RESEARCH NOTE
Familial hypercholesterolemia associated with severe hypoalphalipoproteinemia in a Moroccan family
KARIMA AIT CHIHAB
1,2, RACHID CHATER
1,2, ANA CENARRO
3, ANASS KETTANI
2, SERGIO CASTILLO
3, MOHAMED LOUTFI
1, JOSEP RIBALTA
4, AHMED ADLOUNI
2, MIGUEL POCOVI
3and MARIAME EL MESSAL
1∗
1
Laboratoire 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
2
Laboratoire de Recherche sur les Lipoprot´eines et l’Ath´erosc-l´erose, Facult´e des Sciences Ben M’Sik.
B. P. 7955, Casablanca, Morocco
3
Laboratorio de Investigacion Molecular, Hospital Universitario Miguel Servet. Po Isbel la catolica, 1–3, Zaragaza, Spain
4
Unitat de Recerca de Lipids i Anteriosclerosi, Facultat de Medicina, Universitat Rovira i virgili, Sant Lloren 21. 43201 Reus, Spain
Introduction
Familial hypercholesterolemia (FH) is an autosomal dom- inant genetic disorder characterized by elevated levels of low-density-lipoprotein cholesterol (LDL-C), tendon xan- thomas and increased risk of premature coronary heart dis- ease (CHD). The FH phenotype results from defects in the LDL receptor gene (LDLR), and also defects in other genes like apolipoprotein B (apoB) (familial defective apo B) or proprotein convertase subtilisin / kexin type 9 (PCSK9) (Soria et al. 1989; Abifadel et al. 2003). High-density-lipoprotein cholesterol (HDL-C) levels are significantly reduced in many FH families. However, the metabolic basis of this hypoal- phalipoproteinemia (HALP) has not been clearly understood.
It has been reported that FH heterozygotes with HALP are prone to develop more severe premature artery disease (de Sauvage Nolting et al. 2003). Indeed, the latest guidelines for the diagnosis and management of FH consider levels of HDL-C less than 40 mg / dl as one of the major cardiovascular risk factors in the FH population (Civeira 2004).
In this report, we describe a Moroccan FH family with as- sociated HALP. After screening of the LDLR gene, we iden- tified a novel frameshift mutation in exon 5
of the LDLR gene (756del7). To elucidate the inheritance of the HALP in this family, we analysed some other genes involved in HDL metabolism, such as apoAI, lecithin:cholesterol acyl- transferase (LCAT) and lipoprotein lipase (LPL). We also
*For correspondence. E-mail: elmessal@yahoo.fr.
screened N370S and L444P, the most frequent mutations in the β -glucocerebrosidase gene (GBA) that have been asso- ciated with HALP (Pocovi et al. 1998). This study revealed the IVS3-23C → A mutation in LCAT gene, although it did not appear to cosegregate with HALP phenotype in this family.
Materials and methods
Subjects
At the first consultation, the proband, 15 years old, pre- sented extra vascular lipid deposits and lipid profile charac- teristics of FH homozygotes. However, no evident signs of atherosclerosis had been revealed. Because LDL apheresis is not available in Morocco, a high dose of statin was prescribed to the patient. Proband’s relatives were recruited and eleven were available for clinical examination and blood analyses.
All subjects gave their informed consent prior to their inclu- sion in the study. At the age of 22 years, the proband died by myocardial infarction.
Lipid analysis
We analysed serum TC, TG and HDL-C by enzymatic meth- ods and apo A-I and apo B by an immunoturbidimetric method (Brustolin et al. 1991). We calculated LDL-C by Friedewald formula (Friedewald et al. 1972).
Genetic analyses
DNA isolation:
Genomic DNA from white blood cells was isolated using a salting-out procedure (Miller et al. 1988).
Keywords.
familial hypercholesterolemia; hypoalphalipoproteinemia; LDLR gene; LCAT gene; SSCP; DNA sequencing.
LDLR gene:
We analysed LDLR gene by Southern blot (El Messal et al. 2003) and PCR–SSCP. For SSCP analysis, PCR products were added to 95% formamide bu ff er, denatured at 95
◦C for 5 min, immediately chilled on ice and elec- trophoresed at 1050 V, at 25
◦C or 15
◦C in a MDE gel with or without 5% glycerol, respectively, in 0.6 × TBE bu ff er for 15 h, on an automated DNA sequencer equipped with a water jacket (ALF-Express
TM, Pharmacia Biotech). We sequenced a PCR fragment of LDLR gene (exon 5) using Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer) and a CEQ8000 DNA automated sequencer (Beckman Coul- ter). For mutation confirmation, PCR products were elec- trophoresed at 96 V at room temperature, for 3 h, in 2%
agarose gel in 1 × TAE.
