Impact of \u3cem\u3eMYH6\u3c/em\u3e Variants in Hypoplastic Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain (MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases (P \u3c 1 × 10−5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants (P \u3c 1 × 10−2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β-myosin heavy chain (MYH7) gene in subjects with MYH6 variants (P \u3c 1 × 10−3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo (P \u3c 1 × 10−2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications

The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1′-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783-92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower