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• W3025733573 abstract "HomeCirculation: Genomic and Precision MedicineVol. 13, No. 3Cryptic Splice-Altering Variants in MYBPC3 Are a Prevalent Cause of Hypertrophic Cardiomyopathy LetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toLetterPDF/EPUBCryptic Splice-Altering Variants in MYBPC3 Are a Prevalent Cause of Hypertrophic Cardiomyopathy Luis R. Lopes, MD, PhD Pedro Barbosa, MSc Mario Torrado, PhD Ellie Quinn, BSc, MSc Ana Merino, MD Juan Pablo Ochoa, MD, PhD Joanna Jager, MSc Marta Futema, PhD Maria Carmo-Fonseca, MD, PhD Lorenzo Monserrat, MD, PhD Petros Syrris, PhD Perry M. ElliottMBBS, MD Luis R. LopesLuis R. Lopes Correspondence to: Luis R. Lopes, MD, PhD, Barts Heart Centre, St. Bartholomew’s Hospital, W Smithfield, London EC1A 7BE, United Kingdom. Email E-mail Address: [email protected] https://orcid.org/0000-0002-6408-4667 Inherited Cardiovascular Disease Unit, St Bartholomew’s Hospital, Barts Health NHS Trust (L.R.L., E.Q., P.M.E.). UCL Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, United Kingdom (L.R.L., J.J., M.F., P.S., P.M.E.). Search for more papers by this author , Pedro BarbosaPedro Barbosa https://orcid.org/0000-0002-3892-7640 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina (P.B., M.C.-F.), Universidade de Lisboa, Portugal LASIGE, Faculdade de Ciências (P.B.), Universidade de Lisboa, Portugal. Search for more papers by this author , Mario TorradoMario Torrado Institute of Health Sciences, University of A Coruña (M.T.). Search for more papers by this author , Ellie QuinnEllie Quinn https://orcid.org/0000-0003-3648-6084 Inherited Cardiovascular Disease Unit, St Bartholomew’s Hospital, Barts Health NHS Trust (L.R.L., E.Q., P.M.E.). Search for more papers by this author , Ana MerinoAna Merino Cardiology Department, University Hospital of Burgos (A.M.). Search for more papers by this author , Juan Pablo OchoaJuan Pablo Ochoa https://orcid.org/0000-0002-7966-0313 Cardiology Department, Health in Code, A Coruña, Spain (J.P.O., L.M.) Search for more papers by this author , Joanna JagerJoanna Jager UCL Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, United Kingdom (L.R.L., J.J., M.F., P.S., P.M.E.). Search for more papers by this author , Marta FutemaMarta Futema UCL Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, United Kingdom (L.R.L., J.J., M.F., P.S., P.M.E.). Search for more papers by this author , Maria Carmo-FonsecaMaria Carmo-Fonseca https://orcid.org/0000-0002-3402-7143 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina (P.B., M.C.-F.), Universidade de Lisboa, Portugal Search for more papers by this author , Lorenzo MonserratLorenzo Monserrat https://orcid.org/0000-0001-5776-0623 Cardiology Department, Health in Code, A Coruña, Spain (J.P.O., L.M.) Search for more papers by this author , Petros SyrrisPetros Syrris https://orcid.org/0000-0002-2363-8758 UCL Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, United Kingdom (L.R.L., J.J., M.F., P.S., P.M.E.). Search for more papers by this author and Perry M. ElliottPerry M. Elliott Inherited Cardiovascular Disease Unit, St Bartholomew’s Hospital, Barts Health NHS Trust (L.R.L., E.Q., P.M.E.). UCL Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, United Kingdom (L.R.L., J.J., M.F., P.S., P.M.E.). Search for more papers by this author Originally published12 May 2020https://doi.org/10.1161/CIRCGEN.120.002905Circulation: Genomic and Precision Medicine. 