Mohamed Naceur Slimane

Phenotypic expression of familial hypercholesterolaemia in Central and Southern Tunisia

We studied 14 families with familial hypercholesterolaemia (FH) from Central and Southern Tunisia. Twenty-six living homozygotes were identified in these areas of whom 24 homozygotes and 27 of their obligate heterozygote parents are the subject of this report. Ten of the 14 families are unrelated and in 9 of them there were consanguineous marriages. The mean age of homozygotes was 16 for females (range 2.5-40) and 12.5 for males (range 2-34). All the homozygotes had extensive xanthomatosis and showed variable clinical manifestations of coronary heart disease (CHD). Plasma total and LDL cholesterol levels averaged 18 and 16.9 mmol/l, respectively. Mean high density lipoprotein (HDL) cholesterol values were 0.48 mmol/l for males and 0.70 mmol/l for females. The mean age of the obligate heterozygotes was 44 (range 32-62 years) for mothers and 51 (range 35-80 years) for fathers. None of them had tendon xanthomas, not even the oldest, who was aged 80. Only 5 of the 27 obligate heterozygotes had developed CHD (aged 34-58). Plasma cholesterol levels varied more than twofold (4.1-10 mmol/l) and averaged 6.79 and 7.51 mmol/l for males and females, respectively. LDL cholesterol values were below the age- and sex-related 95th percentile from the Lipid Research Clinics Prevalence Study in 46% of male and 30% of female heterozygotes. The frequency of homozygotes was 1:125,000 and the minimum estimated frequency of heterozygotes was 1:165 in Central and Southern Tunisia. Only Afrikaners in South Africa and French Canadians have such high frequencies of FH.

Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia.

BACKGROUND Abetalipoproteinemia (ABL) and Homozygous Familial Hypobetalipoproteinemia (Ho-FHBL) are rare monogenic diseases characterised by very low plasma levels of cholesterol and triglyceride and the absence or a great reduction of apolipoprotein B (apoB)-containing lipoproteins. ABL results from mutations in the MTP gene; Ho-FHBL may be due to mutations in the APOB gene. METHODS We sequenced MTP and APOB genes in three Tunisian children, born from consanguineous marriage, with very low levels of plasma apoB-containing lipoproteins associated with severe intestinal fat malabsorption. RESULTS Two of them were found to be homozygous for two novel mutations in intron 5 (c.619-3T>G) and in exon 8 (c.923 G>A) of the MTP gene, respectively. The c.619-3T>G substitution caused the formation of an abnormal mRNA devoid of exon 6, predicted to encode a truncated MTP of 233 amino acids. The c.923 G>A is a nonsense mutation resulting in a truncated MTP protein (p.W308X). The third patient was homozygous for a novel nucleotide deletion (c.2172delT) in exon 15 of APOB gene resulting in the formation of a truncated apoB of 706 amino acids (apoB-15.56). CONCLUSIONS These mutations are expected to abolish the apoB lipidation and the assembly of apoB-containing lipoproteins in both liver and intestine.

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

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 degrees 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.

A novel splice site mutation of the LDL receptor gene in a Tunisian hypercholesterolemic family.

BACKGROUND Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in either the low-density lipoprotein receptor, the apolipoprotein B or the proprotein convertase subtilisin/kexin type 9 genes. It is characterized by a high concentration of low-density lipoprotein (LDL), which frequently gives rise to premature coronary disease. In this study, we report a novel splice site mutation of the LDL receptor gene in a Tunisian family. METHODS Seven patients from the family were screened for mutations in the LDLR gene and the apoB gene, using direct sequencing. RT-PCR and study on cultured skin fibroblast were realised to characterize the effect of novel mutation. RESULTS Direct sequencing of the promoter and 18 exons reveals a G>A substitution in the splice site junction of intron 8 (c.1186+1 G>A). Study on cultured skin fibroblasts showed a residual activity of 10% of the LDL receptor. Reverse transcription, amplification and direct sequencing of RNA from patients lymphocytes reveal a deletion of the final 51 bp of exon 8 preserving the reading frame. CONCLUSIONS The study identified a novel splice mutation c.1186+1 G>A in the LDL receptor gene. It causes the utilization of a new cryptic donor splice site 51 bp downstream from the normal site.

