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

Atherosclerosis 222 (2012) 158–166

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Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients Afef Slimani a,∗ , Awatef Jelassi a , Imen Jguirim a , Mohamed Najah a , Lamia Rebhi b , Asma Omezzine b , Faouzi Maatouk c , Khaldoun Ben Hamda c , Maha Kacem d , Jean-Pierre Rabès e , Marianne Abifadel f , Catherine Boileau e,g , Mustapha Rouis h , Mohamed Naceur Slimane a,∗,1 , Mathilde Varret g,1 a

Research Unit of Genetic and Biological Factors of Atherosclerosis, Faculty of Medicine, Monastir 5000, Tunisia URMSP 28/04, Biochemistry Department, Sahloul University Hospital, Sousse, Tunisia c Department of Cardiovascular Diseases, Fattouma Bourguiba Hospital, Monastir 5000, Tunisia d Department of Endocrinology, Farhat Hachad Hospital, Sousse, Tunisia e AP-HP, Hôpital A. Paré, Boulogne-Billancourt, Université Versailles Saint-Quentin-en-Yvelines, France f Faculté de Pharmacie, Université St-Joseph, Beirut, Lebanon g INSERM U698, CHU Xavier Bichat, Université Paris Denis Diderot, France h UMR 7079, Physiology and Physiopathology Université Pierre et Marie Curie, Paris, France b

a r t i c l e

i n f o

Article history: Received 16 October 2011 Received in revised form 9 February 2012 Accepted 9 February 2012 Available online 19 February 2012 Keywords: Familial hypercholesterolemia LDLR gene PCSK9 gene Phenotypic variability

a b s t r a c t 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 LDLcholesterol 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. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Autosomal dominant hypercholesterolemia (ADH, OMIM # 143890) is genetically heterogeneous and associated with

∗ Corresponding authors at: Unit of Research: Genetic and Biologic Factors of Atherosclerosis, Faculty of Medicine, Monastir 5000, Tunisia. Tel.: +216 73 462 200; fax: +216 73 460 737. E-mail addresses: [email protected] (A. Slimani), [email protected] (M.N. Slimane). 1 The authors equally contributed to this work. 0021-9150/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2012.02.018

mutations in the genes encoding the low density lipoprotein receptor (LDLR) (FH, OMIM # 606945) [1], apolipoprotein B (APOB) (FDB, OMIM # 144010) [2], and proprotein convertase subtilisin/kexin type 9 (PCSK9) OMIM # 607786) [3]. Familial hypercholesterolemia (FH) is one of the most common inherited disorders in humans, with a frequency of 1 in 500 for heterozygotes in Western populations, while homozygote forms are rare (1/106 ). It is characterized by a selective increase of lowdensity lipoprotein (LDL) particles in plasma, giving rise to tendon and skin xanthomas, arcus cornea, and premature mortality from cardiovascular complications [1]. A high frequency of FH among the Tunisian population (1/165) has been reported which is related

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to the high level of consanguinity in this population. However, this disease is associated with a moderate clinical and biological expression, especially in Tunisian heterozygous FH [4]. The phenotypic expression of FH, such as the circulating LDLcholesterol level (LDL-C), is influenced by several factors, including the type of mutation (receptor-negative or receptor-defective mutation) [5], the presence of other genetic variants [6], age, gender, dietary habit, body weight and physical activity [7]. Several PCSK9 variants influencing circulating LDL-C levels have been identified [8]. The rs72555377 insertion polymorphism in exon 1 occurs in a nine-leucine repeat sequence located in the signal peptide of PCSK9. The L10 allele, insertion of one Leucine, is associated with lower LDL-C in Caucasian populations [9], while the L11 allele, insertion of two Leucines, is associated with higher LDL-C [10]. The p.Glu670Gln (rs505151), in exon 12 of the PCSK9 gene is responsible for an amino acid change that could potentially be associated with altered PCSK9 activity. Carriers of 670Gln in the general population presented increased plasma TC, LDL-C, and ApoB levels [11], while p.Ile474Val (rs562556) in exon 9 of the PCSK9 gene is associated with approximately 7% lower LDL-cholesterol levels in carriers in a Japanese population [12]. The apoE gene also accounts for a significant fraction of the variation in plasma cholesterol levels in the population [13]. The Apo E2 isoform is associated with lower LDL-C levels except for 5% of E2/E2 carriers who develop dysbetalipoproteinemia (OMIM +107741) [14]. The Apo E4 isoform is associated with elevated total and LDL-C levels [15]. Previous reports from our group identified 7 mutations in the LDLR gene causing FH in the Tunisian population and no APOB mutation [16]. So far, several studies have identified ADH mutations in the PCSK9 gene in other FH populations, but not in the Tunisian population. We present here the phenotypic variability we observed among 56 FH patients from thirteen families carrying different mutations in the LDLR gene and the genetic variants in the PCSK9 and APOE genes that can explain the LDL-cholesterol variability. 2. Materials and methods 2.1. Subjects Fifty-seven subjects from 13 unrelated Tunisian families were analyzed. The diagnosis of FH for probands was made according to the following criteria: LDL-cholesterol values above the 95th percentile when compared to a sex- and age-matched reference population [1], tendon xanthomas in the first decade, history of CHD, and molecular identification of a mutation in the LDLR gene. The control group comprised 100 Tunisian normolipidemic individuals from the same geographic area. This approach was approved by the institutional ethics committee and all patients signed an informed consent form. 2.2. Biochemical analysis Serum total cholesterol (TC), triglyceride (TG) and HDLcholesterol (HDL-C) concentrations were determined at accredited clinical laboratories using routine clinical methods. LDL-cholesterol (LDL-C) concentrations were calculated using the Friedewald equation. Adjusted plasma levels of total-, LDL-, HDL-cholesterol and triglycerides were expressed as the Multiple of Median (MoM) for age and gender of a reference population [1]. 2.3. DNA analysis Genomic DNA was prepared from white blood cells using the salting-out method. LDLR gene was amplified and sequenced as

