Three novel point mutations of the CYP21 gene detected in classical forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) [OMIM 201 910] is a group of autosomal recessive disorders most commonly due to 21-hydroxylase deficiency and presenting with a wide range of clinical manifestations. A limited number of inactivating pseudogene-derived mutations account for the majority of 21-hydroxylase gene ( CYP21) mutations, additional rare mutations can be found in single families and small populations. We found three novel CYP21 mutations in CAH patients suffering from the classical form of the disease, of which one is a frameshift mutation (1353-1354insA) leading to a premature termination codon (K277K, Q228A...E294X), one results in a premature stop codon (2551C>T, R444X), and one is a missense mutation (2609T>C; P463L). The frameshift and premature stop mutations can be predicted to result in a CYP21 protein without any residual enzyme activity. To determine the functional consequences of the P463L mutation, the IN VITRO enzyme activity was studied in COS-7 cells and revealed a reduced 21-hydroxylase activity of 2.6+/-0.8 (SD)% for the conversion of 17-hydroxyprogesterone (17OHP) to 11-deoxycortisol and of 3.0+/-0.5 % for the conversion of progesterone to 11-deoxycorticosterone (DOC). We conclude that functional analyses of unknown mutations provide information on the disease severity and should be always performed when novel CYP21 mutations are detected. Knowledge of the residual 21-hydroxylase function improves both genetic counselling and individual clinical management in CAH patients.     

Three novel CYP21A2 mutations and their protein modelling in patients with classical 21-hydroxylase deficiency from northeastern Iran

OBJECTIVE: Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders frequently caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). We describe three novel CYP21A2 mutations in CAH patients. DESIGN AND METHODS: Sequence analysis of the entire CYP21A2 gene followed by molecular modelling was performed in three unrelated classical CAH patients of northeastern Iranian origin. The active (CYP21A2) and pseudogene (CYP21A1P) alleles were screened for the presence of the new variations in controls. RESULTS: Two novel missense mutations, F404S in exon 9 and T450P in exon 10, were found in homozygous forms in two female patients with a salt-wasting (SW) phenotype. These novel variants were screened by allele-specific polymerase chain reaction (PCR) and excluded in 100 unrelated normal alleles. Prediction of clinical severity, based on molecular modelling and sequence conservation, correlates well with the clinical diagnosis of the patients carrying these mutations. The third novel mutation, a small 10-bp deletion in exon 1, g.19_28del, was found in a female patient with a simple virilizing phenotype in a compound heterozygous form with the common intron 2 splice mutation (IVS2-13A/C>G). This frameshift mutation causes a premature stop codon at amino acid position 48, L48X, resulting in a nonfunctional protein. The CYP21A1P pseudogene alleles were also screened and none of these novel mutations could be detected. CONCLUSIONS: Three novel mutations were found in the CYP21A2 gene and predicted to drastically impair enzyme activity resulting in severe classic CAH. None of these mutations occurs in the CYP21A1P pseudogene.     

H28+C insertion in the CYP21 gene: a novel frameshift mutation in a Brazilian patient with the classical form of 21-hydroxylase deficiency.

In the classical form of 21-hydroxylase deficiency, CYP21- affected genes either carry mutations present in the CYP21P pseudogene (microconversions) or bear a chimeric gene that replaces the active gene as a result of large conversion or deletion mutational events. Previous genotyping of 41 Brazilian patients revealed 64% microconversion, whereas deletions and large gene conversions accounted for up to 21% of the molecular defect. The present paper describes a new mutation disclosed by sequencing an entire gene in which no pseudogene-originated mutation had been found. The patient with the classical form of 21-hydroxylase deficiency is the daughter of a consanguineous marriage, and she is homozygous for a novel frameshift H28+C within exon 1. The mutation causes a stop codon at amino acid 78. Both parents are heterozygous for the mutation as confirmed by allele-specific oligonucleotide PCR. The H28+C is not present in the published CYP21P sequences and is likely to result in an enzyme with no activity.     

