Familial Hypercholesterolemia Associated with Severe Hypoalphalipoproteinemia in a Moroccan Family
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c Indian Academy of Sciences RESEARCH NOTE Familial hypercholesterolemia associated with severe hypoalphalipoproteinemia in a Moroccan family KARIMA AIT CHIHAB1,2, RACHID CHATER1,2, ANA CENARRO3, ANASS KETTANI2, SERGIO CASTILLO3, ∗ MOHAMED LOUTFI1, JOSEP RIBALTA 4, AHMED ADLOUNI2, MIGUEL POCOVI3 and MARIAME EL MESSAL1 1Laboratoire de Biochimie et Biologi`e Molecularie, Groupe de G´en´e tique et Biologie Mol´eculaire, Facult´e des Sciences Ain chock. B. P. 5366, Casablanca, Morocco 2Laboratoire de Recherche sur les Lipoprot´eines et l’Ath´erosc-l´erose, Facult´e des Sciences Ben M’Sik. B. P. 7955, Casablanca, Morocco 3Laboratorio de Investigacion Molecular, Hospital Universitario Miguel Servet. Po Isbel la catolica, 1–3, Zaragaza, Spain 4Unitat de Recerca de Lipids i Anteriosclerosi, Facultat de Medicina, Universitat Rovira i virgili, Sant Lloren 21. 43201 Reus, Spain Introduction screened N370S and L444P, the most frequent mutations in the β-glucocerebrosidase gene (GBA) that have been asso- Familial hypercholesterolemia (FH) is an autosomal dom- ciated with HALP (Pocovi et al. 1998). This study revealed inant genetic disorder characterized by elevated levels of the IVS3-23C→A mutation in LCAT gene, although it did not low-density-lipoprotein cholesterol (LDL-C), tendon xan- appear to cosegregate with HALP phenotype in this family. thomas and increased risk of premature coronary heart dis- ease (CHD). The FH phenotype results from defects in the Materials and methods LDL receptor gene (LDLR), and also defects in other genes like apolipoprotein B (apoB) (familial defective apo B) or Subjects proprotein convertase subtilisin/kexin type 9 (PCSK9)(Soria At the first consultation, the proband, 15 years old, pre- et al. 1989; Abifadel et al. 2003). High-density-lipoprotein sented extra vascular lipid deposits and lipid profile charac- cholesterol (HDL-C) levels are significantly reduced in many teristics of FH homozygotes. However, no evident signs of FH families. However, the metabolic basis of this hypoal- atherosclerosis had been revealed. Because LDL apheresis is phalipoproteinemia (HALP) has not been clearly understood. not available in Morocco, a high dose of statin was prescribed It has been reported that FH heterozygotes with HALP are to the patient. Proband’s relatives were recruited and eleven prone to develop more severe premature artery disease (de were available for clinical examination and blood analyses. Sauvage Nolting et al. 2003). Indeed, the latest guidelines All subjects gave their informed consent prior to their inclu- for the diagnosis and management of FH consider levels of sion in the study. At the age of 22 years, the proband died by HDL-C less than 40 mg/dl as one of the major cardiovascular myocardial infarction. risk factors in the FH population (Civeira 2004). In this report, we describe a Moroccan FH family with as- Lipid analysis sociated HALP. After screening of the LDLR gene, we iden- We analysed serum TC, TG and HDL-C by enzymatic meth- tified a novel frameshift mutation in exon 5 of the LDLR ods and apo A-I and apo B by an immunoturbidimetric gene (756del7). To elucidate the inheritance of the HALP method (Brustolin et al. 1991). We calculated LDL-C by in this family, we analysed some other genes involved in Friedewald formula (Friedewald et al. 1972). HDL metabolism, such as apoAI, lecithin:cholesterol acyl- transferase (LCAT) and lipoprotein lipase (LPL). We also Genetic analyses DNA isolation: Genomic DNA from white blood cells was *For correspondence. E-mail: [email protected]. isolated using a salting-out procedure (Miller et al. 1988). Keywords. familial hypercholesterolemia; hypoalphalipoproteinemia; LDLR gene; LCAT gene; SSCP; DNA sequencing. Journal of Genetics, Vol. 86, No. 2, August 2007 159 Karima Ait Chihab et al. LDLR gene: We analysed LDLR gene by Southern blot (El LCAT gene: We analysed the promoter region, exons and Messal et al. 2003) and PCR–SSCP. For SSCP analysis, PCR exon–intron junctions of LCAT gene by PCR–SSCP and se- products were added to 95% formamide buffer, denatured quenced the PCR fragment (exon 4) as described previously at 95◦C for 5 min, immediately chilled on ice and elec- (Recalde et al. 2002). Mutation confirmation was carried out trophoresed at 1050 V, at 25◦Cor15◦C in a MDE gel with by PCR-restriction enzyme digestion with MspIofexon4 or without 5% glycerol, respectively, in 0.6 × TBE buffer and subsequent 3% Nusieve agarose gel electrophoresis. for 15 h, on an automated DNA sequencer equipped with a water jacket (ALF-ExpressTM, Pharmacia Biotech). We GBA gene: We screened the GBA gene for the presence of sequenced a PCR fragment of LDLR gene (exon 5) using the most frequent mutations, N370S and