an option to all pregnant women from 15 to 22 weeks of preg-
nancy. Women with AFP levels significantly higher or lower than average have a sta-
tistically greater risk for certain birth defects and are offered further testing. MSAFP
screening is capable of identifying 85% of fetuses with spina bifida, 98% with anen-
cephaly, and 20% with Down syndrome. MSAFP screening may also be helpful in
determining the gestational age of the pregnancy, if there is more than one fetus, and
in some cases, fetal death, all of which influence obstetric management.

Amniocentesis
During an amniocentesis, a sam-
ple of the amniotic fluid surrounding
the fetus is obtained. This fluid con-
tains cells from the fetus that can be
cultured, or grown, to determine chro-
mosomal make up, fetal lung maturity,
and other information about the fetus.
Amniocentesis carries a risk of fetal-
death of less than 1%, particularly
when performed by skilled practition-
ers.

Chorionic Villus Sampling (CVS)

In this procedure, a small amount of
tissue is removed from the fetus chorion at
9-10 weeks gestation. The genetic material
of the tissue sample is tested for chromo-
somal abnormalities. The advantage of this
technique is that it can be performed very
early in pregnancy, and the results are
generally available before the end of the
first trimester. The risk of spontaneous
abortion due to the procedure is approxi-
mately 1%.
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Percutaneous Umbilical Blood Sampling (Cordocentesis)
During this procedure blood is obtained directly from the umbilical cord using
ultrasound guidance. Chromosomal and biochemistry tests can be performed on this
pure fetal blood sample. This procedure can be available within minutes to hours.
This relatively new procedure carries has risk of fetal loss somewhere in the range of
1 to 5%.

Indications to invasive methods of prenatal diagnostics:

1. Advanced maternal age (the age of pregnant women is more than 35 years );
2. Existing or suspecting hereditary diseases of parents or a fetus
3. Chromosomal abnormality;
4. Neural tube defect/ abnormal maternal serum (repeated abnormal levels of
αFP (α –fetoprotein) and hCG (human chorionic gonadotropin) );
5. Inborn error of metabolism;
6. Environmental influences on a fetus at first semester of pregnancy (virus infec-
tions, teratogenic drugs, and Roentgen irradiation).
7. Sex determination for some conditions and with the aim of planning family (af-
ter 2nd sex-same offspring).
8. Disease detectable by DNA analysis.

11.6. Principles of Genetic counseling.

Medico-genetic counseling (by P.S. Harper 1984) is a process, as a result of
which patient and their relatives with the hereditary disease risk get information
about consequences of a given disease, probability of its development and inher-
itance, about ways of its prophylaxis.
Genetic counseling involves an attempt by one or more appropriately trained
persons to help the individual or family to:
(1) comprehend the medical facts, including the diagnosis, probable course of
the disorder, and the available management;
(2) appreciate the way heredity contributes to this disorder, and the risk of re-
currence in specified relatives;
(3) understand the alternatives for dealing with the risk of recurrence;
(4) choose the course of action which seems to them appropriate in view of
their risk, their family goals, and their ethical and religious standards, and to act in
accordance with that decision;
(5) to make the best possible adjustment to the disorder in an affected family
member and/or to the risk of recurrence of that disorder.
Methods used by physician-geneticist are strictly specific. The first and the
most important stage of the medico-genetic counseling are a collection of genealogi-
cal information and a graphic picture of it in the family tree. Cytogenetic method (it
studies karyotypes of a proband and relatives), biochemical method (it determinates
amino acids, mono-, polysaccharides, components of lipid metabolism and interme-
diate metabolites) complements clinico-genealogical method. Besides, population-
 100
statistical method and methods of diagnostics with using DNA - probes for revealing
mutant gene plays the greater role.
The following important stage of a physician-geneticist’s work is careful exam-
ination of a patient and family members, who can have symptoms of the same diseas-
es. They are healthy, but they pertain to the group of risk of the given pathology. It is
necessary to exclude their erased or initial forms of disease. If a proband has more
than 5-7 micro abnormalities of development, we suspect the presence of inherited
syndrome or innate pathology. It is important for verification of diagnostics and
prognosis of offspring in the family.
Indications for Genetic Couseling:
1. Known or suspected hereditary disease in a patient or family.
2. Birth defects.
3. Mental retardation.
4. Advanced maternal age.
5. Family history of early onset cancer.
6. Recurrent pregnancy loss.
7. Teratogen exposure.
8. Consaguinity.