High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect

OBJECTIVE: To detect common as well as rare and novel CYP21A mutations in 21-hydroxylase deficiency patients. To estimate the distribution of mutations and compare them with other European studies. To construct haplotypes linked to a recurrent novel mutation. DESIGN: Genetic analysis by sequencing the entire CYP21A2 gene plus Southern blot. PATIENTS: A total of 138 unrelated Spanish patients: 122 nonclassical forms (NCF) and 16 classical forms (CF) were studied. RESULTS: Among the 266 nonrelated mutated alleles; CYP21A2 deletions/conversions and a spectrum of 27 different mutated alleles were found: 15 different single point mutations, 8 nucleotide deletions in exon 3, 3 mutation clusters in exon 6, 9 alleles with more than one mutation, one 21-nucleotide duplication in exon 10, and one allele with CYP21A2 duplicated and both copies mutated. The most frequent mutation in NCF alleles is V281L (71.8%). Among CFs, the most common is I2 g (20%) and Q318X (16%) and rare alleles (21.9%). Six novel causative mutations were found, four associated with CF: I46+1nt, R444X, P463L and M473_R479dup and two associated with NCF: W302 and D322G. The R444X mutation was found in seven unrelated patients and it appeared only once in an ancestral haplotype. In addition, we found a novel single nucleotide polymorphism with a 31.5% frequency for the rare allele. CONCLUSION: A great diversity of haplotypes with a large spectrum of mutated alleles was found. The frequency of the V281L mutation was the highest reported and the relatively high frequency of R444X was the result of a founder effect.     

CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders. The aim of this study was to assess the frequencies of CYP21 mutations and to study genotype-phenotype correlation in a large population of Dutch 21-hydroxylase deficient patients. From 198 patients with 21-hydroxylase deficiency, 370 unrelated alleles were studied. Gene deletion/conversion was present in 118 of the 370 alleles (31.9%). The most frequent point mutations were I2G (28.1%) and I172N (12.4%). Clustering of pseudogene-derived mutations in exons 7 and 8 (V281L-F306 + 1nt-Q318X-R356W) on a single allele was found in seven unrelated alleles (1.9%). This cluster had been reported before in two other Dutch patients and in two patients in a study from New York, but not in other series worldwide. Six novel mutations were found: 995-996insA, 1123delC, G291R, S301Y, Y376X, and R483Q. Genotype-phenotype correlation (in 87 well documented patients) showed that 28 of 29 (97%) patients with two null mutations and 23 of 24 (96%) patients with mutation I2G (homozygous or heterozygous with a null mutation) had classic salt wasting. Patients with mutation I172N (homozygous or heterozygous with a null or I2G mutation) had salt wasting (2 of 17, 12%), simple virilizing (10 of 17, 59%), or nonclassic CAH (5 of 17, 29%). All six patients with mutation P30L, V281L, or P453S (homozygous or compound heterozygous) had nonclassic CAH. The frequency of CYP21 mutations and the genotype-phenotype correlation in 21-hydroxylase deficient patients in The Netherlands show in general high concordance with previous reports from other Western European countries. However, a cluster of four pseudogene-derived point mutations on exons 7 and 8 on a single allele, observed in almost 2% of the unrelated alleles, seems to be particular for the Dutch population and six novel CYP21 gene mutations were found.     

Functional characterization of two novel point mutations in the CYP21 gene causing simple virilizing forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia is a group of autosomal recessive disorders most often caused by deficiency of steroid 21-hydroxylase due to mutations in the CYP21 gene. We studied the functional and structural consequences of two novel missense mutations in the CYP21 gene, detected in two simple virilizing congenital adrenal hyperplasia patients. Both the male and female patient were compound heterozygous for the novel I77T and A434V point mutations, respectively. The in vitro expression analysis in COS-7 cells revealed a reduced 21-hydroxylase activity in the I77T mutant of 3 +/- 2% (sd) for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and of 5 +/- 3% for the conversion of progesterone to 11-deoxycorticosterone. The A434V mutant had a residual enzyme activity of 14 +/- 2% for 17-hydroxyprogesterone and 12 +/- 6% for progesterone. Substrate affinity was similar in the mutants as in the CYP21 wild-type protein, whereas reaction velocity was markedly decreased in both mutants. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. We hypothesize that the I77T mutation markedly decreases the reaction product release and/or substrate entrance to the enzyme's active site, whereas the A434V mutant reduces both the catalytic capacity and reaction velocity. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 21-hydroxylase deficiency and also provides new insights into cytochrome P450 structure-function relationships.     