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previously described [16]. Primers for the 12 PCSK9 exons (coding regions and close flanking intronic parts) were designed with Primer3 online software (http://frodo.wi.mit.edu/). Sequences were performed through a partnership with La PlateForme Séquenc¸age et Génomique (Institut Cochin, Paris, http://cochin.inserm.fr/) Electrophoregrams were analyzed using PolyPhred 5.04 software [17]. 2.4. Direct diagnosis of p.Pro174Ser in PCSK9 gene Exon 3 of PCSK9 gene was amplified by PCR using the following primers: 5 -AGTTGCCCCATGTCGACTAC-3 (forward) and 5 -AAAAAGGGGTCAGTGGAGGT-3 (reverse). The amplification conditions were: 94 ◦ C for 3 min followed by 94 ◦ C/56 ◦ C/72 ◦ C each one for 30 s for 30 cycles and a final extension at 72 ◦ C for 5 min. The PCR fragment was digested with 3U of NCiI restriction enzyme (New England BioLabs). The digestion products (137 bp, 93 pb and 22 pb from the wild type and 159 bp and 93 pb from the mutant allele) were separated on 8% polyacrylamide gel. The bands were visualized on UV transilluminator after staining with ethidium bromide. 2.5. Genotyping of APOE polymorphism All subjects were also genotyped for Apo E polymorphism, as described previously [18]. 2.6. Prediction in silico The causal effect of each new molecular event was estimated with in silico prediction of protein function using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/), SIFT (http://blocks.fhcrc.org/sift/SIFT.html) and Mutation Taster (http://www.mutationtaster.org). The reference sequences used for LDLR were P01130.1 (SwissProt) or NP 000518.1 (NCBI RefSeq) and for PCSK9 was Q8NBP7 (NCBI RefSeq). 2.7. Statistical analyses Statistical analysis was performed using the version 11.0 of SPSS software. We used a non-parametric test (Mann–Whitney test) to compare lipid values, adjusted for age and gender, between carriers and non-carriers of PCSK9 and APOE gene variations. A value of P < 0.05 was considered significant. 3. Results 3.1. Molecular diagnosis of FH Sequencing of the LDLR gene in the 13 probands showed 8 different mutations that were subsequently screened for in each family (Table 1). Of these, one was novel, and consisted of a deletion of an adenine at position 2299 (c.2299delA) and 7 had been reported earlier (c.796G > A/p.Asp266Asn, c.443G > C/p.Cys148Ser, c.267C > G/p.Cys89Trp, c.1477–1479delinsAGAGACA/p.Ser493ArgfsX44, c.1027G > T/p.Gly343Cys, c.1845 + 1G > A and c.1186 + 1G > A/p.Glu380 Gly396del) [16]. The novel mutation (c.2299delA) was located in exon 15 of the LDLR gene. At the protein level it causes the substitution of methionine 767 by cysteine and the appearance of a premature stop codon 21 amino acids downstream, giving rise to a truncated LDL receptor (p.Met767CysfsX21). As expected for a variant encoding a truncated protein, this mutation is predicted to be “probably damaging”, “not tolerated” and “disease causing” using PolyPhen-2 SIFT and

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Table 1 Biological features, LDLR genotypes and population distribution observed in thirteen ADH families. All lipid values are in mmol/L. Family (LDLR mutation) Subjects

Age

Sex

Distribution in Total cholesterol

Family A (p.Met767CysfsX21) I.1 40 F 7.0 I.2 42 M 8.2 M 20.6 II.1 6 14 M 5.0 II.2 9 F 6.0 II.3 Family B (p.Asp266Asn) I.1 53 M 6.1 I.2 51 F 7.6 I.3 55 F 4.0 31 F 12.0 II.1 29 F 4.6 II.2 26 F 6.0 II.3 20 M 5.0 II.4 29 F 3.7 II.5 Family C (p.Ser493ArgfsX44) 47 M 4.1 I.1 I.2 40 F 6.0 II.1 17 F 5.9 M 17.0 II.2 10 II.3 5 M 23.6 7 F 5.9 II.4 Family D (p.Ser493ArgfsX44) 55 M 7.1 I.1 6.0 I.2 45 F II.1 23 F 14.3 M 5.0 II.2 43 II.3 34 M 5.5 Family E (p.Ser493ArgfsX44) I.1 51 M 4.6 23 F 15.1 II.1 F 15.5 II.2 28 Family F (p.Ser493ArgfsX44) 35 F 8.4 I.1 6 M 24.7 II.1 8 F 7.5 II.2 Family G (p.Ser493ArgfsX44) 45 M 6.8 I.1 F 6.7 I.2 37 II.3 3 F 20.5 Family H(p.Ser493ArgfsX44) 38 M 8.9 I.1 11 F 19.7 II.2 Family I (p.Cys148Ser) 40 F 10.2 I.1 42 M 10.0 I.2 10 F 22.0 II.1 13 F 6.6 II.2 M 18.5 II.3 13 II.4 2 M 26.4 Family J (c.1845+1G>A) 42 F 8.4 I.1 27 M 4.6 II.1 Family K (p.Gly343Cys) 88 F 6.9 I.1 56 F 6.4 II.1 44 F 17.6 II.2 Family L (p.Glu380 Gly396del) 72 M 5.7 I.1 62 F 6.9 I.2 33 F 13.4 II.1 16 F 7.0 II.2 36 M 14.0 II.3 Family M (p.Cys89Trp) 60 M 7.7 I.1 66 F 10.5 I.2 63 F 7.6 I.3 31 F 6.9 II.1 14 F 23.4 II.2 24 F 19 II.3

