Identification of CYP21 mutations, one novel, by single strand conformational polymorphism (SSCP) analysis. Mutations in brief no. 218. Online.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal recessive disorder (MIM# 201910) due to mutations in the 21-hydroxylase (CYP21) gene (GDB Accession # M12792). Using our protocol for single strand conformational polymorphism (SSCP) analysis, we have identified two mutations not known to exist in the 21-hydroxylase pseudogene (CYP21P). One mutation involving codon 169, TGC to AC appears to be novel. The 46,XX patient carried the codon 169 mutation on her paternal allele and a large gene deletion/conversion event on her maternal allele. This patient had been referred in the immediate neonatal period for the evaluation of genital ambiguity and had developed hyponatremia and hyperkalemia. The second patient presented with premature pubic hair. She carried R356Q on her maternal allele and V281L on her paternal allele.

17 alpha-hydroxylase/17,20-lyase dysregulation is not caused by mutations in the coding regions of CYP17.

STUDY OBJECTIVE: To determine whether mutations occur in the 17 alpha-hydroxylase/17,20-lyase (CYP17) gene in patients with mild hyperandrogenism. DESIGN: Clinical and molecular genetic study. SETTING: Pediatric endocrine outpatient clinic in an academic research environment. PARTICIPANTS: Girls (n = 11) referred for evaluation of premature pubic hair and adolescent girls (n = 16) referred for evaluation of hirsutism and/or oligomenorrhea. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Mutation detection analysis of the coding regions and intron/exon boundary regions of the CYP17 gene. RESULTS: Two polymorphic nucleotides were identified in the CYP17 gene. No mutations were detected in the 27 subjects. CONCLUSIONS: Mutation detection studies presented herein exclude CYP17 as a candidate gene for premature pubic hair and adolescent hyperandrogenism.

Newborn screening for 21-hydroxylase deficiency: results of CYP21 molecular genetic analysis.

Blood samples for plasma steroid hormone determinations and molecular genotype analysis of the 21-hydroxylase gene (CYP21) were obtained from 15 infants identified through a voluntary newborn screening program. Mutations were identified on both CYP21 alleles in 12 (80%) of 15 infants; all had confirmatory plasma 17-hydroxyprogesterone concentrations > 3500 ng/dl. No patient was found to carry mutations associated with late-onset 21-hydroxylase deficiency. Newborn screening hastened diagnosis in eight infants.

Evidence for a heterozygote advantage in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

21-Hydroxylase deficiency is one of the most common inherited disorders, with carrier frequencies of approximately 10% in all world populations studied to date. The high prevalence of the mutant gene is probably due to a flanking pseudogene serving as a reservoir for mutations. Despite the potential for a high rate of de novo mutations, a founder effect for specific gene conversions is observed in most populations. We hypothesized that there was a survival advantage to 21-hydroxylase heterozygotes, and here we report endocrinological and molecular investigations to test this hypothesis. We defined 28 carriers and 22 mutation-negative controls by molecular genotyping and determined ACTH-stimulated adrenal hormone responses. We found significantly elevated cortisol responses in the carriers compared to controls (30 min cortisol levels: normal, 24.2 +/- 4.6 micrograms/dL; carrier, 28.1 +/- 4.2 micrograms/dL; P < 0.005). Cortisol has a crucial role in maintaining homeostasis, influencing differentiation, suppressing inflammation, and effecting cross-talk among the immune, nervous, and endocrine systems. The brisk cortisol response we have documented in carriers of 21-hydroxylase may enable a rapid return to homeostasis in response to infectious, inflammatory, or other environmental stresses and may protect from inappropriate immune responses, such as autoimmune diseases.

Hyperandrogenism and manifesting heterozygotes for 21-hydroxylase deficiency.

