LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues. We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues. Next generation sequencing (NGS) broadly refers to the new wave of DNA sequencing technologies that have emerged in the post-Sanger sequencing era (1Grada A. Weinbrecht K. Next-generation sequencing: methodology and application.J. Invest. Dermatol. 2013; 133: e11Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 2Metzker M.L. Sequencing technologies - the next generation.Nat. Rev. Genet. 2010; 11: 31-46Crossref PubMed Scopus (4997) Google Scholar). NGS platforms provide massively parallel sequencing of millions of DNA fragments, which enables the rapid sequencing of whole genomes in less than a day and at a fraction of the cost compared with Sanger sequencing. The recent clinical application of NGS has revolutionized the ability to rapidly develop molecular diagnoses in inherited disease (3Chang, F., Li, M. M. Clinical application of amplicon-based next-generation sequencing in cancer. Cancer Genet. Epub ahead of print. October 11, 2013; doi;10.1016/j.cancergen.2013.10.003.Google Scholar, 4Sikkema-Raddatz B. Johansson L.F. de Boer E.N. Almomani R. Boven L.G. van den Berg M.P. van Spaendonck-Zwarts K.Y. van Tintelen J.P. Sijmons R.H. Jongbloed J.D. et al.Targeted next-generation sequencing can replace Sanger sequencing in clinical diagnostics.Hum. Mutat. 2013; 34: 1035-1042Crossref PubMed Scopus (209) Google Scholar), especially monogenic diseases (2Metzker M.L. Sequencing technologies - the next generation.Nat. Rev. Genet. 2010; 11: 31-46Crossref PubMed Scopus (4997) Google Scholar, 5Yang Y. Muzny D.M. Reid J.G. Bainbridge M.N. Willis A. Ward P.A. Braxton A. Beuten J. Xia F. Niu Z. et al.Clinical whole-exome sequencing for the diagnosis of mendelian disorders.N. Engl. J. Med. 2013; 369: 1502-1511Crossref PubMed Scopus (1411) Google Scholar, 6Katsanis S.H. Katsanis N. Molecular genetic testing and the future of clinical genomics.Nat. Rev. Genet. 2013; 14: 415-426Crossref PubMed Scopus (267) Google Scholar). Furthermore, the cost of NGS is rapidly decreasing, and has made tangible the prospect of incorporating genome-based diagnosis into medical care. In this regard, the US Food and Drug Administration recently approved Illumina's MiSeqDx for NGS applications in the clinical setting (7Collins F.S. Hamburg M.A. First FDA authorization for next-generation sequencer.N. Engl. J. Med. 2013; 369: 2369-2371Crossref PubMed Scopus (149) Google Scholar). These developments are relevant for genetic dyslipidemias, as the comprehensive detection of genome-wide variation opens up new approaches to further characterize the polygenic basis of complex metabolic traits (8Panoutsopoulou K. Tachmazidou I. Zeggini E. In search of low-frequency and rare variants affecting complex traits.Hum. Mol. Genet. 2013; 22: R16-R21Crossref PubMed Scopus (64) Google Scholar, 9Kilpinen H. Barrett J.C. How next-generation sequencing is transforming complex disease genetics.Trends Genet. 2013; 29: 23-30Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Clinically, the identification of causative genetic mutations in patients with suspected familial hypercholesterolemia (FH) is a criterion for diagnosing “definite FH” or “probable FH” in two widely used clinical algorithms (10Heath K.E. Humphries S.E. Middleton-Price H. Boxer M. A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom.Eur. J. Hum. Genet. 2001; 9: 244-252Crossref PubMed Scopus (95) Google Scholar, 11Fouchier S.W. Defesche J.C. Umans-Eckenhausen M.W. Kastelein J.P. The molecular basis of familial hypercholesterolemia in The Netherlands.Hum. Genet. 2001; 109: 602-615Crossref PubMed Scopus (178) Google Scholar). Also, screening for causative mutations in candidate genes in lipolysis for the diagnosis of familial chylomicronemia is supplanting traditional biochemical diagnostic methods, such as LPL activity in plasma collected postheparin infusion (12Rahalkar A.R. Giffen F. Har B. Ho J. Morrison K.M. Hill J. Wang J. Hegele R.A. Joy T. Novel LPL mutations associated with lipoprotein lipase deficiency: two case reports and a literature review.Can. J. Physiol. Pharmacol. 2009; 87: 151-160Crossref PubMed Scopus (64) Google Scholar, 13Johansen C.T. Hegele R.A. Genetic bases of hypertriglyceridemic phenotypes.Curr. Opin. Lipidol. 2011; 22: 247-253Crossref PubMed Scopus (96) Google Scholar). It is not presently clear whether molecular diagnosis will be clinically important for the >20 other monogenic disorders of lipid and lipoprotein metabolism whose molecular basis has been solved (11Fouchier S.W. Defesche J.C. Umans-Eckenhausen M.W. Kastelein J.P. The molecular basis of familial hypercholesterolemia in The Netherlands.Hum. Genet. 