Two prevalent CYP17 mutations and genotype-phenotype correlations in 24 Brazilian patients with 17-hydroxylase deficiency

We performed molecular genetic analysis of 24 subjects from 19 families with 17-hydroxylase deficiency in Brazil. Of 7 novel CYP17 mutations, 2 (W406R and R362C) account for 50% and 32% of the mutant alleles, respectively. Both mutations were completely inactive when studied in COS-7 cells and yeast microsomes; however, phenotypic features varied among subjects. Some 46,XY individuals with these genotypes had ambiguous genitalia, and other subjects had normal blood pressure and/or serum potassium. We found mutations W406R and R362C principally in families with Spanish and Portuguese ancestry, respectively, suggesting that two independent founder effects contribute to the increased prevalence of 17-hydroxylase deficiency in Brazil. Mutations Y329D and P428L retained a trace of activity, yet the two individuals with these mutations had severe hypertension and hypokalemia. The 46,XX female with mutation Y329D reached Tanner stage 5, whereas the 46,XY subject with mutation P428L remained sexually infantile. The severity of hypertension, hypokalemia, 17-deoxysteroid excess, and sex steroid deficiency varied, even among patients with completely inactive CYP17 protein(s). Spontaneous sexual development occurred only in 46,XX females with partial deficiencies. We conclude that other factors, in addition to CYP17 genotype, contribute to the phenotype of individual patients with 17-hydroxylase deficiency.     

Analysis of CYP21A2 gene mutation and phenotype in patients with 21-hydroxylase deficiency

<正>Objective To investigate the spectrum of CYP21A2gene mutation and the correlation between genotype and phenotype in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas. Methods Genomic DNA was extracted from the peripheral blood samples of the proband. Locus-specific PCR,direct sequencing of PCR     

新复合杂合突变致非经典型21-羟化酶缺陷症

目的分析1例不典型表型的21-羟化酶缺陷症（210HD）患者的诊断过程和分子遗传学资料。方法根据患者临床资料、激素测定及影像学资料确诊，PCR产物直接测序方法检测CYP21基因突变。结果患者为老年女性，以高血压就诊；基础激素测定示孕酮、睾酮、雄烯二酮、空腹17-羟孕酮等高于正常水平；双侧肾上腺结节样增生；快速ACTH兴奋试验显示，激发后17-羟孕酮水平为68．3μg／L。基因测序发现，CYP21基因编码区C1187T（R356W）杂合突变，合并启动子区域C-125T，G-112A，T-109C三个位点相联杂合突变，该复合杂合突变类型尚未见文献报道。结论CYP21基因编码区C1187T杂合突变合并启动子区域C-125T，G-112A，T-109C三位点相联杂合突变可能与不典型表现的210HD的发生有关。     

Variable ACTH-stimulated 17-hydroxyprogesterone values in 21-hydroxylase deficiency carriers are not related to the different CYP21 gene mutations