2020;13:e002905The yield of genetic testing in hypertrophic cardiomyopathy (HCM) is only 40%, even in patients with family histories of the disease. This may be caused by a high prevalence of nongenetic phenocopies or complex genetic mechanisms but may also reflect the inability of conventional diagnostic sequencing to detect intronic variants distant from the essential donor/acceptor dinucleotides with the potential to disrupt splicing (also known as cryptic splice mutations). The availability of whole genomes from the genome aggregation database (gnomAD; https://gnomad.broadinstitute.org) and novel prediction tools has improved the capacity for analyzing and interpreting deep intronic sequences.In this study, we performed large-scale unbiased screening of intronic variants in MYBPC3 in 1644 unrelated and consecutive patients with HCM (49.5±15.6 years old at diagnosis, 1103 [67.1%] male, 1000 white [60.8%], 156 Asian [9.5%], and 75 black [4.6%]). All patients gave written informed consent and the study was approved by the regional ethics committee (15/LO/0549). Eight hundred seventy-four probands were screened with next-generation sequencing of the whole-genomic region of 41 genes, and 770 probands were screened using whole-exome sequencing including a ≈100 bp intronic region beyond the intron-exon boundaries. Sequencing, variant calling, filtering, and annotation were as previously described.1,2 To prioritize intronic variants with an impact on splicing, a deep-learning approach, SpliceAIv1.3 was applied (https://github.com/Illumina/SpliceAI)3 with a very stringent threshold (≥0.9). To predict branchpoint disruptions, LabBranchoR (https://github.com/jpaggi/labranchor) was run. To evaluate the functional consequences of a novel variant, expression of mRNA was analyzed using blood samples, according to published methods.4 The data that support the findings of this study are available from the corresponding author upon reasonable request.Five hundred forty-six patients (33.2%) had coding (or canonical splicing) variants with minor allele frequency <1×10−4 in MYH7, MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, and ACTC1. Four variants with a very high (≥0.90) SpliceAI score were detected: 3 located in intron 13 (c.1224-52G>A [0.99; N=18], c.1224-80G>A [0.94; N=3], and c.1224-21A>G [0.90; N=1]) and another, c.906-36G>A (0.91; N=2), in intron 9 (Figure [A]). These 4 variants were present in 24/1644 (2.2% of otherwise mutation-negative patients). All are predicted to cause a cryptic splice site and an expansion to a larger subsequent (micro)exon, in turn leading to a frameshift and stop codon, as previously shown for c.1224-52G>A, c.1224-80G>A, and c.906-36G>A with RNA assays.5–7Download figureDownload PowerPointFigure. Bioinformatic analysis, co-segregation studies and RNA assays of cryptic splice-altering variants in MYBPC3.A, MYBPC3 deep intronic variants c.1224-52G>A and c.1224-80G>A generate cryptic splice sites within intron 13 with expansion of exon 14. c.906-36G>A generates a cryptic splice site within intron 9 with expansion of exon 10. Score refers to spliceAI score. B, Pedigrees demonstrating co-segregation of the MYBPC3 c.1224-52G>A variant with HCM phenotype in 3 families. Circles: women; squares: men. +: mutation-positive; −: mutation-negative; colored symbols: affected (hypertrophic cardiomyopathy [HCM]). C, Pedigrees demonstrating co-segregation of the MYBPC3 c.1898-23A>G variant with HCM phenotype in 2 previously “mutation-negative” families. Circles: women; squares: men. +, mutation-positive; −, mutation-negative; colored symbols: affected (HCM). D, RT-PCR analysis of the ectopic expression of MYBPC3 in the blood of controls (lanes 1–2) and index patients carrying the c.1898-23A>G variant (lane 3 and lane 4). Negative controls were loaded in lane 5 (-RT) and 6 (nontemplate). L-100 bp DNA ladder. The lower bands (368 bp, MYBPC3) correspond to the normal mRNA, and the longer bands (473 bp, IR19-MYBPC3) correspond to a mis-spliced intron 19-retained transcript.Eighteen patients had c.1224-52G>A (1.1% compared with minor allele frequency in GnomAD of 2.8×10−5 (odds ratio, 197.9 [95% CI, 66.9–585.6]; P≤0.0001). The variants c.1224-80G>A, c.906-36G>A, and c.1224-21A>G were not found in GnomAD. Co-segregation for c.1224-52G>A was demonstrated in 3 families (Figure [B]).Of the 24 index patients carrying these 4 variants, age of diagnosis was 10 to 72 years, 16 (67%) were male and 6 were Asian (25% versus 9.5% in the overall