Targeted next-generation sequencing to diagnose disorders of HDL cholesterol
2015; Elsevier BV; Volume: 56; Issue: 10 Linguagem: Inglês
10.1194/jlr.p058891
ISSN1539-7262
AutoresN. Sadananda Singh, Jia Nee Foo, Meng Tiak Toh, Lubomira Cermakova, Laia Trigueros‐Motos, Teddy Chan, Herty Liany, Jennifer A. Collins, Sima Gerami, Roshni R. Singaraja, Michael R. Hayden, Gordon A. Francis, Jiří Fröhlich, Chiea Chuen Khor, Liam R. Brunham,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoA low level of HDL cholesterol (HDL-C) is a common clinical scenario and an important marker for increased cardiovascular risk. Many patients with very low or very high HDL-C have a rare mutation in one of several genes, but identification of the molecular abnormality in patients with extreme HDL-C is rarely performed in clinical practice. We investigated the accuracy and diagnostic yield of a targeted next-generation sequencing (NGS) assay for extreme levels of HDL-C. We developed a targeted NGS panel to capture the exons, intron/exon boundaries, and untranslated regions of 26 genes with highly penetrant effects on plasma lipid levels. We sequenced 141 patients with extreme HDL-C levels and prioritized variants in accordance with medical genetics guidelines. We identified 35 pathogenic and probably pathogenic variants in HDL genes, including 21 novel variants, and performed functional validation on a subset of these. Overall, a molecular diagnosis was established in 35.9% of patients with low HDL-C and 5.2% with high HDL-C, and all prioritized variants identified by NGS were confirmed by Sanger sequencing. Our results suggest that a molecular diagnosis can be identified in a substantial proportion of patients with low HDL-C using targeted NGS. A low level of HDL cholesterol (HDL-C) is a common clinical scenario and an important marker for increased cardiovascular risk. Many patients with very low or very high HDL-C have a rare mutation in one of several genes, but identification of the molecular abnormality in patients with extreme HDL-C is rarely performed in clinical practice. We investigated the accuracy and diagnostic yield of a targeted next-generation sequencing (NGS) assay for extreme levels of HDL-C. We developed a targeted NGS panel to capture the exons, intron/exon boundaries, and untranslated regions of 26 genes with highly penetrant effects on plasma lipid levels. We sequenced 141 patients with extreme HDL-C levels and prioritized variants in accordance with medical genetics guidelines. We identified 35 pathogenic and probably pathogenic variants in HDL genes, including 21 novel variants, and performed functional validation on a subset of these. Overall, a molecular diagnosis was established in 35.9% of patients with low HDL-C and 5.2% with high HDL-C, and all prioritized variants identified by NGS were confirmed by Sanger sequencing. Our results suggest that a molecular diagnosis can be identified in a substantial proportion of patients with low HDL-C using targeted NGS. A low concentration of plasma HDL cholesterol (HDL-C) is one of the most common lipid abnormalities and an important risk factor for CVD. Low HDL-C predicts increased CVD risk, even among patients with aggressively treated LDL cholesterol (LDL-C) (1.Yusuf S. Hawken S. Ounpuu S. Dans T. Avezum A. Lanas F. McQueen M. Budaj A. Pais P. Varigos J. et al.Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study.Lancet. 