The currently used cutoff level for ACTH-stimulated 17- hydroxyprogesterone (17OHP) for the diagnosis of the nonclassical (NC) form of 21-hydroxylase deficiency (21OHD), established before molecular studies, is based on the mean + 2 SD of 17OHP levels of obligate heterozygotes. However, carriers of CYP21 mutations present variable ACTH-stimulated 17OHP levels, ranging from normal values up to 30 nmol/liter. The aim of this study was to determine whether ACTH-stimulated 17OHP levels in obligate carriers for 21OHD would be correlated with the impairment of the enzyme activity caused by these mutations, which would affect the 17OHP cutoff level for the diagnosis of the NC form. Fifty-nine parents of patients with the classical and NC forms of 21OHD had their DNA screened for the mutations found in the index case and were divided into three mutation groups according to the impairment of enzyme activity (A = 0%, B = 3%, and C > 20%). All parents carried mutations in one allele (29 of group A, 9 of group B, and 21 of group C). Blood samples were collected at baseline condition and 60 min after ACTH (250 microg i.v.) to measure 17OHP levels. The levels among groups A, B, and C were compared using the Kruskall Wallis test. ACTH-stimulated 17OHP levels identified 39% of the carriers (9 in group A, 2 in group B and 12 in group C). The mean +/- SD basal 17OHP levels in groups A, B, and C were: 2.94 +/- 1.89, 1.77 +/- 0.81 and 3.90 +/- 2.43 nmol/liter, respectively (P > 0.05) and for ACTH-stimulated levels were 12.6 +/- 7.2, 13.2 +/- 12.9 and 16.8 +/- 7.8 nmol/liter, respectively (P > 0.05). Two carriers presented ACTH-stimulated 17OHP levels between 30 and 45 nmol/liter and their entire CYP21 sequencing revealed only one mutation in heterozygous state indicating that the current cutoff level might overestimate the diagnosis of the NC form. We conclude that the variable ACTH-stimulated 17-OHP levels in carriers are not related to CYP21 gene mutations with different impairment of enzyme activity.     

Multicentric study of Brazilian patients with 21-hydroxylase deficiency: a genotype-phenotype correlation

We analyzed the clinical and molecular data of 205 patients with the three different clinical forms of 21-hydroxylase deficiency, in whom the clinical and molecular diagnosis were already defined. The most frequent mutations were I2 splice in the salt wasting form, I172N in the simple virilizing and V281L in the nonclassical form, presenting similar frequencies as those observed in other populations. We found a lower frequency of 21-hydroxylase gene deletion, similar to that previously identified in Argentinean and Mexican populations. Five new mutations were described in our population: G424S, H28+C, Ins 1003 1004 A, R408C and IVS2-2A>G. The genotype was classified in three groups according to the impairment of enzymatic activity observed in vitro, Group A: 0-2%, Group B: 3-7% and Group C: >20%. Group A mutations correlated with the salt wasting form, the Group B with simple virilizing form and Group C with the non classical form. The severity of genotype showed a positive correlation with higher 17OH-progesterone and testosterone levels. The I2 splice mutation in homo or hemizygosis confers classical form phenotype with both salt wasting and simple virilizing forms, precluding the prediction of the clinical form through genotype in pre and neonatal diagnosis. The good genotype-phenotype correlation in patients with 21-hydroxylase deficiency shows the usefulness of genotype to predict the clinical form for genetic counseling, prenatal diagnosis and to confirm neonatal screening diagnosis, except in cases with I2 splice mutation.     

Clinical phenotype and mutation spectrum of the CYP21A2 gene in patients with steroid 21-hydroxylase deficiency

Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.     

'Cryptic' form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in the Yugoslav population

Five individuals with the asymptomatic, 'non-classical', 'cryptic' form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (21-OH) deficiency from 5 unrelated families were discovered during hormonal studies and HLA-typing performed in a series of 24 families with CAH due to 21-OH deficiency. Four of the 5 individuals with the 'cryptic' form of CAH belong to families where the index case was a patient with the classical form of CAH due to 21-OH deficiency. The fifth one originated from a family where the index case was a girl with the 'non-classical', 'late-onset' form of the disease. All the 5 individuals had no clinical symptoms in spite of clearcut biochemical signs of 21-OH deficiency: increased 17-OH-progesterone (17-OHP), dehydroepiandrosterone and androstenedione levels, particularly after ACTH-stimulation. The 17-OHP response upon ACTH stimulation of heterozygotes for this 'non-classical' form of 21-OH deficiency did not differ from the response of heterozygous individuals for the classical form of the disease. The results of this study confirm the hypothesis that individuals with the 'cryptic' form of CAH due to 21-OH deficiency are genetic compounds bearing one allele for the severe, classical form, and on the homologous locus, another one for the mild 'non-classical' form of CAH due to 21-OH deficiency. Their genotype was 21-OH severe/21-OH mild.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel missense mutation, GLY424SER, in Brazilian patients with 21-hydroxylase deficiency.

