Mutation screening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis. (1/305)

21-hydroxylase deficiency is a recessively inherited disorder of steroidogenesis, resulting from mutations in the CYP21 gene. This 3.5 kb gene and a highly related CYP21P pseudogene reside on tandemly duplicated 30 kb segments of DNA in the class III HLA region, and the great majority of pathogenic mutations result from sequence exchanges involving the duplicated units. We now describe a comprehensive survey of CYP21 mutations in the British population, encompassing a screen for 17 different mutations in a total of 284 disease chromosomes. The most common mutations were as follows: large scale deletions/conversions (45% of the affected chromosomes), the intron 2 splice mutation (30.3%), R357W (9.8%), and I172N (7.0%). Mutations were detected in over 92% of the chromosomes examined, suggesting that accurate DNA based diagnosis is possible in most cases using the described strategy. In order to extend highly accurate prenatal diagnosis to all families where samples are available from a previously affected child, we have developed a linkage analysis approach using novel, highly informative microsatellite markers from the class III HLA region.  (+info)

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli. (2/305)

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.  (+info)

Congenital adrenal hyperplasia: not really a zebra. (3/305)

Congenital adrenal hyperplasia was once considered a rare inherited disorder with severe manifestations. Mild congenital adrenal hyperplasia, however, is common, affecting one in 100 to 1,000 persons in the United States and frequently eluding diagnosis. Both classic and nonclassic forms of the disease are caused by deficiencies in the adrenal enzymes that are used to synthesize glucocorticoids. The net result is increased production from the adrenal gland of cortisol precursors and androgens. Even mild congenital adrenal hyperplasia can result in life-threatening sinus or pulmonary infections, orthostatic syncope, shortened stature and severe acne. Women with mild congenital adrenal hyperplasia often present with hirsutism, oligomenorrhea or infertility. Congenital adrenal hyperplasia is diagnosed by demonstration of excess cortisol precursors in the serum during an adrenal corticotropic hormone challenge. Diagnosis of congenital adrenal hyerplasia in fetuses that are at risk for congenital adrenal hyperplasia can be determined using human leukocyte antigen haplotype or by demonstration of excess cortisol precursors in amniotic fluid. Treatment includes carefully monitored hormone replacement therapy. Recognition of the problem and timely replacement therapy can reduce morbidity and enhance quality of life in patients that are affected by congenital adrenal hyperplasia.  (+info)

Tracing past population migrations: genealogy of steroid 21-hydroxylase (CYP21) gene mutations in Finland. (4/305)

The genealogic origin of steroid 21-hydroxylase gene (CYP21) mutations and associated haplotypes was determined in 74 unrelated Finnish families with CYP21 deficiency (congenital adrenal hyperplasia, CAH). These families account for two thirds (85/119) of all diagnosed patients of Finnish descent found in this country. We recently demonstrated that multiple founder mutations each associated with a particular haplotype can be found in Finland. Interestingly, some of the haplotypes were identical to those observed in various European populations, whereas others have not been described elsewhere, indicating a local and perhaps a more recent origin. In the present report we show that each of the major founder haplotypes originates from a particular geographic region of Finland. Thus many local genetic isolates are to be expected in Finland. Our finding is in a clear contrast to the genetic diseases known as the 'Finnish disease heritage', in which only one mutation usually predominates. Some of the CYP21 haplotypes proved very informative for analysis of the history of the Finnish population. For example, the origin of one frequent haplotype was shown to cluster in a region assumed by archaeological data to be a major site of immigration by settlers of either Scandinavian or Baltic origin during the first centuries AD. As this haplotype is frequent in many European patient populations, we provide independent genetic evidence of this Iron Age immigration. On the other hand, another frequent haplotype found solely in Finland reflects a more recent (post 15th century) settlement expansion. Consequently, well characterised and sufficiently frequent autosomal gene markers can provide useful information on migrations both between and within populations.  (+info)

Modular variations of the human major histocompatibility complex class III genes for serine/threonine kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and tenascin TNX (the RCCX module). A mechanism for gene deletions and disease associations. (5/305)