cohort; P=0.009); the remainder were white. Nineteen (79%) had a family history of sudden death and/or HCM. Maximal wall thickness varied between 13 and 30 mm and 6 (25%) had left ventricular outflow tract obstruction. Seven were deemed to be at high sudden death risk or already had an implantable cardioverter-defibrillator at baseline.Finally, a novel variant, c.1898-23A>G with a low SpliceAI score (0.04, GnomAD minor allele frequency 0.000005) segregated with the phenotype in 2 “mutation-negative” families (Figure [C]). These families were enrolled and studied with whole-exome sequencing with the initial aim of novel gene discovery, and this variant was the only suitable candidate found. We have then searched for this variant in the first cohort of 874 patients and found it in an additional proband. As shown in Figure [D], the MYBPC3 RT-PCR amplification of total RNA showed an additional longer 473 bp band detected in the probands’ RNA. Sanger sequencing revealed that these longer bands contained the complete sequence of intron 19. This mis-spliced transcript introduces a premature termination codon in intron 19 and is expected to cause nonsense-mediated mRNA decay, leading to haploinsufficiency. Given the low SpliceAI score for this variant, we sought to understand the mechanism that could explain mis-splicing; we have found a high probability of branchpoint disruption (probability of position being a branchpoint reduced by 60%).Two recent studies of small cohorts of mutation-negative cases (465 and 936 probands) described 8 novel cryptic splice-altering variants in MYBPC3, with a prevalence of 9% and 6.5%, respectively. In our study, the prevalence was only 2.2% in otherwise mutation-negative patients. The difference may be explained by the very stringent criteria employed and the fact that whole-exome sequencing coverage is limited to ≈100 bp from the exon-intron boundary, meaning that deeper intronic variants might have been missed. None of our candidate variants were described in a previous analysis of MYBPC3 splice variants at noncanonical splice sites, which used both a splice prediction tool and a mini-gene assay to identify variants that alter MYBPC3 splicing.8If the variant calling in our cohort was limited to conventional splice-sites, all the families with cryptic splice-altering variants would have been considered mutation-negative. One variant in particular (c.1224-52G>A) was unusually common (1.1%). The cause for the high frequency is uncertain but seems unlikely to be a recent founder effect, considering the heterogeneous geographic origin of the patients. As a comparison, the causal HCM variant considered as the most common to date (MYBPC3 p.Arg502Trp) has an estimated prevalence of 1.4% to 2.0% (95% CI).9 Patients with these particular cryptic splicing intronic variants in MYBPC3 showed a higher prevalence of Asian ethnicity; this finding needs to be replicated in other populations.Sequencing of deep intronic regions of MYBPC3 increases the yield of genetic testing and thus improves counseling and evaluation of families with HCM. New phenotype-modifying genetic therapies tailored for splicing altering variants are being tested in animal models; as such, this increase in yield might in the future be translated into tailored therapy.Nonstandard Abbreviations and AcronymsHCMhypertrophic cardiomyopathySources of FundingDr Lopes is a recipient of a Medical Research Council (MRC) Clinical Academic Research Partnership (CARP) award. P. Barbosa is supported by an FCT (Fundacão para a Ciência e Tecnologia, Portugal) Fellowship SFRH/BD/137062/2018. Dr Torrado is funded by a grant (GRC ED431C 2018/38) from the Autonomic Government of Galicia, Spain. J. Jager and M. Futema are funded by the Fondation Leducq Transatlantic Networks of Excellence Program grant (no. 14 CVD03). This work was funded by the British Heart Foundation Program Grant RG/15/8/31480 and the National Institute for Health Research University College London Hospitals Biomedical Research Centre.DisclosuresDr Monserrat is shareholder in Health in Code. The other authors report no conflicts.FootnotesFor Sources of Funding and Disclosures, see page 167.Correspondence to: Luis R. Lopes, MD, PhD, Barts Heart Centre, St. Bartholomew’s Hospital, W Smithfield, London EC1A 7BE, United Kingdom. Email luis.