Elsevier

Atherosclerosis

Volume 154, Issue 3, 15 February 2001, Pages 557-565
Atherosclerosis

Fh-Souassi: a founder frameshift mutation in exon 10 of the LDL-receptor gene, associated with a mild phenotype in Tunisian families

https://doi.org/10.1016/S0021-9150(00)00572-4Get rights and content

Abstract

Familial hypercholesterolemia (FH) has a higher prevalence in central Tunisia together with a milder clinical expression than in western countries. The molecular basis of FH in Tunisia remains unknown. Our aim was to identify FH-causing mutations in three unrelated families (21 subjects) from the area of Souassi (central Tunisia). In probands with a presentation of homozygous FH, the promoter and 18 exons of the low density lipoprotein (LDL)-receptor gene were sequenced in both orientations. A novel complex frameshift mutation was identified in exon 10, nucleotides 1477–1479 (TCT) at Serine 472 were replaced by an insertion of seven nucleotides (AGAGACA), producing a premature termination codon 43 amino acids downstream. Binding of 125I-labelled LDL at 4°C to cultured fibroblasts from two probands showed <2% normal LDL-receptor activity. AvaII digestion of PCR amplified genomic DNA identified this unique mutation in all families; homozygotes n=11, heterozygotes n=10. All mutation carriers shared the same haplotype (7 RFLPs), suggesting that they had a common ancestor. Despite high plasma LDL levels (m=16.0±3.0 mmol/l) and extravascular cholesterol deposits, most homozygotes were diagnosed after puberty and had a delayed onset of cardiovascular complications. Moreover, most heterozygotes were free of clinical signs and had plasma LDL cholesterol in the normal range (4.7±1.3 mmol/l) without taking any lipid-lowering medication. This mild clinical phenotype which contrasted with the severity of the mutation, could not be explained by specific apolipoprotein E or lipoprotein lipase alleles.

Introduction

Familial hypercholesterolemia (FH) is a dominantly inherited disorder of lipoprotein metabolism characterised by hypercholesterolemia, xanthomas and premature coronary heart disease [1]. The disease is caused by absent or impaired function of cell surface low density lipoprotein (LDL)-receptors that play a major role in the catabolism of LDL, the major cholesterol carrier in human plasma. Numerous mutations at various sites in the LDL-receptor gene have been reported, including nonsense and missense mutations, deletions and insertions affecting the synthesis, post-translational processing, ligand binding activity or internalisation and recycling of the LDL receptor [2], [3].

Most populations in the western world have a prevalence of heterozygous FH of approximately one person in 500, with in most instances high allelic heterogeneity. However, in other populations, a few mutations have a high prevalence as the result of a founder effect. Examples include the Lebanese Christian Arabs [4] French Canadians [5] the Finns [6], and South African Afrikaners [7]. We reported earlier a high frequency of FH among Tunisian population (1/165) which is related to inter-marriage and consanguinity within families [8]. Moreover, we observed that Tunisian patients with a clinical diagnosis of homozygous FH have total and LDL-cholesterol concentrations similar to those of FH homozygotes in other parts of the world, however with a delayed onset of cardiovascular disease (CVD). In addition, their obligate heterozygous parents have plasma total and LDL-cholesterol much lower than their counterparts in western countries and are free of extravascular cholesterol deposits (particularly tendon xanthomas) or premature CVD. This remarkable variation in the severity of the disease could result either from the expression of specific alleles at the LDL-receptor locus, or from other genetic and/or environmental causes of hypercholesterolemia in Tunisia. Mild functional defects have been described to account for a milder clinical presentation [9], [10], [11], [12], [13]. However, Sun et al. [14] have reported a similar clinical pattern in heterozygous FH from China, independent of the type of mutation. In addition, environmental factors have been suspected to modulate the expression of the same mutations, in Cuban subjects carrying the FH-Afrikaner-2 mutation [15] or in Chinese subjects living either in China or Pacific Canada [16]. Systematic studies of FH in Tunisians have not been conducted. Our report represents the start of a comprehensive effort to identify mutations in the LDL receptor gene that cause FH.

Here, we describe the characterisation of a novel frameshift mutation found in the LDL-receptor gene in three unrelated families originating from the area of Souassi.

Section snippets

Subjects

Probands were defined by at least one young individual (age <30 years) presenting with multiple planar, tuberous or tendinous xanthoma and plasma LDL cholesterol above 10 mmol/l. Relatives with a similar clinical presentation were considered as clinical homozygotes. Family members, who had given informed consent for the study, were examined for the presence of extravascular cholesterol deposits (xanthomas or corneal arcus) and cardiovascular risk factors (obesity, BMI >30; diabetes, plasma

Results

Direct sequencing of the promoter, 18 exons and their flanking sequences of the LDL receptor gene in three unrelated probands showed only one novel frameshift mutation in exon 10. This mutation (FH-Souassi) was a deletion/insertion in which the codon Serine 472 (TCT) was deleted while 7 bp (AGAGACA) were inserted (Fig. 1). The mutation caused a frameshift introducing 43 new amino-acids and a premature TGA-Stop codon at residue 514 within the epidermal growth-factor precursor homology domain.

Discussion

In the present study, we report a novel frameshift mutation in exon 10 of the LDL-receptor gene, in three unrelated families living in the same area of Souassi, central Tunisia. This mutation, found homozygous in 11 hypercholesterolemic patients, is a complex deletion/insertion removing three nucleotides (TCT) encoding for Serine 472 and inserting 7 bp (AGAGACA). Interestingly the inserted nucleotides correspond to an imperfect repeat of the complementary strand in this region (GAGACA) of exon

Acknowledgements

The authors wish to dedicate this work to Dr Pousse for his sincere clinical involvement during this investigation. This work was supported by grants from the Secrétariat d'Etat à la Recherche Scientifique et Technique (Tunisia), INSERM (France) and British Council (Tunisia). We are grateful to Professor G.R. Thompson (Hammersmith Hospital, London), Professor J.C. Fruchart (Institut Pasteur, Lille), Professor E. Clauser and Professor G. Béreziat (Hôpital Saint-Antoine, Paris) for their

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