Fluorescence In Situ Hybridization (FISH)

By: Clare O'Connor, Ph.D. (Biology Department, Boston College) © 2008 Nature Education
Citation: O'Connor, C. (2008) Fluorescence in situ hybridization (FISH). Nature
Education 1(1):171

Cytogeneticists can now go "FISH-ing" for chromosomal abnormalities, which are deletions and
duplications that can cause disease. How exactly does FISH work?
Aa Aa Aa
Cytogenetics entered the molecular era with the introduction of in situ hybridization, a procedure
that allows researchers to locate the positions of specific DNA sequences on chromosomes.
Since the first in situ hybridization experiments in 1969 (Gall & Pardue, 1969), many variations
of the procedure have been developed, and its sensitivity has increased enormously. Today,
most in situ hybridization procedures use fluorescent probes to detect DNA sequences, and the
process is commonly referred to as FISH (fluorescence in situ hybridization). A variety of FISH
procedures are available to cytogeneticists, who use them to diagnose many types of
chromosomal abnormalities in patients. The success of FISH, and all other methods of in
situ hybridization, depends on the remarkable stability of the DNA double helix.
In Situ Hybridization Is Used to Localize DNA Sequences on Chromosomes
In 1953, James Watson and Francis Crick described the extensive network of hydrogen bonds
that hold together the two antiparallel strands in the DNA double helix (Watson & Crick, 1953).
Today, even schoolchildren know that adenine on one DNA strand binds to thymine on
the complementary DNA strand, and that cytosine likewise binds to guanine. Because of the
many hydrogen bonds formed between these bases, the double helix is a remarkably stable
structure. Moreover, if the hydrogen bonds that hold the helix together are broken with heat or
chemicals, the helix is able to re-form when conditions become more favorable. This ability of
the DNA helix to re-form, or renature, provides the basis for molecular hybridization.
In molecular hybridization, a labeled DNA or RNA sequence is used as a probe to identify or
quantify the naturally occurring counterpart of the sequence in a biological sample. In the 1960s,
researchers Joseph Gall and Mary Lou Pardue realized that molecular hybridization could be
used to identify the position of DNA sequences in situ (i.e., in their natural positions within
a chromosome). In fact, in 1969, the two scientists published a landmark paper demonstrating
that radioactive copies of a ribosomal DNA sequence could be used to detect complementary
DNA sequences in the nucleus of a frog egg. Since those original observations, many
refinements have increased the versatility and sensitivity of the procedure to the extent that in
situ hybridization is now considered an essential tool in cytogenetics.
Fluorescent Probes Are Introduced

Figure 1: Principles of fluorescence in situ hybridization (FISH).