Premature adrenarche and functional adolescent androgen excess are common disorders which may evolve into polycystic ovary syndrome (PCOS). In all three disorders, ACTH-stimulated 17-hydroxyprogesterone concentrations are often somewhat elevated. To determine the role of 21-hydroxylase (CYP21) gene mutations in these disorders, we performed molecular genotype analysis on 48 children and adolescents referred for evaluation of hyperandrogenic findings and diagnosed as having premature adrenarche or functional androgen excess. For comparison, DNA samples from 80 healthy adults were genotyped. Seventeen of the 48 hyperandrogenic patients were found to be heterozygotic carriers of CYP21 mutations. The frequency of heterozygosity was significantly greater among symptomatic patients (35%) than among the healthy controls (6%), P < 0.001. Seven mutation-positive patients (50%) and only one mutation-negative patient had ACTH-stimulated 17-hydroxyprogesterone concentrations typical for heterozygotic carriers of 21-hydroxylase deficiency, 400-1000 ng/dl. The significant difference in heterozygote frequency between symptomatic patients and healthy controls suggests that heterozygosity for 21-hydroxylase deficiency may be associated with premature adrenarche and functional adolescent hyperandrogenism. Longitudinal studies are necessary to determine if heterozygosity for 21-hydroxylase deficiency predicts risk for PCOS.

Changes in abundance of IgG 2a mRNA in the nucleus and cytoplasm of a murine B-lymphoma before and after fusion to a myeloma cell.

Changes in IgG mRNA half-life, transcription and nuclear and cytoplasmic abundance were studied in two cell lines which contain an identical Ig gamma 2a heavy chain but which differ in its expression. The A20.2J mouse lymphoma expresses about equal amounts of Ig gamma 2a secretory- and membrane-specific mRNAs whereas in the AXJ hybrids, resulting from the fusion of A20.2J with the J558L myeloma, the secretory-specific form dominates. Further evidence of dominance of the myeloma phenotype was seen in the large changes in mRNA abundance and nuclear accumulation as well as in a small increase in Ig gamma 2a mRNA half-lives for both secretory and membrane forms. Contributing to the observed > 100-fold increase in the ratio of secretory vs membrane forms of the Ig gamma 2a heavy chain in the AXJ hybrids are both a 10-fold decrease in the production of the membrane form by post-transcriptional RNA processing events and a approximately 6-7-fold decrease in the nuclear to cytoplasmic ratio for the Ig secretory gamma 2a and kappa light chain RNAs. Differential RNA accumulation in the nucleus in the lymphoma cell therefore contributes to the differential expression of Ig secretory mRNA.

Changes in secreted uterine proteins associated with embryo implantation in the mouse.

A dual-label ratio method was used in conjunction with two-dimensional polyacrylamide gel electrophoresis to measure the relative changes in rates of production of individual secreted proteins by mouse uteri at the start of the process of decidualization. A characteristic pattern of differential changes in the rate of synthesis and secretion of the proteins was found to be associated with development of a positive Pontamine Blue reaction at the site of embryo implantation. These changes were compared with those associated with development of experimentally induced deciduomata and although the patterns were similar, presumably reflecting common processes in transformation of the endometrium, there was preferential enhancement of a subset of small (Mr 14,000-20,000) acidic proteins in the authentic implantation sites. It is suggested that this embryo-dependent modification of constitutive changes associated with decidualization reflects a form of embryo-maternal signal-response mechanism that may be important for the process of implantation in mice.

Synthesis and secretion of stage-specific proteins by peri-implantation mouse embryos.

Blastocysts were recovered from intact mice at various times on the fourth and fifth days of pregnancy and incubated in vitro with 35S-methionine. Labeled proteins synthesized by the embryos and secreted into the medium were separated in two-dimensions on polyacrylamide gels by electrophoresis and localized by fluorography. The array of proteins synthesized and secreted by late stage blastocysts was found to be qualitatively and quantitatively different from those released by embryos at earlier stages of development. Similar changes were also observed in secreted proteins when delayed-implanting embryos were reactivated after an injection of estrogen. Furthermore, there was a temporal correlation between the appearance of certain proteins secreted by the embryos and changes in specific proteins synthesized and released by the uterus. It is suggested that these various secreted proteins constitute a signal-response mechanism that is important for the process of embryo implantation in mice.