Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.     

Predisposition for de novo gene aberrations in the offspring of mothers with a duplicated CYP21A2 gene

CONTEXT: Although CYP21A2 de novo mutations are assumed to account for 1 to 2% of congenital adrenal hyperplasia (CAH) alleles and CYP21 genotyping has been done worldwide, there are only a few well-documented cases of CYP21A2 de novo mutations. The majority of these are deletions resulting from unequal crossings over owing to misalignment of homologous chromosomes during meiosis. Whereas so far, only heterozygous deletions of the CYP21A1P pseudogene were seen as premutations for de novo aberrations, the present report addresses such a predisposing role for parental duplicated CYP21A2 genes. SUBJECTS AND METHODS: As part of routine diagnostic procedures, CYP21 genotyping has been performed in two unrelated female CAH index patients and in their clinically asymptomatic parents and siblings. RESULTS: Both patients have inherited the paternal Intron2splice mutation and have harbored a de novo gene aberration (large deletion and I271N/exon 4) on their maternal haplotype. Surprisingly, both mothers were carriers of rare duplicated CYP21A2 haplotypes carrying CAH alleles, which were not detected in the daughters. Among 133 CAH alleles that were detected in patients and that could be traced to the respective family members by genotyping, these two de novo aberrations (representing 1.5% of 133 traced CAH alleles) were the only ones identified. CONCLUSION: Because both de novo CYP21A2 gene aberrations so far identified in our laboratory occurred in the gametes of mothers carrying rare duplicated CYP21A2 haplotypes, we hypothesize that duplicated CYP21A2 genes could predispose for de novo mutations in the offspring, which is of relevance for prenatal CYP21 genotyping and genetic counseling.     

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.     

A rare duplicated 21-hydroxylase haplotype and a de novo mutation: a family analysis

21-Hydroxylase (21-OH) genotyping was performed in clinically unaffected family members of a congenital adrenal hyperplasia (CAH) index patient (Prader stage 3), who is a compound heterozygous carrier of the I172N (exon 4) and the intron2 splicing mutations. Whereas the latter mutation could be traced to the father, the exon 4 aberration represents a de novo mutation (accounting for 1% of CAH alleles) harbored on an unaffected allele, which was inherited from the mother. Although clinically and biochemically unaffected, the patient's brother was found to be compound heterozygous for intron2splice (paternal allele) and Q318X in exon 8 (maternal allele). As shown by PCR-based sequence and Southern blot analysis, the maternal haplotype, inherited by the brother, has a duplicated CYP21B (functional) gene, one of which carries a Q318X mutation. This duplicated Q318X-affected haplotype is the first of its kind among 800 alleles screened for 21-OH deficiency in our laboratory and has to date been reported only in three Swedish CAH patients, all of them bearing an intron2splice and a Q318X mutation. This family analysis highlights the complexity of the CYP21/CYP21P(pseudogene) loci and the difficulties of 21-OH genotyping.     

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.     

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.     

CYP21A2 Gene Mutations in Congenital Adrenal Hyperplasia: Genotype?phenotype correlation in Turkish children

Background: Congenital adrenal hyperplasia (CAH) due 21−hydroxylase deficiency (21−OHD) is a common autosomal recessive disorder. It is caused by defects in the CYP21A2 gene.Objective: Our aim was to determine the frequency of common gene mutations and to evaluate genotype−phenotype correlations in Turkish 21−OHD patients.Methods: Molecular analysis of the CYP21A2 gene was performed for the detection of the eight most common point mutations [p.P30L, IVS2−13C>G (IVS−2), p.I172N, exon 6 mutation cluster (p.I236N, p.V237E, p.M239K), p.V281L, p.Q318X, p.R356W, 8−bp−deletion], of large deletion and conversion by southern blotting, allele specific semi−quantitative PCR/enzyme restriction method and sequencing, in 56 patients with 21−OHD, from 52 families.Results: Disease−causing mutations were identified in 77 out of 91 alleles (84.6%) of the patients. Mutations were found in 34 of 43 alleles (79.1%) in salt wasting (SW; n=26), 32 of 36 alleles (88.8%) in simple virilizing (SV; n=24) and 11 of 12 alleles (91.6%) in non−classical (NC; n=6) form of CAH. The most frequent mutations were IVS−2 (22.0%), large conversion (14.3%), p.I172N (9.9%) p.R356W (8.8%), and large deletion (6.6%). In the SW form, the most frequent genotypes were homozygous for IVS−2 (11.5%) and homozygous for large conversion of the gene (11.5%). In the SV form, the most frequent genotype was homozygous for IVS−2 (20%), followed by compound heterozygous for p.I172N/8−bp del (10%). Homozygous for p.V281L (16.7%) was most common in NC. In most cases there was good correlation between genotype and phenotype. In the SW and NC forms, genotypes of all the patients correlated with their phenotypes. Conclusions: This is the first comprehensive study on the molecular basis of CAH patients in the Turkish population. Based on these results, we propose a modified screening strategy to facilitate molecular testing of CAH patients in our population.Conflict of interest:None declared.     

Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders. CAH is most often caused by deficiency of steroid 21-hydroxylase. The frequency of CYP21-inactivating mutations and the genotype-phenotype relationship were characterized in 155 well defined unrelated CAH patients. We were able to elucidate 306 of 310 disease-causing alleles (diagnostic sensitivity, 98.7%). The most frequent mutation was the intron 2 splice site mutation (30.3%), followed by gene deletions (20.3%), the I172N mutation (19.7%) and large gene conversions (7.1%). Five point mutations were detected that have not been described in other CAH cohorts. Genotypes were categorized in 4 mutation groups (null, A, B, and C) according to their predicted functional consequences and compared to the clinical phenotype. The positive predictive value for null mutations (ppv(null)) was 100%, as all patients with these mutations had a salt-wasting phenotype. In mutation group A (intron 2 splice site mutation in homozygous or heterozygous form with a null mutation), the ppv(A) to manifest with salt-wasting CAH was 90%. In group B predicted to result in simple virilizing CAH (I172N in homozygous or compound heterozygous form with a more severe mutation), ppv(B) was 74%. In group C (P30L, V281L, P453S in homozygous or compound heterozygous form with a more severe mutation), ppv(C) was 64.7% to exhibit the nonclassical form of CAH, but 90% when excluding the P30L mutation. Thus, in general, a good genotype-phenotype relationship is shown in patients with either the severest or the mildest mutations. A considerable degree of divergence is observed within mutation groups of intermediate severity. As yet undefined factors modifying 21-hydroxylase gene expression and steroid hormone action are likely to account for these differences in phenotypic expression.     

Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalomas

OBJECTIVE: The aims of the present study were (a) to examine the occurrence of 21-hydroxylase gene (CYP21) mutations in patients with unilateral and bilateral adrenal incidentalomas and (b) to correlate the results of mutation screening with hormonal parameters of 21-hydroxylase deficiency. DESIGN: The frequency of the eight commonly occurring CYP21 mutations in blood DNA samples of 19 patients with bilateral, as well as in blood and tumoral tissue DNA samples of 31 patients with unilateral adrenal incidentalomas, was determined. In all patients, hormonal evaluation for 21-hydroxylase deficiency was performed using measurements of basal and ACTH-stimulated plasma 17-hydroxyprogesterone (17-OHP) concentrations. METHODS: Blood and tumoral DNA samples were analyzed by allele-specific PCR for the detection of the eight commonly occurring CYP21 mutations (deletion/large gene conversion, intron 2 splicing, Ile172Asn, exon 6 cluster, Val281Leu, Leu307insT, Gln318Stop and Arg356Trp mutations). Plasma 17-OHP concentrations were measured by radioimmunoassay. RESULTS: Of the 19 patients with bilateral adrenal incidentalomas, one patient had homozygous (Val281Leu) and three patients had heterozygous germline CYP21 mutations (Val281Leu in two cases and Arg356Trp in one case). Heterozygous germline CYP21 mutations were also detected in five of the 31 patients with unilateral adrenal incidentalomas (Ile172Asn in three cases and Val281Leu in two cases). Mutation screening of tumoral DNA in unilateral incidentalomas showed the presence of corresponding germline mutations but no additional somatic mutations were found. ACTH-stimulated plasma 17-OHP concentrations were above 1500 ng/dl in all patients with bilateral incidentalomas who had homozygous and heterozygous CYP21 mutations, but heterozygous carriers with unilateral incidentalomas had highly variable ACTH-stimulated plasma 17-OHP levels (between 111 and 1705 ng/dl). CONCLUSIONS: These results suggest a similar frequency of germline CYP21 mutations in patients with bilateral and unilateral adrenal incidentalomas (21.1% and 16.1% respectively). Therefore, it cannot be ruled out that, in at least some patients, CYP21 mutations may play a role in the pathomechanism of bilateral and unilateral adrenal incidentalomas. However, the lack of clear association of CYP21 mutations with increased ACTH-stimulated plasma l7-OHP response, especially in patients with unilateral incidentalomas, suggests that the effect of CYP21 mutations on adrenocortical tumor formation may also involve mechanism(s) independent of ACTH-induced changes in 17-OHP secretion.     

