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ONLINE MUTATION REPORT

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CYS127S (FH-Kairouan) and D245N (FH-Tozeur)
mutations in the LDL receptor gene in Tunisian families
with familial hypercholesterolaemia
M N Slimane, S Lestavel, V Clavey, F Maatouk, M H Ben Fahrat, J C Fruchart,
M Hammami, P Benlian
.............................................................................................................................

J Med Genet 2002;39:e74(http://www.jmedgenet.com/cgi/content/full/39/11/e74)

F
amilial hypercholesterolaemia (FH, MIM 143890) is a
Key points
dominantly inherited metabolic disorder caused by
various mutations of the LDL receptor (LDL-R) gene
causing delayed clearance of serum low density lipoproteins • The objective of the study was to characterise further the
(LDL). The prevalence of homozygous FH has been estimated spectrum of LDL receptor (LDL-R) gene mutations causing
to be 1 in a million in European and North American familial hypercholesterolaemia (FH) in central and
southern Tunisia.
populations.1 In central and southern Tunisia, the prevalence
of homozygous FH is approximately seven-fold higher.2 The • DNA sequencing of the promoter and exonic regions of
minimal estimated frequency of heterozygotes is about the LDL-R gene was performed in 15 clinically
1/160.2 These frequencies are as high as those found in popu- diagnosed FH patients from two unrelated families
lations with a high degree of inbreeding, such as Afrikaners in attending the lipid clinic in Monastir. Segregation
South Africa,3 Christian Lebanese,4 Finns,5 and French analysis was performed in relatives. Functional studies
Canadians.6 In these populations, only a limited number of of LDL-R activity on primary cell cultures from skin fibro-
LDL-R gene mutations cause FH in the majority of affected blasts were undertaken.
cases as the result of a founder effect. Because of numerous • Two missense mutations in the LDL receptor gene were
consanguineous unions in rural areas in Tunisia, it can be pre- identified. One is a novel mutation replacing G with C
dicted that the number of mutations causing FH may be lim- at nucleotide 443 (443 G>C) causing a substitution of
ited. Moreover, we had reported that heterozygous FH might cysteine for serine at codon 127 in exon 4. This muta-
be misdiagnosed through classical symptom assessment tion, named C127S or FH-Kairouan, creates a recogni-
because of mild clinical expression. Therefore, Tunisian tion site for MnlI and was detected in three homozygotes
patients with FH would be easier to identify by direct and six heterozygotes from the same family. The second
genotyping of frequent disease causing mutations. We have mutation replaces G with A at nucleotide 796 (796
recently identified one founder frameshift mutation (FsI472; G>A) and causes a substitution of aspartic acid for
del TCT, ins AGAGACA, →43 aa-term) in five families asparagine at codon 245 in exon 5. This mutation,
originating from the coastal region of Monastir in Tunisia.7 named D245N or (FH-Tozeur) and involving a CpG
The aim of this study was to characterise further the spec- dinucleotide, has been reported in a Dutch patient. In
the homozygotes, both mutations were associated with
trum of LDL receptor gene mutations in the Tunisian popula-
a severe clinical expression and abolished LDL receptor
tion. In order to investigate the influence of potential modifier
activity in cultured fibroblasts obtained from skin biopsy.
alleles on phenotypic expression of FH, apoE polymorphism8
Heterozygous carriers of either of the two mutations had
and common LPL gene variants9–11 were analysed. Here we
significantly increased plasma LDL cholesterol, two adult
describe two novel missense mutations in the LDL-R gene
patients being clinically symptomatic.
causing FH in two unrelated families from central and south-
ern Tunisia. • Both of these LDL-R gene mutations enrich the spectrum
of mutations causing FH in Tunisia. Owing to a high
level of inbreeding in the Tunisian population, these
findings should provide useful diagnostic tools and
MATERIALS AND METHODS
allow further studies on genotype/phenotype
Patients
correlations.
Probands were defined by at least one person (aged <30
years) presenting with multiple planar, tuberous, or tendinous
xanthomas and plasma LDL cholesterol >400 mg/dl. Family
members, who had given informed consent for the study, were Genetic analysis
examined for the presence of extravascular deposits (xantho- Selective polymerase chain reaction (PCR) amplification of the
mas or corneal arcus), cardiovascular risk factors, and signs of promoter region and of the entire coding sequence of the
cardiovascular disease. Plasma lipids (TC, TG) and HDL chol- LDL-R gene (18 exons) was performed on genomic DNA from
esterol were measured by enzymatic and precipitation probands, as previously described.1 Automated sequencing of
methods as previously described.2 LDL cholesterol was purified PCR products (Sephadex G50 spin columns, Boe-
calculated by the Friedewald formula. According to these cri- hringer Mannheim, Germany) was performed on both sense
teria, FH was identified in two unrelated families: family A and antisense strands using Taq polymerase and fluorescent
included nine subjects from the Kairouan area (central dinucleotides according to the supplier’s instructions (Applied
Tunisia) and family B included six subjects from the Tozeur Biosystems, Perkin Elmer, St Quentin en Yvelines, France).12
area (southern Tunisia). When a mutation was detected, another sequencing reaction

