Ann Lab Med.  2025 Jan;45(1):96-100. 10.3343/alm.2024.0201.

Reanalysis of Next-generation Sequencing Data in Patients With Hypertrophic Cardiomyopathy: Contribution of Spliceogenic MYBPC3 Variants in an Italian Cohort

• Caroselli S
• Fabiani M
• Micolonghi C
• Savio C
• Tini G
• Musumeci B
• Pagannone E
• Germani A
• Libi F
• Visco V
• Pizzuti A
• Autore C
• Petrucci S
• Rubattu S
• Piane M

• KMID: 2563861
• DOI: http://doi.org/10.3343/alm.2024.0201

Abstract

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of diseasecausing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40–60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the in-silico detection of intronic variants in MYBPC3 that may escape detection by algorithms normally used with tagged diagnostic panels. We included 142 HCM probands with negative results in Illumina TruSight Cardio panel analysis, including exonic regions of 174 cardiomyopathy genes. Raw data were re-analyzed using existing bioinformatics tools. The spliceogenic variant c.1224-80G > A was detected in three patients (2.1%), leading us to reconsider their molecular diagnosis. These patients showed late onset and mild symptoms, although no peculiar phenotypic characteristics were shared. Collectively, rare spliceogenic MYBPC3 variants may play a role in causing HCM, and their systematic detection should be performed to provide more comprehensive solutions in genetic testing using multigenic panels.

Keyword

High-throughput nucleotide sequencing; Hypertrophic cardiomyopathy; Introns; Loss of function mutation; RNA splicing

Figure

• 

  Fig. 1 Parallel multistep workflow for diagnostic setting. Left side (first approach): development and application of an in-silico hotspot for previously reported PVs; right side (second approach): in-silico filtering and in-vitro analysis of unknown variants. Abbreviations: PVs, pathogenic variants; IGV, Integrative Genomics Viewer; ACMG, American College of Medical Genetics and Genomics.

• 

  Fig. 2 MYBPC3 exon 14 splicing. (A) Schematic representation of the nucleotide sequences of exon 13, intron 13, and exon 14. Canonical splicing of intron 13 with the juxtaposition of the 3′-exon 13/5′-exon 14 junction is shown above (green lines). The variant c.1224-80G>A (in red) creates a cryptic splice acceptor site in intron 13 (dinucleotide “AG”), which leads to the juxtaposition of 3′-exon 13 with a locus upstream of 5′-exon 14 (bottom, red lines), inserting 78 intronic nucleotides into the transcript. (B) Representation of aligned NGS reads (top) and Sanger electropherogram (bottom) corresponding to the heterozygous variant c.1224-80G>A (arrow). Abbreviation: NGS, next-generation sequencing.

Reference

References

1. Maron BJ, Maron MS. 2013; Hypertrophic cardiomyopathy. Lancet. 381:242–55. DOI: 10.1016/S0140-6736(12)60397-3. PMID: 22874472.

2. Zampieri M, Salvi S, Fumagalli C, Argirò A, Zocchi C, Del Franco A, et al. 2023; Clinical scenarios of hypertrophic cardiomyopathy-related mortality: relevance of age and stage of disease at presentation. Int J Cardiol. 374:65–72. DOI: 10.1016/j.ijcard.2022.12.056. PMID: 36621577.

3. Arbelo E, Protonotarios A, Gimeno JR, Arbustini E, Barriales-Villa R, Basso C, et al. 2023; 2023 ESC guidelines for the management of cardiomyopathies. Eur Heart J. 44:3503–626. DOI: 10.1093/eurheartj/ehad194. PMID: 37622657.

4. Mazzarotto F, Olivotto I, Boschi B, Girolami F, Poggesi C, Barton PJR, et al. 2020; Contemporary insights into the genetics of hypertrophic cardiomyopathy: toward a new era in clinical testing? J Am Heart Assoc. 9:e015473. DOI: 10.1161/JAHA.119.015473. PMID: 32306808. PMCID: PMC7428545. PMID: b884d942541041a89eac8d3be5f77b5f.