Molecular genetic analysis of Tunisian patients with a classic form of 21-hydroxylase deficiency: identification of four novel mutations and high prevalence of Q318X mutation

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders mainly due to defects in the steroid 21-hydroxylase (CYP21) gene. To determine the mutational spectrum in the Tunisian CAH population, the CYP21 active gene was analyzed in 51 unrelated patients using our cascade strategy (digestion by restriction enzyme, sequencing). All patients had a classical form of 21-hydroxylase deficiency. Mutations were detected in over 94% of the chromosomes examined. The most frequent mutation in the Tunisian CAH population was found to be Q318X, with large prevalence (35.3%), in contrast to 0.5-13.8% described in other series. Incidence of other mutations does not differ, as previously described: large deletions (19.6%), mutation in intron 2 (17.6%), and I172N (10.8%). Four novel mutations were found in four patients with the salt-wasting form. These four novel mutations include three point mutations that have not been reported to occur in the CYP21P pseudogene: R483W, W19X, 2669insC, and one small conversion of DNA sequence from exon 5 to exon 8. Our results have shown a good genotype/phenotype correlation in the case of most mutations. This is the first report of screening for mutations of 21-hydroxylase gene in the Tunisian population and even in the Arab population.     

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.     

Salt-wasting congenital adrenal hyperplasia: detection and characterization of mutations in the steroid 21-hydroxylase gene, CYP21, using the polymerase chain reaction.

We have characterized mutations in the steroid 21-hydroxylase gene (CYP21) in salt-wasting congenital adrenal hyperplasia (SW-CAH) subjects, healthy control subjects, and affected sibling pairs with SW-CAH. To identify point mutations in CYP21, we have used an improved polymerase chain reaction methodology that allows analysis of the entire CYP21 gene. In addition, we have used polymerase chain reaction to search for abnormally spliced mRNAs resulting from putatively abnormal CYP21 genes transfected into COS1 cells. We found that all 26 SW-CAH subjects from whom DNA could be completely analyzed, had mutations that could account for the 21-hydroxylase enzyme deficiency. These mutations included CYP21 gene deletion, conversion to the inactive CYP21P form, point mutations leading to amino acid substitutions or stop codons, small gene deletions, and a point mutation in intron-2 that leads to an abnormally spliced mRNA. The point mutation in intron-2 was directly shown to activate a cryptic splice site 19 basepairs from exon-3 of CYP21 and thereby cause a reading frame mutation. This CYP21 mutation was frequently found in our white SW-CAH subjects, while the frequency of this mutation was extremely low in a racially matched control population. Furthermore, affected sibling pairs shared this mutation in all cases examined. The results presented should have important applications for the prenatal diagnosis of CAH.     

Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)     

Increased prevalence of heterozygous 21-OH germline mutations in patients with adrenal incidentalomas

OBJECTIVE: As a result of the widespread use and the enhanced quality of high-resolution radiological techniques [computed tomography (CT), magnetic resonance imaging (MRI)] a high frequency (4-10%) of adrenal incidentalomas has been detected in the general population. It is still debated whether undiagnosed 21-hydroxylase (21-OH) deficiency, accounting for more than 90% of congenital adrenal hyperplasia (CAH) cases, predisposes for adrenal tumours. We therefore performed an analysis of the prevalence of 21-OH germline mutations in patients with non-functional adrenal incidentalomas. SUBJECTS AND METHODS: Fifty Austrian patients with non-functional adrenal adenomas detected by CT for unrelated reasons were screened by PCR-based sequencing for the most common point mutations and by Southern blot analysis for large gene deletion/conversion events of the 21-OH gene. RESULTS: Heterozygosity for large gene conversions was shown in 5 (10%), for Q318 point mutations in 2 (4%) and for the Intron2splice mutation in 1 (2%) of the 50 patients with adrenal adenomas. One (2%) patient (70 years of age), identified to have a chimeric CYP21AB gene with a junction site before Intron 2 on one and a large (30 kb) deletion on the other allele, was diagnosed to be affected by CAH. CONCLUSION: 21-OH mutation screening indicates a higher frequency of classic CAH carriers (16%) and of manifest CAH (2%) due to 21-OH-deficiency among patients with adrenal adenomas than in the general population (1-2% carrier frequency for classic CAH).     

An androgen receptor gene mutation (E653K) in a family with congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency as well as in partial androgen insensitivity

An androgen receptor (AR) variant (E653K) was found in two unrelated Swedish families. One family had two girls affected with congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency. The girls, who showed mild virilization in relation to their CYP21 genotype, had inherited the AR gene mutation from their father, who showed no symptoms of androgen insensitivity. The other family had a boy with partial androgen insensitivity and ambiguous genitalia, and he had inherited the AR gene mutation from his mother. The mutant receptor showed a transactivating capacity in the same range as the normal receptor at high concentrations of ligand (1 and 10 nM dihydrotestosterone), but absent or reduced transactivation at low levels (0.01 and 0.1 nM). The receptor variant was not found among 250 additional unselected Swedish men. Sequencing of the AR gene in five unrelated CAH girls with the I172N mutation in CYP21 and minimal virilization did not reveal any additional deviations from the normal reference sequence. In addition, there was no difference in lengths of the polymorphic CAG repeat in the AR gene between CAH girls with the I172N mutation who showed minimal and severe virilization, and we found no evidence of skewed X-inactivation. We conclude that AR gene mutations or polymorphisms are not a common factor influencing the degree of hyperandrogenic symptoms displayed by CAH girls, and that the AR E653K mutation is compatible with normal genital development, although it can cause genital malformations in susceptible individuals.     

Carrier frequency of congenital adrenal hyperplasia (21-hydroxylase deficiency) in a middle European population

Based on newborn screening data, the carrier frequency of congenital adrenal hyperplasia (CAH) in the general population has been estimated to be 1:55. The higher CAH frequency (particularly of milder forms of the disease) reported for certain populations including Yugoslavs (1.6%) relates to population genetic and hormonal data. However, so far, true carrier frequency for CAH due to 21-OH deficiency has not been determined by comprehensive mutation analysis of the 21-OH gene (CYP21A2) in an unselected European population. This study used CYP21A2 genotyping (sequence/Southern blot analysis) to determine CAH carrier frequency in a middle European (Austrian) population. The study included 100 migrants from the former Yugoslavia and 100 individuals of non-Yugoslavian origin. None of these individuals showed clinical hyperandrogenism or had a family history of CAH. Genotyping 400 unrelated alleles from 200 clinically unaffected individuals, this study revealed a carrier frequency of 9.5%, including so-called "classic" (5.5%) and "nonclassic" (4%) CYP21A2-gene aberrations. The observed heterozygosity for CAH in Yugoslavs was not different (P = 0.8095) from that in non-Yugoslavs. In conclusion, the observed CAH carrier frequency of 9.5% suggests a higher prevalence of CAH heterozygosity in a middle European population than hitherto estimated independently of the individuals' Yugoslav or non-Yugoslav origin.     

