ALU transposition induces familial hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy (LVH) in the absence of predisposing cardiovascular conditions. Pathogenic variants in at least 16 cardiac sarcomeric genes have been implicated in HCM, most of which act in a dominant‐negative fashion. However loss‐of‐function (haploinsufficiency) is the most common disease mechanism for pathogenic variants in MYBPC3, suggesting that MYBPC3 complete deletion may play a role in HCM pathogenesis. Here, we investigate MYBPC3 complete deletion as a disease mechanism in HCM by analyzing two unrelated patients with confirmed diagnosis of HCM that tested negative by Sanger sequencing analysis. Methods MYBPC3 complete deletion was investigated by Multiplex ligation‐dependent probe amplification (MLPA) and microarray analyses. The mechanism of deletion was investigated by interrogating the SINEBase database. Results Patient‐1 was diagnosed with nonobstructive HCM in his mid‐40s while undergoing assessment for palpitations, and patient‐2 with obstructive HCM in his late‐20s while undergoing systolic heart murmur assessment for an unrelated illness. MLPA testing revealed a heterozygous deletion of all MYBPC3 exons in both patients. Subsequent microarray testing confirmed these deletions which extended beyond the 5′ and 3′ ends of MYBPC3. Genomic assessment suggested that these deletions resulted from Alu/Alu‐homologous recombination. Conclusion Our results demonstrate that haploinsufficiency resulting from MYBPC3 complete deletion, potentially mediated by Alu recombination, is an important disease mechanism in cardiomyopathy and emphasizes the importance of copy number variation analysis in patients clinically suspected of HCM.

(haploinsufficiency) is the most common disease mechanism for pathogenic variants in MYBPC3, suggesting that MYBPC3 complete deletion may play a role in HCM pathogenesis. Here, we investigate MYBPC3 complete deletion as a disease mechanism in HCM by analyzing two unrelated patients with confirmed diagnosis of HCM that tested negative by Sanger sequencing analysis. Methods: MYBPC3 complete deletion was investigated by Multiplex ligationdependent probe amplification (MLPA) and microarray analyses. The mechanism of deletion was investigated by interrogating the SINEBase database. Results: Patient-1 was diagnosed with nonobstructive HCM in his mid-40s while undergoing assessment for palpitations, and patient-2 with obstructive HCM in his late-20s while undergoing systolic heart murmur assessment for an unrelated illness. MLPA testing revealed a heterozygous deletion of all MYBPC3 exons in both patients. Subsequent microarray testing confirmed these deletions which extended beyond the 5′ and 3′ ends of MYBPC3. Genomic assessment suggested that these deletions resulted from Alu/Alu-homologous recombination. Conclusion: Our results demonstrate that haploinsufficiency resulting from MYBPC3 complete deletion, potentially mediated by Alu recombination, is an important disease mechanism in cardiomyopathy and emphasizes the importance of copy number variation analysis in patients clinically suspected of HCM.

K E Y W O R D S
cardiomyopathy, copy number, deletion, MYBPC3

| INTRODUCTION
Hypertrophic cardiomyopathy (HCM) is characterized by the presence of left ventricular hypertrophy (LVH) in the absence of predisposing cardiovascular or systemic conditions (Cirino & Ho, 2008). HCM is inherited in an autosomal-dominant manner and pathogenic variants in at least 16 genes, the majority of which are missense that act in a dominant-negative manner, have been implicated (Cirino & Ho, 2008). One of the principal genes in HCM is myosin-binding protein C, cardiac (MYBPC3: OMIM_600958), which plays a structural role and modulates myocardial contraction. About 70% of pathogenic variants in MYBPC3 are nonsense/frameshift (Carrier, Schlossarek, Willis, & Eschenhagen, 2010) suggesting haploinsufficiency as the pathogenic mechanism for MYBPC3. Although partial deletions in MYBPC3 have been implicated in HCM (Chanavat et al., 2012;Lopes et al., 2015), a complete deletion has not been reported before. We present two HCM patients with a complete deletion of MYBPC3 and investigated the potential origin of these deletions.

| Ethical compliance
All procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients in accordance with routine ethical and clinical standards. Additional informed consent was obtained for identifying information included in this article.

| RESULTS AND DISCUSSION
Patient-1 is a 64-year-old male who was diagnosed with nonobstructive HCM at age 46 ( Figure S1). His echocardiogram revealed moderately dilated left and right atria, and magnetic resonance imaging showed severe asymmetric LVH and interventricular septal wall thickness. Electrocardiogram (ECG) revealed atrial flutter and ST-T wave abnormalities ( Figure S2).
To our knowledge, this is the first report of patients where a complete deletion of MYBPC3 has contributed to the development of HCM. Despite recent reports of the use of high-throughput next-generation sequencing for copy number variation (CNV) analysis (Lopes et al., 2015), because this approach had not yet been optimized in our laboratory, deletions were detected by MLPA and confirmed by microarray. Our data suggest that MYBPC3 complete deletion, potentially mediated by Alu recombination is an important disease mechanism in cardiomyopathy and emphasize the importance of CNV analysis in patients clinically suspected of HCM, especially patients who tested negative by SS or other methods not optimized for CNV detection.