(a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization,
the DNA probe is labeled by various means, such as nick translation, random primed labeling,
and PCR. Two labeling strategies are commonly used: indirect labeling (left panel) and direct
labeling (right panel). For indirect labeling, probes are labeled with modified nucleotides that
contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to
contain a fluorophore. (c) The labeled probe and the target DNA are denatured. (d) Combining
the denatured probe and target allows the annealing of complementary DNA sequences. (e) If the
probe has been labeled indirectly, an extra step is required for visualization of the nonfluorescent
hapten that uses an enzymatic or immunological detection system. Whereas FISH is faster with
directly labeled probes, indirect labeling offers the advantage of signal amplification by using
several layers of antibodies, and it might therefore produce a signal that is brighter compared
with background levels.
© 2005 Nature Publishing Group Speicher, M. R. et al. The new cytogenetics: blurring the
boundaries with molecular biology. Nature Reviews Genetics 6, 784 (2005). All rights
reserved.
Soon after Gall and Pardue's work, fluorescent labels quickly replaced radioactive labels in
hybridization probes because of their greater safety, stability, and ease of detection (Rudkin &
Stollar, 1977). In fact, most current in situ hybridization is done using FISH procedures (Trask,
2002; Speicher & Carter, 2005). Detecting a DNA sequence can be compared to looking for a
needle in a haystack, with the needle being the DNA sequence of interest and the haystack being
a set of chromosomes. This search is made much easier if the investigator has a powerful
"magnet"—in this case, a fluorescent copy of the DNA sequence of interest. Hybridization
occurs when the "magnet" meets the "needle"; this requires both a probe and a target, as shown
in Figure 1. In the figure, the probe sequence, often a piece of cloned DNA, is shown in red. The
target DNA—chromosomes on a glass slide—is shown in blue (in the right column). Hydrogen
bonds that join the two strands of the DNA helix are represented by black lines.
The first step in the process is to make either a fluorescent copy of the probe sequence (Figure
1b, middle column) or a modified copy of the probe sequence that can be rendered fluorescent
later in the procedure (Figure 1b, left column). Next, before any hybridization can occur, both
the target and the probe sequences must be denatured with heat or chemicals (Figure 1c).
This denaturation step is necessary in order for new hydrogen bonds to form between the target
and the probe during the subsequent hybridization step. The probe and target sequences are then
mixed together (Figure 1d), and the probe specifically hybridizes to its complementary sequence
on the chromosome. If the probe is already fluorescent (middle column), it will be possible to
detect the site of hybridization directly. In other cases (left column), an additional step may be
needed to visualize the hybridized probe. Hybrids formed between the probes and their
chromosomal targets can be detected using a fluorescent microscope.
When investigators design a FISH experiment, they need to consider whether the sensitivity and
resolution needed for the experiment lie within the technical limits of fluorescence microscopy.
Sensitivity depends on the light-gathering ability of the particular microscope, which determines
whether small target sequences, which are more difficult to see than large target sequences, can
be detected. Resolution refers to the ability to distinguish between two points along the length of
a chromosome. Ultimately, light microscopy cannot resolve objects that are separated by less
than 200–250 nm, the lower limit of the visible light spectrum. With these technical limits in
mind, investigators also need to consider the conformation of DNA within the
chromosome. Metaphase chromosomes are thousands of times more compacted
than interphase chromosomes, which in turn are at least ten times more compacted than naked
DNA. (Remember that one 3.4 nm turn of the DNA helix corresponds to 10 base pairs of DNA.)