A previous screening of 17 mutations in 130 Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency did not identify mutations in 20% of the alleles. To diagnose these alleles we sequenced the entire CYP21 gene of one Mulatto patient with the simple virilizing form, who had only the R356W mutation in a heterozygous state. We identified a heterozygous G-A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region where glycine is conserved in at least 4 species. This novel mutation eliminates 1 of the restriction sites of the BanI enzyme, which made its screening possible for the whole series. The G424S mutation was found in a compound heterozygous state in 5 families; 4 presented the simple virilizing form, and 1 presented the nonclassical form. Interestingly, 3 of 5 families have a Mulatto origin. This mutation was not identified in 118 CYP21 alleles of normal individuals, ruling out the possibility of a polymorphism, or in 80 pseudogenes, indicating a casual mutagenic event and not a microconversion event. All patients with the G424S mutation presented CYP21P and C4A gene deletions and human leukocyte antigen DR17 on the same haplotype, suggesting a linkage disequilibrium and a probable founder effect. Search for the G424S mutation in other populations will reveal whether it is restricted to the Brazilian patients or if it has a wider ethnic distribution.     

Cryptic 21-hydroxylase deficiency in families of patients with classical congenital adrenal hyperplasia

Serum androgens and 17-hydroxyprogesterone concentrations and HLA genotypes were determined in 124 families of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH). In 8 pedigrees, we discovered 16 pubertal or postpubertal family members of either sex who had biochemical evidence of 21-hydroxylase deficiency but were without clinical symptoms of excess virilism, amenorrhea, or infertility. We designated these family members as individuals with cryptic 21-hydroxylase deficiency. Within each generation, the family members with cryptic 21-hydroxylase deficiency were HLA identical. It is proposed that these family members are genetic compounds, having 21-hydroxylase deficiency as a result of two recessive gene defects: 1) a severe 21-hydroxylase gene defect present in the index case with classical CAH (21-OHCAH) and 2) a mild 21-hydroxylase gene defect (21-OHCRYPTIC). Thus, the CAH genotype in the family members with cryptic 21-hydroxylase deficiency is 21-OHCAH/21-OHCRYPTIC. Lod score analysis for linkage between the cryptogenic 21-OH trait and HLA gave a combined Lod score for males and females of theta = 0.00 of 3.409. Close genetic linkage between HLA and 21-OHCRYPTIC was thus established. This study provides support for the previously reported heterogeneity of 21-hydroxylase deficiency which may result from allelic variability at the locus for steroid 21-hydroxylase.     

Extraadrenal steroid 21-hydroxylase activity in a woman with congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency

The 21-hydroxylation of plasma progesterone (P) has been demonstrated in pregnant, nonpregnant, and adrenalectomized women and in men. The fractional conversion of plasma progesterone to deoxycorticosterone (DOC), [rho]P-DOC BU, among those subjects was 0.009 +/- 0.001 (mean +/- SEM, n = 32). The [rho]P-DOC BU in a woman with congenital adrenal hyperplasia due to apparent adrenal steroid 21-hydroxylase deficiency was 0.010 when she was taking cortisone acetate, and the [rho]P-DOC BU determined when she was not taking cortisone acetate was 0.012. Moreover, the value computed for the fractional conversion of 17 alpha-hydroxyprogesterone to 11-deoxycortisol in this woman (0.004) was similar to that observed in a woman with normal adrenal function (0.005). Therefore, extraadrenal 21-hydroxylase activity in a woman with nonsalt-losing congenital adrenal hyperplasia due to 21-hydroxylase deficiency was similar to that found in persons with normal adrenal function.