A Novel Loss of Function Mutation of PCSK9 Gene in White Subjects With Low-Plasma Low-Density Lipoprotein Cholesterol

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1 A Novel Loss of Function Mutation of PCSK9 Gene in White Subjects With Low-Plasma Low-Density Lipoprotein Cholesterol Tommaso Fasano, Angelo B. Cefalù, Enza Di Leo, Davide Noto, Daniela Pollaccia, Letizia Bocchi, Vincenza Valenti, Renato Bonardi, Ornella Guardamagna, Maurizio Averna, Patrizia Tarugi Objectives The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African- Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. Methods and Results We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest ( 5 th ) percentile, 44 in the highest ( 95 th ) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delg) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. Conclusions We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals. (Arterioscler Thromb Vasc Biol. 2007;27: ) Key Words: familial hypobetalipoproteinemia hypocholesterolemia loss of function mutation PCSK9 gene Familial hypobetalipoproteinemia (FHBL) is a codominant disorder characterized by very low levels of plasma low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apob). The frequency of subjects with the clinical diagnosis of heterozygous FHBL has been estimated to be 1:500/1: FHBL heterozygotes may be asymptomatic (they are often identified through population screening for low-plasma cholesterol) 2 or have clinical manifestations (fatty liver disease and intestinal fat malabsorption), which require medical intervention. 3 5 FHBL may or may not be linked to the APOB gene. Most mutations in APOB gene prevent the complete translation of apob mrna, leading to truncated apobs of various size that may or may not be secreted into the plasma. 5,6 Only a single amino acid substitution (R463W) has been reported so far as the cause of FHBL. 7 In some kindred, FHBL is linked to loci on chromosomes 3 (3p21) and 13 (13q) but the genes involved have not been identified yet. 8 Recently, PCSK9 gene has emerged as an important candidate gene for FHBL. This gene encodes a cholesterol regulated proprotein convertase that is a member of the subtilisin/kexin type 9 serine protease subfamily of proprotein convertases. In 2003 Abifadel et al 9 reported that some missense mutations in PCSK9 cause a new form of autosomal dominant hypercholesterolemia, an observation confirmed by others In mice the overexpression of wild-type PCSK9, as well as of mutant PCSK9 associated with hypercholesterolemia, resulted in a marked reduction of hepatic LDLreceptor (LDL-r) protein leading to hypercholesterolemia Missense mutations of PCSK9 associated with hypercholesterolemia in humans would increase the capacity of PCSK9 to limit the number of LDL-r, thus representing Original received October 20, 2006; final version accepted November 30, From the Department of Biomedical Sciences (T.F., E.D., L.B., P.T.), University of Modena and Reggio Emilia, Italy; Department of Clinical Medicine and Emerging Diseases (A.B.C., D.N., D.P., V.V., M.A.), University of Palermo, Italy; Division of Gastroenterology and Nutrition (R.B.), Molinette Hospital, Torino, Italy; Department of Pediatrics (O.G.), University of Torino, Italy. T.F. and A.B.C. contributed equally to this work. Correspondence to Patrizia Tarugi, PhD, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, I Modena, Italy. tarugi@unimore.it; or Maurizio Averna, MD, Department of Clinical Medicine and Emerging Diseases, University of Palermo, Italy via del Vespro 141 I Palermo. avernam@unipa.it 2007 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at DOI: /01.ATV f 677

2 678 Arterioscler Thromb Vasc Biol. March 2007 gain of function mutations. In sharp contrast, mice with targeted inactivation of PCSK9 gene (pcsk9 / ) have an increased number of LDL-r in the liver, increased removal of plasma LDL, and reduced plasma LDL-C. 16 In 2005 Cohen et al 17 found that 2 inactivating mutations of PCSK9 (Y142X and C679X), present in 2% to 2.6% of blacks of the Dallas Heart Study, were associated with 30% to 40% reduction of plasma LDL-C. They proposed the concept that in human loss of function mutations of PCSK9 would increase the number of liver LDL-r and the receptor mediated uptake and catabolism of plasma LDL, as observed in pcsk9 / mice. These loss of function mutations confer substantial protection against coronary heart disease. 