2004; 364: 937-952Abstract Full Text Full Text PDF PubMed Scopus (8412) Google Scholar, 2.Boekholdt S.M. Arsenault B.J. Hovingh G.K. Mora S. Pedersen T.R. Larosa J.C. Welch K.M. Amarenco P. Demicco D.A. Tonkin A.M. et al.Levels and changes of HDL cholesterol and apolipoprotein A-I in relation to risk of cardiovascular events among statin-treated patients: a meta-analysis.Circulation. 2013; 128: 1504-1512Crossref PubMed Scopus (147) Google Scholar, 3.Barter P. Gotto A.M. LaRosa J.C. Maroni J. Szarek M. Grundy S.M. Kastelein J.J. Bittner V. Fruchart J.C. Treating to New Targets Investigators. HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events.N. Engl. J. Med. 2007; 357: 1301-1310Crossref PubMed Scopus (1295) Google Scholar). HDL-C levels are highly heritable (4.Goode E.L. Cherny S.S. Christian J.C. Jarvik G.P. de Andrade M. Heritability of longitudinal measures of body mass index and lipid and lipoprotein levels in aging twins.Twin Res. Hum. Genet. 2007; 10: 703-711Crossref PubMed Scopus (60) Google Scholar) and display both locus and allelic heterogeneity, with multiple variants in several genes leading to very high or very low HDL-C. The genetic architecture of HDL-C is notable among polygenic traits in that rare variants with presumed large effect sizes are present in a substantial proportion of patients with very low or very high HDL-C, usually in the same genes that cause extremely rare Mendelian disorders of HDL-C (5.Cohen J.C. Kiss R.S. Pertsemlidis A. Marcel Y.L. McPherson R. Hobbs H.H. Multiple rare alleles contribute to low plasma levels of HDL cholesterol.Science. 2004; 305: 869-872Crossref PubMed Scopus (901) Google Scholar, 6.Tietjen I. Hovingh G.K. Singaraja R. Radomski C. McEwen J. Chan E. Mattice M. Legendre A. Kastelein J.J. Hayden M.R. Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT.Biochim. Biophys. Acta. 2012; 1821: 416-424Crossref PubMed Scopus (41) Google Scholar, 7.Frikke-Schmidt R. Nordestgaard B.G. Stene M.C. Sethi A.A. Remaley A.T. Schnohr P. Grande P. Tybjaerg-Hansen A. Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease.JAMA. 2008; 299: 2524-2532Crossref PubMed Scopus (393) Google Scholar, 8.Kiss R.S. Kavaslar N. Okuhira K. Freeman M.W. Walter S. Milne R.W. McPherson R. Marcel Y.L. Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1139-1145Crossref PubMed Scopus (52) Google Scholar, 9.Berge K.E. Leren T.P. Mutations in APOA-I and ABCA1 in Norwegians with low levels of HDL cholesterol.Clin. Chim. Acta. 2010; 411: 2019-2023Crossref PubMed Scopus (15) Google Scholar, 10.Candini C. Schimmel A.W. Peter J. Bochem A.E. Holleboom A.G. Vergeer M. Dullaart R.P. Dallinga-Thie G.M. Hovingh G.K. Khoo K.L. et al.Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol.Atherosclerosis. 2010; 213: 492-498Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11.Alrasadi K. Ruel I.L. Marcil M. Genest J. Functional mutations of the ABCA1 gene in subjects of French-Canadian descent with HDL deficiency.Atherosclerosis. 2006; 188: 281-291Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12.Motazacker M.M. Peter J. Treskes M. Shoulders C.C. Kuivenhoven J.A. Hovingh G.K. Evidence of a polygenic origin of extreme high-density lipoprotein cholesterol levels.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1521-1528Crossref PubMed Scopus (42) Google Scholar). This suggests that it may be possible to establish a molecular diagnosis in many patients with extreme HDL-C levels. However, detection of the specific molecular abnormality in patients with extreme HDL-C is rarely performed outside of specialized research laboratories because of the cost, complexity, and time required to do so, and uncertainty regarding the clinical utility of establishing a molecular diagnosis. The development of next-generation sequencing (NGS) technologies has created new opportunities for the routine use of sequencing in clinical medicine. Targeted NGS panels have been effectively used for newborn carrier screening and for the diagnosis of inherited cardiomyopathy, hereditary cancers, and other conditions (13.Rehm H.L. Disease-targeted sequencing: a cornerstone in the clinic.Nat. Rev. Genet. 