TG

HDL-cholesterol

LDL-cholesterol

Molecular status

Reference population*

Heterozygous (%)†

0.7 1.0 1.3 0.6 0.7

1.1 0.8 2.6 1.0 1.6

5.1 6.9 15.8 3.1 4.0

+/− +/− +/+ +/− +/−

>95th >95th >95th 70th >95th

75–90 25–75

2.1 1.1 0.7 0.9 0.9 0.8 0.5 1.3

0.6 0.6 1.5 0.5 0.6 1.7 0.7 0.6

4.6 6.5 2.1 11.3 3.6 3.9 4.0 2.0

+/− +/− +/− +/+ +/− +/− +/− −/−

73th >95th <5th >95th 78th 89th 88th 18th

25–75 75–90 <10

0.9 0.5 0.9 0.6 1.9 0.4

0.3 0.8 0.5 0.4 0.3 0.6

3.7 5.0 5.0 16.9 22.9 4.3

+/− +/− +/− +/+ +/+ +/−

48th 78th >95th >95th >95th >95th

10–25 25–75 25–75

1.9 0.7 0.8 3.9 2.8

1.0 1.0 0.8 1.2 1.0

5.1 4.6 13.1 1.2 2.9

+/− +/− +/+ +/− +/−

87th 89th >95th <5th 31th

25–75 25–75

1.0 0.9 1.9

1.3 0.7 1.1

2.8 14.0 13.6

+/− +/+ +/+

25th >95th >95th

3.3 1.8 1.0

1.0 0.5 1.3

6.1 21.5 5.6

+/− +/+ +/−

>95th >95th >95th

1.1 0.8 2.1

0.6 0.9 0.8

5.4 5.4 18.8

+/− +/− +/+

>95th >95th >95th

25–75 25–75

0.8 1.0

1.2 0.5

6.7 18.7

+/− +/+

>95th >95th

75–90

1.2 0.7 1.5 0.9 1.2 1.9

0.6 1.1 0.3 1.1 0.7 0.7

9.6 4.0 21.4 4.0 17.5 25.4

+/− +/− +/+ +/− +/+ +/+

>95th 60th >95th >95th >95th >95th

>90 10–25

0.8 2.2

1.3 1.0

6.9 3.2

+/− +/−

>95th 57th

75–90 10–25

1.1 1.0 1.0

1.2 1.5 1.0

5.2 4.4 16.1

+/− +/− +/+

52th 61th >95th

25–75 10–25

0.91 0.6 1.5 1.5 0.9

0.9 0.9 0.8 1.0 0.7

4.4 6.0 13.2 5.3 13.1

+/− +/− +/+ +/− +/+

73th >95th >95th >95th >95th

25–75 25–75

1.5 2.5 1.4 1.3 1.4 2.1

0.9 1.3 1.1 1.2 0.5 0.6

4.5 8.1 5.6 5.1 19.1 17.5

+/− +/− +/− +/− +/+ +/+

71th >95th 89th >95th >95th >95th

10–25 75–90 25–75 25–75

Homozygous (%)‡

75–90 25–75 25–75

<10 25–75 25–75 25–75

25–75 75–90 25–75

10–25 <10 10–25 < 10 25–75 25–75 25–75 75–90 >90

25–75

25–75

75–90 >90 25–75 > 90

25–75

10–25 75–90 10–25

25–75 25–75

* Percentile for LDL levels, adjusted for age and sex, computed from a reference population (Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic basis of inherited diseases, sixth edition; 1989. Vol. I. McGraw-Hill, Inc., New York, pp. 1215–1250). † Range of LDL levels (%), adjusted for age and sex, computed from the values of the 38 ADH heterozygous subjects of this study (Fig. 1). ‡ Range of LDL levels (%), adjusted for age and sex, computed from the values of the 18 ADH homozygous subjects of this study (Fig. 1).