18 Other mutations causing non conservative amino acid substitutions in PCSK9 were found to be associated with a significant, although less pronounced, reduction of plasma LDL-C and to an increased response to statin therapy In the present study we report the identification of a novel loss of function mutation of PCSK9 gene in a subject with the clinical diagnosis of heterozygous FHBL and in 2 hypocholesterolemic blood donors deriving from a white population. Materials and Methods Human Subjects Written informed consent was obtained from all subjects investigated. The study was approved by the institutional human investigation committee of each participating institution. FHBL Subjects We investigated 61 unrelated white subjects with the clinical diagnosis of FHBL who had been admitted to divisions of gastroenterology/hepatology for the presence of fatty liver and mild but persistent elevation of serum liver enzymes. 3,4 These subjects were recruited in various geographic area of Italy. In all of them, the criteria for the diagnosis of FHBL were the plasma level of LDL-C and apob below 5th percentile of the distribution in the population and the exclusion of secondary forms of hypolipidemia. The systematic sequence of APOB gene revealed that 35 of them were carriers of pathogenic mutations of this gene (33 were heterozygous for mutations predicted to result in truncated apob and 2 were heterozygous for the R463W substitution). Eight subjects were found to be heterozygous for novel nonconservative amino acid substitutions in apob with unknown functional effect. The remaining 18 subjects, in whom no mutations in APOB gene were found, were included in the present study under the designation of APOB negative FHBL subjects. Hypocholesterolemic A group of 7000 white blood donors (recruited in the Blood Banks of the Hospitals in the city of Palermo, Sicily) were screened for low-plasma cholesterol during routine laboratory tests. This screening identified 548 subjects with plasma cholesterol below the 5th percentile (3.31 mmol/l) of the general population. From this group of hypocholesterolemic subjects we recruited 102 individuals (27 7 years of age) with the lowest plasma cholesterol values (below 2 nd percentile of the population). None of these subjects were found to be carriers of truncated apobs detectable in plasma, as revealed by plasma apob immunoblot. The sequence of exons 1 to 25 of APOB gene in all these subjects showed that none of them carried mutations resulting in short truncated apobs that are not secreted into the plasma. These blood donors represent a selected population of healthy adults, who are subjected to strict clinical and laboratory monitoring. Subjects from the General Population The sequence variants of PCSK9 gene found in FHBL subjects and hypocholesterolemic blood donors were screened in 42 subjects with the lowest ( 5 th ) percentile, 44 subjects with the highest ( 95 th ) percentile, and 100 subjects with average plasma cholesterol, deriving from the population of the district of Ventimiglia di Sicilia. The adult population of this district in Sicily has been undergoing study for cardiovascular risk factors since A database of this population (622 males and 729 females) containing information regarding family history, dietary habits, physical activity, lifestyle, clinical examination, anthropometric measurements, and laboratory parameters is available. 22,23 In addition, the PCSK9 gene variants were screened in 40 consecutive patients with primary hypercholesterolemia attending the Lipid Clinic at the University of Palermo. Laboratory Investigations Plasma lipids and lipoproteins were measured by routine procedures. The sequence of APOB, PCSK9, and LDL-r genes was performed as previously described. 5,24 The sequence of PCSK9 gene was performed in all APOB negative FHBL patients (18 subjects) and in all hypocholesterolemic blood donors (102 subjects). PCSK9 variants found by systematic sequencing of PCSK9 gene in these subjects were screened in the other population samples by direct sequencing of the appropriate amplicons. APOE genotype was performed as reported. 25 Statistical Analysis Statistical analysis was performed by using the SPSS 13.0 (SPSS Inc, Chicago, Ill) program. Differences in the distribution of categorical variables were assessed by 2 test. Differences between groups for continuous variables were assessed by Mann-Whitney test. Results Analysis of PCSK9 gene in APOB Negative FHBL Subjects We defined APOB negative FHBL subjects as those patients in whom no mutations of APOB gene were found. We cannot rule out the possibility that these subjects have mutations in APOB gene that have not been detected by our sequencing procedure (ie, large deletions/insertions or mutations located in introns and regulatory sequences). However, we have not been able to conduct co-segregation analysis in the kindred of these subjects because of the small number of family