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Figure 1 Family A. (A) Partial LDL-R gene sequence of exon 4 from a control subject (Normal) and from proband II.6 (Mutant). (B)
Representative experiment of specific binding and internalisation of 125I-LDL at 37°C by fibroblasts from proband II.6, his mother I.1, and a
normolipidaemic subject (Normal) (mean of triplicates). (C) Pedigree of family A. (D) Mutation detection by MnlI restriction of PCR products
from exon 4 in sibs from generation II (lanes 1-7) and a control subject (N).

was performed both on genomic DNA from a relative and from RESULTS
a new PCR product from the proband. Screening relatives for DNA and functional analyses
the newly identified mutation was performed after digestion We analysed two FH probands belonging to two unrelated
of PCR products with the appropriate enzyme (Appligène, families A and B living in central and southern Tunisia,
Illkirch, France). The resulting fragments were size separated respectively. Both probands were born to consanguineous par-
by electrophoresis on a 2% agarose gel. The Arg3500Gln ents (figs 1 and 2).
mutation of apoB was not found in any of the patients Proband II.6 from family A was found to be genetically
analysed, using the method described by Hansen et al.14 ApoE homozygous for a novel missense mutation at codon 127 in
E2, E3, and E4 alleles were determined by allele specific oligo- exon 4 (fig 1A). A base substitution (TGT to TCT) caused an
nucleotide hybridisation (Innogenetics, Gent, Belgium). Com- amino acid change from cysteine to serine, and was
mon LPL mutations (Asp9Asn), (Asn291Ser), and designated C127S or FH-Kairouan.
(Ser447Stop) were detected by enzymatic restriction of PCR Specific binding and internalisation of LDL at 37°C by
products as described previously.9–11 fibroblasts of proband II.6 from family A was less than 2% of
the activity found in normal cells from a normolipidaemic
LDL-R activity donor (fig 1B). This proband displayed similar results (data
Analysis of LDL-R activity was performed on cultured skin not shown) for degradation at 37°C. As expected, cultured
fibroblasts obtained after skin biopsy from proband II.6 and cells from his mother (I.1) showed half of normal LDL-R
his mother I.1 from family A, from proband II.1 from family B, activity.
and from a healthy donor. Cells were cultured in Dulbecco’s The C127S mutation created a new restriction site for MnlI
Modified Eagle Medium (DMEM) supplemented with 10% (CCTC), allowing rapid detection of the mutation in all family
fetal calf serum (vol/vol). Induction of the expression of maxi- members. After agarose gel electrophoresis of MnlI digested
mal LDL-R activity was obtained by a 48 hour incubation of PCR products from exon 4, a 192 bp fragment characterised
the cultured fibroblasts in lipoprotein deficient serum. Native the normal allele, while a shorter 174 bp fragment character-
LDL used in these studies was isolated from normolipidaemic ised the mutant allele (fig 1C, D). This method helped to diag-
subjects. Binding, uptake, and degradation of 125I-LDL by cul- nose unequivocally all carriers of the mutation in family A.
tured skin fibroblasts were determined as described The second proband II.1 from family B was found to be
elsewhere.15 Only results of binding and internalisation at genetically homozygous for a missense mutation at codon 245
37°C are shown as data representative of two independent of exon 5 (fig 2A). A base substitution (GAT to AAT) caused an
experiments performed in triplicate. amino acid change from aspartic acid to asparagine. Genomic

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Figure 2 Family B. (A) Partial LDL-R gene sequence of exon 5 from a control subject (Normal) and from proband II.1 (Mutant). (B)
Representative experiment of specific binding and internalisation of 125I-LDL at 37°C by fibroblasts from proband II.1 and from a
normolipidaemic subject (Normal) (mean of triplicates). (C) Pedigree of family B.

