In search of a genetic explanation for LDLc variability in an FH family: common SNPs and a rare mutation in MTTP explain only part of LDL variability in an FH family

We previously identified a highly consanguineous familial hypercholesterolemia (FH) family demonstrating segregation of the JD Bari mutation in the LDL receptor as well as a putative cholesterol-lowering trait. We aimed to identify genes related to the latter effect. LDL cholesterol (LDLc) values were normalized for FH affectation status, age, and gender. Using genome-wide SNP data, we examined whether known SNPs gleaned from a genome-wide association study could explain the variation observed in LDLc. Four individuals with markedly reduced LDL levels underwent whole exome sequencing. After prioritizing all potential mutations, we identified the most promising candidate genes and tested them for segregation with the lowering trait. We transfected a plasmid carrying the top candidate mutation, microsomal triglyceride transfer protein (MTTP) R634C, into COS-7 cells to test enzymatic activity. The SNP score explained 3% of the observed variability. MTTP R634C showed reduced activity (49.1 nmol/ml) compared with the WT allele (185.8 nmol/ml) (P = 0.0012) and was marginally associated with reduced LDLc in FH patients (P = 0.05). Phenotypic variability in a FH pedigree can only partially be explained by a combination of common SNPs and a rare mutation and a rare variant in the MTTP gene. LDLc variability in FH patients may have nongenetic causes. We previously identified a highly consanguineous familial hypercholesterolemia (FH) family demonstrating segregation of the JD Bari mutation in the LDL receptor as well as a putative cholesterol-lowering trait. We aimed to identify genes related to the latter effect. LDL cholesterol (LDLc) values were normalized for FH affectation status, age, and gender. Using genome-wide SNP data, we examined whether known SNPs gleaned from a genome-wide association study could explain the variation observed in LDLc. Four individuals with markedly reduced LDL levels underwent whole exome sequencing. After prioritizing all potential mutations, we identified the most promising candidate genes and tested them for segregation with the lowering trait. We transfected a plasmid carrying the top candidate mutation, microsomal triglyceride transfer protein (MTTP) R634C, into COS-7 cells to test enzymatic activity. The SNP score explained 3% of the observed variability. MTTP R634C showed reduced activity (49.1 nmol/ml) compared with the WT allele (185.8 nmol/ml) (P = 0.0012) and was marginally associated with reduced LDLc in FH patients (P = 0.05). Phenotypic variability in a FH pedigree can only partially be explained by a combination of common SNPs and a rare mutation and a rare variant in the MTTP gene. LDLc variability in FH patients may have nongenetic causes. Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by a high LDL cholesterol (LDLc) content in the serum. FH occurs in one in 250−500 in heterozygous (HTZ) form and one in a million in homozygous (HMZ) form (1Otani K. Takeuchi M. Kaku K. Haruki N. Yoshitani H. Eto M. Tamura M. Okazaki M. Abe H. Fujino Y. et al.Evidence of a vicious cycle in mitral regurgitation with prolapse: secondary tethering attributed to primary prolapse demonstrated by three-dimensional echocardiography exacerbates regurgitation.Circulation. 2012; 126: S214-S221Crossref PubMed Scopus (25) Google Scholar). In most cases, the disorder results from mutations in the LDL receptor (LDLR) gene. Mutations in the APOB-100 gene and the recently identified proprotein convertase subtilisin/kexin type 9 (PCSK9) gene result in phenocopies of the disease (2Austin M.A. Hutter C.M. Zimmern R.L. Humphries S.E. Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review.Am. J. Epidemiol. 2004; 160: 407-420Crossref PubMed Scopus (478) Google Scholar). Over a thousand different mutations in LDLR have been recorded to date. Owing to the high content of LDL in the blood, this disorder predisposes patients to early atherosclerosis, premature coronary heart disease, cerebrovascular accidents, calcification of cardiac valves leading to their dysfunction, and even early death. It is thought that at least 5% of all early myocardial infarctions are in FH individuals. Although FH is caused by mutations in single genes, individuals with FH show striking phenotypic variability. LDLc levels and age of onset of cardiovascular disease are highly variable (3Jansen A.C.M. van Wissen S. Defesche J.C. Kastelein J.J.P. Phenotypic variability in familial hypercholesterolaemia: an update.Curr. Opin. Lipidol. 