Rapid screening method to detect mutations in CYP21, the gene for 21‐hydroxylase

To facilitate a rapid and practical molecular diagnosis of 21‐hydroxylase deficiency (21‐OHD), we developed a polymerase chain reaction (PCR) test in which only the 21‐OH gene (CYP21) is amplified. We applied the test to diagnose 23 patients with salt‐wasting type of 21‐OHD. The upstream and downstream sequences of CYP21 have been specifically amplified by using a primer set containing the 8‐bp deletion sequence of exon 3, which is distinct from its pseudogene CYP21P. The amplified PCR products were further subjected to mutation detection by restriction analysis: E1PL by AciI, I2g by PstI, E63a by DraIII, E7VL by ApaLI, E8non by PstI, and E8RW by AciI. To detect delections and/or gene conversions occurring on exon 3, we used the method described by Rumsby and Honour [1990: J Med Genet 27:676–678]. Our method is able to elucidate 8 common CYP21 mutations by using only 3 primer pairs and 4 restriction enzymes. The overall detection ratio of abnormal haplotypes by this method was over 95%, indicating that our method is practical and useful, particularly for carrier detection. Am. J. Med. Genet. 94:28–31, 2000. © 2000 Wiley‐Liss, Inc.     

A rapid screening for steroid 21‐hydroxylase mutations in patients with congenital adrenal hyperplasia

Steroid 21‐hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH). CAH due to 21‐hydroxylase deficiency is divided into three classes: salt‐wasting (classical), non‐classical and simple virilizing, reflecting different degrees of clinical severity. Using polymerase chain reaction (PCR) and allele‐specific oligonucleotide hybridisation (ASO), we screened the DNA of 62 Caucasian CAH families (heterozygous parents and children) for 14 different and frequently‐found CYP21‐mutations (HGMD). Of the 62 patients (21 males, 41 females), 26 females and 11 males had the classical or salt‐wasting form, 3 females and 1 male had the non‐classical form and 14 females and 7 males had simple virilizing CAH. More than 60% of the patients were compound‐heterozygous. We found the mutations on 110 alleles (out of 124 alleles). There were 30 CYP21 gene deletions/conversions, 3 substitutions (P30L) in exon 1, 30 splice mutations (c.93‐13A/C>G) in intron 2, 26 point mutations (I172N) in exon 4, one cluster of mutations (I236N, V237E, M239K) in exon 6, 8 mutations (V281L and 1760‐1761insT) in exon 7, and 8 nonsense (Q318X) and 4 missense (R356W) mutations in exon 8. Our study supports the case for using this rapid technique for CAH‐family screening as long as alleles from both affected patients and parents are screened in parallel. Hum Mutat 13:505, 1999. © 1999 Wiley‐Liss, Inc.     

A novel frameshift mutation (141delT) in exon 1 of the 21‐hydroxylase gene (CYP21) in a patient with the salt wasting form of congenital adrenal hyperplasia

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. Hum Mutat 14:90–91, 1999. © 1999 Wiley‐Liss, Inc.     

Splicing mutation in CYP21 associated with delayed presentation of salt-wasting congenital adrenal hyperplasia

Patients with salt-wasting congenital adrenal hyperplasia (SW-CAH) most commonly carry an A-G transition at nucleotide 656 (nt 656 A→G), causing abnormal splicing of ex-ons 2 and 3 in CYP21, the gene encoding active steroid 21-hydroxylase. Affected infants are severely deficient in cortisol and aldo-sterone, and usually come to medical attention during the neonatal period. We report on 2 affected boys, homozygous for the nt 656 mutation, who thrived in early infancy, but suffered salt-wasting crises unusually late in infancy, at 3.5 and 5.5 months, respectively. Laboratory studies at presentation showed hyponatremia, hyperkalemia, dehydration, and acidosis; serum aldo-sterone was low in spite of markedly elevated plasma renin activity. Basal 17-hydroxyprogesterone levels were only moderately elevated, yet the stimulated levels were more typical of severe, classic CAH due to 21-hydroxylase deficiency. Genomic DNA from the patients was analyzed. Southern blot showed no major deletions or rearrangements. CYP21 -specific amplification by polymerase chain reaction, coupled with allele-specific hybridization using wild-type and mutant probes at each of 9 sites for recognized disease-causing mutations, revealed a single, homozygous mutation in each patient: nt 656 A→G. These results were confirmed by sequence analysis. We conclude that the common nt 656 A→G mutation is sometimes associated with delayed phenotypic expression of SW-CAH. We speculate that variable splicing of the mutant CYP21 may modify the clinical manifestations of this disease. © 1995 Wiley-Liss, Inc.     