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Mutation Taster, respectively. And this novel deletion was absent in normolipidic control subjects (n = 100). In family A carrying this mutation, a history of hypercholesterolemia and severe CHD was noted. Both parents had dyslipidemia, hypertension, diabetes and a history of cardiovascular events. The homozygous proband (A.II.1) presented, at 6 years old, severe hypercholesterolemia (Table 1) and cutaneous and tendon xanthomas. His electrocardiogram stress test revealed myocardial ischemia and echo Doppler cardiac test showed hypertrophy of the left ventricle. 3.2. Phenotypic variability Biochemical and molecular specifications of the 57 subjects are shown in Table 1. LDL-Cholesterol values adjusted for age and gender according to a reference population [1] were low for several cases, particularly for three heterozygous FH subjects who presented values under the 30th percentile of the reference population. To evaluate the variability in lipid levels among the FH homozygotes (N = 18) and the FH heterozygotes (N = 38) we determined the distribution of the total- and LDL-C values adjusted for age and gender, expressed as the Multiple of Median, in each FH group (Supplemental Fig. 1). 3.2.1. Homozygous FH patients In the homozygous group, we observed a wide distribution of total- and LDL-C MoM values with two subjects showing extreme values (Supplemental Fig. 1A). At the low-end of this distribution (<10%), the woman B.II.1 presented the moderate clinical phenotype without tendon xanthomas at 20 years of age while all other homozygous patients in this study showed tendon xanthomas that appeared before 20. She had 40% stenosis of the aortic root while homozygous FH subjects usually present severe stenosis of up to 90%. For the last 11 years she has been treated with atorvastatin (40 mg/day) resulting in a substantial reduction (36%) of TotalC, not commonly observed for homozygous FH subjects. At the molecular level she was homozygous for the p.Asp266Asn missense mutation in the LDLR gene, and the 125 I-LDL binding, endocytosis, and degradation assays performed on fibroblasts showed a <2% LDL receptor activity [19]. At the high-end of this distribution (<90%), the boy I.II.4 presented very high LDL-C values (Table 1). He presented a severe clinical phenotype with extensive xanthomatosis and coronary heart disease (CHD). At the molecular level he was homozygous for the p.Cys148Ser missense mutation in the LDLR gene, like his sister I.II.1 and his brother I.II.3 (Table 1). Interestingly, these two sibs, with lower LDL-C than their brother, exhibited a similar severe clinical phenotype with tendon xanthomas and CHD. 3.2.2. Heterozygous FH patients In the heterozygous group, we observed a wide distribution of total- and LDL-C MoM values with six subjects with extreme values (Supplemental Fig. 1B). At the low-end of this distribution (<10%), the three subjects B.I.3, D.II.2, and E.I.1 presented low LDL-C levels for FH heterozygotes (Table 1). Compared to a reference population [1], subjects B.I.3 and D.II.2 are biologically defined as hypocholesterolemic subjects with a LDL-C under the 5th percentile while they were heterozygous carriers of the p.Ser493ArgfsX44 and p.Asp266Asn mutations in the LDLR gene, respectively. At the highend of this distribution (> 90%), the three subjects F.II.2, I.I.1 and I.II.2 presented high LDL-C levels for FH heterozygotes, (Table 1). At the molecular level, subject F.II.2 (MoM = 2.19, Median 1.4) carried the p.Ser493ArgfsX44 mutation in the LDLR gene like 13 other subjects from this study (Table 1) who presented an LDL-C MoM ranging from 0.53 to 1.97. Subjects I.I.1 and I.II.2 (MoM = 2.91 and

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2.14, Median 1.4) carried the p.Cys148Ser mutation in the LDLR gene, also carried by subject I.I.2 (MoM = 1.1). 3.3. Genetic factors that modify the severity of FH In order to find the molecular factors at the origin of the observed clinico/biological variability among FH patients from this study, we sequenced the 12 exons of PCSK9 and genotyped the apoE isoforms for all subjects (Supplemental Table). 3.3.1. Polymorphisms in PCSK9 and APOE genes and circulating lipid levels Among the 53 FH patients genotyped for the p.Leu21dup/tri PCK9 polymorphism (rs72555377), the L10 and L11 alleles were found for 19 (4 L10/L10, 15 L9/L10) and 3 (L9/L11) FH patients, respectively. In this sample, the frequency of the L10 and L11 alleles were 21.7% and 2.8% respectively. Among the 49 FH patients genotyped for rs562556, the p.474Val allele was found with a frequency of 5.1% (1 Val/Val, 3 Ile/Val). Among the 51 FH patients genotyped for rs505151, the p.670Gln allele was found with a frequency of 15.7% (4 Gln/Gln, 8 Glu/Gln). Among the 49 FH patients genotyped for the apo E isoforms, the E2 and E4 alleles were found with a frequency of 5.3% (5 E3/E2) and 2.1% (2 E3/E4) respectively. In this sample of the Tunisian population all these alleles were in Hardy–Weinberg equilibrium. We evaluated the effect of each allele of these PCSK9 and APOE polymorphisms on circulating lipid levels adjusted for age and gender (values expressed as Multiple of Median) in this sample of heterozygous FH Tunisian patients (Table 2). None of the observed differences were statistically significant. 3.3.2. A new PCSK9 variant In family B we identified a novel missense variation in exon 3 of the PCSK9 gene (Fig. 2). It consists in the substitution of a Cytosine (C) by a Thymine (T) at nucleotide 520 (c.520C > T). At the protein level, it causes a change from Proline (CCC) to Serine (TCC), and was noted p.Pro174Ser. Prediction analysis using the Poly-Phen, SIFT and Mutation Taster tools indicated that this variation was “probably damaging”, “tolerated” and “disease causing”, respectively. This novel variant of the PCSK9 gene can be rapidly detected by NCiI digestion and was not found in 100 Tunisian controls, thus indicating that this PCSK9 variation is not a frequent polymorphism. Family B carried also the p.Asp266Asn mutation in the LDLR gene (Fig. 1). The proband B.II.1 was homozygous for both the LDLR mutation (p.Asp266Asn) and the PCSK9 variation (p.Pro174Ser) and presented the clinico/biological phenotype of FH heterozygotes as argued above, thus indicating that this new PCSK9 variation is able to reduce the severity of FH, acting as a loss-of-function variant. Subjects B.II.2, B.II.3 and B.II.4 were found to be homozygous for the PCSK9 variant and heterozygous for the LDLR mutation and presented LDL-C levels below the 90th percentile (Table 1, Fig. 1). Moreover, the father B.I.1, heterozygous for both the PCSK9 variant and the LDLR mutation, also presented LDL-C levels below the 75th percentile. Interestingly, the aunt (B.I.3) also heterozygous for both the PCSK9 variant and the LDLR mutation, was biologically defined as a hypocholesterolemic subject with an LDL-C under the 5th percentile as argued above. Therefore, the loss-of-function effect of this new PCSK9 variant is observed for the homozygous FH proband and for the 5 related heterozygous FH subjects. However, the mother B.I.2, heterozygous for both the PCSK9 variant and the LDLR mutation, presented a classical biological phenotype of FH heterozygotes. 3.3.3. Known PCSK9 and APOE variants In addition to this novel variation in the PCSK9 gene, we reported in family C three heterozygous carriers of the L11 allele of the