2002; 13: 165-171Crossref PubMed Scopus (76) Google Scholar). The mutational heterogeneity of FH explains some of the phenotypic variation found among FH homozygotes in whom a strong correlation is found between residual receptor activity and severity of the disease (4Kotze M.J. De Villiers W.J. Steyn K. Kriek J.A. Marais A.D. Langenhoven E. Herbert J.S. Graadt Van Roggen J.F. Van der Westhuyzen D.R. Coetzee G.A. Phenotypic variation among familial hypercholesterolemics heterozygous for either one of two Afrikaner founder LDL receptor mutations.Arterioscler. Thromb. 1993; 13: 1460-1468Crossref PubMed Scopus (104) Google Scholar, 5Sprecher D.L. Hoeg J.M. Schaefer E.J. Zech L.A. Gregg R.E. Lakatos E. Brewer Jr., H.B. The association of LDL receptor activity, LDL cholesterol level, and clinical course in homozygous familial hypercholesterolemia.Metabolism. 1985; 34: 294-299Abstract Full Text PDF PubMed Scopus (57) Google Scholar). However, the correlation between mutation type and LDL levels is complex. Significant variations in LDLc levels that cannot be explained by gender and age differences are seen even in individuals carrying exactly the same mutation. Studies have shown that while the mutation type and residual activity of the mutated LDLR can explain some of the variability in cholesterol and LDL levels, factors such as age, gender, and apoE genotype also have significant effects (6Huijgen R. Fouchier S.W. Denoun M. Hutten B.A. Vissers M.N. Lambert G. Kastelein J.J. Plasma levels of PCSK9 and phenotypic variability in familial hypercholesterolemia.J. Lipid Res. 2012; 53: 979-983Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Bertolini S. Cantafora A. Averna M. Cortese C. Motti C. Martini S. Pes G. Postiglione A. Stefanutti C. Blotta I. et al.Clinical expression of familial hypercholesterolemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype.Arterioscler. Thromb. Vasc. Biol. 2000; 20: E41-E52Crossref PubMed Google Scholar). In a study of 177 untreated FH patients in the United Kingdom, Humphries et al. (8Humphries S.E. Whittall R.A. Hubbart C.S. Maplebeck S. Cooper J.A. Soutar A.K. Naoumova R. Thompson G.R. Seed M. Durrington P.N. et al.Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk.J. Med. Genet. 2006; 43: 943-949Crossref PubMed Scopus (227) Google Scholar) demonstrated a large range of total cholesterol levels ranging from just above 5 mmol/l to over 15 mmol/l. Mutations in genes that are known to reduce LDLc, particularly APOB mutations known to cause hypobetalipoproteinemia but also mutations in PCSK9, APOE, and ANGPTL3 were found to reduce LDLc in some FH patients (9Huijgen R. Sjouke B. Vis K. de Randamie J.S.E. Defesche J.C. Kastelein J.J.P. Hovingh G.K. Fouchier S.W. 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: 448-455Crossref PubMed Scopus (31) Google Scholar, 10Leren T.P. Berge K.E. Identification of mutations in the apolipoprotein B-100 gene and in the PCSK9 gene as the cause of hypocholesterolemia.Clin. Chim. Acta. 2008; 397: 92-95Crossref PubMed Scopus (11) Google Scholar, 11Slimani A. Jelassi A. Jguirim I. Najah M. Rebhi L. Omezzine A. Maatouk F. Hamda K.B. Kacem M. Rabès J-P. et al.Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients.Atherosclerosis. 2012; 222: 158-166Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 12Rabacchi C. Bigazzi F. Puntoni M. Sbrana F. Sampietro T. Tarugi P. Bertolini S. Calandra S. Phenotypic variability in 4 homozygous familial hypercholesterolemia siblings compound heterozygous for LDLR mutations.J. Clin. Lipidol. 2016; 10: 944-952.e1Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) These and other studies (13Arca M. Jokinen E. Low density lipoprotein receptor mutations in a selected population of individuals with moderate hypercholesterolemia.Atherosclerosis. 1998; 136: 187-194Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) demonstrate that the large variability in phenotypic FH is influenced by both genetic and environmental modifiers. Several large pedigrees segregating LDLR mutations have been described in which some carriers of an LDLR mutation have untreated normal cholesterol levels, suggesting the existence of a gene with a cholesterol-lowering effect. Hobbs et al. (14Hobbs H.H. Leitersdorf E. Leffert C.C. Cryer D.R. Brown M.S. Goldstein J.L. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors.J. Clin. Invest. 1989; 84: 656-664Crossref PubMed Scopus (113) Google Scholar), the first to suggest the existence of such a gene, described a 9-year-old male who was HMZ for a point mutation changing Ser156 to Leu, and whose plasma cholesterol level was higher than 500 mg/dl. The proband's mother, although HTZ for this mutation, had an LDLc level in the 28th percentile for the population. Further genotyping of the family identified the mutant gene in HTZ form in 17 of the mother's relatives, five of whom had normal LDLc values. The inheritance pattern of the cholesterol-suppressing trait was consistent with single gene-dominant transmission (14Hobbs H.H. Leitersdorf E. Leffert C.C. Cryer D.R. Brown M.S. Goldstein J.L. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors.J. Clin. Invest. 1989; 84: 656-664Crossref PubMed Scopus (113) Google Scholar). Through linkage analysis, the LDLR locus, apoB-100, and apoE were excluded as potential cholesterol-lowering genes (14Hobbs H.H. Leitersdorf E. Leffert C.C. Cryer D.R. Brown M.S. Goldstein J.L. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors.J. Clin. Invest. 1989; 84: 656-664Crossref PubMed Scopus (113) Google Scholar). In a subsequent study, the same group tested the kinetics of apoB-containing particles. They demonstrated higher than normal catabolism of LDL particles, suggesting that the cholesterol-lowering effect could be due to either decreased secretion of apoB-containing lipoproteins or enhanced clearance of LDL precursor lipoproteins (15Vega G.L. Hobbs H.H. Grundy S.M. Low density lipoprotein kinetics in a family having defective low density lipoprotein receptors in which hypercholesterolemia is suppressed.Arterioscler. Thromb. 1991; 11: 578-585Crossref PubMed Google Scholar). Sass et al. (16Sass C. Giroux L.M. Ma Y. Roy M. Lavigne J. Lussier-Cacan S. Davignon J. Minnich A. Evidence for a cholesterol-lowering gene in a French-Canadian kindred with familial hypercholesterolemia.Hum. Genet. 1995; 96: 21-26Crossref PubMed Scopus (18) Google Scholar) described a French Canadian FH family with a 5 kb deletion in the LDLR, resulting in complete loss of function of the allele (17Ma Y.H. Betard C. Roy M. Davignon J. Kessling A.M. Identification of a second “French Canadian” LDL receptor gene deletion and development of a rapid method to detect both deletions.Clin. Genet. 1989; 36: 219-228PubMed Google Scholar). Part of the cholesterol-lowering effect could be attributed to an apoE2 allele, but could not explain it in full, leading the authors to conclude that the existence of other cholesterol-lowering genes is highly likely. The above two examples that in some FH a strong effect of cholesterol lowering can the In a we described a large and highly FH family family with segregation of the JD Bari mutation H. B. A. L. S. H. R. S. et cholesterol-lowering gene to J. Genet. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The LDLR mutation in this family in the and causes defective receptor with of LDLR activity A. H. L. R. M. Leitersdorf E. of familial hypercholesterolemia in Genet. PubMed Scopus (31) Google Scholar, M.S. Goldstein J.L. of a mutant of with a in the of low density Full Text PDF PubMed Scopus Google Scholar). FH mutation carriers in family had normal or even low LDL it was that this family for one or genes that LDLc, for the high cholesterol in carriers of the FH mutation H. B. A. L. S. H. R. S. et cholesterol-lowering gene to J. Genet. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The mutation the cholesterol-lowering effect in this family not been but was analysis, to the of we to a cholesterol-lowering gene this The of the study was to identify the putative mutation in family common cholesterol we SNP to genotype the pedigree and a score for on genome-wide association After this to explain most of the observed variability in LDLc, we exome to for rare that explain the observed cholesterol-lowering effect. were of family of whom individuals were identified the and for of whom was A. H. L. R. M. Leitersdorf E. of familial hypercholesterolemia in Genet. PubMed Scopus (31) Google Scholar). The study was by the of the in with the their to in the who underwent an form for the of the pedigree were the was S. B. K. L. M.A. J. P. de M.J. et a for association and linkage J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. C.C. S. S.M. J.J. to the of and PubMed Scopus Google Scholar) to a genotyping than a variant than and allele above We also with and from (P LDLc values were to for the known LDLR mutation by the individuals into on the genotype HMZ HTZ and and from the LDLc of the was by the J. a for genome-wide trait J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar), which a that can for of between study We that to we to the one were in the We also an association with results to by We a score SNPs found to LDLc in a large genome-wide association study (P S. S. S. A. J. S. et and of associated with lipid Genet. PubMed Scopus Google Scholar). SNPs