Steroid 21‐hydroxylase deficiency: Mutational spectrum in Denmark,three novel mutations,and in vitro expression analysis

We have investigated 68 unrelated 21‐hydroxylase deficient Danish patients, representing 136 alleles, and determined the mutational spectrum of the CYP21 gene. The most frequent mutations detected were deletion of CYP21 and the splice mutation in intron 2 (I2‐splice). Segregation analysis showed evidence of a de novo mutation in each of two patients. Three novel mutations were detected: G64E in exon 1, Q262X in exon 7, and A362V in exon 8. G64E and A362V were introduced in the CYP21 cDNA by in vitro site‐directed mutagenesis, and the two corresponding proteins were transiently expressed in COS‐7 cells. The activity of 21‐hydroxylase was determined using the two hormone substrates 17‐hydroxyprogesterone and progesterone. The analysis showed no enzyme activity for any of the substrates, a result that correlates well with the severity of the patients' disease. Hum Mutat 13:482–486, 1999. © 1999 Wiley‐Liss, Inc.     

Functional characterization of three CYP21A2 sequence variants (p.A265V,p.W302S,p.D322G) employing a yeast co‐expression system

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. © 2008 Wiley‐Liss, Inc.     

21-羟化酶缺乏症家系的基因突变研究

目的通过对3个21-羟化酶缺乏症家系的21-羟化酶基因(steroid21-hydroxylase gene,CYP21)直接测序研究,探讨家系中该基因突变类型。方法收集4例患者及其部分家系成员外周血,提取基因组DNA,PCR扩增CYP21基因后直接测序。结果CYP21基因序列分析共检测到6种突变类型。家系1中患者CYP21基因存在4种杂合突变:clusteE6、Q318X、A391T、P459H,其中前3种突变串联排列于同一条染色体上,P459H突变目前国内外尚未见报道,A391T为罕见突变;家系2中患者CYP21基因存在clusteE6、R483W两种杂合突变,其中R483W为罕见突变类型;家系3中患者第4外显子存在I172N纯合突变。结论在3个21-羟化酶缺乏症家系中共检测出6种突变类型,其中P459H为新发现的突变,A391T、R483W为罕见突变。虽然导致21-羟化酶缺乏症的突变主要是一些从假基因(CYP21P)转位到CYP21的序列,但随机突变也是21-羟化酶缺乏症的原因。     

CYP21A2 mutation update: Comprehensive analysis of databases and published genetic variants

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroidogenesis. Disorders in steroid 21‐hydroxylation account for over 95% of patients with CAH. Clinically, the 21‐hydroxylase deficiency has been classified in a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non‐classical. Known allelic variants in the disease causing CYP21A2 gene are spread among different sources. Until recently, most variants reported have been identified in the clinical setting, which presumably bias described variants to pathogenic ones, as those found in the CYPAlleles database. Nevertheless, a large number of variants are being described in massive genome projects, many of which are found in dbSNP, but lack functional implications and/or their phenotypic effect. In this work, we gathered a total of 1,340 GVs in the CYP21A2 gene, from which 899 variants were unique and 230 have an effect on human health, and compiled all this information in an integrated database. We also connected CYP21A2 sequence information to phenotypic effects for all available mutations, including double mutants in cis. Data compiled in the present work could help physicians in the genetic counseling of families affected with 21‐hydroxylase deficiency.     

Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V

Kharrat M, Trabelsi S, Chaabouni M, Maazoul F, Kraoua L, Ben Jemaa L, Gandoura N, Barsaoui S, Morel Y, M’rad R, Chaabouni H. Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V. Steroid 11β‐hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β‐hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β‐hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population.     

Molecular analysis of CYP21A2 can optimize the follow‐up of positive results in newborn screening for congenital adrenal hyperplasia

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17‐hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3–7%, C > 20%. Twenty‐one children with neonatal symptoms and/or 17OHP levels > 80 ng/ml carried A genotypes, except two virilized girls (17OHP < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP > 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false‐positives and help distinguish between clinical forms of CAH.     