Apo B gene:
We screened for R3500Q and R3531C muta- tions in apoB gene as described by Rab`es et al. (1997).
Apo E gene:
We determined the apoE genotype by PCR–
restriction analysis with HhaI as described by Hixon and Vernier (1990).
Apo AI gene:
We analysed the promoter region, exons and exon–intron junctions of apoAI gene by PCR–SSCP (Re- calde et al. 2002).
LCAT gene:
We analysed the promoter region, exons and exon–intron junctions of LCAT gene by PCR–SSCP and se- quenced the PCR fragment (exon 4) as described previously (Recalde et al. 2002). Mutation confirmation was carried out by PCR-restriction enzyme digestion with MspI of exon 4 and subsequent 3% Nusieve agarose gel electrophoresis.
GBA gene:
We screened the GBA gene for the presence of the most frequent mutations, N370S and L444P, by PCR and restriction enzyme digestion with XhoI and NciI, respectively (Beutler et al. 1990; Tsuji et al. 1987).
LPL gene:
We analysed the promoter region, exons and 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).
Results
Family analysis
The biochemical and clinical features of the proband and his relatives are presented in table 1. The proband (II-5) and his sisters II-7 and II-8 (figure 1) showed biochemical and clin- ical features of homozygous FH. Among the other recruited relatives, only the proband’s sisters II-2 and II-6 would be 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.
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 of the examined subjects showed signs of ischaemia in elec- trocardiograms or cardiac scans. Surprisingly, the three ho- mozygous FH patients had a very low concentration of HDL- C and apo AI, never previously reported in FH subjects.
Also, except I-1 and I-4, the other relatives showed a de- crease in HDL-C levels, but not to the extent observed in homozygous subjects. After an irregular observance of high dose lipid lowering treatment, the proband died by myocar- dial infarction.
FH molecular analysis
Southern blot analysis did not reveal any major defects in LDLR gene. However, LDLR gene PCR-SSCP analysis of all studied subjects revealed two abnormal SSCP profiles in exon 5, when compared with the healthy subject pattern: an heterozygous pattern for the parents, II-2, II-3, II-6, and III- 1, and an homozygous profile for the proband, II-7, and II-8.
Concerning the subjects I-1, I-4, and II-4, they presented a normal SSCP profile. DNA sequencing of PCR products of the proband, II-7, and II-8 subjects showed a novel deletion of 7 bp at nucleotide 756 of cDNA, 756del7, in homozygous state. This frameshift mutation creates a stop codon at posi- tion 241, and would give rise to a truncated protein (data not shown). The confirmation of the heterozygous status in the
putative heterozygotes by PCR and electrophoresis showed two bands: normal allele (180 bp) and mutant allele (173 bp). The genotype of the studied subjects is indicated in fig- ure 1. In the same way, we analysed 107 healthy subjects for the presence of the identified mutation. None of them were carrier of the 756del7 mutation in the LDLR gene.
We screened the proband and his relatives for R3500Q and R3531C mutations in the apoB gene, but none of them had either of these two FDB-causing mutations. We also analysed the apoE genotype, in order to assess its contribu- tion to FH phenotype. All subjects of the studied family were homozygous for the E3 allele.
HALP molecular analysis
The entire apoAI, LCAT and LPL genes were screened, and
the two most frequent mutations in GBA gene, N370S and
L444P, were analysed. PCR–SSCP of LCAT gene of the
proband and his parents showed a heterozygous pattern in
the father, when compared with the healthy control subject
and the heterozygous IVS3-23C → A positive control sam-
ple. DNA sequencing confirmed the presence of the IVS3-
23C → A mutation, located 22 bases upstream of the acceptor
splicing site of intron 3 of LCAT gene. The screening of
IVS3-23C → A in the rest of relatives, by restriction analysis
with MspI, showed heterozygosity in II-2, II-3, II-4 and III-1 subjects, and homozygosity in I-4 subject.
The cosegregation analysis (figure 1) showed a non- cosegregation of the IVS3-23C → A mutation with the HALP phenotype: the proband II-5, II-7, and II-8 subjects, with se- vere HALP, and I-2 and II-6 subjects, with moderate HALP, did not carry the LCAT mutation. However, the subject I-4, with normal HDL-C levels, was homozygous for this muta- tion.
