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.

To investigate the effects of sex hormones on ethanol (EtOH)-induced bone loss, female Sprague-Dawley rats were fed control or EtOH-containing diets (12 g/kg/day) by intragastric infusion. After 3 weeks, rats receiving EtOH had significant decreases in tibial trabecular and total bone mineral density, induction of receptor activator of nuclear factor-〉 ligand (RANKL) mRNA expression, and enhanced bone resorption, all of which were prevented by treatment with 17␤-estradiol (E 2 ). The addition of progesterone did not enhance the beneficial effect of E 2 alone. Consistent with our in vivo findings, EtOH stimulated RANKL mRNA expression in cultured primary osteoblasts, and this expression was blocked by 4-methylpyrazole. Acetaldehyde also induced RANKL expression. Class 1 alcohol dehydrogenase was found to be expressed and EtOHinducible in cultured osteoblasts, whereas CYP2E1 was undetectable. We found that EtOH induced phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducers and activators of transcription 3 (STAT3). E 2 and the mitogenactivated protein kinase kinase inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) blocked ERK and STAT3 phosphorylation and blocked RANKL induction. Moreover, E 2 completely blocked EtOH-induced osteoclastogenesis in a primary osteoblast and osteoclast precursor coculture system. The E 2 effects were estrogen receptor-mediated. Therefore, E 2 prevents EtOH-induced bone loss by opposing the induction of RANKL mRNA in osteoblasts and ethanol-induced osteoclastogenesis, through opposing effects on sustained ERK signaling.

In many black African populations, the capacity for CYP2D6-dependent drug metabolism is generally reduced. A specific variant of the CYP2D6 gene (CYP2D6*17) that carries three functional mutations (T107I, R296C, and S486T) has been found to be present in Zimbabwean subjects with impaired CYP2D6-dependent hydroxylase activity. To evaluate whether the CYP2D6*17 allele was the major cause behind the decreased rate of drug metabolism and to examine the role of the different mutations, CYP2D6 cDNAs containing all eight combinations of the mutations were created. Expression of the cDNAs in COS-1 cells revealed that the CYP2D6 17 enzyme displayed only 20% of the wild-type (CYP2D6 1) activity, whereas the T107I substitution on its own had no significant effect on enzyme function. Expression in yeast showed that the three possible single amino-acid mutant CYP2D6 variants all had properties similar to CYP2D6 1 when the kinetics of bufuralol hydroxylation was examined. However, enzymes containing both the T107I and R296C mutations exhibited a more than 5-fold higher K(m) for bufuralol than the wild-type enzyme, whereas the S486T mutation was of little importance. In contrast, when codeine was used as a substrate, the T107I substitution alone was sufficient to cause a significant increase in the apparent K(m), indicating a differential effect for this substitution depending on the CYP2D6 substrate. In conclusion, the CYP2D6*17 allele represents the first human cytochrome P450 polymorphic variant in which a combination of substitutions is required to alter the enzyme's catalytic properties and is the first case in which a decreased CYP2D6 activity, as monitored in vivo, has been documented to be caused by an enzyme with altered affinity for CYP2D6 substrates.