DNA sequencing in both parents found them to be hetero- LDL cholesterol level and she was obese (BMI=33.2). The
zygous for this mutation. The mutation was designated D245N mother of the proband II.1 (family B) had xanthelasma but
and named FH-Tozeur. Proband II.1 from family B had less was free from CAD at the age of 46.
than 2% of normal LDL-R activity in cultured fibroblasts (fig
2B). DISCUSSION
Two unrelated FH families originating from central and
Genotype-phenotype relationship southern Tunisia were found with missense mutations of the
In three homozygotes carrying the C127S mutation from LDL-R gene.
family A, levels of plasma LDL cholesterol varied from 746 to The C127S mutation in exon 4, designated FH-Kairouan,
966 mg/dl with a mean of 855 mg/dl (fig 1C). All had extensive changed a highly conserved cysteine in the fourth of the seven
xanthomatosis and CAD. They were all carriers of genotype tandem cysteine rich repeats involved in the structure of the
E3E3 for apoE and of wild type for the D9N and N291S ligand binding domain.17 Most missense mutations located in
variants of the LPL gene. Only proband II.6 was a this region have been reported to produce a class 1 (null allele)
heterozygous carrier of the Stop447 mutation, suggesting that or a class 2 (transport defective) type of defect.1 Here, LDL-R
common modifier alleles were not contributing to plasma activity was abolished in cultured fibroblasts from the
lipoprotein levels.16 In contrast, plasma LDL cholesterol in proband and was significantly reduced in his mother. All car-
proband II.1 from family B, who was homozygous for the riers of the mutation had significant hypercholesterolaemia in
D245N mutation, was only 428 mg/dl (fig 2C). Proband B was the family. Therefore, this mutation was found to be responsi-
a carrier of wild type alleles for apoE and for the LPL gene. This ble for a profound alteration in LDL-R function and an FH
woman was free of xanthomatosis at the age of 20, but she had phenotype in this family from central Tunisia.
40% stenosis of the aortic root. These results suggested The second mutation, D245N in exon 5, designated
dissimilarity in clinical expression between homozygotes FH-Tozeur, changed a highly conserved residue located at the
carrying different mutations. C-terminal end in the sixth of the seven cystein rich repeats
Heterozygotes carrying one copy of either of the LDL-R gene that form the binding site for apoB. This mutation, which
mutations (C127S or D245N) had significantly increased occurs on a CpG dinucleotide, was previously reported in a
plasma LDL cholesterol. Four of them (AI.2, AII.1, AII.5, and Dutch patient,18 with no mention of any functional conse-
AII.7) were heterozygous carriers of the E4 allele. Four C127S quence in vitro. In addition, a mutation involving the same
carrier heterozygotes (AI.2, AII.1, AII.6, and AII.7) were also codon (D245E), FH-Cincinnati-1, was reported in an Ameri-
carriers of the LPL Stop447 mutation. The mother of proband can homozygous FH patient,1 which produces a class 2B phe-
II.6 (family A) had coronary artery disease (CAD) and high notype. Here less than 2% of the normal LDL-R activity was

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found in cultured fibroblasts from the proband. Hypercholes- Kerkeni, and C Morel for their technical assistance and S Braham for
terolaemia segregated dominantly with an allelic dosage preparing the manuscript.

J Med Genet: first published as 10.1136/jmg.39.11.e74 on 1 November 2002. Downloaded from http://jmg.bmj.com/ on July 10, 2023 by guest. Protected by copyright.
effect, confirming this mutation as the cause of FH in this
family from southern Tunisia. .....................
Genotype-phenotype relationships were analysed in ho- Authors’ affiliations
mozygotes and heterozygotes. In three homozygotes carrying M N Slimane, M Hammami, Laboratoire de Biochimie, Faculté de
the C127S mutation, very high levels of plasma LDL Médecine, 5019 Monastir, Tunisia
cholesterol were associated with extensive xanthomatosis and S Lestavel, V Clavey, J C Fruchart, Unité INSERM U545, Institut
CAD. In contrast, plasma LDL cholesterol in the proband who Pasteur de Lille, Université de Lille 2, 59019 Lille, France
F Maatouk, M H Ben Fahrat, Service de Cardiologie, Centre
was homozygous for the D245N mutation was nearly two Hospitalier Universitaire, 5019 Monastir, Tunisia
times lower, with no xanthomatosis; however, she had 40% P Benlian, Département de Biologie Moléculaire, Hôpital Saint Antoine,
stenosis in the aortic root. 75571 Paris, France
These differences in the LDL cholesterol levels between the
Correspondence to: Dr M N Slimane, Laboratoire de Biochimie, Faculté
two probands support the fact that other genes with lipid de Médecine, 5019 Monastir, Tunisia; [email protected]
lowering effects could interact with the phenotypic expression
of the mutation. Recently, Deiana et al19 reported that the mean
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