for the we linkage in The of SNP was effect and was The score for was in as the of the SNP effects for all effect by that The score was to the LDLc level in R. Using whole exome we from of FH family were HTZ for the JD Bari mutation, but had normal LDLc levels, suggesting that had a cholesterol-lowering trait and one had a high cholesterol level as for an FH family was and the were and to for was a and we to that the was to the to the with a of The of the of and the of than or to were on and SNPs were with the between 4 and than and between two SNPs than 5 SNPs with allele in were SNPs were also LDLc was compared between carriers and of candidate COS-7 cells demonstrate microsomal triglyceride transfer protein (MTTP) levels and were for the COS-7 cells as a cells MTTP and COS-7 cells were in of of and of at and 5% test for the dominant effect of the R634C mutation, we transfected cells with WT and mutated plasmid and MTTP activity. was to the and in of we of for with the as described by the of the were in and of the large MTTP was in relation to a values for MTTP were normalized the by a gene COS-7 and cells were transfected the as described by the of were for the plasmid WT plasmid mutated MTTP and plasmid to The of MTTP was by sequencing. of transfected COS-7 and cells were for protein and MTTP activity were cells were for to MTTP and expression was on a MTTP activity was an activity to the a method for transfer activity by the in that occurs as a lipid is from to was to MTTP activity between patients underwent of the on a with an the and were = = of and were for the of on the was on for The results were into the to the = After the LDLc values to age and gender, we a genome-wide association the of J. a for genome-wide trait J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). The results showed with LDLc at genome-wide significant values the associated were found to be on to the known LDLR mutation. we the LDLc to the known LDLR genome-wide associated SNPs could be in whether the in LDLc values could be at least in by the effect of genes we the score for in the pedigree to the effect in a S. S. S. A. J. S. et and of associated with lipid Genet. PubMed Scopus Google Scholar). of the LDLR LDLc values on the score correlation between SNP and LDLc values = = However, we the two individuals with an strong cholesterol-lowering the explained 3% of the = = may suggest that the can explain of the variability in LDLc, results are by the strong effects of some individuals the effect may be by a rare variant in a gene a cholesterol-lowering effect. We whole exome of individuals with in an to a rare variant that could explain the cholesterol-lowering effect. We whole exome on FH family SNPs as described in the and exome were on the to for both and inheritance The individuals HMZ variation at the same the this SNPs on and S. L. A. P. method and for PubMed Scopus Google Scholar, P. S. the effects of on protein function the PubMed Scopus Google and SNPs were The individuals HTZ variation at the same the variation at that this we a total of of which were to be The individuals have either type of variation or at the same while the this we of which were to be of the individuals have type of variation at the same while the the the total of were it was by or that be The of result to candidate genes that the of inheritance and a in cholesterol total of LDLc genes were from PubMed and and were with the of genes from the the variations of that in both were to be were between at least two individuals but not with the variations were five the of H. M. G. PubMed Scopus Google Scholar) a variant in the MTTP was the to be cholesterol MTTP for a protein in the and secretion of apoB-containing lipoproteins in the and Mutations in MTTP are the of a rare autosomal disorder characterized by the of apoB-containing lipoproteins as of both and N. The of the microsomal triglyceride transfer protein in 2000; 20: PubMed Scopus Google Scholar). of a LDLc with individuals also demonstrated strong association with MTTP M. genome-wide genes, and PubMed Scopus Google Scholar). It be that this was not seen in suggesting that the SNP effect is of the candidate mutations that the of with and are is the of candidate mutations five potential two MTTP and are MTTP is a candidate it is known to cause lowering of LDLc. LDLR is the JD Bari mutation known to cause group in a with and are is the of candidate mutations five potential two MTTP and are MTTP is a candidate it is known to cause lowering of LDLc. LDLR is the JD Bari mutation known to cause group The mutation was only in with an allele of and and the population of a effect on the gene or