2001; 109: 602-615Crossref PubMed Scopus (178) Google Scholar), particularly as feasibility and efficiency via Sanger sequencing have limited the broad integration of clinical resequencing for dyslipidemia patients. However, NGS now presents a tool to evaluate the effectiveness of genome-based diagnosis in monogenic dyslipidemias and address clinical feasibility and applicability, as well as potential ethical concerns that come with genome-wide variant detection. Here, we have designed and evaluated a targeted resequencing panel for monogenic dyslipidemias termed LipidSeq. Our objective was to utilize an NGS-based approach to facilitate molecular diagnosis of dyslipidemias in patient samples studied at the Blackburn Cardiovascular Genetics Laboratory with the intention of replacing existing Sanger sequencing-based methods. Our laboratory performs molecular diagnosis of largely clinical samples from patients covering a range of dyslipidemias that are characterized by: 1) very high levels of LDL cholesterol, including FH and related conditions; 2) very low levels of LDL cholesterol, including abetalipoproteinemia and hypobetalipoproteinemia (HBL); 3) very high levels of HDL cholesterol; 4) very low levels of HDL cholesterol, including Tangier disease and familial deficiencies of apoA-1 and LCAT; and 5) very high levels of TG, including familial chylomicronemia (14Fu J. Kwok S. Sinai L. Abdel-Razek O. Babula J. Chen D. Farago E. Fernandopulle N. Leith S. Loyzer M. et al.Western Database of Lipid Variants (WDLV): a catalogue of genetic variants in monogenic dyslipidemias.Can. J. Cardiol. 2013; 29: 934-939Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The laboratory also receives samples for molecular diagnosis of miscellaneous dyslipidemias, as well as monogenic forms of diabetes, such as lipodystrophy syndromes (15Hegele R.A. Joy T.R. Al-Attar S.A. Rutt B.K. Lipodystrophies: windows on adipose biology and metabolism.J. Lipid Res. 2007; 48: 1433-1444Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Vigouroux C. Caron-Debarle M. Le Dour C. Magre J. Capeau J. Molecular mechanisms of human lipodystrophies: from adipocyte lipid droplet to oxidative stress and lipotoxicity.Int. J. Biochem. Cell Biol. 2011; 43: 862-876Crossref PubMed Scopus (106) Google Scholar), and mature onset diabetes of the young (MODY) (17Hattersley A. Bruining J. Shield J. Njolstad P. Donaghue K.C. The diagnosis and management of monogenic diabetes in children and adolescents.Pediatr. Diabetes. 2009; 10: 33-42Crossref PubMed Scopus (166) Google Scholar). Our aims were to: 1) determine the accuracy of NGS compared with traditional Sanger sequencing with respect to variant discovery in monogenic dyslipidemias and related metabolic disorders; 2) evaluate the reproducibility of variant discovery between samples; and 3) assess the potential diagnostic utility of targeted high-throughput sequencing technology in the clinic. We used the Nextera Custom Enrichment kit (Illumina, San Diego, CA) to capture genomic regions corresponding to 73 genes (supplementary Table I) and 178 SNPs (supplementary Table II) associated with clinical dyslipidemias and related metabolic disorders, comprising 689 kb of was also for of of the 73 targeted genes was as well as into the and an kb of and of We also SNPs that were based on to polygenic for LDL and HDL and TG, based on genome-wide of lipid traits K. M. J.P. S. et clinical and of for blood 2010; PubMed Scopus Google Scholar). SNPs were a on the variant of were from the of the D. M. B. et and Res. 2013; PubMed Scopus Google and were to the (Illumina, San Diego, In 84 patient samples were in this The of the patients whose genomic DNA was in the of Sanger sequencing C.T. Wang J. H. R.A. et of rare variants in genes identified by genome-wide of Genet. 2010; PubMed Scopus Google Scholar, C.T. Wang J. R.A. M.W. M. M. et of rare variants in candidate genes in patients with Genet. PubMed Scopus Google Scholar). The of the samples that were including genomic DNA samples and whole DNA The samples were from patients with a range of including with FH molecular were FH patients with mutations in the candidate FH genes and were recently with and were with other rare monogenic phenotypes including with familial lipodystrophy with and with FH patients were as FH based on the Lipid to with LipidSeq (11Fouchier S.W. Defesche J.C. Umans-Eckenhausen M.W. Kastelein J.P. The molecular basis of familial hypercholesterolemia in The Netherlands.Hum. Genet. 