members. The mean plasma levels of total cholesterol, LDL-C, and apob of these patients ( mmol/l, mmol/l, and mg/dl, respectively) were similar to those found in FHBL subjects heterozygous for pathogenic mutations of APOB gene (supplemental Table I, available online at Two APOB negative FHBL subjects were 2/ 2 homozygotes and two were 2/ 3 heterozygotes; their plasma levels of total cholesterol, LDL-C, and apob did not differ from the mean values found in the whole FHBL group (supplemental Table I). The results of the analysis of PCSK9 gene in APOB negative FHBL patients are shown in the Table. One patient (FHBL- 57) was heterozygous for a novel single nucleotide deletion in exon 1 (c.202delg), which causes a frameshift in mrna, leading to a premature stop codon (Ala68fsLeu82X). The predicted product of this mrna is a short peptide of 81 amino acids. The carrier of this mutation was a 34-year-old white overweight male (body mass index 30 kg/m 2 ) who had been referred to the clinic for fatty liver. His plasma lipids are

3 Fasano et al Inactivating Mutation of PCSK9 in Hypocholesterolemia 679 PCSK9 Gene Variants Found in APOB Negative FHBL Patients and in Hypocholesterolemic Minor Allele Frequency Variation (cdna) Location Consequences (Protein) FHBL Subjects Hypocholesterolemic c.42_43ctgins Exon 1 L15_L16insL c.137g T Exon 1 R46L c.158c T Exon 1 A53V c.202delg Exon 1 Ala68fsLeu82X c.230c T Exon 2 T77I c.341t C Exon 2 V114A c.1327g A Exon 8 A443T c.1420a G Exon 9 I474V c.1564g A Exon 10 A522T c.1847c T Exon 11 P616L c.2009a G Exon 12 E670G shown in the Figure. Proband s mother (deceased at the age of 59 for stroke) had a plasma lipid profile consistent with the clinical diagnosis of FHBL; however, she did not carry mutations in either APOB or PCSK9 gene. The proband s 67-year-old father was also overweight (body mass index 27 kg/m 2 ), had diabetes, and had fatty liver. His plasma cholesterol was within the normal range; however, plasma LDL-C was lower than that found in the control group and close to 10 th percentile value. He was a carrier of the PCSK9 mutation. No mutations were found in APOB and LDL-r genes. Also the proband s 62-year-old paternal aunt was a carrier of the PCSK9 mutation. She was obese (body mass index 35 kg/m 2 ) and had plasma LDL-C level lower than in normolipidemic controls ( mmol/l) (supplemental Table II). Also, the proband s 3-year-old son was a mutation carrier. Proband s family had lived for several generations in a district of Southern Italy, close to the island of Sicily. Pedigree of family FHBL-57. The notation under each subject was listed in the following order: pedigree number, total plasma cholesterol (TC, mmol/l), plasma LDL cholesterol (LDL- C, mmol/l), apob level (mg/dl), and APOE genotype. Subjects with missing plasma lipid levels did not participate. Among APOB negative FHBL subjects we found two carriers of the R46L variant (Table), a rare amino acid substitution previously reported to be associated with mildly reduced plasma LDL-C level Analysis of PCSK9 Gene in Hypocholesterolemic In view of the geographic origin of the carriers of PCSK9 mutation, we thought that the most appropriate control population for the search of this novel mutation was that living in the same geographic area. For this reason we sequenced PCSK9 gene in 102 hypocholesterolemic blood donors recruited in Sicily, whose mean levels of plasma total cholesterol and LDL-C ( mmol/l and mmol/l, respectively) were similar to those found in APOB negative FHBL patients (supplemental Tables I and II). The sequence of PCSK9 gene in these individuals led to the identification of 2 apparently unrelated carriers of the Ala68fsLeu82X mutation (Table). The plasma lipids levels of these subjects (BD-575 and BD-445) were: total cholesterol 2.15 and 2.53 mmol/l; triglycerides 0.42 and 1.83 mmol/l; LDL-C 0.62 and 0.96 mmol/l; high-density lipoprotein cholesterol 1.34 and 0.75 mmol/l; and apob 28 and 40 mg/dl, respectively. These subjects were healthy and had no clinical or laboratory signs of liver disease; the other routine laboratory tests were normal. Because these blood donors were identified by a code number only (as enforced by Italian law), we have not been able to enroll their relatives to assess the co-segregation of PCSK9 mutation with the low plasma LDL-C phenotype in their families. In the group of hypocholesterolemic blood donors, we found 4 carriers of the R46L and 1 carrier of A443T substitutions, reported to be associated with reduced plasma LDL-C, 19 as well as 4 carriers of nonconservative amino acid substitutions not reported previously (Table). The plasma lipid profiles of these carriers are shown in supplemental Table III.