L444P,byPCRand Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer) restriction enzyme digestion with XhoIandNciI, respectively and a CEQ8000 DNA automated sequencer (Beckman Coul- (Beutler et al. 1990; Tsuji et al. 1987). ter). For mutation confirmation, PCR products were elec- trophoresed at 96 V at room temperature, for 3 h, in 2% LPL gene: We analysed the promoter region, exons and agarose gel in 1× TAE. exon–intron junctions of LPL gene by direct sequencing in an ABIPRISM 3100 Genetic Analyser. Sequence of primers used were reported previously by Abifadel et al. (2004). Apo B gene: We screened for R3500Q and R3531C muta- apoB et al tions in gene as described by Rab`es . (1997). Results Apo E gene: We determined the apoE genotype by PCR– Family analysis restriction analysis with HhaI as described by Hixon and The biochemical and clinical features of the proband and his Vernier (1990). relatives are presented in table 1. The proband (II-5) and his sisters II-7 and II-8 (figure 1) showed biochemical and clin- Apo AI gene: We analysed the promoter region, exons and ical features of homozygous FH. Among the other recruited exon–intron junctions of apoAI gene by PCR–SSCP (Re- relatives, only the proband’s sisters II-2 and II-6 would be calde et al. 2002). heterozygous FH patients (table 1), according to proband’s Figure 1. Pedigree of the analysed individuals indicating LDL-C levels, FH genotype, HDL-C levels and HALP genotype. Proband (deceased) is indicated by an arrow; wt, wild type allele; mut, mutant allele. LDL-C and HDL-C levels were obtained before any medical treatment. 160 Journal of Genetics, Vol. 86, No. 2, August 2007 FH associated with severe hypoalphalipoproteinemia Table 1. Clinical and biochemical features of the proband and his relatives. TC HDL-C LDL-C TG Apo AI Apo B FH Subjects Sex Age (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) EVLD CAD diagnosis I-1 Female 46 166 40 115 53 ND ND No No Non FH I-2 Female 40 220 23 173 116 89 117 No No Non FH (Mother) I-3 Male 50 279 24 221 174 84 167 No No Non FH (Father) I-4 Female 28 184 47 124 66 ND ND No No Non FH II-2 Female 22 438 38 368 190 143 222 No No Heterozygous FH II-3 Female 21 173 24 133 83 71 99 No No Non FH II-4 Male 31 101 29 61 57 ND ND No No Non FH II-5 Male 15 837 3 821 67 ND ND PCX, Deceased Homozygous (Proband) TX, LX, by FH 20 (462) (15) (416) (156) (51) (264) AC myocardial infarction II-6 Female 17 248 30 194 126 83 128 No No Heterozygous FH II-7 Female 15 736 16 693 136 61 394 PCX, Ischemia Homozygous TX, AC FH II-8 Female 10 722 14 682 141 52 409 PCX, No Homozygous TX, AC FH III-1 Female 10 213 35 152 131 101 111 No No Non FH TC, total cholesterol; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; TG, triglycerides; ND, not determined; EVLD, extra vas- cular lipid deposits; PCX, planar cutaneous xanthomas; TX, tendon xanthomas; LX, left xanthelasma; AC, arcus cornealis; CAD, coronary artery disease. Lipid values are obtained before any medical treatment. Lipid values of the proband after high dose of lipid lowering treatment are given in parentheses. The clinical diagnosis of the proband’s relatives is based on ”proband’s relative diagnosis” (Civeira 2004). relative diagnostic criteria (Civeira 2004). Except II-7, none putative heterozygotes by PCR and electrophoresis showed of the examined subjects showed signs of ischaemia in elec- two bands: normal allele (180 bp) and mutant allele (173 trocardiograms or cardiac scans. Surprisingly, the three ho- bp). The genotype of the studied subjects is indicated in fig- mozygous FH patients had a very low concentration of HDL- ure 1. In the same way, we analysed 107 healthy subjects for C and apo AI, never previously reported in FH subjects. the presence of the identified mutation. None of them were Also, except I-1 and I-4, the other relatives showed a de- carrier of the 756del7 mutation in the LDLR gene. crease in HDL-C levels, but not to the extent observed in We screened the proband and his relatives for R3500Q homozygous subjects. After an irregular observance of high and R3531C mutations in the apoB gene, but none of them dose lipid lowering treatment, the proband died by myocar- had either of these two FDB-causing mutations. We also dial infarction. analysed the apoE genotype, in order to assess its contribu- tion to FH phenotype. All subjects of the studied family were FH molecular analysis homozygous for the E3 allele. Southern blot analysis did not reveal any major defects in LDLR gene. However, LDLR gene PCR-SSCP analysis of HALP molecular analysis all studied subjects revealed two abnormal SSCP profiles in exon 5, when compared with the healthy subject pattern: an The entire apoAI, LCAT and LPL genes were screened, and heterozygous pattern for the parents, II-2, II-3, II-6, and III- the two most frequent mutations in GBA gene, N370S and 1, and an homozygous profile for the proband, II-7, and II-8.