The frequent variations of human complement component C4 gene size and gene numbers, plus the extensive polymorphism of the proteins, render C4 an excellent marker for major histocompatibility complex disease associations. As shown by definitive RFLPs, the tandemly arranged genes RP, C4, CYP21, and TNX are duplicated together as a discrete genetic unit termed the RCCX module. Duplications of the RCCX modules occurred by the addition of genomic fragments containing a long (L) or a short (S) C4 gene, a CYP21A or a CYP21B gene, and the gene fragments TNXA and RP2. Four major RCCX structures with bimodular L-L, bimodular L-S, monomodular L, and monomodular S are present in the Caucasian population. These modules are readily detectable by TaqI RFLPs. The RCCX modular variations appear to be a root cause for the acquisition of deleterious mutations from pseudogenes or gene segments in the RCCX to their corresponding functional genes. In a patient with congenital adrenal hyperplasia, we discovered a TNXB-TNXA recombinant with the deletion of RP2-C4B-CYP21B. Elucidation of the DNA sequence for the recombination breakpoint region and sequence analyses yielded definitive proof for an unequal crossover between TNXA from a bimodular chromosome and TNXB from a monomodular chromosome.  (+info)

Single-nucleotide polymorphisms in intron 2 of CYP21P: evidence for a higher rate of mutation at CpG dinucleotides in the functional steroid 21-hydroxylase gene and application to segregation analysis in congenital adrenal hyperplasia. (6/305)

BACKGROUND: Intron 2 of CYP21, the functional steroid 21-hydroxylase gene contains several single-nucleotide polymorphisms (SNPs). We tested the hypothesis that intron 2 of the pseudogene, CYP21P, might also be polymorphic and provide markers for segregation analysis of this region of the genome, including observable markers for segregation analysis of CYP21 gene deletions. A comparison of SNPs in both genes might provide insights into the rates of mutation in these duplicated genes. METHODS: After amplification with PCR, we examined restriction site polymorphisms in intron 2 of CYP21P in 24 members of the parental generation of the Centre d'Etude du Polymorphisme Humain families and selected offspring. RESULTS: Intron 2 of CYP21P contains frequent SNPs around nucleotide 398 and nucleotide 509, which can be typed by PCR/restriction enzyme digestion with HaeIII. Of the 48 CYP21P alleles examined, 44 could be characterized unambiguously. Of these 44 alleles, 4 were deleted, and the frequencies of restriction at the polymorphic HaeIII sites were 20 of 40 at nucleotide 398 and 30 of 40 at nucleotide 509. Both polymorphisms result from C-->T transitions that occur at CpG dinucleotides. The frequencies of C at these nucleotides in CYP21P are significantly higher than at the corresponding nucleotides in CYP21 of the same individuals (P <0.01). CONCLUSION: These data suggest that these CpG dinucleotides are more frequently mutated in CYP21 than in CYP21P, and that several mutations at CpG dinucleotides in the coding regions of CYP21 might result from CpG instability rather than the more usually proposed mechanism of gene conversion. These frequent SNPs provide useful markers for studying both allelic segregation of CYP21, particularly for chromosomes with known CYP21 deletions, and for investigating the origin of these polymorphisms.  (+info)

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells. (7/305)

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3beta-hydroxysteroid dehydrogenase (3betaHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with 'reporter cells' containing the glucocorticoid receptor (GR) and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3betaHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.  (+info)

Mutation analysis in patients with congenital adrenal hyperplasia in the Spanish population: identification of putative novel steroid 21-hydroxylase deficiency alleles associated with the classic form of the disease. (8/305)

Steroid 21-hydroxylase deficiency, due to the genetic impairment of the CYP21 gene, is a major cause of congenital adrenal hyperplasia (CAH). In about 80% of the cases, the defect is related with the transfer of deleterious point mutations from the CYP21P pseudogene to the active CYP21 gene. Sixteen different point mutations have been searched for in 60 Spanish patients with the classic form of CAH and 171 unaffected family members, using selective amplification of the CYP21 gene followed by allele-specific oligonucleotide hybridization (PCR-ASOH) and sequencing analysis. While 31.9% of the disease alleles carry CYP21 deletions or large gene conversions, around 58% of the alleles carry single point mutations. Corresponding segregation of mutations was found in every case indicating that none of them has apparently appeared de novo. The most frequent mutations found in our sample are i2G, V281L, R356W, Q318X, P453S and F306+t, with rates of 30, 14.2, 10, 9.2, 9.2 and 7. 5%, respectively. We found similar frequencies for the A and C polymorphism at position 656 (40 and 31.5%, respectively) in wild-type alleles for the i2G mutation. Around 10% of the alleles, for which no mutations were identified by searching for the sixteen previously known mutations, are currently being sequenced and new possible mutations and polymorphisms have been identified.  (+info)