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Table 2 Plasma lipid levels, adjusted for age and gender (values expressed as Multiple of Median: MoM), in different genotypic groups of heterozygous FH Tunisian patients. Values are given as medians (25th to 75th interquartile range). Genotype

Total-cholesterol (MoM)

Triglycerides (MoM)

LDL-cholesterol (MoM)

HDL-cholesterol (MoM)

L9/L9 L9/L10 and L10/L10 p value

20 15

N

1.27 (1.04–1.7) 1.36 (1.12–1.5) 0.97

0.75 (0.60–1.39) 1.05 (0.83–1.17) 0.27

1.45 (1.12–2.04) 1.39 (1.22–1.87) 0.65

0.8 (0.61–1.13) 0.72 (0.61–0.92) 0.58

Glu670/Glu670 Glu670/Gln670 and Gln676/Gln670 p value

26 8

1.32 (1.10–1.52) 1.23 (0.98–1.82) 0.83

0.91 (0.62–1.19) 1.04 (0.62–1.19) 0.76

1.39 (1.24–1.96) 1.40 (0.85–1.91) 0.65

0.80 (0.61–1.13) 0.68 (0.61–0.86) 0.44

Ile474/Ile474 Ile474/Val474 p value

30 3

1.29 (1.07–1.61) 1.46 (1.17–1.75) 0.39

1.00 (0.62–1.33) 0.51 (0.43–0.97) 0.079

1.40 (1.13–1.96) 1.83 (1.29–1.97) 0.51

0.78 (0.61–1.12) 0.77 (0.61–1.23) 0.85

Apo E3/E3 Apo E3/E2 p value

25 4

1.29 (1.09–1.57) 1.23 (1.05–1.41) 0.75

0.86 (0.61–1.22) 0.97 (0.56–1.58) 0.92

1.39 (1.12–1.92) 1.33 (1.13–1.72) 0.78

0.79 (0.62–1.12) 0.79 (0.65–1.12) 0.84

p.Leu21dup/tri PCSK9 polymorphism (C.I.2, C.II.3 and C.II.4) (Fig. 2). The L11 allele was not found in 100 Tunisian normocholesterolemic and normotriglyceridemic controls indicating that it is not a frequent polymorphism. Three members of family C (C.I.2, C.II.1 and C.II.2) were heterozygous for the p.Ile474Val polymorphism of PCSK9. In family C, the FH-causing mutation is a frameshift in exon 10 of LDLR gene, p.Ser493ArgfsX44, that was identified only in Tunisian FH subjects [23]. None of the PCSK9 variants or APOE alleles are associated, in this family, with a modified expression of the disease. In families D and E, the FH-causing mutation is also the p.Ser493ArgfsX44 variant in the LDLR gene. Heterozygous carriers, D.II.2, D.II.3 and E.I.1, presented a normal biological phenotype (LDL-cholesterol level <5th and at the 31st and 25th percentiles respectively, Table 1). The L10 allele of the p.Leu21dup (rs72555377) PCSK9 polymorphism cannot be associated with lower plasma LDL-C levels in family D since it is carried by subject D.II.2 but not by subject D.II.3. And subject D.I.1 presenting a classical FH biological phenotype (LDL-cholesterol level at the 87th percentile, Table 1) also carried the L10 PCSK9 allele (Fig. 2). Nor can the p.Gln670 allele of rs505151 be associated with increased plasma LDL-C levels in families D and E since it is carried by subjects D.II.2 and E.I.1 but not by subject D.II.3, and family members D.I.1 and D.I.2 presenting a classical FH biological phenotype (LDL-cholesterol level at the 87th and 89th percentile respectively, Table 1) also carried the p.Gln670 PCSK9 allele (Fig. 2).

In family I, the FH-causing mutation is the p.Cys148Ser mutation in the LDLR gene. Heterozygous carriers, I.I.2 and I.II.2, and homozygous carrier I.II.4 presented extreme values of plasma LDL-cholesterol when compared to the present sample of Tunisian FH patients (Table 1). The p.Gln670 allele of rs505151 cannot be associated with increased plasma LDL-cholesterol levels in family I since it is carried by subject I.I.2 but not by subjects I.II.2 and I.II.4. And family members I.I.1, I.II.1 and I.II.3 presenting a classical FH biological phenotype also carried the p.Gln670 PCSK9 allele Likewise, the apo E4 allele cannot be associated with increased plasma LDL-cholesterol levels in family I since it is carried by subjects I.I.2 and I.II.2 but not by subject I.II.4 (Fig. 3). 4. Discussion In this study, we report the molecular analysis of the LDLR, PCSK9 and APOE genes in 56 Tunisian FH patients from 13 families and we identified two novel mutations in the LDLR and PCSK9 genes. In family A, we identified a novel frameshift mutation (c.2299delA, p.Met767CysfsX21) which occurs in the glycosylation domain. Expression of the mutant gene and functional studies of the encoded receptor have not been performed, but the pathogenic significance of this mutation is favored by several arguments. Indeed, cosegregation of the mutant allele with the FH phenotype has been demonstrated in this family, and it introduces a premature stop codon resulting in a truncated protein.