When all these factors are considered together, investigators typically expect to obtain resolution
in the range of megabases for positions on metaphase chromosomes and resolution in the range
of tens of thousands of kilobases for interphase chromosomes.
Using FISH to Identify the Positions of Genes

Figure 2: Cytogenetic analyses of sequence-integrated clones.

(a) Using FISH, fluorescent signals are observed at cytogenetic bands (grey) where fragments of
a sequence-tagged bacterial artificial chromosome hybridize (red). (b) A clone selected on the
basis of band location is used in FISH analysis to map the breakpoint of a translocation involving
chromosomes 11 and 19 in a patient with multiple congenital malformations and mental
retardation. The clone spans the breakpoint on chromosome 19; thus, the red signal is split
between the derivative 11 and derivative 19 chromosomes and is present on the normal
chromosome 19.
© 2001 Nature Publishing Group Cheung, V. G. et al. Integration of cytogenetic landmarks
into the draft sequence of the human genome. Nature 409, 954 (2001). All rights
reserved.
FISH provides a powerful tool for identifying the location of a cloned DNA sequence on
metaphase chromosomes. Figure 2a shows the results of a typical FISH experiment, in which a
cloned DNA sequence was hybridized to normal metaphase chromosomes. Red bands are
detected at hybridization sites on two homologous chromosomes, which can be identified by
their characteristic banding patterns. Closer examination shows that each red band actually
consists of two spots, corresponding to the two sister chromatids in a mitotic chromosome. A
skilled cytogeneticist would be able to use these hybridization data together with the banding
pattern to place the probe sequence within a few megabases of other known genes on the
chromosome.
Historically, FISH and other in situ hybridization results played a primary role in mapping genes
on human chromosomes. Results from these experiments were collected and compiled in
databases, and this information proved useful during the annotation phase of the Human Genome
Project (HGP). Now that the HGP is complete, investigators rarely use in situ hybridization
simply to identify the chromosomal location of a human gene. (In species for which
the genome has not been sequenced, however, FISH and related in situ hybridization methods
continue to provide important data for mapping the positions of genes on chromosomes.)
Currently, human FISH applications are principally directed toward clinical diagnoses.
Diagnosing Chromosomal Abnormalities Using Karyotypes and FISH
FISH and other in situ hybridization procedures are important in the clinical diagnosis of various
chromosomal abnormalities, including deletions, duplications, and translocations. Figure 2b
shows one example in which investigators used FISH together with standard karyotyping to
analyze a patient translocation. The hybridization probe corresponded to a segment of
chromosome 19 that was suspected to include the translocation breakpoint. Three areas of
hybridization are apparent in the fluorescent image. One spot corresponds to the patient's normal
copy of chromosome 19 (nl19), and the other two spots correspond to the altered, or derived
(der), versions of chromosomes 11 and 19 that were produced during the translocation. Thus,
investigators were able to use the data both to narrow down the breakpoint region on
chromosome 19 and to identify the second chromosome involved in the translocation.
The hybridization probe used in Figure 2b was one of thousands of bacterial artificial
chromosome (BAC) clones from the HGP that have been made available to the scientific
community. Today, cytogeneticists are able to use extensive HGP clone resources to precisely
identify the sites of chromosomal rearrangements that appear in karyotypes. In fact, a consortium
of scientists has mapped over 7,000 DNA clones from the HGP to specific bands on human
chromosomes (BAC Research Consortium, 2001). At least one clone is available for every
megabase segment of chromosomal DNA. (The only exception is the Y chromosome, because it
is relatively gene-poor.)
Using Collections of FISH Probes to “Paint” Entire Chromosomes