2001; 109: 602-615Crossref PubMed Scopus (178) Google Scholar). patients in this plasma patients a approved by the at DNA was in of The and of genomic DNA samples was by on a and a The DNA was to a of and a DNA samples were to a of the was at the the Nextera Custom Enrichment samples were with to the designed of with and to the were in with the DNA used the and used the was the were the kit on a samples were in of of was on to a on the (Illumina, San Diego, CA) to the was in as a for Sequencing was in and either in or sequencing were for and as and variant were a designed in sequencing were from and to the human of two were used to including a to mutations and of a variant detection tool was used to variants to a and These were to by identification of variants in regions for Sanger sequencing were in and variant reports for were in for in variants identified of regions were in was K. M. H. of genetic variants from high-throughput sequencing Res. 2010; PubMed Scopus Google Scholar). and made to human were from to facilitate including 2013; the and Sequencing and from and Variants were also compared the Database an Variants were into 1) variants in of 2) clear variants two of an 3) variants in the or or 4) variants in with on or variants. variants were as and or were as or either a or not Novel variants were as in either or and in were identified in of samples and were sequencing variants were not in were in are as between were was as a of samples high as by a of of which of were and of a of an a of of which of were and of a of for sequencing of identified and of are based on of identified and of are based on with with with samples with samples of identified and of are based on in a new samples with samples was well on the The of identified was in and in as would be samples was variation samples two samples a of of two samples a of of of between and for samples in were of the Our capture were designed to of approach in a of in and in The of with a was in and in the of with a was of and of detection was at was that in based on a in and of in and These were for the in and sequenced. A of with that the of with was in and in and a of were with including of in and in We additional sequencing to assess the concordance between we from genomic DNA and from The NGS samples well, a of of which of were and of a of performance were genomic versus DNA with of versus Table The of for a was corresponding to genomic DNA the of was corresponding to a DNA Sequencing was also genomic versus DNA samples the in genomic DNA versus DNA samples was versus other of were genomic DNA compared between genomic and DNA DNA DNA with with with in a new were in of with an concordance rate of genomic DNA with concordance compared with The of was genomic versus DNA samples the of was lower genomic versus DNA samples was by variants that were in either the or than variants that were identified in samples with (supplementary Table Furthermore, the of variants were in regions of lower clinical including the or These regions provide limited diagnostic as on and is less clear in to variants. The concordance rate was genomic DNA samples and DNA samples These that concordance genomic DNA is to DNA is for sequencing and the identification of clinically relevant of variants identified in by to a variant identified and between the and to a variant identified in samples was or the variant was in either the or refers to and to a variant identified and between the and to a variant identified in samples was or the variant was in either the or in a new refers to and variants. The two were of samples that were previously Sanger in the Sanger sequencing (8Panoutsopoulou K. Tachmazidou I. Zeggini E. In search of low-frequency and rare variants affecting complex traits.Hum. Mol. Genet. 2013; 22: R16-R21Crossref PubMed Scopus (64) Google Scholar). was compared for and rare variants in from genes in kb and additional with monogenic phenotypes were to genes kb to kb polygenic or patients with candidate mutations R.A. genetic and clinical Rev. Genet. 2009; 10: PubMed Scopus Google Scholar), Sanger sequencing identified variants including variants and corresponding to a of were identified by the including that were or by Sanger and identified by the Sanger we compared for samples for a of SNPs from of plasma C.T. Hegele R.A. Genetic bases of hypertriglyceridemic phenotypes.Curr. Opin. Lipidol. 