Apolipoprotein E Knockout Mice Over-Expressing Human Tissue Inhibitor of Metalloproteinase 1 Are Protected against Aneurysm Formation but Not against Atherosclerotic Plaque Development

Aims: We investigated the effect of plasma levels of human tissue inhibitor of metalloproteinase (hTIMP)-1 on arterial lesion development and aneurysm formation in apolipoprotein-E-deficient mice (ApoE–/–). Methods: Control and transgenic mice were fed either a chow diet or a high-fat diet for 90 and 180 days. Results: hTIMP-1 has a tendency to decrease atherosclerotic lesions, but did not attain significance (approximately 6% reduction in hTIMP-1+/+, p = 0.075, and approximately 4% in hTIMP-1+/0, p = 0.088 vs. control). Immunohistological and histological analyses revealed a reduction in macrophage accumulation (23% of control in hTIMP+/0, p = 0.065, and 49% of control in hTIMP+/+, p < 0.05) but not in collagen degradation within the lesion in transgenic mice. Moreover, elastin degradation in sites of pseudo-microaneurysms was reduced in transgenic mice (37% of control in hTIMP-1+/0, p < 0.05, and 50% of control in hTIMP-1+/+, p < 0.05). DNA array analysis of matrix metalloproteinase (MMP) expression followed by real-time PCR quantification revealed a significant up-regulation of MMP-3, MMP-12 and MMP-13 in arterial lesions of ApoE–/– mice fed a high-fat diet in comparison with the same mice fed a chow diet. Conclusion: These data show that hTIMP-1 reduces aneurysm formation in ApoE–/– mice but does not protect them against the development of arterial lesions.

Moderate phenotypic expression of familial hypercholesterolemia in Tunisia.

BACKGROUND Autosomal Dominant Hypercholesterolemia (ADH) is an autosomal dominant disease caused by mutations in the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Xanthomas and coronary heart diseases (CHD) at an early age are the major clinical manifestations of the disease. METHODS 16 families with familial hypercholesterolemia from different regions in Tunisia participated in the study. Mutations within the LDLR gene were screened through DNA sequencing. Lipids values were measured by standard enzymatic methods. RESULTS We present here thirty five homozygotes and fifty six heterozygotes. Homozygotes presented extensive xanthomatosis, variable clinical manifestations of CHD, and total cholesterol levels in males and females of 17.26+/-4.18 and 17.64+/-2.59 mmol/L respectively. HDL-cholesterol levels were 0.62+/-0.24 and 1.00+/-0.61 mmol/L for males and females, respectively. None of the heterozygotes had tendon xanthomas (except for one female aged 62), eight had corneal arcus, and nine developed CHD mean between 46 and 88 years old. Total cholesterol levels in males and females ranged from 4.60 to 8.90 and from 4.30 to 10.50 mmol/L, respectively. CONCLUSION Tunisian FH heterozygotes are characterized by a moderate clinical and biological expression of the disease.

PPAR-β/δ activation promotes phospholipid transfer protein expression

The peroxisome proliferator-activated receptor (PPAR)-β/δ has emerged as a promising therapeutic target for treating dyslipidemia, including beneficial effects on HDL cholesterol (HDL-C). In the current study, we determined the effects of the PPAR-β/δ agonist GW0742 on HDL composition and the expression of liver HDL-related genes in mice and cultured human cells. The experiments were carried out in C57BL/6 wild-type, LDL receptor (LDLR)-deficient mice and PPAR-β/δ-deficient mice treated with GW0742 (10mg/kg/day) or a vehicle solution for 14 days. GW0742 upregulated liver phospholipid transfer protein (Pltp) gene expression and increased serum PLTP activity in mice. When given to wild-type mice, GW0742 significantly increased serum HDL-C and HDL phospholipids; GW0742 also raised serum potential to generate preβ-HDL formation. The GW0742-mediated effects on liver Pltp expression and serum enzyme activity were completely abolished in PPAR-β/δ-deficient mice. GW0742 also stimulated PLTP mRNA expression in mouse J774 macrophages, differentiated human THP-1 macrophages and human hepatoma Huh7. Collectively, our findings demonstrate a common transcriptional upregulation by GW0742-activated PPAR-β/δ of Pltp expression in cultured cells and in mouse liver resulting in enhanced serum PLTP activity. Our results also indicate that PPAR-β/δ activation may modulate PLTP-mediated preβ-HDL formation and macrophage cholesterol efflux.