2013; 14: 295-300Crossref PubMed Scopus (296) Google Scholar), but to date have not been established for disorders of HDL-C. The objective of this study was to evaluate the accuracy and diagnostic yield of a targeted NGS panel to establish the molecular diagnosis of abnormal HDL-C in a range of patients with extreme HDL phenotypes visiting a specialty lipid clinic. Our results indicate that this approach can reliably and accurately identify pathogenic variants in a substantial proportion of patients with low levels of HDL-C. We recruited consecutive patients with HDL-C levels below the 10th percentile or greater than the 90th percentile using age- and gender-adjusted population data from the Lipid Research Clinics (14.Heiss G. Johnson N.J. Reiland S. Davis C.E. Tyroler H.A. The epidemiology of plasma high-density lipoprotein cholesterol levels. The Lipid Research Clinics Program Prevalence Study. Summary.Circulation. 1980; 62: IV116-IV136Crossref PubMed Scopus (108) Google Scholar), hereafter referred to as "extreme HDL-C," regardless of other lipid parameters, from the Healthy Heart Program Prevention Clinic at St. Paul's Hospital, Vancouver, Canada, a large specialty lipid clinic serving a multi-ethnic patient population. We did not attempt to exclude patients with potential secondary causes of low HDL-C (e.g., hypertriglyceridemia) or high HDL-C (e.g., alcohol use). Related individuals were not specifically recruited. Patients were excluded if they could not speak English or were unable or unwilling to provide written informed consent. As a positive control, we included patients in whom a molecular diagnosis of a Mendelian disorder of HDL had previously been established by Sanger sequencing (15.Brunham L.R. Kang M.H. Van Karnebeek C. Sadananda S.N. Collins J.A. Zhang L.H. Sayson B. Miao F. Stockler S. Frohlich J. et al.Clinical, biochemical, and molecular characterization of novel mutations in ABCA1 in families with Tangier disease.JIMD Rep. 2015; 18: 51-62Crossref PubMed Scopus (17) Google Scholar). Clinical data were abstracted from the patients' medical records. In cases where multiple sets of laboratory values were available, we used the most recent values. All subjects provided written informed consent. This study was approved by the Clinical Research Ethics Board of the University of British Columbia. DNA was isolated from venous blood samples using DNeasy kits (Qiagen) or saliva using Oragene kits (DNA Genotek) and quantified using the Quant-iT PicoGreen assay (Life Technologies). Sequencing libraries were prepared according to the manufacturer's instructions (Illumina, San Diego, CA). In-solution hybridization capture was performed with SeqCap EZ library reagents (Roche-Nimblegen). Captured DNA was quantified by the KAPA system (Kapa Biosystems) and diluted to a final concentration of 5 nM. Sequencing was performed on an Illumina MiSeq instrument in 2 × 151 bp mode. Reads were mapped to the reference human genome (hg19) using Burrows-Wheeler alignment tool v0.5.9 in paired read mode (maximal exact match algorithm) and processed following the recommended guidelines by the Genome Analysis Toolkit "Best Practices for Variant Calling" (available at http://www.broadinstitute.org/genome-analysis-toolkit). Variants were called using the Genome Analysis Toolkit v3 Unified Genotyper. We applied the following variant-level and genotype-level quality filters for variant calling: quality by depth (QD) < 2.0; root mean square of mapping quality (MQ) < 40.0; rank sum test of mapping quality (MQRankSum) < −12.5; rank sum test of read position (ReadPosRankSum) < −8.0, <20 times base coverage; <Q20 genotype quality; and allele balance <0.15 for heterozygous calls and <0.85 for homozygous calls. All single nucleotide variants and indels that passed these quality control filters were annotated with the SIFT, Polyphen 2 [HumDiv], Polyphen 2 [HumVar], PROVEAN, and Condel tools, and matched against public databases of variants (1000 Genomes, HapMap, and National Heart, Lung, and Blood Institute exome variant server). We then prioritized variants in HDL genes according to the following criteria: 1) variants that are reported to be disease-causing in the Human Gene Mutation Database (HGMD) (16.Stenson P.D. Mort M. Ball E.V. Howells K. Phillips A.D. Thomas N.S. Cooper D.N. The Human Gene Mutation Database: 2008 update.Genome Med. 2009; 1: 13Crossref PubMed Scopus (686) Google Scholar); 2) disruptive variants [nonsense, splice-site (two nucleotides on either side of the intron/exon boundary) and frameshift] that are novel or rare [minor allele frequency (MAF) <1% in public databases and MAF <4% in study samples]; and 3) novel or rare (MAF <1%) missense variants that were predicted to be deleterious by all of SIFT, Polyphen 2 [HumDiv], Polyphen 2 [HumVar], PROVEAN, and Condel. Variants that met these criteria were validated by bidirectional Sanger sequencing of PCR amplicons (primer sequences available on request). ABCA1 cDNA sequences were cloned into the pCDNA3.1 vector and the W590L and R2200* mutations generated by site-directed mutagenesis. Immunoblotting and immunofluorescence in transiently transfected HEK293 cells were carried out as previously described (15.Brunham L.R. Kang M.H. Van Karnebeek C. Sadananda S.N. Collins J.A. Zhang L.H. Sayson B. Miao F. Stockler S. Frohlich J. et al.Clinical, biochemical, and molecular characterization of novel mutations in ABCA1 in families with Tangier disease.JIMD Rep. 2015; 18: 51-62Crossref PubMed Scopus (17) Google Scholar). Cholesterol efflux assays were performed, as previously described (15.Brunham L.R. Kang M.H. Van Karnebeek C. Sadananda S.N. Collins J.A. Zhang L.H. Sayson B. Miao F. Stockler S. Frohlich J. et al.Clinical, biochemical, and molecular characterization of novel mutations in ABCA1 in families with Tangier disease.JIMD Rep. 2015; 18: 51-62Crossref PubMed Scopus (17) Google Scholar), in transiently transfected HEK293 cells. Data represent the mean ± SD of at least three independent experiments, each performed in triplicate. LCAT assays were performed as recommended by the manufacturer (Calbiochem) using 5 μl of human plasma. The fluorescent intensities at 390 nm and 470 nm were measured at time = 0, 2, 4, and 8 h. LCAT activity was calculated as the rate of change in the ratio of fluorescent intensities at 470 nm and 390 nm. Data represent the mean ± SD of three independent experiments, each performed in triplicate. Results are presented as mean ± SD. Differences between groups were compared with two-tailed Student's t-test, or one-way ANOVA for three or more groups, or for comparison between proportions, chi-squared test. Calculations were performed in GraphPad Prism software. P < 0.05 was considered statistically significant. We generated a customized NGS panel to capture the exons, intron/exon boundaries, and flanking untranslated regions (UTRs) of 26 genes with known roles in plasma lipid metabolism based on data from the HGMD and manual curation of the literature (Table 1). We included genes in which rare variants are known to cause highly penetrant effects on plasma levels of HDL-C, LDL-C, TG, and lipoprotein (a), as well as on the response to lipid-lowering therapy. We generated a probe library using Nimblegen SeqCap technology to capture these targets.TABLE 1List of genes sequencedPhenotypeGeneMIM NumberDiseaseLocusNumber of ExonsLow HDL-CABCA1600046Tangier disease9q31.150Low HDL-CAPOA1107680ApoA-I deficiency11q23-q245Low HDL-CAPOA2107670ApoA-II deficiency1q23.34Low HDL-CLCAT606967Familial LCAT deficiency/fish eye disease16q22.16Low HDL-CNPC1607623Niemann-Pick disease18q11.227Low HDL-CPLTP172425—20q13.1217High HDL-CCETP118470—16q2117High HDL-CGALNT2602274—1q41-q4216High HDL-CLIPG603684—18q21.111High HDL-CSCARB1601040—12q24.3113High LDL-CABCG5605459Sitosterolemia2p2116High LDL-CABCG8605460Sitosterolemia2p2113High LDL-C/low LDL-CAPOB107730FH2p24-p2329High LDL-CLDLR606945FH19p13.218High LDL-CLDLRAP1605747FH1p36-p3513High LDL-C/low LDL-CPCSK9607786FH1p32.312Low LDL-CMTTP157147Abetalipoproteinemia4q2419High TGAPOA5606368—11q234High TGAPOC2608083Chlyomicronemia19q13.24High TGAPOE107741Dysbetalipoproteinemia19q13.24High TGGPIHBP1612757—8q24.34High TGLMF1611761—16p13.320High TGLPL609708Chlyomicronemia8p2210Low TG/high HDL-CAPOC3107720—11q23.34High lipoprotein (a)LPA152200—6q2640Statin myopathySLCO1B1604843—12p15The MIM number is from the Online Mendelian Inheritance in Man database. FH, familial hypercholesterolemia. Open table in a new tab The MIM number is from the Online Mendelian Inheritance in Man database. FH, familial hypercholesterolemia. To assess the accuracy and diagnostic yield of this panel, we sequenced 141 patients with extreme levels of HDL-C, including 64 patients with extremely low HDL-C and 77 patients with extremely high HDL-C. This patient population was hypothesized to harbor a substantial percentage of damaging variants in HDL-related genes (5.Cohen J.C. Kiss R.S. Pertsemlidis A. Marcel Y.L. McPherson R. Hobbs H.H. Multiple rare alleles contribute to low plasma levels of HDL cholesterol.Science. 2004; 305: 869-872Crossref PubMed Scopus (901) Google Scholar, 1.Yusuf S. Hawken S. Ounpuu S. Dans T. Avezum A. Lanas F. McQueen M. Budaj A. Pais P. Varigos J. et al.Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study.Lancet. 2004; 364: 937-952Abstract Full Text Full Text PDF PubMed Scopus (8412) Google Scholar, 2.Boekholdt S.M. Arsenault B.J. Hovingh G.K. Mora S. Pedersen T.R. Larosa J.C. Welch K.M. Amarenco P. Demicco D.A. Tonkin A.M. et al.Levels and changes of HDL cholesterol and apolipoprotein A-I in relation to risk of cardiovascular events among statin-treated patients: a meta-analysis.Circulation. 2013; 128: 1504-1512Crossref PubMed Scopus (147) Google Scholar, 3.Barter P. Gotto A.M. LaRosa J.C. Maroni J. Szarek M. Grundy S.M. Kastelein J.J. Bittner V. Fruchart J.C. Treating to New Targets Investigators. HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events.N. Engl. J. Med. 2007; 357: 1301-1310Crossref PubMed Scopus (1295) Google Scholar, 4.Goode E.L. Cherny S.S. Christian J.C. Jarvik G.P. de Andrade M. Heritability of longitudinal measures of body mass index and lipid and lipoprotein levels in aging twins.Twin Res. Hum. Genet. 