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Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (CYP21A2) deficiency is the commonest inborn error in steroid hormone biosynthesis. Functional in vitro assessment of mutant activity generally correlates well with clinical phenotype and therefore has contributed greatly to phenotype prediction in this CAH variant. Three CYP21A2 sequence variants (g.1641C>T, p.A265V; g.1752G>C, p.W302S; and g.2012A>G, p.D322G) identified in patients with non-classic and simple virilizing CAH were characterized using a yeast co-expression system and a computational three-dimensional CYP21A2 model. Computational analysis of the mutants in the three-dimensional structural model predicted no relevant effect of p.A265V, while p.W302S and p.D322G were predicted to impact significantly on enzyme function. Consistent with these findings, in vitro mutant analysis revealed enzyme activity similar to wild-type for p.A265V, whereas p.W302S and p.D322G exerted activities compatible with simple virilizing and non-classical CAH, respectively. The results indicate that p.A265V is an allelic variant rather than a disease-causing amino acid change, whilst p.W302S and p.D322G could be confirmed as functionally relevant mutations. These findings emphasize the value of in vitro functional analysis of sequence variations in predicting genotype-phenotype correlations and disease severity., ((c) 2008 Wiley-Liss, Inc.)

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent inherited defect of fatty acid oxidation, with a significant morbidity and mortality in undiagnosed patients. Adverse outcomes can effectively be prevented by avoiding metabolic stress and following simple dietary measures. Therefore, prospective newborn screening (NBS) is being proposed for this condition. However, technical validation of MCADD population screening and assessment of its overall benefit require broadening of the as-yet-scarce knowledge of the MCADD genetic heterogeneity unraveled by NBS and its phenotypic consequences. Here, we describe the entire spectrum of sequence variations occurring in newborns with MCADD in the population of Bavaria, Germany, in relation to the biochemical phenotype. Among 524,287 newborns, we identified 62 cases of MCADD, indicating a birth incidence of 1 in 8,456. In all of the 57 newborns available for analysis, two alterations within the MCADD gene (ACADM) were identified. The most prevalent alteration c.985A>G (Lys329Glu) occurred in 27 (47%) newborns in the homozygous and in 18 (32%) in the heterozygous state (63% of defective alleles). The mild folding variant c.199T>C (Tyr67His) was identified in nine individuals, six of them being compound heterozygous with c.985A>G (Lys329Glu). Neither of the prevalent alterations were found in the remaining nine newborns. A total of 18 sequence variations were identified; 13 of them were novel: eight missense mutations, one nonsense mutation, two splice variants, and two small deletions. The remaining five were previously reported in MCADD patients. The ACADM heterogeneity uncovered was larger as anticipated from previous c.985A>G (Lys329Glu) carrier screening data. In addition, we show that MCADD appears to occur as frequently in Turkish newborns as in the native German population. Our data validate that biochemical NBS for MCADD is a highly specific procedure for disease detection, with the identification of a significant share of milder biochemical phenotypes, such as c.199T>C (Tyr67His). These show statistically lower acylcarnitine markers, allowing us to distinguish subgroups within the spectrum of ACADM sequence variations that correlate to biochemical MCADD disease expression. Our data might provide technical and medical guidance for decision making in the worldwide efforts to introduce MCADD population screening.

X-linked adrenal hypoplasia congenita (AHC) is a rare developmental disorder associated with primary adrenal insufficiency and combined primary and secondary male hypogonadism. It is caused by deletions or mutations of the NR0B1 (DAX1) gene encoding DAX1, an atypical orphan member of the nuclear receptor superfamily. The continuous molecular genetic analysis of male patients with primary adrenal insufficiency revealed 13 novel mutations within the coding region of the NR0B1 gene which are predicted to inactivate the DAX1 function. These were three nonsense mutations (c.312C>A, p.Cys104X, c.670C>T, p.Gln224X; and c.873G>A, p.Trp291X), five duplications (c.269_270dup, c.421_422dup, c.895_896dup, c.989dup, c.999_1000dup), and five deletions (c.483del, c.745_746del, c.734_740del, c.1092del, and c.1346del). All of the mutations resulted in a premature stop codon destroying the ligand binding domain of the predictive DAX1 protein.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disease with a wide range of clinical manifestation. In 90-95% of the cases it is caused by 21-hydroxylase deficiency (OMIM #201910) due to mutations of the CYP21 gene (GDB Accession #M12792). In most cases the CYP21-inactivating point mutations are transferred by apparent gene conversions from CYP21P to CYP21. In only a few cases point mutations have been described, which are not present in the pseudogene. Using Southern blot analysis and DNA sequencing we have identified a novel mutation (141delT) of the CYP21 gene in a patient suffering from the salt wasting form of CAH. This results in a premature termination of a truncated protein at amino acid position 51 (L51X), which is likely to result in an enzyme with no activity. This novel mutation has not been reported to occur in the CYP21P alleles and it was not found in the CYP21P alleles in this CAH family.