I

? 1

2

3

LDLR c.796 G>A (p.Asp266Asn) G (Asp) II 1

2

3

4

5

A (Asn)

PCSK9 rs72555377 (p.Leu21dup) c.520C>T (p.Pro174Ser) L9 C (Pro)

L10 T (Ser)

Fig. 1. Genotypes for LDLR and PCSK9 gene variations in family B. Blackened symbols indicate homozygous affected members, half-blackened symbols indicate heterozygotes affected members (with LDL-cholesterol level above the 70th percentile of an age- and gender-matched reference population, Table 1), clear symbols indicate unaffected members.

D

C

E ?

I 1

1

2

1

2

2

LDLR c.1477-1479delinsAGAGACA (p.Ser493ArgfsX44) delinsAGAGACA(ArgfsX44 g )

PCSK9 rs72555377 (p.Leu21dup/tri) L9

L10

L11

rs562556 A>G ( pp.Ile474Va Ile474Val) l) A(Ile)

G(Val)

II 1

2

3

4

1

2

3

1

2

rs505151 A>G (p.Glu670Gln) A (Glu)

G (Gln)

E3

E2

APOE

A. Slimani et al. / Atherosclerosis 222 (2012) 158–166

WT ((Ser))

Fig. 2. Genotypes for LDLR, PCSK9 and APOE gene variations in families C, D and E. Blackened symbols indicate homozygous affected members, half-blackened symbols indicate heterozygous affected members (with LDL-cholesterol level above the 70th percentile of an age- and gender-matched reference population, Table 1), clear symbols indicate unaffected members.

163

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A. Slimani et al. / Atherosclerosis 222 (2012) 158–166

LDLR c.443G>C (p.Cys148Ser) C (Cys)

G(Ser)

PCSK9 rs72555377 (p.Leu21dup)

I 1

L10

L9

2

rs505151 A>G (p.Glu670Gln) A (Glu)

G (Gln)

E3

E4

APOE

II 1

2

3

4

Fig. 3. Genotypes for LDLR, PCSK9 and APOE gene variations in family I. Blackened symbols indicate homozygous affected members, half-blackened symbols indicate heterozygous affected members (with LDL-cholesterol level above the 70th percentile of an age- and gender-matched reference population, Table 1), clear symbols indicate unaffected members.

Among the 56 Tunisian FH patients of this study (38 heterozygotes and 18 homozygotes), plasma levels of total- and LDL-C varied widely indicating the existence of FH-modifier factors. For several FH subjects, the discrepancy between the clinico/biological and molecular phenotype observed reveals the existence of factors that either decrease or increase the severity of the disease. In a previous study, we identified one of these factors as the traditional Tunisian diet which is enriched in polyunsaturated fats [4]. This type of diet has been shown to have long-term beneficial therapeutic effects by reducing the incidence of recurrent cardiovascular events. In the present study, we show that 24% of the subjects heterozygous for a mutation in the LDLR gene have LDL-cholesterol levels under the 60th percentile of an age- and gender-matched reference population (9 out of 38, Table 1). Other normocholesterolemic subjects carriers of a mutation in the LDLR gene have been reported [20–22]. And, a cholesterol-lowering gene was mapped to chromosome 13, but remains unidentified [23]. In order to identify the putative molecular actors underlying the phenotypic variability observed among the FH subjects of this sample, the PCSK9 gene was screened and the apo E genotypes were determined. Among the 13 unrelated FH probands of this study, the frequency of the L10, L11, p.474Val and p.670Gln PCSK9 alleles were similar to those reported in previous studies (dbSNP: www.ncbi. nlm.nih.gov/projects/SNP/). The frequency of the E2 and E4 APOE alleles ranges from 1.6 to 13.1% and from 8.2 to 31%, respectively [13]. The frequency of the apo E2 allele, among the 13 unrelated FH probands of this study, is within the reported ranges, the frequency of the E4 allele is lower (3.8%) than/those reported in other populations, very probably because of the small size of the sample. The association of the L10 PCSK9 allele with lower LDLcholesterol levels was reported in a Caucasian population [9] as well as in a sample of FH subjects carrying the same mutation

within the LDLR gene, p.C681X [24], but not found in the sample of Tunisian FH patients reported here. This discordance may be due to the type of LDLR mutation since our sample of FH patients is not homogeneous at the molecular level. To clarify this point, we also performed the analysis on a sample of patients with the same LDLR mutation, p.Ser493ArgfsX44 (families C–H), but did not find any significant association. An alternative explanation for this discordance could be the existence of another variant, in linkage disequilibrium with the PCSK9 L10 allele, with an effect on LDLcholesterol levels. To clarify this point, we also performed the analysis for FH subjects without any other PCSK9 alleles such as p.474Val. and p.670Gln or APOE alleles E2 and E4, and still did not observe a significant association. The same observation was reported by Pisciotta et al. [25]. The association of the p.Gln670 PCSK9 allele with higher LDL-cholesterol levels was reported in the Lipoprotein Coronary Atherosclerosis Study (LCAS) population [10] as well as in a Chinese Population [11], in men, but not women, from an European population [26] and in a sample of middle aged subjects from Italy [27], but not in the sample of Tunisian FH patients reported here. This discordance was also observed in the Dallas Heart Study [28] as well as in healthy men in the UK [29]. The association of the p.Val474 PCSK9 allele with lower LDL- Cholesterol levels was reported in a Japanese population [18], but not in the sample of Tunisian FH patients reported here. A similar discordance was observed in other previously reported populations [27–29]. The association of the apo E2 allele with lower LDL-Cholesterol levels reported in a large number of studies (OMIM +107741) was not found here in our sample of Tunisian FH patients. This discordance may be due to the small size of the sample analyzed here. At the level of the whole sample of FH subjects, the PCSK9 and APOE variants studied here appears to be without any effect on plasma lipid levels, but with the inherent limitation of the small sample size.