2007; 10: 703-711Crossref PubMed Scopus (60) Google Scholar, 5.Cohen J.C. Kiss R.S. Pertsemlidis A. Marcel Y.L. McPherson R. Hobbs H.H. Multiple rare alleles contribute to low plasma levels of HDL cholesterol.Science. 2004; 305: 869-872Crossref PubMed Scopus (901) Google Scholar, 6.Tietjen I. Hovingh G.K. Singaraja R. Radomski C. McEwen J. Chan E. Mattice M. Legendre A. Kastelein J.J. Hayden M.R. Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT.Biochim. Biophys. Acta. 2012; 1821: 416-424Crossref PubMed Scopus (41) Google Scholar, 7.Frikke-Schmidt R. Nordestgaard B.G. Stene M.C. Sethi A.A. Remaley A.T. Schnohr P. Grande P. Tybjaerg-Hansen A. Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease.JAMA. 2008; 299: 2524-2532Crossref PubMed Scopus (393) Google Scholar, 8.Kiss R.S. Kavaslar N. Okuhira K. Freeman M.W. Walter S. Milne R.W. McPherson R. Marcel Y.L. Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1139-1145Crossref PubMed Scopus (52) Google Scholar, 9.Berge K.E. Leren T.P. Mutations in APOA-I and ABCA1 in Norwegians with low levels of HDL cholesterol.Clin. Chim. Acta. 2010; 411: 2019-2023Crossref PubMed Scopus (15) Google Scholar, 10.Candini C. Schimmel A.W. Peter J. Bochem A.E. Holleboom A.G. Vergeer M. Dullaart R.P. Dallinga-Thie G.M. Hovingh G.K. Khoo K.L. et al.Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol.Atherosclerosis. 2010; 213: 492-498Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11.Alrasadi K. Ruel I.L. Marcil M. Genest J. Functional mutations of the ABCA1 gene in subjects of French-Canadian descent with HDL deficiency.Atherosclerosis. 2006; 188: 281-291Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12.Motazacker M.M. Peter J. Treskes M. Shoulders C.C. Kuivenhoven J.A. Hovingh G.K. Evidence of a polygenic origin of extreme high-density lipoprotein cholesterol levels.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1521-1528Crossref PubMed Scopus (42) Google Scholar). The clinical characteristics of these patients are shown in Table 2. Patients with low HDL-C also had lower total plasma cholesterol and LDL-C, as well as higher TGs, higher BMI, and a higher frequency of diabetes mellitus, consistent with previous observations (17.Juren A.J. Sarwal G. Al-Sarraf A. Vrablik M. Chan D. Humphries K.H. Frohlich J.J. Low prevalence of type 2 diabetes mellitus among patients with high levels of high-density lipoprotein cholesterol.J. Clin. Lipidol. 2013; 7: 194-198Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Most patients in both groups were of self-reported European ancestry.TABLE 2Characteristics of patients with extreme HDL-CHigh HDL-C PatientsLow HDL-C PatientsPNumber7764—Age (mean ± SD)58.4 ± 1053.4 ± 130.01Gender (% male)26.079.7<0.0001TC (mean ± SD, mmol/l)5.87 ± 1.34.15 ± 1.2<0.0001HDL-C (mean ± SD, mmol/l)2.47 ± 0.40.66 ± 0.2<0.0001LDL-C (mean ± SD, mmol/l)3.06 ± 1.12.13 ± 0.9<0.0001TG (mean ± SD, mmol/l)0.88 ± 0.43.88 ± 3.4<0.0001ApoA-I (mean ± SD, g/l)2.14 ± 0.31.07 ± 0.2<0.0001BMI (mean ± SD, kg/m2)25.0 ± 1629.7 ± 6<0.0001CAD (%)6.512.50.2Diabetes mellitus (%)0.027.0<0.0001Current smoker (%)4.814.10.07Lipid lowering medication (%)59.562.90.6Self-reported ancestryEuropean (%)81.871.80.3Asian (%)11.714.10.3Other/not reported (%)6.514.10.3TC, total cholesterol; CAD, coronary artery disease. P values represent two-tailed t-test for comparison of means and chi-squared test for comparison of proportions. Open table in a new tab TC, total cholesterol; CAD, coronary artery disease. P values represent two-tailed t-test for comparison of means and chi-squared test for comparison of proportions. We performed