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

Human gene copy number spectra analysis in congenital heart malformations

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency “spectra” to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways

Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms

BACKGROUND: Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. METHODS AND FINDINGS: Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. CONCLUSIONS: In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma

Structure and function of hepatic cytochromes P450 : Implications for drug development

Prediction of human drug metabolism in vivo by use of in vitro systems is of immense importance in drug development since it allows avoidance of drugs coming out on the market that are subjected to interindividual variability in metabolism or causes drug-drug interactions, both of which in turn can lead to adverse drug reactions and result in non-optimal treatment. In the present investigations the importance of P450 haplotype on substrate specificity was studied as well as a comparison of different in vitro systems for optimal prediction of in vivo drug metabolism. Regions important for membrane interactions of one form of P450 were elucidated in order to engineer a soluble form of the enzyme of use for crystallization studies. A new CYP2D6 allele (CYP2D6*17) common in Black African populations carrying three mutations Thrl07IIe, Arg296Cys and Ser486Thr was investigated by recombinant expression and the enzyme characterized using CYP2D6 specific substrates. It was found that both the Thrl07IIe, Arg296Cys substitutions were required in order to yield an enzyme with altered substrate specificity in a manner seen in vivo, emphasizing the necessity to consider haplotypes rather than single SNPs for functional analyses of many polymorphic genes. Various in vitro systems like homology models of P450 enzymes, recombinant enzymes, liver microsomes, hepatocyte primary cultures and liver slices were compared with respect to predictability of in vivo metabolism of 3 different drugs in the EUROCYP project where I was responsible for the recombinant enzyme systems using yeast. The expression systems were optimized for nine different human CYPs. It was found that the recombinant system under-predicted the overall clearance of almokalant, mainly being a substrate for UDP-glucuronosyltransferases. For carbamazepine, only CYP3A4 among the 9 different forms of P450 examined was active. The addition of cytochrome b5 lead to a overestimated clearance putting emphasis on the composition of cofactors when dealing with CYP3A4. For selegiline we found that, in contrast to previous studies, CYP2B6 and CYP2C19 were the most active enzymes, but some contribution from CYP1A2 and CYP3A4 was also predicted. Scaling up our recombinant data to the human situation yielded values slightly higher but not significantly different from the in vivo clearance. Hence, the recombinant system correctly predicted selegiline to be a high clearance drug. It was concluded that the recombinant system was of good value for determination of the enzyme specificity but not for in vivo clearance of drug candidates. The role of the hydrophobic NH2-terminal of rat CYP2E1 for mitochondrial targeting and membrane interactions was studied by expressing NH2-terminally truncated variants of this enzyme in Saccharomyces cerevisiae. Six variants were expressed along with the wt CYP2E1, four were truncated at position 29, 64, 82 or 95 (delta95-2E1), respectively and one variant had Leu to Asp amino acid exchanges at positions 90 and 91 (delta82mut-2E1). With the exception of delta95-2E1 and delta82mut-2E1, all variants were active in NADPH supported chlorzoxazone 6-hydroxylation, but when the artificial electron donor cumene hydroperoxide was used, also these were active. A region between as 82 and 95 was found to be critical for mitochondrial import of CYP2E1. It was found that delta82mut-2E1 was soluble and catalytically active in phosphate buffer and could be used for crystallization studies. It is concluded that the B-helix of CYP2E1 and possibly other CYPs is important for mitochondrial targeting as well as for membrane association where interactions of ionic nature occur