2011; 22: 247-253Crossref PubMed Scopus (96) Google Scholar), which been previously in SNPs to be including the and SNPs also in that DNA with to samples at the we concordance for variants samples from patients with potential monogenic Sanger sequencing identified including variants and corresponding to were by the were by the and were by the Sanger sequencing as the the high accuracy by the to clinically relevant variants in samples for We an additional genomic DNA samples from patients with of monogenic or polygenic a of samples (supplementary Table Sequencing in samples were as in the samples In we identified variants including and and variants. of variants were the and variants were and including and variants. In we which to with variants. including of which were and were We focused on the ability of LipidSeq panel to genetic variants to patient clinical phenotypes the patients sequencing this in which we detection based on the disease existing the detection dyslipidemia patients previously for candidate mutations are not FH patients previously for candidate mutations are not in a new In samples from and we rare variants 73 candidate genes in patients while patients rare variants. patients with suspected FH in candidate sequencing been rare variants in candidate FH genes, including in in and in (supplementary Table Furthermore, of patients rare variants in candidate monogenic genes, which in in and in and (supplementary Table the patients for rare variants in monogenic genes, were of rare variants in polygenic genes, including in and in (supplementary Table of two patients with suspected rare variants in the candidate (supplementary Table The patients studied variants in known lipodystrophy the patient was for a variant in of (supplementary Table patients were for the of low-frequency variants in dyslipidemia genes in the LipidSeq A of FH patients previously to be at candidate FH genes was also the LipidSeq rare variants additional with potential to the dyslipidemia phenotypes (supplementary Table In the performance of NGS-based dyslipidemia resequencing we 1) the Nextera kit with the NGS sequencing in an of with an for of 2) variant detection accuracy was in samples with known mutations that been Sanger 3) DNA variant detection to Sanger sequencing and thus presents a for NGS genomic DNA is not and 4) in samples from individuals with a of dyslipidemias, including and in which sequencing been were identified as at candidate variant likely affecting the patient which were with Sanger sequencing. Clinical implementation of LipidSeq has clinical resequencing LipidSeq to and patient DNA samples compared with the of than this to a of screening with Sanger sequencing. a more limited the resequencing of the causative genes for and can including and variant from whole exome sequencing can we patient samples for the dyslipidemia genes and other related metabolic genes at a cost of which was the cost of Sanger sequencing the candidate FH genes whole exome sequencing on the is the cost of the LipidSeq panel based on samples with a of patient samples in to the NGS a more and approach for clinical resequencing in monogenic These with the concordance in variant detection with Sanger sequencing that LipidSeq has the potential to replace Sanger sequencing in the clinic. The LipidSeq panel also genetic that help the of candidate dyslipidemia patients. the LipidSeq panel genes that at in metabolic associated with dyslipidemias, we low-frequency variants that a in the clinical two patients with FH variants in FH genes, were of rare variants in the genes and (supplementary Table rare variants in and were in FH patients previously for candidate mutations (supplementary Table Genetic variation in and has been associated with while has recently as a for LDL S. P. S. and genetic LDL and for and disease Lipid Res. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). However, rare variation in has not been to LDL rare variants in genes not be of clinical such be important for future into the polygenic of dyslipidemias, particularly in patients are in known Furthermore, as the panel is limited to genes in lipoprotein this targeted approach more than the of variants from the that would be by whole exome of In to on rare variant we also designed the LipidSeq panel to 178 SNPs to polygenic genetic sequencing were designed of the SNPs in and regions that would not have been exome sequencing potential clinical we used the variants associated with a lipid identified the K. M. J.P. S. et clinical and of for blood 2010; PubMed Scopus Google Scholar). SNPs were based on the with lipid the SNPs previously associated with plasma were into a were for HDL and LDL These were designed to the polygenic of complex blood lipid traits and by SNPs as for the of candidate variation in for disease or plasma lipid a recent by et S. R. M. P. K. F. F. et of lipoprotein to patients with polygenic and monogenic familial a 2013; Full Text Full Text PDF PubMed Scopus Google a LDL in FH patients from FH patients. of polygenic with