high-throughput sequencing of this cohort to a mean depth of 1,559 ± 290 reads per base after quality filtering. Ninety-three percent of target bases were covered by 20 or more reads. Ninety-four percent of targeted exons had ≥50% of bases covered with an average of 15 or more reads. The mean GC (guanine-cytosine) content of exons with <50% of bases covered with 15 or more reads was 70.8 ± 4% compared with 52.0 ± 8% for exons with ≥50% of bases covered with 15 or more reads, suggesting that local genomic features interfered with the capture of these loci. We identified 668 variants that met quality filtering criteria, including 357 exonic variants, 37 intronic variants, 212 3′UTR variants, 37 5′UTR variants, 18 variants downstream of the 3′UTR, and 7 variants upstream of 5′UTR. We then prioritized these variants (supplementary Fig. 1) based on established medical genetics recommendations (18.Richards C.S. Bale S. Bellissimo D.B. Das S. Grody W.W. Hegde M.R. Lyon E. Ward B.E. Molecular Subcommittee of the ACMG Laboratory Quality Assurance Committee ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007.Genet. Med. 2008; 10: 294-300Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar), as follows. Variants known to be disease-causing for abnormalities of HDL-C in HGMD were designated as "pathogenic." Novel or rare variants (MAF <1% in public databases and MAF 1 variant. Variants in the ABCA1 gene were most frequent, followed by variants in LCAT. No variants that met our prioritization criteria were identified in APOA2, GALNT2, APOC3, or PLTP. Twenty-one of the prioritized variants we identified were novel, including ten novel variants in ABCA1, three novel variants in LCAT, two novel variants in each of APOA1, NPC1, LIPG, and one novel variant in each of CETP and SCARB1 (Table 3).TABLE 3Pathogenic and probably pathogenic variants identified in patients with extreme HDL-CRef. Seq.Nuc. ChangeAmino Acid ChangersID/NovelHGMDFunctional PredictionMAFPhenotypeLow HDL genesABCA1NM_005502c.1196T>Cp.Val399Alars9282543:GDMTolerated0.002LowABCA1NM_005502c.1755C>Ap.Asp585GluNovel—Damaging—LowABCA1NM_005502c.1769G>Tp.Trp590LeuNovelDMDamaging—LowABCA1NM_005502c.1913G>Ap.Arg638GlnNovelDMDamaging—LowABCA1NM_005502c.2328G>Cp.Lys776Asnrs138880920DPDamaging0.003HighABCA1NM_005502c.2540C>Tp.Pro847LeuNovel—Damaging—LowABCA1NM_005502c.3338delTp.Gln980Serfs*9Novel———LowABCA1NM_005502c.3121C>Gp.Leu1041Valrs192935024—Damaging0.001HighABCA1NM_005502c.3541C>Ap.Ala1046Asprs141021096DMDamaging—LowABCA1NM_005502c.3946C>Tp.Ser1181Phers76881554DMDamaging0.0006HighABCA1NM_005502c.4449delCp.Leu1484Cysfs*17Novel———LowABCA1NM_005502c.4939C>Tp.Thr1512MNovelDMDamaging—LowABCA1NM_005502c.5039G>Ap.Arg1680Glnrs150125857DMDamaging0.0002LowABCA1NM_005502c.5550G>Tp.Tryp1699Cysrs146934490DMDamaging—LowABCA1NM_005502c.5449C>Tp.Arg1817*Novel—Damaging—LowABCA1NM_005502c.5702_5703dupGAp.Ile1902Glufs*12Novel———LowABCA1NM_005502c.7002C>Tp.Arg2200*Novel—Damaging—LowABCA1NM_005502c.6730G>Ap.Val2244Ilers144588452DMTolerated0.0004LowAPOA1NM_000039c.138C>Tp.Arg34*Novel—Damaging—LowAPOA1NM_000039c.382T>Ap.Lys131Metrs4882—Damaging—LowAPOA1NM_000039c.391_393delp.Lys131delNovel———LowLCATNM_000229c.451C>Tp.Thr147Ilers121908050DMDamaging—LowLCATNM_000229c.487G>Ap.Arg159GlnNovelDMDamagingLowLCATNM_000229c.694T>Ap.Ser232Thrrs4986970DMTolerated0.0084BothLCATNM_000229c.1043C>Ap.Thr348IleNovel—Damaging—LowLCATNM_000229c.1103G>Tp.Gly368ValNovelDMDamaging—LowNPC1NM_000271c.665A>Gp.Asn222Serrs55680026DMTolerated0.003BothNPC1NM_000271c.3308G>Tp. Gly1012CysNovel—DamagingLowNPC1NM_000271c.3689T>Cp.Leu1230SerNovel—Damaging—HighHigh