Figure 3: Spectral karyotyping and multicolor-FISH paint each human chromosome in

one of 24 colors.
Cytogenetic localization of DNA sequences with fluorescence in situ hybridization (FISH).
© 2014 Panels a) and b) modified from © 2000 Cambridge University Press. McNeil, N. &
Ried, T. Novel molecular cytogenetic techniques for identifying complex chromosomal
rearrangements: technology and applications in molecular medicine. Expert Reviews in
Molecular Medicine (online: September 2000). All rights reserved.
The detection of chromosome rearrangements with site-specific probes (Figure 2b) can be a
lengthy endeavor, especially if complex rearrangements have occurred or if the rearranged
regions are difficult to identify by their banding patterns in a karyotype. Fortunately,
cytogeneticists now have the option of using multifluor FISH, or spectral karyotyping, to quickly
scan a set of metaphase chromosomes for potential rearrangements (Speicher et al., 1996;
Schrock et al., 1996). Multifluor FISH generates a karyotype in which each chromosome appears
to be painted with a different color. Each "paint" is actually a collection of hybridization probes
for sequences that span the length of a particular chromosome.
With multifluor FISH, investigators first prepare a collection of DNA sequences to be used as
probes for each chromosome. In Figure 3a, the probe chromosomes have been physically
separated from one another by flow cytometry. (Today, investigators would probably use
commercially available DNA collections for each chromosome.) In the next step, the DNA
samples are labeled with combinations of fluorochromes that produce a unique color for each
chromosome. (The Cot-1 DNA step in the figure removes repetitive DNA sequences [e.g.,
centromeric DNA] that would bind to all chromosomes.) The fluorescent hybridization probes
are then combined with and hybridized to metaphase chromosomes. Figure 3b shows images of
interphase and metaphase chromosomes as they would appear through a microscope after
hybridization. To human eyes, several of the metaphase chromosomes appear to have the same
color, but digital processing of the image would distinguish spectral differences between the
chromosomes. A normal human chromosome (Figure 3b) will have a uniform color along its
length, but a rearranged chromosome will have a striped appearance.
Although chromosome paints allow rapid assessment of large chromosomal changes in
metaphase spreads, the resolution of the method is limited. Thus, while chromosome
painting allows investigators to quickly identify chromosomes involved in translocations and to
identify large deletions and/or duplications, small deletions and duplications will not be
detectable. If investigators need more detailed information about the actual sequences involved
in chromosomal rearrangements, they need to follow up with site-specific probes, as previously
described (Figure 2).
Using FISH to Analyze Interphase Chromosomes
 Figure 4: Using FISH to detect chromosomal abnormalities in interphase nuclei.
(a) The duplication of a small portion of chromosome 17 that causes Charcot-Marie-Tooth
syndrome is evident from the appearance of three, rather than two, red signals in this nucleus.
The green spots mark a sequence outside the duplication. (b) The translocation that creates a
fusion of the BCR (on chromosome 22) and ABL (on chromosome 9) genes in the Philadelphia
chromosome is evident from the close juxtaposition of one pair of green and red signals. These
signals were generated using FISH probes for sequences located near these two genes,
respectively. der(22) is the Philadelphia chromosome. Only the relevant portions of the normal
and abnormal chromosomes are shown in the diagram below each panel.
© 1986 Cold Spring Harbor Laboratory Press Modified with permission from Gray, J.
W., et al. Flow karyotyping and sorting of human chromosomes. Cold Spring Harbor
Symposia on Quantitative Biology 51, 141–149 (1986). All rights reserved. Plot courtesy of
Ger van den Engh, Institute of Systems Biology. All rights reserved.
Figure Detail
Since the introduction of FISH, cytogeneticists have been able to analyze interphase
chromosomes as well as the metaphase chromosomes used in karyotypes (Trask, 2002). This
offers a real practical advantage, in that cells do not need to be cultured for several days or weeks
before chromosomes can be prepared for analysis. In addition, FISH can be used to analyze
chromosomes from specimens such as solid tumors, which are of great clinical interest but do not
divide frequently. Another useful feature of FISH is that researchers are able to simultaneously
monitor multiple sites if the hybridization probes have been labeled with different fluorophores.
Figure 4 shows two examples of how interphase FISH can be used to diagnose chromosome
abnormalities. Figure 4a shows an interphase nucleus from a patient with Charcot-Marie-
Tooth disease (CMT) type 1A (Lupski et al., 1991). CMT type 1A is a relatively common
neurological condition caused by a duplication in a gene on chromosome 17 that encodes one of
the proteins in the myelin sheath that surrounds nerve axons. In Figure 4a, the patient's cell has
been hybridized with a red-labeled probe corresponding to a sequence within the duplicated
region, along with a green probe corresponding to a sequence on chromosome 17 that lies
outside of the duplicated region. From the two green signals, it is possible to locate two copies of
chromosome 17 within the nucleus. One chromosome has the normal configuration, while the
second, der(17), contains the duplicated region, which is evident from two nearby red signals.
The figure also serves to illustrate another important feature of interphase FISH. Because
interphase chromatin is about 10,000 times less compacted than mitotic chromatin, it is possible
to resolve the duplicated regions on der(17) as discrete points. This small duplication would have
been difficult to resolve in mitotic chromosomes.
Figure 4b shows a FISH analysis that was used to detect the presence of a chromosomal
translocation in a patient suffering from chronic myelogenous leukemia (Tkachuk et al., 1990).
In most cases of this disease, a segment of chromosome 9 that contains the ABL proto-
oncogene fuses with the breakpoint cluster region (BCR) on chromosome 22 during a reciprocal
translocation. The derived chromosome 22, or der(22), also known as the Philadelphia
chromosome, contains a BCR-ABL fusion gene in which the powerful BCR promoter drives
synthesis of the ABL oncogene transcript, leading to cancer. Figure 4b demonstrates that BCR-
ABL fusions can be readily identified by FISH when a green-labeled hybridization probe
flanking BCR is applied together with a red-labeled probe flanking ABL. In this image, the
normal copies of chromosomes 9 and 22 are detected as red and green spots, respectively. On the
other hand, the Philadelphia chromosome is visible as a complex fused spot, which appears to
have a central yellow region with red and green subregions on either side. (In fluorescence
microscopy, yellow is indicative of very close proximity of red and green probes, such that they
appear to overlap.) The intricate substructure of the fused spot is detectable in interphase
chromosomes, but it would not be resolved in a similar FISH analysis of metaphase
chromosomes. Thus, two-color interphase FISH provides a sensitive method for analyzing
chromosome fusion events without the need for a prior cell culture.
Another research application of interphase FISH makes use of chromosome-specific paints to
obtain information about the organization of chromosomes within the nucleus. Figure 2a (upper
left) shows an interphase nucleus that has been stained with chromosome-specific paints. One
can see from the figure that the chromosomes occupy distinct territories within the nucleus. By
creatively combining chromosome-specific probes with gene-specific probes and antibodies,
investigators can use FISH to provide exciting new insights about nuclear architecture.