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However, at the individual level, some of the PCSK9 and APOE alleles can explain a part of the observed variability in the biological expression of the disease. We identified a novel missense mutation in PCSK9 (c.520C > T, p.Pro174Ser) in family B. It is located in the catalytic domain responsible for the autocatalytic processing of the convertase. Six members of family B presented reduced LDL-C levels with respect to their LDLR genotype, that can be explained by the presence of this new mutation in the PCSK9 gene. This lowering effect of a PCSK9 mutation on baseline LDL-C levels in FH patients seems specific to this new mutation since it was not observed for another loss of function mutation, the p.Arg46Leu, screened among 1130 unrelated FH subjects [30]. Several loss of function mutations in the PCSK9 gene were reported in hypocholesterolemic subjects and associated with an increased response to statin therapy [31–33]. In agreement with these reports, subject B.II.1, homozygous for both the new putative loss of function mutation in PCSK9 and a LDLR mutation, presented an increased response to atorvastatin when compared to homozygous FH subjects. Although these clinical and biological observations are the first evidence for assigning a loss of function role to the p.P174S PCSK9 mutation, in vitro experiments are required to definitively conclude. On the other hand, Pisciotta et al. reported rare missense mutations of PCSK9 that may worsen the clinical phenotype of patients carrying LDLR mutations [34]. The association of the L11 PCSK9 allele with dyslipidemia was reported in two French Canadian families with FCHL and in one French Canadian woman and her father with hypercholesterolemia [35]. The three FH subjects reported here with the L11 PCSK9 allele do not present increased LDL cholesterol levels when compared to other FH subjects. It should be noted, however, that subject C.II.3 is also heterozygous for the E2 allele of the APOE gene that is reported to have a LDL-cholesterol reducingeffect. Thus, this latter may hide the possible LDL-cholesterol increasing-effect of the L11 PCSK9 allele. Finally, subject C.I.2 presented a slightly reduced, 78th percentile LDL-cholesterol that can be attributable to the presence of either/both the p.474Val PCSK9 allele and the E2 allele of the APOE gene despite presence of the L11 PCSK9 allele. Taken together, data from family C are not in favor of a LDL-cholesterol increasing-effect for the L11 PCSK9 allele. The normal biological phenotype of the heterozygote FH subjects C.I.1 (LDL-cholesterol at the 48th percentile), D.II.2 (LDLcholesterol <5th percentile), D.II.3 (LDL-cholesterol at the 31st percentile) and E.I.1 (LDL-cholesterol at the 25th percentile) was not explained in this study, nor was the worsened biological phenotype of the homozygous FH subject I.II.4 and of the heterozygous FH subjects F.II.2, I.I.1 and I.II.2. Thus, altogether these observations suggest the existence of other FH-modifier factors that remain to be identified. As recently shown, five different mutations in the APOB gene, resulting in truncated apo B, are responsible for a 56% reduction in LDL-cholesterol in 6.7% of a sample of FH subjects with LDL-cholesterol below the 50th percentile for age and gender from The Netherlands [36]. The searching for FH-modifier mutations within genes associated with a hypocholesterolemic phenotype, such as APOB and ANGPTL3 genes, should be performed in the Tunisian FH population.

5. Conclusion In conclusion, the genetic variations in PCSK9 and APOE genes analyzed in this study may explain a part of the variable phenotypic expression observed in Tunisian FH patients at the individual level but not in the whole sample.