11.7. Genetic Engineering

Gene engineering can be defined as the science dealing with the correction of
nature's mistakes at their source, the DNA molecule, comprising the gene. Through
it's techniques, genetic engineering carries out the deliberate modification of specific
genes or implants desired new genes into chromosomes. The genetic or biochemical
techniques by which genetic information contained in two or more different DNA
molecules could be combined in one unit fall within the score of genetic engineering.
It can also be defined as the science of adding, removing or replacing genetic units in
order to achieve permanent and heritable changes in plants or animals for the benefit
of mankind.
Terms such as 'gene surgery', 'gene therapy', 'gene manipulation', 'gene
transplantation', ,gene intervention' and 'algeny' have been synonymously used
with genetic engineering. A detailed knowledge of the molecular nature of the gene
and the ability to manipulate cells of higher organisms as well as bacteria, may
eventually allow the possibility of genetic engineering, i.e., the artificial production
of new varieties of animals or plants by altering the genotype of existing varieties
without the slow hit-and-miss of selective breeding. For humans it is only a distant
future possibility and is based on the work of similar nature on lower organisms es-
pecially microorganisms. Artificially induced mutations by man in the strict sense are
also covered under genetic engineering.

11.8. Gene Therapy

Gene therapy is a way to correct the defective genes that are the cause of disease
development. When the genes are altered proteins are not able to function normally
 101
and as a result of this, defects can occur. Current gene therapy is still being
experimented with, but in some cases it is very effective.
Genes are carried on chromosomes and are the basic physical and functional
parts of hereditary. When there is a genetic disorder, gene therapy can help fix the
problem either permanently or at least temporarily. The most common form of gene
therapy is to insert a gene into a nonspecific place to replace a malfunctioning gene.
Another method is gene swapping, where an abnormal gene is replaced by a normal
gene. Genes could also be repaired through "selective reverse mutation" which
returns the gene to it's original function. The degree to which a gene is turned on or
off can also be altered.
Gene therapy works on the principle belief that a virus genome can be
manipulated to remove disease causing genes and new therapeutic genes can be
inserted in their place. These new genes are called gene therapy vectors.

Gene therapy using an Adenovirus vector. A new gene is inserted into an

adenovirus vector, which is used to introduce the modified DNA into a human cell. If
the treatment is successful, the new gene will make a functional protein.
A few of the different viruses used as gene therapy vectors are: Retroviruses -
A class of viruses that can create double-stranded DNA copies of their original RNA
genomes. Theses copies of its genomes can be mixed into the chromosomes of "host"
cells. HIV is a type of retrovirus. Adenoviruses - A class of viruses with double-
stranded DNA genome that cause respiratory, intestinal, and eye infections in
humans. The common cold is an adenovirus. Adeno-associated viruses - A class of
small, single-stranded DNA viruses that can insert their genetic material at a specific
site on chromosome 19. (chromosome 19 represents about 2% of the human genome
and contains about 1,500 genes. Some of the genes included are genes that code for
insulin-dependent diabetes, myotonic dystrophy, migraines, and inherited high blood
cholesterol). A class of double-stranded DNA viruses that infect a particular cell type,
neurons, called Herpes simplex viruses is another common virus used in gene
therapy. It is the virus that causes cold sores.
Major advancements have been made in gene therapy. There are many new
discoveries in helping cure and treat diseases that claim millions of lives. Some of the
disease that have cures or treatments because of gene therapy include: Parkinson's,
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Huntington's, Cystic Fibrosis, Some cancers, "Bubble Boy" syndrome and sickle cell.
With technology jumping ahead, maybe someday there will be a cure for every life
threatening disease.