Matrix metalloproteinase-1 and matrix metalloproteinase-12 gene polymorphisms and the risk of ischemic stroke in a Tunisian population

Matrix metalloproteinases (MMPs) play an important role in early atherosclerosis, extracellular matrix remodeling, plaque rupture and myocardial infarction. MMP gene polymorphisms contribute to the risk of developing cardiovascular diseases. In this study, we investigated, for the first time, the association between MMP-1-16071G/2G, MMP-12 -82A/G and MMP-12 1082A/G genotypes and haplotypes and the risk of ischemic stroke (IS) among patients with type 2 diabetes mellitus (T2DM). To examine whether these genetic polymorphisms are associated with susceptibility to IS, 196 patients with IS and 192 controls were examined by PCR-based RFLP. When the analyses were adjusted for multiple risk factors, no interaction between T2DM and MMP-1-1607 1G/2G polymorphism on the risk of ischemic stroke was found (p=0.074). However, MMP-12 polymorphisms genotypes were associated with the higher risk of IS in diabetic patients compared with total patients. The -82G-1082G haplotype of MMP-12 polymorphisms was associated with higher risk of ischemic stroke in diabetic patients [AOR=2.33; 95% CI (1.25-3.62), P=0.032]. These findings showed that there was an important joint effect of the MMP-12 polymorphisms and T2DM on the risk of IS and therefore it can be considered as a potential marker of cerebrovascular disorders in diabetic patients.

Limited mutational heterogeneity in the LDLR gene in familial hypercholesterolemia in Tunisia.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In previous studies, we have identified novel mutations in Tunisian FH families. In this study, we have extended our investigation to additional families. Five unrelated probands were screened for mutations in the LDLR and APOB genes, using direct sequencing and enzymatic restriction. We identified two novel LDLR mutations: a missense mutation in exon 7: p.Gly343Cys (c.1027G>T), and a nonsense mutation in exon 17: p.Lys816X (c.2446A>T). Using the PolyPhen and SIFT prediction computer programs the p.Gly343Cys is predicted to have a deleterious effect on LDL receptor activity. The missense mutation we found in exon 3, p.Cys89Trp (c.267C>G), has previously been identified in patients from United Kingdom and Spain, and is reported here for the first time in the Tunisian population. Finally, the framshift mutation in exon 10, p.Ser493ArgfsX44, is reported here for the fourth and fifth time in Tunisian families. The latter is the most frequent FH-causing mutation in Tunisia. These LDLR gene mutations enrich the spectrum of mutations causing FH in the Tunisian population. The framshift mutation, p.Ser493ArgfsX44, seems to be a founder mutation in this population.

Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients

BACKGROUND Autosomal dominant hypercholesterolemia (ADH) is commonly caused by mutations in the low-density lipoprotein (LDL) receptor gene (LDLR), in the apolipoprotein B-100 gene (APOB), or in the proprotein convertase subtilisin kexine 9 gene (PCSK9). ADH subjects carrying a mutation in LDLR present highly variable plasma LDL-cholesterol (LDL-C). This variability might be due to environmental factors or the effect of some modifying genes such as PCSK9 and APOE. AIMS We investigated the molecular basis of thirteen Tunisian ADH families and attempted to determine the impact of PCSK9 and APOE gene variations on LDL-cholesterol levels and on the variable phenotypic expression of the disease. METHODS AND RESULTS Fifty six subjects were screened for mutations in the LDLR gene through direct sequencing. The causative mutation was found to segregate with the disease in each family and a new frameshift mutation, p.Met767CysfsX21, was identified in one family. The distribution of total- and LDL-cholesterol levels, adjusted for age and gender, among homozygous and heterozygous ADH patients varied widely. Within seven families, nine subjects presented low LDL-cholesterol levels despite carrying a mutation in the LDLR gene. To identify the molecular actors underlying this phenotypic variability, the PCSK9 gene was screened using direct sequencing and/or enzymatic restriction analysis, and the apo E genotypes were determined. A new missense variation (p.Pro174Ser) in the PCSK9 gene was identified and characterized as a new putative loss-of-function mutation. CONCLUSION Genetic variations in PCSK9 and APOE genes could explain only part of the variability observed in the phenotypic expression in Tunisian ADH patients carrying mutations in the LDLR gene. Other genetic variants and environmental factors very probably act to fully explain this phenotypic variability.