不同类型21-羟化酶缺乏症的临床表型和基因型对比研究

目的对3种不同类型21-羟化酶缺乏症临床特点进行对比研究.方法9例失盐型患者,26例单纯男性化型患者和8例非经典型患者基因组DNA用特定引物特异性扩增CYP21,用特定限制性内切酶消化后经琼脂糖凝胶电泳鉴定突变.回顾性分析临床和生化检查特点.结果(1)三种临床表现类型间,初诊年龄、初诊时骨龄、身高;反应21-羟化酶活性的17-OHP,ACTH及24hrUFC;失盐表现等均存在有统计学意义的差异,P＜0.05.(2)8/9例失盐型患者是引起酶活性严重下降的基因型,20例能分型的单纯男性化型患者中16例是引起酶活性中度下降的基因型,8例非经典型全部为酶活性轻度下降的基因型.基因型构成比在三种临床表型间存在有统计学意义的差异,x2=39.039,P=0.000.结论(1)21-羟化酶缺乏症三种亚型临床表现具有各自特征.(2)基因型构成在三种21-羟化酶缺乏症临床分型间存在差异,基因型决定临床表型.     

Clinical and genetic analysis of patients with X‐linked hyper‐IgM syndrome

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency (21‐OHD) is a common autosomal recessive disorder caused by mutations in the CYP21A2 gene. The carrier frequency of CYP21A2 mutations has been estimated to be 1:25 to 1:10 on the basis of newborn screening. The main objective of this study was to determine the carrier frequency in the Cypriot population of mutations in the CYP21A2 gene. Three hundred unrelated subjects (150 males and 150 females) from the general population of Cyprus were screened for mutations in the CYP21A2 gene and its promoter. The CYP21A2 genotype analysis identified six different mutants and revealed a carrier frequency of 9.83% with the mild p.Val281Leu being the most frequent (4.3%), followed by p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) and p.Met283Val (0.17%). The notable high CYP21A2 carrier frequency of the Cypriot population is one of the highest reported so far by genotype analysis. Knowledge of the mutational spectrum of CYP21A2 will enable to optimize mutation detection strategy for genetic diagnosis of 21‐OHD not only in Cyprus, but also the greater Mediterranean region.     

CYP21 mutations and congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder caused mainly by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene on chromosome 6p21.3. The wide range of CAH phenotypes is associated with multiple mutations known to affect 21-hydroxylase enzyme activity. To date, 56 different CYP21 mutations have been reported, mostly point mutations, but small deletions or insertions have been described too, as well as complete gene deletions. Fifteen mutations, constituting 90-95% of alleles, are derived from intergenic recombination of DNA sequences between the CYP21 gene and the highly homologous CYP21P pseudogene, while the remaining are spontaneous mutations. A reliable and accurate detection of CYP21 mutations is not only important for clinical diagnosis, but also for carrier detection as there is a high variability in the basal level of 17-hydroxyprogesterone between normal and heterozygous individuals. Several strategies based on polymerase chain reaction (PCR)-driven amplification with allele-specific oligonucleotides to the CYP21 gene have been developed. It has been demonstrated that one reaction for PCR amplification of the CYP21 gene and the chimeric CYP21P/CYP21 gene using mixed primers in combination with nested PCR and single-strand conformation polymorphism is considered highly efficient and accurate for molecular diagnosis of CAH due to 21-hydroxylase deficiency.     

Identification of CYP21 mutations,one novel,by single strand conformational polymorphism (SSCP) analysis

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. © 1998 Wiley‐Liss, Inc.     

Prenatal diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) in Croatia

We report on the prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase in 20 at-risk pregnancies (16 salt-wasting and 4 simple virilizing families). We have diagnosed 3 affected fetuses (2 males and 1 female), 3 healthy homozygotes (2 males and 1 female), and 14 healthy heterozygotes (7 females and 7 males). These data were collected over 4 years. In 16 fetuses, the diagnosis was made with measurements of 17-hydroxyprogesterone (17-OHP) and Δ-4-androstenedione (Δ) in amniotic fluid (AF), human leukocyte antigen (HLA) typing of amniotic cells, as well as karyotypes between the 16th and 18th weeks of gestation. In 4 fetuses, DNA analysis of amniotic cells was also performed. In 3 pregnancies in which affected fetuses were suspected (on the basis of HLA typing and measurements of 17-OHP and Δ concentrations in AF), the fetuses were electively aborted between the 17th to 19th weeks of gestation by parental decision. In all aborted fetuses, diagnosis was confirmed with HLA typing, autopsy findings of hyperplastic adrenal glands, and ambiguous genitalia in female fetuses. Postnatal diagnosis was confirmed in healthy fetuses with HLA typing and serum measurements of 17-OHP concentrations, and in 4 of them with DNA analysis. In 3 of the 4 families, DNA analyses revealed the following mutations: in Family 1, the index case mutation was Intron 2, Exon 3/Exon 6, and the fetus was Normal/Exon 6; in Family 2, the index case mutation was Ex1 Int2 Ex3/Int2, and the fetus was Ex1 Int2 Ex3/Normal; and in Family 3, the index case mutation was Ex8₃₅₆/Ex8₃₅₆, and the fetus was Ex8₃₅₆/Normal. We also report one case of prenatal diagnosis and treatment. Dexamethasone 0.5 mg BID (20 μg/kg/d) was given starting at 6th week of gestation. Prenatal diagnosis suggested, but did not prove, that the female fetus was a heterozygote as the fetus lacked the paternal mutation Ex8₃₁₈. No mutation was found in the mother. The fetus, the mother, and the affected sib shared a haplotype, further suggesting heterozygosity. The unaffected status was confirmed postnatally. Am J. Med. Genet. 72:302–306, 1997. © 1997 Wiley-Liss, Inc.     