Discussion
In this study, we describe a Moroccan family with a combined phenotype of FH and HALP. The novel LDLR gene mutation identified in this family, 756del7, induces a frameshift with creation of a stop codon at position 241 and it would cause a class 1 LDL receptor defect. This 756del7 mutation is likely to be the FH causing mutation, as nei- ther any other LDLR gene mutation nor the two most fre- quent FDB-causing mutations, R3500Q and R3531C, have been identified in this family. Moreover, the 756del7 mu- tation has not been detected in a population of 107 healthy controls, suggesting that it is not a polymorphism, but a dis- ease causing mutation. The homozygous carriers of 756del7 mutation are severely a ff ected: the proband died prematurely by CHD, his two sisters are a ff ected by severe hypercholes- terolemia and one of them shows ischemia complications.
Four out of six heterozygotes have LDL-C levels in nor- mal range. Several examples of FH with low LDL-C have been reported and the lack of correlation between phenotype and genotype has been explained by environmental (Sun et al. 1994), and / or genetic factors (Sass et al. 1995). For the family reported here, environmental factors could explain in part the interindividual phenotype variation (data not shown).
The presence of homozygous apoE3 genotype in all the het- erozygotes of 756del7 mutation would exclude this gene in FH phenotype suppression, but does not exclude the probable presence of other genetic factors.
All carriers of the 756del7 mutation present severe HALP, particularly pronounced in the homozygotes. Signifi- cantly reduced plasma levels of HDL-C have been reported in many FH families but the mechanism of this HALP is poorly understood. Kinetic studies suggest that the catabolism of HDL-apo AI is increased in both homozygous and heterozy- gous FH patients, which is thought to be unrelated to the magnitude of hypercholesterolemia (Fr´enais et al. 1999). In our FH / HALP subjects, an interaction between LDL and HDL metabolism is likely to occur, since we observed an important di ff erence between homozygous and heterozygous FH patients regarding their HDL-C levels, and also found that the decrease of LDL-C in the proband after lipid low- ering therapy was accompanied by a large increase of HDL- C (table 1). Other explanations have been put forward for the reduced HDL-C levels in lacking functional LDLR back- ground, such an enhanced activity of cholesteryl ester trans-
fer protein (Gu´erin et al. 1994) or overexpression of SR-BI, a hepatic receptor for HDL (Wang et al. 1998). Variations in any gene involved in HDL metabolism, like mutations in LPL gene, could also be related to HALP associated with FH (Pimstone et al. 1995). In our studied family, no muta- tions were found in the LPL gene or in apoAI. Also, N370S and L444P, the most frequent mutations in GBA gene, as- sociated with HALP, were not found. However, we identi- fied the IVS3-23 C → A mutation in LCAT gene, previously described in Spanish heterozygous HALP subjects (Recalde et al. 2002). By introducing a new acceptor splicing dinu- cleotide AG, this mutation could partially induce an aberrant processing of LCAT mRNA and be responsible for the HALP phenotype (Recalde et al. 2002). However, the noncosegre- gation of IVS3-23 C → A mutation and the reduced HDL-C levels observed in our Moroccan family is not in accordance with a causal e ff ect of this mutation.
It has been reported that FH heterozygotes with reduced HDL-C levels are prone to develop more severe premature artery disease when compared with FH heterozygotes with normal HDL-C levels (de Sauvage Nolting et al. 2003). In our studied family, all FH heterozygotes are asymptomatic for myocardial ischaemia in electrocardiograms and cardiac scans. However, before drawing any conclusions about the increased susceptibility to atherosclerosis due to the combi- nation of HALP and FH phenotypes in the studied family, more cardiovascular analyses need to be performed.
In conclusion, the 756del7 novel mutation in LDLR gene appears to be the FH causing mutation in the studied fam- ily. While the correlation between genotype and phenotype is clear in homozygotes, it is less evident in heterozygotes for FH. These results show the improved diagnostic preci- sion obtained by introducing genetic diagnosis in FH fami- lies with identified mutation (Civeira 2004), even when the mutation spectrum of the LDLR gene in Moroccan popula- tion seems to be heterogeneous (El Messal et al. 2003, 2006;
Chater et al. 2006). Finally, further studies are needed to elucidate the molecular basis of the HALP observed in the studied family and the role of the reduced HDL-C levels in the development of atherosclerosis in a FH context.
Acknowledgements
We are indebted to all members of the studied family for their coop- eration. Our thanks to Dr Assali (cardiologist, Larache, Morocco) for his collaboration. We also thank the group of Prof. C. Boileau (INSERM U383) for their help with the LPL gene study. This work was supported in part by “Action integree Maroco-Espagnole no.
54/PR/99” and a grant of “Fondo de Investigaciones Sanitarias FIS PI031106, Spain”.
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