Additional Applications of FISH in the Clinic and Research Laboratory

Exciting new applications of FISH that extend its range continue to be developed. For example,
cytogeneticists now use comparative genomic hybridization to detect quantitative differences,
like copy number variations, in the chromosomes of their patients. Recently, investigators have
also been able to increase the resolution of FISH by using stretched chromatin fibers (Parra &
Windle, 1993) or microarrays as the target. With tools such as these, cytogenetics has been able
to move from studying whole chromosomes on the macroscopic scale, to studying the DNA of
which these chromosomes consist.
Mendelian Genetics: Patterns of Inheritance and Single-Gene Disorders
By: Heidi Chial, Ph.D. (Write Science Right) © 2008 Nature Education
Citation: Chial, H. (2008) Mendelian genetics: Patterns of inheritance and single-gene
disorders. Nature Education 1(1):63
What can Gregor Mendel’s pea plants tell us about human disease? Single gene disorders, like
Huntington’s disease and cystic fibrosis, actually follow Mendelian inheritance patterns.
Aa Aa Aa
Mendel's studies of inheritance patterns in pea plants are a solid foundation for our current
understanding of single-gene diseases in humans. Also called Mendelian or monogenic diseases,
these kinds of diseases are caused by mutations in one gene, and they sometimes run in
families. Pedigree analyses of large families with many affected individuals can be used to
determine whether a disease-associated gene is located on an autosome or on a sex chromosome,
and whether the related disease phenotype is dominant or recessive.
Autosomal Recessive Single-Gene Diseases
Autosomal recessive single-gene diseases occur only in individuals with two mutant alleles of
the disease-associated gene. Remember, for any given gene, a person inherits one allele from his
or her mother and one allele from his or her father. Therefore, individuals with an autosomal
recessive single-gene disease inherit one mutant allele of the disease-associated gene from each
of their parents. In pedigrees of families with multiple affected generations, autosomal recessive
single-gene diseases often show a clear pattern in which the disease "skips" one or more
generations.
Phenylketonuria (PKU) is a prominent example of a single-gene disease with an autosomal
recessive inheritance pattern. PKU is associated with mutations in the gene that encodes
the enzyme phenylalanine hydroxylase (PAH); when a person has these mutations, he or she
cannot properly manufacture PAH, so he or she is subsequently unable to break down the amino
acid phenylalanine, which is an essential building block of dietary proteins. As a result,
individuals with PKU accumulate high levels of phenylalanine in their urine and blood, and this
buildup eventually causes mental retardation and behavioral abnormalities.
The PKU-associated enzyme deficiency was determined biochemically in the 1950s—long
before the PAH-encoding gene was mapped to human chromosome 12 and cloned in 1983.
Specifically, Dr. Willard Centerwall, whose child was mentally handicapped, developed the first
diagnostic test for PKU in 1957. Called the "wet diaper" test, Centerwall's test involved adding a
drop of ferric chloride to a wet diaper; if the diaper turned green, the infant was diagnosed with
PKU. The wet diaper test was used to reliably test infants at eight weeks after birth; by this time,
however, infants who were affected by PKU had already often suffered irreversible brain
damage.
Thus, in 1960, Dr. Robert Guthrie, whose niece suffered from PKU and whose son was also
mentally handicapped, established a more sensitive method for detecting elevated phenylalanine
levels in blood, which permitted a diagnosis of PKU within three days after birth. Guthrie's test
used bacteria that were unable to make their own phenylalanine as messengers to report high
blood levels of phenylalanine in an infant's blood sample obtained via heel prick. With Guthrie's
method, the phenylalanine-deficient bacteria were grown in media together with a paper disk
spotted with a drop of the infant's blood. If the phenylalanine levels in the blood were high, the
bacteria would grow robustly, and a diagnosis of PKU could be made. Through the ability to