gene We tested FH individuals from other pedigrees R634C in MTTP was in family LDLc for all family were normalized to the percentile in the same FH affectation status, age, and gender. for FH affectation status, the family were into two HTZ for the LDLR mutation and WT for the of was into two MTTP HTZ and and for the effect of MTTP mutation on LDLc levels in the was for all family and the age of whom the correlation between age and cholesterol is The values for a in LDLc levels between carriers and of the R634C MTTP mutation LDLR mutation were = and = for the and values not = due to the are of an an to show the of the MTTP R634C mutation was shown in MTTP activity in transfected was reduced by the R634C mutation (49.1 for mutated and = MTTP activity not from that in In the level of activity was than in which MTTP activity levels were between the mutated and suggesting that was in between the MTTP mutations in may cause significant only in the test the that mutation have a dominant we transfected cells with WT and mutated The activity of the mutated cells was to the we could not demonstrate a dominant effect with the mutation that MTTP are known to cause test for the effect or the R634C MTTP mutation on was by F. L. F. Y. T. M. of in patients with a Full Text Full Text PDF PubMed Scopus Google Scholar) in HTZ LDLR mutation were HTZ for the R634C MTTP mutation and were significant between the two to a of a significant in and that the R634C MTTP mutation is not associated with significant of the it may be to MTTP that not cause content by and in MTTP mutation carriers and at results in of results in of in a FH is a genetic in which the function of LDLR is of LDLc from the into the LDLc in some of the FH family described in this study were than In to explain this observed we first at the score for LDLc in the After individuals with strong cholesterol-lowering effects were from the analysis, the score could explain of the observed variability in LDLc. whether a rare variant was a in LDLc, we exome in a for a rare variant with a strong effect. mutation in the MTTP gene, not previously described in was FH patients carrying this MTTP mutation demonstrated LDLc values than both SNP score and the rare variant could at only of the variability in LDLc observed in the LDLc variability in pedigree may only be explained by SNP score or rare It is that LDLc variability in FH patients may be and also and environmental MTTP is in the and a in lipoprotein the of and between H. triglyceride transfer Acta. PubMed Scopus Google Scholar). MTTP is to have an a and a J. P. triglyceride transfer protein and in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. P. J. et of a for the and secretion of Biol. PubMed Scopus Google Scholar). The with the of the with both protein and and the both the and the of MTTP J. P. triglyceride transfer protein and in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. P. K. triglyceride transfer a PubMed Scopus Google Scholar). The R634C mutation is in the and activity we that the of a by a with the of the mutation, the activity. in in transfected cells demonstrated a significant in the mutated allele compared with the WT gene. The levels that the in activity was not due to levels of in the transfected cells but was the result of either activity or reduced protein for MTTP mutation were not to have a significant We tested the that the R634C mutation may have a dominant effect but could not demonstrate such an effect. is that in FH and the markedly LDLc at even a effect have a significant effect on that MTTP activity are to These have particularly M. transfer protein in Opin. Lipidol. PubMed Scopus (46) Google Scholar). of in FH patients carrying the MTTP mutation and significant differences in either or plasma It is that the effect of the mutation is not associated with in suggesting that such not result from of MTTP activity. In in this study of a large consanguineous FH family a cholesterol-lowering we to genetic of the FH trait. SNP score could explain only 3% of the observed variability in LDLc. in the for rare identified an R634C MTTP mutation that LDLc in HTZ We showed that this mutation MTTP activity in transfected COS-7 and that this is not associated with a an in the that an FH can be by MTTP mutations as well as by common However, at explain only of the observed variability in LDLc among FH causes may be for the observed LDLc variability. These may as well as environmental with familial hypercholesterolemia genome-wide association study homozygous heterozygous LDL cholesterol LDL receptor microsomal triglyceride transfer protein

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