sequencing of monogenic dyslipidemia genes provide additional that be in patients. the the of the that is by a rare variant of in a dyslipidemia Also, are of patients with monogenic forms of dyslipidemia in the is not by a rare of by a high polygenic variants of underlie the S. R. M. P. K. F. F. et of lipoprotein to patients with polygenic and monogenic familial a 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). The LipidSeq approach has detection with targeted resequencing which the potential for more comprehensive that and rare variation as the next in polygenic We also the of DNA samples in the NGS LipidSeq approach, samples from patients be to or with and in to DNA We objective in DNA compared with genomic as by and an of sequencing the concordance between genomic and DNA samples as compared with Sanger sequencing was a with the a clear between genomic and DNA based on NGS performance DNA at a the of genomic DNA and thus as an DNA high molecular genomic DNA is not The of LipidSeq largely from the approach to genomic which genetic variation at known dyslipidemia Also, to the of new genes in lipoprotein metabolism by whole exome sequencing of of individuals has been further that the whole exome approach not be for clinical of patients with monogenic dyslipidemias K. J.P. R. C. C. S. J. et and familial Engl. J. Med. 2010; PubMed Scopus Google Scholar, M. K. D. A. K. M. L. et of a with hypercholesterolemia associated with the Mutat. 2013; 34: PubMed Scopus Google Scholar, S.W. B. A. B. O. et sequencing and clinical disease as Biol. 2013; PubMed Scopus Google Scholar). the of variant detection the of a molecular diagnosis less in to an whole exome sequencing mutations that have clear on patient phenotypes are likely to be the of variants of is a targeted approach than the whole exome sequencing be to genes in metabolic of whole exome the LipidSeq approach has the of lower cost and LipidSeq was NGS the US Food and Drug Administration approved authorization for the MiSeqDx (7Collins F.S. Hamburg M.A. First FDA authorization for next-generation sequencer.N. Engl. J. Med. 2013; 369: 2369-2371Crossref PubMed Scopus (149) Google Scholar). the and of NGS technology and the for clinical NGS for of the LipidSeq panel that are NGS approaches to screening for we low or at the and other as well as regions in These regions were the of variant However, regions are largely and are of less for mutations B. Johansson L.F. de Boer E.N. Almomani R. Boven L.G. van den Berg M.P. van Spaendonck-Zwarts K.Y. van Tintelen J.P. Sijmons R.H. Jongbloed J.D. et al.Targeted next-generation sequencing can replace Sanger sequencing in clinical diagnostics.Hum. Mutat. 2013; 34: 1035-1042Crossref PubMed Scopus (209) Google Scholar). NGS-based approaches not and and to Chen K. L. parallel sequencing approaches for of Mol. Biol. PubMed Scopus Google Scholar). Our a variation tool the further of the performance of this tool is implementation in clinical ethical have emerged incidental genomic and the to which detection are to the particularly and clinically actionable mutations are be to the patient L.G. and for the integration of massively parallel genomic sequencing into clinical from the Med. 14: Full Text Full Text PDF PubMed Scopus Google Scholar, L.G. I. S.E. R. Next-generation sequencing in the are we Rev. Genet. PubMed Scopus Google Scholar, M.L. S. C.T. genomic disease of 2013; PubMed Scopus Google Scholar). In to this the of Genetics and identified genes associated with diseases for which incidental genomic be to patients Berg K.E. et for of incidental in clinical exome and Med. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). the genes on the LipidSeq panel the known FH genes and the LipidSeq approach patient to incidental genomic to In we report the comprehensive targeted NGS approach for molecular diagnoses the of monogenic The panel performs well, with high concordance in samples with known mutations based on Sanger sequencing and a high detection rate of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, targeted NGS of will help to provide a more focused picture of monogenic and polygenic contributors that underlie and will not provide incidental clinically actionable variants in that disease A to on the and of variants based on clinical as comprehensive genomic variation in dyslipidemia patients to be we to into the of variants the dyslipidemia The especially the to in this with of Genetics and familial hypercholesterolemia familial lipodystrophy genetic genome-wide hypobetalipoproteinemia mature onset diabetes of the young next generation sequencing whole

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