Identification of heterozygotic carriers of 21-hydroxylase deficiency: Sensitivity of ACTH stimulation tests

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. To ascertain carrier status, adrenocorticotropin (ACTH) stimulation tests are often used. To determine the sensitivity of ACTH stimulation to detect heterozygotes and to correlate stimulated 17-hydroxyprogesterone responses with molecular genotype, we compared molecular genetic analysis of the 21-hydroxylase (CYP21) gene with 17-hydroxyprogesterone responses at 30 min in 51 individuals. Molecular genotype analysis and ACTH stimulation tests were performed in healthy volunteers (n = 20) and relatives of patients with congenital adrenal hyperplasia (n = 31). Polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, allele-specific oligonucleotide hybridization (ASOH) analysis, and restriction fragment length polymorphism (RFLP) analysis were utilized to screen for 14 CYP21 mutations which account for >90% of the mutations associated with 21-hydroxylase deficiency. Molecular genotype analysis classified 28 individuals as heterozygotic carriers and 23 individuals as normal for all mutations tested. As a group, the heterozygotes had significantly greater stimulated 17-hydroxyprogesterone responses at 10 and 30 min (P < 0.0005). However, on an individual basis, 14/28 (50%) genotyped heterozygotic carriers had stimulated 17-hydroxyprogesterone concentrations, 17-hydroxyprogesterone/cortisol ratios, and 17-hydroxyprogesterone incremental elevations indistinguishable from the genotyped normal individuals. Thus, a normal 17-hydroxyprogesterone response to ACTH stimulation testing does not exclude carrier status for 21-hydroxylase deficiency. Molecular genotype analysis is a more reliable method to determine 21-hydroxylase heterozygotes. Am. J. Med. Genet. 76:337–342, 1998. © 1998 Wiley-Liss, Inc.     

Structural analysis of the chimeric <Emphasis Type="Italic">CYP21P/CYP21</Emphasis> gene in steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed “apparent gene conversion,” from the neighboring CYP21P pseudogene. A chimeric CYP21P/CYP21 gene with its 5′ end corresponding to CYP21P and 3′ end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with −300 nucleotides of the 5′ head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21P and CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes. Received: March 13, 2002 / Accepted: June 17, 2002     

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.     

Mutation screening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis

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.     

Characterization of novel missense mutations in <Emphasis Type="Italic">CYP21</Emphasis> causing congenital adrenal hyperplasia

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is the most common inherited disorder of steroid metabolism, with an incidence of 1/10,000 in the general Caucasian population. Although most patients carry a deletion of the CYP21 gene or any of nine pseudogene-derived point mutations, the number of reported rare mutations continues to increase, and consist today of more than 80 different point mutations. In this study, we report the characterization of four additional missense mutations in CYP21. Two of these, L166P and A391T, are novel missense mutations, whereas the R479L and R483Q mutations have been detected previously. Functional assays of mutagenized CYP21 were performed in transiently transfected mammalian cells in vitro, and enzymatic ability of substrate conversion of the two natural substrates of CYP21-17-hydroxyprogesterone and progesterone-was determined. All mutants displayed reduced in vitro enzyme activities compared with wild type, but to different extents, corresponding to clinical phenotypes that span the whole spectrum of disease severity. Functional studies are important to further establish the relationships between genotype and clinical phenotype as well as in vitro CYP21 activity in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. This has relevance for diagnosis, prognosis, and genetic counseling for affected families.