5. Walsh R, Buchan R, Wilk A, John S, Felkin LE, Thomson KL, et al. 2017; Defining the genetic architecture of hypertrophic cardiomyopathy: re-evaluating the role of non-sarcomeric genes. Eur Heart J. 38:3461–8. DOI: 10.1093/eurheartj/ehw603. PMID: 28082330. PMCID: PMC5837460.

6. Torrado M, Maneiro E, Lamounier Junior A, Fernández-Burriel M, Sánchez Giralt S, Martínez-Carapeto A, et al. 2022; Identification of an elusive spliceogenic MYBPC3 variant in an otherwise genotype-negative hypertrophic cardiomyopathy pedigree. Sci Rep. 12:7284. DOI: 10.1038/s41598-022-11159-y. PMID: 35508642. PMCID: PMC9068804. PMID: c868467e243b4073b28e6489e0a7d28c.

7. Janin A, Chanavat V, Rollat-Farnier PA, Bardel C, Nguyen K, Chevalier P, et al. 2020; Whole MYBPC3 NGS sequencing as a molecular strategy to improve the efficiency of molecular diagnosis of patients with hypertrophic cardiomyopathy. Hum Mutat. 41:465–75. DOI: 10.1002/humu.23944. PMID: 31730716.

8. Singer ES, Crowe J, Holliday M, Isbister JC, Lal S, Nowak N, et al. 2023; The burden of splice-disrupting variants in inherited heart disease and unexplained sudden cardiac death. NPJ Genom Med. 8:29. DOI: 10.1038/s41525-023-00373-w. PMID: 37821546. PMCID: PMC10567745. PMID: 8fed1ba26d23493d97359693ba5f0785.

9. Lopes LR, Barbosa P, Torrado M, Quinn E, Merino A, Ochoa JP, et al. 2020; Cryptic splice-altering variants in MYBPC3 are a prevalent cause of hypertrophic cardiomyopathy. Circ Genom Precis Med. 13:e002905. DOI: 10.1161/CIRCGEN.120.002905. PMID: 32396390.

10. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. 2011; Integrative genomics viewer. Nat Biotechnol. 29:24–6. DOI: 10.1038/nbt.1754. PMID: 21221095. PMCID: PMC3346182.

11. SpliceAI Lookup. Broad Institute. https://spliceailookup.broadinstitute.org/#. Updated on March 29, 2024.

12. Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, Darbandi SF, Knowles D, Li YI, et al. 2019; Predicting splicing from primary sequence with deep learning. Cell. 176:535–48.e24. DOI: 10.1016/j.cell.2018.12.015. PMID: 30661751.

13. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. 2015; Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 17:405–24. DOI: 10.1038/gim.2015.30. PMID: 25741868. PMCID: PMC4544753.

14. Ustianenko D, Weyn-Vanhentenryck SM, Zhang C. 2017; Microexons: discovery, regulation, and function. Wiley Interdiscip Rev RNA. 8:0.1002/wrna.1418. DOI: 10.1002/wrna.1418. PMID: 28188674. PMCID: PMC5863539.

15. Carrier L, Bonne G, Bährend E, Yu B, Richard P, Niel F, et al. 1997; Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy. Circ Res. 80:427–34. DOI: 10.1161/01.res.0000435859.24609.b3. PMID: 9048664.

16. Oakley CE, Hambly BD, Curmi PMG, Brown LJ. 2004; Myosin binding protein C: structural abnormalities in familial hypertrophic cardiomyopathy. Cell Res. 14:95–110. DOI: 10.1038/sj.cr.7290208. PMID: 15115610.

17. Sterner DA, Berget SM. 1993; In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit. Mol Cell Biol. 13:2677–87. DOI: 10.1128/MCB.13.5.2677. PMID: 7682652. PMCID: PMC359639.

18. Carlo T, Sterner DA, Berget SM. 1996; An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon. RNA. 2:342–53. DOI: 10.7717/peerj.14824/fig-3. PMID: 8634915. PMCID: PMC1369377.

19. Frank-Hansen R, Page SP, Syrris P, McKenna WJ, Christiansen M, Andersen PS. 2008; 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. 16:1062–9. DOI: 10.1038/ejhg.2008.52. PMID: 18337725.