HDL genesCETPNM_000078c.118+1-118+4delGTAAsplice siteNovelDM——HighCETPNM_000078c.1376A>Gp.Asp459Glyrs2303790DMDamaging0.0062HighLIPGNM_006033c.716T>Cp.Ile239ThrNovelDMDamaging—HighLIPGNM_006033c.1069G>Tp.Glu273*Novel—DamagingHighLIPGNM_006033c.1426C>Tp.Arg476Trprs117623631DMDamaging0.003HighSCARB1NM_001082959c.715G>Ap.Gly239ArgNovel—Damaging—LowMAF is based on 1000 Genomes CEU (Utah residents with ancestry from northern and western Europe) data. Functional prediction based on SIFT. DM, disease-causing mutation in HGMD; DP, disease-associated polymorphism in HGMD; Ref. Seq., reference sequence; Nuc., nulceotide; rsID, reference SNP identification. Phenotype refers to whether the variant was detected in a patient (or patients) with low HDL-C, high HDL-C, or in both groups of patients. Open table in a new tab MAF is based on 1000 Genomes CEU (Utah residents with ancestry from northern and western Europe) data. Functional prediction based on SIFT. DM, disease-causing mutation in HGMD; DP, disease-associated polymorphism in HGMD; Ref. Seq., reference sequence; Nuc., nulceotide; rsID, reference SNP identification. Phenotype refers to whether the variant was detected in a patient (or patients) with low HDL-C, high HDL-C, or in both groups of patients. Of note, three patients with extremely low HDL-C were found to harbor homozygous or compound heterozygous mutations in the APOA1, ABCA1, and LCAT genes (Table 4). This included a 43-year-old female subject [patient identification number (ID) 114] with extremely low HDL-C (0.1 mmol/l) and ApoA-I (0.05 g/l), found to be homozygous for a novel premature truncation in the APOA1 gene (p.Arg34*); a 21-year-old male subject (patient ID 18301) with near undetectable HDL-C (0.1 mmol/l) found to be compound heterozygous for one known disease-causing mutation in ABCA1 (p.Trp590Leu) and one novel premature truncation in ABCA1 (p.Arg2200*); and a 21-year-old male subject (patient ID 34) with extremely low HDL-C (0.1 mmol/l) and less severely reduced ApoA-I levels (0.9 g/l), found to be homozygous for a novel predicted deleterious missense variant in LCAT (p.Thr348Ile), as well as heterozygous for a previously identified variant in ABCA1 (p.Val2244Ile) (19.Probst M.C. Thumann H. Aslanidis C. Langmann T. Buechler C. Patsch W. Baralle F.E. Dallinga-Thie G.M. Geisel J. Keller C. et al.Screening for functional sequence variations and mutations in ABCA1.Atherosclerosis. 2004; 175: 269-279Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These results established molecular diagnoses of the rare Mendelian disorders ApoA-I deficiency, Tangier disease, and familial LCAT deficiency, respectively, in these three individuals. Another individual (patient ID 35) who is the sibling of patient ID 34 and had a less dramatic reduction in HDL-C (0.5 mmol/l) was found to be heterozygous for the same LCAT variant (p.Thr348Ile), establishing a gene-dosage relationship for this variant with HDL-C levels.TABLE 4Individuals with a new molecular diagnosis of HDLPatient IDGenderAgeTCHDL-CLDL-CTGApoA-IGeneTypeVariantLow HDL-C patients114F433.480.102.302.490.05APOA1Homo.p.Arg34*18301M211.800.101.360.850.27ABCA1Cmpd. het.p.Arg2200*pTrp590Leu34F212.300.101.701.100.90LCATHomo.p.Thr348IleABCA1Het.p.Val2244Ile13M635.180.333.732.441.04ABCA1Het.p.Arg638Gln96M593.700.36–4.060.89LCATHet.p.Arg159Gln41M623.870.392.282.651.15LCATHet.p.Ala165ThrABCA1Het.p.Arg1680Gln28M413.100.401.303.000.60APOA1Het.p.Lys131delLCATHet.p.Ser232Thr4F673.150.441.7
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