[email protected]netReferences1. Lopes LR, Syrris P, Guttmann OP, O’Mahony C, Tang HC, Dalageorgou C, Jenkins S, Hubank M, Monserrat L, McKenna WJ, et al.. Novel genotype-phenotype associations demonstrated by high-throughput sequencing in patients with hypertrophic cardiomyopathy.Heart. 2015; 101:294–301. doi: 10.1136/heartjnl-2014-306387CrossrefMedlineGoogle Scholar2. Lopes LR, Futema M, Akhtar MM, Lorenzini M, Pittman A, Syrris P, Elliott PM. Prevalence of TTR variants detected by whole-exome sequencing in hypertrophic cardiomyopathy.Amyloid. 2019; 26:243–247. doi: 10.1080/13506129.2019.1665996CrossrefMedlineGoogle Scholar3. Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, Darbandi SF, Knowles D, Li YI, Kosmicki JA, Arbelaez J, Cui W, Schwartz GB, et al.. Predicting splicing from primary sequence with deep learning.Cell. 2019; 176:535–548.e24. doi: 10.1016/j.cell.2018.12.015CrossrefMedlineGoogle Scholar4. Singer ES, Ingles J, Semsarian C, Bagnall RD. Key value of RNA analysis of MYBPC3 splice-site variants in hypertrophic cardiomyopathy.Circ Genom Precis Med. 2019; 12:e002368. doi: 10.1161/CIRCGEN.118.002368LinkGoogle Scholar5. Bagnall RD, Ingles J, Dinger ME, Cowley MJ, Ross SB, Minoche AE, Lal S, Turner C, Colley A, Rajagopalan S, et al.. Whole genome sequencing improves outcomes of genetic testing in patients with hypertrophic cardiomyopathy.J Am Coll Cardiol. 2018; 72:419–429. doi: 10.1016/j.jacc.2018.04.078CrossrefMedlineGoogle Scholar6. Janin A, Chanavat V, Rollat-Farnier PA, Bardel C, Nguyen K, Chevalier P, Eicher JC, Faivre L, Piard J, Albert E, et al.. Whole MYBPC3 NGS sequencing as a molecular strategy to improve the efficiency of molecular diagnosis of patients with hypertrophic cardiomyopathy.Hum Mutat. 2020; 41:465–475. doi: 10.1002/humu.23944CrossrefMedlineGoogle Scholar7. Frank-Hansen R, Page SP, Syrris P, McKenna WJ, Christiansen M, Andersen PS. Micro-exons of the cardiac myosin binding protein C gene: flanking introns contain a disproportionately large number of hypertrophic cardiomyopathy mutations.Eur J Hum Genet. 2008; 16:1062–1069. doi: 10.1038/ejhg.2008.52CrossrefMedlineGoogle Scholar8. Ito K, Patel PN, Gorham JM, McDonough B, DePalma SR, Adler EE, Lam L, MacRae CA, Mohiuddin SM, Fatkin D, et al.. Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing.Proc Natl Acad Sci U S A. 2017; 114:7689–7694. doi: 10.1073/pnas.1707741114CrossrefMedlineGoogle Scholar9. Whiffin N, Roberts AM, Minikel E, Zappala Z, Walsh R, O’Donnell-Luria AH, Karczewski KJ, Harrison SM, Thomson KL, Sage H, et al.. Using high-resolution variant frequencies empowers clinical genome interpretation and enables investigation of genetic architecture.Am J Hum Genet. 2019; 104:187–190. doi: 10.1016/j.ajhg.2018.11.012CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByHolliday M, Singer E, Ross S, Lim S, Lal S, Ingles J, Semsarian C and Bagnall R (2021) Transcriptome Sequencing of Patients With Hypertrophic Cardiomyopathy Reveals Novel Splice-Altering Variants in MYBPC3, Circulation: Genomic and Precision Medicine, 14:2, Online publication date: 1-Apr-2021. June 2020Vol 13, Issue 3Article InformationMetrics Download: 1,109 © 2020 The Authors. Circulation: Genomic and Precision Medicine is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited and is not used for commercial purposes.https://doi.org/10.1161/CIRCGEN.120.002905PMID: 32396390 Originally publishedMay 12, 2020 KeywordsgeneticsMYBPC3intronscryptic splice sitescardiomyopathy, hypertrophicPDF download SubjectsClinical StudiesEtiologyCardiomyopathyGene Expression and RegulationGenetics" @default.
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