4 680 Arterioscler Thromb Vasc Biol. March 2007 Population Screening for PCSK9 Sequence Variants We next screened individuals from the general population for the amino acid variants of PCSK9 found in FHBL subjects and hypocholesterolemic blood donors. The mean levels of plasma lipids in normolipidemic subjects and in subjects with the lowest (below 5 th percentile) and highest (above 95 th percentile) plasma cholesterol levels are shown in supplemental Table II. None of the subjects deriving from the general population was found to carry the Ala62fsLeu82X mutation or the amino acid substitutions (R46L, T77I, V114A, A443T, A522T, P616L) found in FHBL patients and/or in hypocholesterolemic blood donors. The same was also true for the group of 40 patients with primary hypercholesterolemia attending the Lipid Clinic (supplemental Table IV). Discussion PCSK9 Is a Plausible Candidate Gene in FHBL The sequence of PCSK9 gene in 18 APOB negative FHBL subjects led to the identification of an FHBL proband heterozygous for a novel single nucleotide deletion in exon 1 (c.202delg) causing frameshift and the occurrence of a premature termination codon in mrna (Ala68fsLeu82X). The predicted translation product of this mrna is a short peptide of 81 amino acids devoid of function, which most likely is not produced because of the rapid degradation of the corresponding mrna (nonsense-mediated mrna decay) as demonstrated for the nonsense mutation Y142X found in blacks. 26 The plasma levels of LDL-C and apob in the proband heterozygous for Ala68fsLeu82X was similar to that found in our FHBL patients heterozygous for APOB gene mutations and in APOB negative FHBL subjects. However, the analysis of available family members revealed that in the 2 other adult carriers of the mutation (the father and paternal aunt of the proband) the level of plasma LDL-C and apob were reduced to a much lesser extent as compared with the proband. On the assumption that proband s father was a carrier of other genetic defects producing an elevation of plasma LDL-C, we sequenced the LDL-r and APOB genes. These analyses, however, were negative, suggesting that in the father and possibly in the paternal aunt of the proband other factors (such as diabetes, overweight, and age) might have masked the LDL-lowering effect of PCSK9 mutation. A large variability in plasma LDL-C level (from the 5th to the 50th percentile of the population values) has been previously reported in carriers of the 2 nonsense mutations of PCSK9 (Y142X and C679X) found in blacks of the Dallas Heart Study. 17 Loss of Function Mutation Ala68fsLeu82X in Population On the assumption that the Ala68fsLeu82X in PCSK9 was indeed the cause of low LDL-C, we addressed the question whether this mutation was also present in healthy hypocholesterolemic subjects of the general population. The sequence of PCSK9 gene in hypocholesterolemic blood donors (with plasma LDL-C similar to those found in APOB negative FHBL patients) revealed that 2 subjects were heterozygous for the Ala68fsLeu82X mutation; this mutation was not found in the other population samples we investigated. This observation supports the concept that the Ala68fsLeu82X mutation is indeed the cause of a substantial reduction of plasma LDL-C and apob, similar to that observed in FHBL carriers of APOB gene mutations. Rare Amino Acid Variants in PCSK9 The sequence of PCSK9 gene in APOB negative FHBL patients and in hypocholesterolemic blood donors revealed the presence of carriers of rare nonconservative amino acid substitutions that have been associated with variable reduction of LDL-C in other populations. Four hypocholesterolemic blood donors and 2 APOB negative FHBL patients were found to be heterozygous for R46L substitution, which was not found in the other population samples we examined. Carriers of this mutation identified in white subjects of Dallas Heart Study have lower-plasma LDL-C (15% reduction). 17 Recently, Berge et al 19 reported 3 kindred including overall 14 carriers of R46L, whose mean plasma LDL-C was significantly lower than the corresponding value in noncarrier family members. Functional analysis of R46L substitution indicated that cells expressing this mutation had an increased LDL binding to cell surface LDL-r and increased LDL internalization with respect to cells expressing the wild-type PCSK9, 27 thus providing an explanation for the reduced plasma LDL-C found in carriers of this variant. In any case, the moderate reduction of plasma LDL-C found in carriers of R46L substitution does not account for the very low LDL-C level found in our APOB negative FHBL patients and hypocholesterolemic blood donors, thus suggesting that this variant probably plays an ancillary role in determining plasma LDL-C levels in these subjects. The same consideration applies to the A443T substitution found in 1 hypocholesterolemic blood donor, as this amino acid variant, previously reported in subjects from the Dallas Heart Study, appears to have a minute effect (2% LDL-C reduction) in heterozygous state. 19 The other 4 amino acid substitutions (T77I, V114A, A522T, and P616L) found in hypocholesterolemic blood donors have not been reported previously. Threonine, valine, and alanine, at positions 77, 114, and 522, respectively, are conserved among mammalian species and are located in highly conserved regions. This suggests that their non conservative substitution might have some biological effect. In conclusion, we identified a novel inactivating mutation of PCSK9 as a rare cause of low-plasma LDL-C and apob levels in a group of APOB negative FHBL patients. Two carriers of this mutation have been discovered among 102 white hypocholesterolemic blood donors. We also found rare amino acid variants of PCSK9 in FHBL patients and in hypocholesterolemic blood donors; some of these variants were previously reported to be associated with reduced plasma LDL-C level, thus indicating that they might reduce PCSK9 function. Sources of Funding This work was supported by a grant from Telethon Onlus (project no. GGP05042) to P.T.

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