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Acknowledgments We are indebted to family members for their cooperation. This work was supported by grants from the Tunisian and French Ministries of Higher Education and Scientific Research (Project PHC-Utique 10G0812 du CMCU, comité mixte pour la coopération universitaire Franco-Tunisien) and from L’Agence Nationale de la Recherche (ANR-08-GENO-002-01). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2012.02.018. References [1] Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 2863–913. [2] Kane JP, Havel RJ. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 2717–52. [3] Abifadel M, Varret M, Rabès J-P, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003;34:154–6. [4] Jelassi A, Slimani A, Jguirim I, et al. Moderate phenotypic expression of familial hypercholesterolemia in Tunisia. Clin Chim Acta 2010;411:735–8. [5] Guardamagna O, Restagno G, Rolfo E, et al. The type of LDLR gene mutation predicts cardiovascular risk in children with familial hypercholesterolemia. J Pediatr 2009;155:199–204. [6] Bertolini S, Pisciotta L, Di Scala L, et al. Genetic polymorphisms affecting the phenotypic expression of familial hypercholesterolemia. Atherosclerosis 2004;174:57–65. [7] Jansen AC, van Wissen S, Defesche JC, Kastelein JJ. Phenotypic variability in familial hypercholesterolemia: an update. Curr Opin Lipidol 2002;13:165–71. [8] Abifadel M, Rabès JP, Devillers M, et al. Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease. Hum Mutat 2009;30(4):520–9. [9] Yue P, Averna M, Lin X, Schonfeld G. The c.43 44insCTG variation in PCSK9 is associated with low plasma LDL-cholesterol in a Caucasian population. Hum Mutat 2006;27:460–6. [10] Chen SN, Ballantyne CM, Gotto Jr AM, et al. A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis. J Am Coll Cardiol 2005;45:1611–9. [11] Aung LHH, Yin R-X, Miao L, et al. The proprotein convertase subtilisin/kexin type 9 gene E670G polymorphism and serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Lipids Health Dis 2011;10:2–15. [12] Miyake Y, Kimura R, Kokubo Y, et al. Genetic variants in PCSK9 in the Japanese population: Rare genetic variants in PCSK9 might collectively contribute to plasma LDL cholesterol levels in the general population. Atherosclerosis 2008;196:29–36. [13] Eichner JE, Dunn ST, Perveen G, et al. Apolipoprotein E polymorphism and cardiovascular disease: a HuGE review. Am J Epidemiol 2002;155:487–95. [14] Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J Biol Chem 1982;257:2518–21. [15] Hatters DM, Peters-Libeu CA, Weisgraber KH. Apolipoprotein E structure: insights into function. Trends Biochem Sci 2006;31:445–54. [16] Jelassi A, Jguirim I, Najah M, et al. Limited mutational heterogeneity in the LDLR gene in familial hypercholesterolemia in Tunisia. Atherosclerosis 2009;203:449–53. [17] Stephens M, Sloan JS, Robertson PD, Scheet P, Nickerson DA. Automating sequence-based detection and genotyping of SNPs from diploid samples. Nat Genet 2006;38:375–81. [18] Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990;31:545–8. [19] Slimane MN, Lestavel S, Clavey V, et al. CYS127S (FH-Kairouan) and D245N (FH-Tozeur) mutations in the LDL receptor gene in Tunisian families with familial hypercholesterolaemia. J Med Genet 2002;39:e74. [20] Hobbs HH, Leitersdorf E, Leffert CC, et al. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors. J Clin Invest 1989;84:656–64. [21] Ekström U, Abrahamson M, Florén C-H, et al. An individual with a healthy phenotype in spite of a pathogenic LDL receptor mutation C240F. Clin Genet 1999;55:332–9. [22] Tosi I, Toledo-Leiva P, Neuwirth C, Naoumova RP, Soutar AK. Genetic defects causing familial hypercholesterolaemia: identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic. Atherosclerosis 2007;194:102–11.

166

A. Slimani et al. / Atherosclerosis 222 (2012) 158–166

[23] Knoblauch H, Muller-Myhsok B, Busjahn A, et al. A cholesterol lowering gene maps to chromosome 13.q. Am J Hum Genet 2000;66:157–66. [24] Abifadel M, Rabès JP, Jambart S, et al. The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene. Hum Mutat 2009;30:E682–91. [25] Pisciotta L, Sallo R, Rabacchi C, et al. Leucine 10 allelic variant in signal peptide of PCSK9 increases the LDL cholesterol-lowering effect of statins in patients with familial hypercholesterolemia. Nutr Metab Cardiovasc Dis 2011, September 13 [Epub ahead of print]. [26] Evans D, Beil FU. The E670G SNP in the PCSK9 gene is associated with polygenic hypercholesterolemia in men but not in women. BMC Med Genet 2006; 7:66. [27] Norata GD, Garlaschelli K, Grigore L, et al. Effects of PCSK9 variants on common carotid artery intima media thickness and relation to ApoE alleles. Atherosclerosis 2010;208:177–82. [28] Kotowski IK, Pertsemlidis A, Luke A, et al. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am J Hum Genet 2006;78:410–22. [29] Scartezini M, Hubbart C, Whittall RA, et al. The PCSK9 gene R46L variant is associated with lower plasma lipid levels and cardiovascular risk in healthy U.K. men. Clin Sci (Lond) 2007;113:435–41.

[30] Strøm TB, Holla ØL, Cameron J, Berge KE, Leren TP. Loss-of-function mutation R46L in the PCSK9 gene has little impact on the levels of total serum cholesterol in familial hypercholesterolemia heterozygotes. Clin Chim Acta 2010;411(3–4):229–33. [31] Berge KE, Ose L, Leren TP. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy. Arterioscler Thromb Vasc Biol 2006;26:1094–100. [32] Davignon J, Dubuc G. Statins and ezetimibe modulate plasma proprotein convertase subtilisin kexin-9 (PCSK9) levels. Trans Am Clin Climatol Assoc 2009;120:163–73. [33] Thompson JF, Hyde CL, Wood LS. Comprehensive whole genome and candidate gene analysis for response to statin therapy in the Treating to New Targets (TNT) cohort. Circ Cardiovasc Genet 2009;2:173–81. [34] Pisciotta L, Oliva CP, Cefalu AB, et al. Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia. Atherosclerosis 2006;186:433–40. [35] Abifadel M, Bernier L, Dubuc G, et al. A PCSK9 variant and familial combined hyperlipidaemia. J Med Genet 2008;45:780–6. [36] Huijgen R, Sjouke B, Vis K, et al. Genetic variation in APOB PCSK9, and ANGPTL3 in carriers of pathogenic autosomal dominant hypercholesterolemic mutations with unexpected low LDL-Cl Levels. Hum Mutat 2012;33:448–55.