discover that their child had PKU at such an early age, parents became able to respond
immediately by feeding their child a modified diet low in proteins and phenylalanine, thereby
allowing more normal cognitive development. Guthrie's test continues to be used today, and the
practice of obtaining an infant's blood sample via heel prick is now used in numerous additional
diagnostic tests.
Several other human diseases, including cystic fibrosis, sickle-cell anemia, and oculocutaneous
albinism, also exhibit an autosomal recessive inheritance pattern. Cystic fibrosis is associated
with recessive mutations in the CFTR gene, whereas sickle-cell anemia is associated with
recessive mutations in the beta hemoglobin (HBB) gene. Interestingly, although
individuals homozygous for the mutant HBB gene suffer from sickle-cell
anemia, heterozygous carriers are resistant to malaria. This fact explains the higher frequency of
sickle-cell anemia in today's African Americans, who are descendants of a group that had an
advantage against endemic malaria if they carried HBB mutations. Finally, oculocutaneous
albinism is associated with autosomal recessive mutations in the OCA2 gene. This gene is
involved in biosynthesis of the pigment melanin, which gives color to a person's hair, skin, and
eyes.
Autosomal Dominant Single-Gene Diseases
Autosomal dominant single-gene diseases occur in individuals who have a single mutant copy of
the disease-associated gene. In this case, the presence of a single nonmutant or "wild-type" copy
of the gene is not enough to prevent the disease. Individuals can inherit the mutant copy of the
disease-associated gene from either an affected mother or an affected father.
Huntington's disease, a progressive neurodegenerative disorder, is a well-known example of an
autosomal dominant single-gene disease; most individuals with a single copy of the mutant
huntingtin gene (HTT) will have Huntington's disease later in life. Typically, autosomal
dominant diseases affect individuals in their early years and prevent them from living past
infancy or childhood, which in turn precludes these individuals from reproducing and potentially
passing on the mutation to their offspring. In the case of Huntington's disease, however, the late
onset of the disorder means that many affected individuals have already had children before they
are even aware that they carry the mutation.
Disease-associated changes in the huntingtin gene consist of a special type of mutation called
triplet repeats; these mutations are simply extra repetitions of the three-base DNA sequence
CAG. The number of CAG repeats in a mutated huntingtin gene determines the age at which a
person will develop Huntington's disease, as well as how severe the condition will be. Genetic
tests can be used to determine how many CAG repeats are in an individual's huntingtin gene,
thereby providing a highly accurate assessment of the individual's disease risk. Because affected
parents have a 50% chance of passing a mutant copy of the huntingtin gene on to each of their
offspring, children of people with Huntington's disease are often faced with the dilemma of
whether to undergo such testing. Genetic testing can either provide immediate relief in knowing
that one is free from the disease, or the confirmation that one will certainly suffer from the
condition at some point in the future.
Myotonic dystrophy, familial hypercholesterolemia, neurofibromatosis, and polycystic kidney
disease serve as additional examples of autosomal dominant single-gene diseases. Myotonic
dystrophy is associated with dominant mutations in the dystrophia myotonica protein kinase
(DMPK) gene; familial hypercholesterolemia is associated with dominant mutations in both the
low-density lipoprotein receptor (LDLR) gene and the apolipoprotein B (APOB) gene; and
neurofibromatosis is associated with dominant mutations in the neurofibromin (NF1) gene.
Autosomal dominant polycystic kidney disease can be caused by mutations in either the
polycystic kidney disease 1 (PKD1) gene or the polycystic kidney disease 2 (PKD2) gene;
the PKD1 gene is located on human chromosome 16, whereas the PKD2 gene is located on
human chromosome 4.
Table 1. Examples of Several Human Diseases, Their Modes of Inheritance, and the
Associated Genes

