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.     

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.     

Genetic diagnosis of 21‐hydroxylase deficiency: DGGE‐based mutation scanning of CYP21

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21‐hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported, and still novel mutations are detected. This makes genetic diagnosis as well as carrier detection of 21‐hydroxylase deficiency a complicated matter. We developed a simple nonradioactive assay based on the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the CYP21 gene. DGGE allows a fast scanning of PCR‐amplified segments of genes for the presence or absence of any single base pair alterations. We have performed this technique on the coding sequence and intron‐exon junctions of CYP21. Our results emphasize that this procedure constitutes a fast and reliable approach when performing diagnosis of 21‐hydroxylase deficiency. Hum Mutat 13:385–389, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) occurs worldwide. The most common mutations in the CYP21A2 gene in 716 unrelated patients were analyzed and the mutations were grouped by ethnicity, as defined through self-declaration corroborated by review of pedigrees extending to two or three generations. Prevalent allelic mutations and genotypes were found to vary significantly among ethnic groups, and the predominance of the prevalent mutations and genotypes in several of these populations was significant. There are ethnic-specific mutations in the CYP21A2 gene. A large deletion is prevalent in the Anglo-Saxons; a V281L (1685 G to T) mutation is prevalent in Ashkenazi Jews; an R356W (2109 G to A) mutation is prevalent in the Croatians; an IVS2 AS -13 (A/C to G) mutation is prevalent in the Iranians and Yupik-speaking Eskimos of Western Alaska; and a Q318X (1994 C to T) mutation is prevalent in East Indians. Genotype/phenotype non-correlation was seen when at least one IVS2 AS -13 (A/C to G) mutation in the CYP21A2 gene was present.     

先天性肾上腺皮质增生症患者21-羟化酶基因启动子区域突变的初步研究

目的研究先天性肾上腺皮质增生症（ｃｏｎｇｅｎｉｔａｌａｄｒｅｎａｌｈｙｐｅｒｐｌａｓｉａ,ＣＡＨ）患者２１－羟化酶基因启动子区域的突变。方法用ＰＣＲ、ＳＳＣＰ、内切酶酶谱分析及测序分析方法对１２例ＣＡＨ患者的２１－羟化酶基因启动子区域进行研究。结果１２例患者中有６例出现异常ＳＳＣＰ条带,其中１例在ＣＫ－２（－１０１）结合区域内的ＫｐｎⅠ内切酶识别位点及其－２０１处的ＴａｑⅠ内切酶识别位点存在突变,并经测序证实。结论在ＣＡＨ患者－２１羟化酶基因启动子区域存在突变,可能为ＣＡＨ发病机理之一。     

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.     

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.     

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.     

A genetic epidemiology study of congenital adrenal hyperplasia in Italy

Congenital adrenal hyperplasia due to 21‐hydroxylase deficiency (21OHD‐CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD‐CAH neonatal screening is based on 17‐hydroxyprogesterone (17OHP) serum levels, showing high type I error rate and low sensitivity to mild CAH forms. Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD‐CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy. These findings provide an updated fine‐grained picture of 21OHD‐CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.     

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.     

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.     

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。     

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.     

Molecular and structural analysis of two novel StAR mutations in patients with lipoid congenital adrenal hyperplasia

Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) cause lipoid congenital adrenal hyperplasia. We report a novel homozygous splice site mutation (IVS1 + 2T --> G) in STAR in two sisters (46XY, 46XX) who presented with primary adrenal insufficiency at birth and a novel homozygous R182H missense mutation in the putative lipid transfer domain of StAR in a phenotypic female (46XY) with adrenal failure and a parotid tumor. These cases highlight the importance of StAR-dependent steroidogenesis during fetal development and early infancy and of the critical functional role of R182 in cholesterol transport.     

Genetic linkage and HLA association in congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Twenty-eight families of patients with congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency were studied to evaluate the specific HLA linkage relationship and HLA antigen association to the 21-OH deficiency gene. Genotype assignment, based on hormonal studies (ACTH stimulation) and HLA genotyping, correlated very well (p less than 0.01) in 23 unaffected sibs of children with 21-OH deficiency further supporting the genetic linkage of the 21-OH deficiency gene to the HLA complex. One family was informative for the placement of the 21-OH deficiency gene outside the HLA complex on the HLA-DR locus side. In this family HLA-A, B, C, DR, MT, MB, and glyoxylase typing and mixed lymphocyte culture was performed. An association of 21-OH deficiency and the HLA-A3 antigen was noted in the 28 families. This association is not secondary to the association of the 21-OH deficiency gene with HLA-BW47.     

Amniotic 17-α hydroxyprogesterone and HLA typing for the prenatal diagnosis of 21-α hydroxylase deficiency — congenital adrenal hyperplasia

We have investigated a family with one child affected with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Prenatal determination of 17-α hydroxyprogesterone (17OHP) in amniotic fluid (AF) and HLA typing of amniotic fibroblasts from a pregnancy at risk showed that the fetus was not affected. A healthy cousin with HLA haplotypes identical to those of the proposita (only one being identical by descent) had a normal plasma level of 17OHP. The prenatal diagnosis of a fetus affected with 21-hydroxylase deficiency CAH may be established by the determination of 17OHP in AF. This is a relatively quick procedure that can be confirmed by the HLA genotype, and is mandatory in families with a parent homozygous for an HLA haplotype and in certain recombinant haplotypes in the fetus.