Identification of an elusive spliceogenic MYBPC3 variant in an otherwise genotype-negative hypertrophic cardiomyopathy pedigree

Abstract

The finding of a genotype-negative hypertrophic cardiomyopathy (HCM) pedigree with several affected members indicating a familial origin of the disease has driven this study to discover causative gene variants. Genetic testing of the proband and subsequent family screening revealed the presence of a rare variant in the MYBPC3 gene, c.3331−26T>G in intron 30, with evidence supporting cosegregation with the disease in the family. An analysis of potential splice-altering activity using several splicing algorithms consistently yielded low scores. Minigene expression analysis at the mRNA and protein levels revealed that c.3331−26T>G is a spliceogenic variant with major splice-altering activity leading to undetectable levels of properly spliced transcripts or the corresponding protein. Minigene and patient mRNA analyses indicated that this variant induces complete and partial retention of intron 30, which was expected to lead to haploinsufficiency in carrier patients. As most spliceogenic MYBPC3 variants, c.3331−26T>G appears to be non-recurrent, since it was identified in only two additional unrelated probands in our large HCM cohort. In fact, the frequency analysis of 46 known splice-altering MYBPC3 intronic nucleotide substitutions in our HCM cohort revealed 9 recurrent and 16 non-recurrent variants present in a few probands (≤ 4), while 21 were not detected. The identification of non-recurrent elusive MYBPC3 spliceogenic variants that escape detection by in silico algorithms represents a challenge for genetic diagnosis of HCM and contributes to solving a fraction of genotype-negative HCM cases.

Identification of Two Homozygous Variants in MYBPC3 and SMYD1 Genes Associated with Severe Infantile Cardiomyopathy

“…Around 75% of these variants were found to be loss-of-function and resulted in absent or truncated proteins with impaired ability to bind to other sarcomeric proteins, being the main reason for the development of hypertrophic cardiomyopathy in both pediatric and adult populations [ 44 , 45 ]. To date, this specific MYBPC3 c.1224-52 G>A (IVS13-52 G>A) variant has only been identified in adult patients with hypertrophic cardiomyopathy containing a heterozygous variant or unknown heterozygosity [ 17 , 18 , 19 , 20 , 21 ] (summarized in Table 3 ). This specific truncation occurs in the immunoglobulin-type C2 domain, which forms a compact globular structure.…”

“…Intronic variants in MYBPC3 in the context of hypertrophic cardiomyopathy have also been identified [ 14 , 15 , 16 ] and, in the last few years, have focused on one specific intronic MYBPC3 variant c.1224-52G>A (IVS13-52G>A) [ 17 , 18 , 19 , 20 , 21 ]. Specifically, Bagnall et al reported on a cohort of unrelated gene-elusive probands with HCM that had undergone genome sequencing, in which they identified the intronic MYBPC3 variant c.1224-52G>A in two adult male family members, as well as an adult proband from another family [ 17 ].…”

“…Interestingly, co-segregation of this variant with HCM was demonstrated in three families [ 20 ]. Finally, in the most recent study published by Torrado et al, they analyzed a large HCM cohort in which they identified 12 adult carriers of this specific MYBPC3 c.1224-52G>A variant [ 21 ]. Thus, to date, this variant has been identified independently and in conjunction with other variants in adult patients with hypertrophic cardiomyopathy [ 17 , 18 , 19 , 20 , 21 , 22 ].…”

The role of noncoding genetic variants in cardiomyopathy

“…RNA analyses were performed to confirm aberrant splicing through the inclusion of cryptic exons in cardiomyocytes from patient-derived induced pluripotent stem cells (iPSC-CMs) and in a myectomy sample from one affected relative of the proband (c.1928−569G > T only). In addition, the role of one MYBPC3 intronic variant (c.3331−26T > G) was found to account for a genotype-negative proband in a family with a history of HCM ( 71 ). This variant segregates with two diseased descendants of the proband and it was found in unrelated HCM patients.…”

Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy

“…Currently, the common method for gene sequencing is next-generation sequencing (NGS), but most of the gene panels and exome assays are targeted to coding regions and canonical splice sites, whereas introns and promoter regions are ignored. On the other hand, there is increasing evidence that variants in non-coding regions can contribute to the disease, mostly by altering splicing [ 4 , 5 , 6 , 7 , 8 , 9 ]. A whole-gene sequence is usually obtained only by whole-genome sequencing, which is more expensive than target gene analysis.…”