Type of Inheritance Example Gene Responsible

Autosomal recessive Phenylketonuria Phenylalanine hydroxylase (PAH)

Cystic fibrosis Cystic fibrosis conductance

regulator (CFTR)

Sickle-cell anemia Beta hemoglobin (HBB)

Oculocutaneous albinism OCA2

Autosomal dominant Huntington's disease Huntingtin (HTT)

X Chromosome–Linked Recessive Single-Gene Diseases

Single-gene diseases that involve genes found on the sex chromosomes have somewhat different
inheritance patterns than those that involve genes found on a person's autosomes. The reason for
these differences lies in the genetic distinction between males and females. Recall that females
have two copies of the X chromosome, and they receive one copy from each parent. Therefore,
females with an X chromosome-linked recessive disease inherit one copy of the mutant gene
from an affected father and the second copy of the mutant gene from their mother, who is most
often a carrier (heterozygous) but who might be affected (homozygous). Males, on the other
hand, have only one copy of the X chromosome, which they always receive from their mother.
Therefore, males with an X chromosome-linked disease always receive the mutant copy of the
gene from their mother. Moreover, because men don't have a second copy of the X chromosome
to potentially "cancel out" the negative effects of X-linked mutations, they are far more likely
than women to be affected by X chromosome-linked recessive diseases.
The blood-clotting disorder hemophilia A is one of several single-gene diseases that exhibit an X
chromosome-linked recessive pattern of inheritance. Males who have a mutant copy of the factor
VIII gene (F8) will always have hemophilia. In contrast, women are rarely affected by this
disease, although they are most often carries of the mutated gene. Duchenne muscular dystrophy
is another example of a single-gene disease that exhibits an X chromosome-linked recessive
inheritance pattern. This condition is associated with mutations in the dystrophin gene (DMD).
X Chromosome–Linked Dominant Single-Gene Diseases
Few dominantly inherited forms of human disease are X chromosome linked. Females with an X
chromosome-linked dominant disease can inherit the mutant gene from either an affected mother
or an affected father, whereas males always inherit such diseases from an affected mother.
Examples of X chromosome-linked dominant diseases are rare, but several do exist. For instance,
dominant mutations in the phosphate-regulating endopeptidase gene (PHEX), which resides on
the X chromosome, are associated with X-linked dominant hypophosphatemic rickets. Similarly,
Rett syndrome, a neurodevelopmental disease, is associated with dominant mutations in the
methyl-CpG-binding protein 2 gene (MECP2). Rett syndrome almost exclusively affects
females, because male embryos with a dominant mutation in the MECP2 gene rarely survive.
Y Chromosome–Linked Single-Gene Disease
Like X-linked dominant diseases, Y chromosome-linked diseases are also extremely rare.
Because only males have a Y chromosome and they always receive their Y chromosome from
their father, Y-linked single-gene diseases are always passed on from affected fathers to their
sons. It makes no difference whether the Y chromosome-linked mutation is dominant or
recessive, because only one copy of the mutated gene is ever present; thus, the disease-associated
phenotype always shows.
One example of a Y-linked disorder is nonobstructive spermatogenic failure, a condition that
leads to infertility problems in males. This disorder is associated with mutations in the ubiquitin-
specific protease 9Y gene (USP9Y) on the Y chromosome.
Using the Human Genome Sequence to Study Disease
With the complete sequence of the human genome in hand, scientists are now poised to match
monogenic disease phenotypes to their corresponding genes. By analyzing complex pedigrees,
geneticists can correlate changes in gene sequence with particular disease states. After all, once a
disease-associated change in the DNA sequence of a gene is identified, it is much easier to
determine how the structure of the corresponding gene product (protein) might be changed in a
manner that alters its biological function. The nature of disease-associated changes in protein
structure and function can in turn enhance our ability to design drugs that effectively and
specifically target mutant proteins.
Recent estimates predict that the human genome includes 25,000 protein-encoding genes.
Although 1,822 of the protein-encoding genes in humans are estimated to be associated with
monogenic disease, the identities of more than 1,500 of these genes remain unknown, largely
because many of these single-gene diseases are rare and occur in small numbers of families
(Antonarakis & Beckmann, 2006). Referred to as "orphan" diseases, these relatively uncommon
disorders receive much less research funding than more common diseases, which are often
considered a better investment by funding agencies and biopharmaceutical companies. However,
many of the common diseases exhibit a more complex inheritance pattern and are associated
with mutations in multiple genes (in other words, these conditions are polygenic). As a result,
research efforts have begun to shift from a focus on monogenic disease to a focus on polygenic
disease, which can involve complex interactions between genes and the environment that are not
easily interpreted.
From Monogenic to Oligogenic Disease: Modifier Genes
In recent years, a number of diseases initially characterized as monogenic have been shown to be
caused or modified by an additional gene or genes. These diseases have been categorized as
"oligogenic" rather than "polygenic," because they involve only a relatively small number of
genes. For example, cystic fibrosis is typically characterized as a single-gene disease associated
with recessive mutations in the CFTR gene. However, more extensive studies of CFTR mutations
in larger and more diverse populations have shown that mutations in additional genes could
perhaps modulate the severity and type of disease-related phenotypes (Figure 1).
Collectively, studies of disease that start with the discovery of a single disease-associated gene
can provide an invaluable opportunity to expand our knowledge of more complex oligogenic
links through the discovery of additional causative or modifying genes.
Figure 1: Complexity in monogenic diseases.
Mutations in CFTR almost always cause the CF phenotype. Owing to modification effects by
other genetic factors, the presence and nature of mutations at the CFTR locus cannot predict what
the phenotypic manifestation of the disease will be. Therefore, although CF is considered a
Mendelian recessive disease, the phenotype in each patient depends on a discrete number of
alleles at different loci. Meconium ileus describes the obstruction at birth of the small and/or
large intestine (ileus) with the first fecal excretion (meconium). (NB. CFTR = cystic fibrosis
transmembrane conductance regulator, CFM1 = cystic fibrosis modifier, HLA-II = MHC class II
antigen, MBL2 = mannose-binding lectin (protein C) 2, NOS1 = nitric oxide synthase
1, TGFB1 = transforming growth factor-a1, and TNFA = tumour necrosis factor-a encoding
gene.)
© 2002 Nature Publishing Group Badano, J. L. et al. Beyond Mendel: an evolving view of
human genetic disease transmission. Nature Review Genetics 3, 780 (2002). All rights
reserved.