Analysis of mutations causing steroid 21-hydroxylase deficiency

Steroid 21-hydroxylase deficiency is the most frequent cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol. Mutations causing this disorder have been characterized by hybridization analysis of patient DNA samples using cDNA and oligonucleotide probes, and by cloning and sequencing of mutant 21-hydroxylase (CYP21B) genes. About 20% of mutant alleles carry a 30 kilobasepair deletion that includes the 3' end of the CYP21A pseudogene, the C4B complement gene, and the 5' end of CYP21B, leaving behind a single CYP21A-like gene that is not functional. Non-deletional mutations include a nonsense mutation at codon 318 that is associated with severe disease and missense mutations at codons 172 (isoleucine to asparagine) and 281 (valine to leucine) that are respectively associated with intermediate and mild deficiency states. All of these alleles have apparently resulted from gene conversion events that have transferred deleterious mutations from the CYP21A pseudogene to CYP21B. Thus, recombinations between CYP21A and CYP21B probably account for the majority of 21-hydroxylase deficiency alleles.     

Steroid 21-hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype-phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia

Objective To report genotype–phenotype correlation in a large cohort of patients. Context Study of the CYP21A2 gene in 866 unrelated chromosomes of 21‐hydroxylase deficiency in Argentinean patients with classic and nonclassic (NC) forms of congenital adrenal hyperplasia (CAH). Methods Eleven most common mutations were analysed by allele‐specific polymerase chain reaction, restriction fragment length polymorphism (RFLP) or southern blot analysis. Gene sequencing was performed when no mutation was detected in one allele or the genotype–phenotype correlation was lacking. Results The 11‐most‐common‐mutation screening allowed for the detection of 88·1% of affected alleles (80·3% in the NC and 95·2% in the classic forms). p.V281L, IVS2‐13A/C>G (In2) and gene deletions and large gene conversions were the most prevalent mutations. In2 (35·2%) in salt wasting (SW), p.I172N (37·3%) in simple virilizing and p.V281L (54·1%) in NC CAH were the most prevalent mutations within the clinical forms. In 7/15 p.P30L mutation alleles, a chimeric CYP21A1P/CYP21A2 gene [PromCYP21A1P; p.P30L] was detected, while 6/15 represented a single‐nucleotide substitution, and in 2/15 linkage with mutations, p.[P30L; V281L] and [p.P30L; IVS2‐13A/C > G; p.Q318X] was found. In two SW patients, a novel nonsense mutation, p.Q41X, was observed. In three p.V281L mutation patients, the phenotype was more severe than predicted by genotype. Sequence analysis revealed an intronic alteration in the allele carrying the p.V281L mutation [IVS2 + 5G > A; p.V281L]. An aberrant splicing in this p.V281L mutated allele explains the clinical phenotype. Conclusions A high percentage of CYP21A2 affected alleles is detected by the 11‐mutation screening study. Genotype–phenotype correlation was high, but when the phenotype is more severe than predicted by genotype, presence of two alterations in one allele should be ruled out.     

Mutation in the CYP21B gene (Ile-172----Asn) causes steroid 21-hydroxylase deficiency.

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. It results from a deficiency in a specific cytochrome P450, P450c21 (P450XXIA). The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) are located in the HLA complex on chromosome 6p. Many mutant alleles are associated with deletions of CYP21B; we report the cloning and characterization of a nondeleted mutant CYP21B gene. This mutant gene is expressed on transfection into mouse Y1 adrenal cells, producing mRNA levels similar to those seen after transfection of the normal CYP21B gene. In codon 172 of the mutant gene, the normal codon ATC, encoding isoleucine, has been changed to AAC, encoding asparagine. This mutation is normally present in the CYP21A pseudogene, so that it may have been transferred to the mutant CYP21B gene by gene conversion. Hybridization of oligonucleotide probes corresponding to this and two other mutations normally present in CYP21A demonstrated that 4 out of 20 patients carried the codon 172 mutation; in one of these patients, the mutation was present as part of a larger gene conversion involving at least exons 3-6. Gene conversion may be a frequent cause of 21-hydroxylase deficiency alleles due to the presence of six chi-like sequences (GCTGGGG) in the CYP21 genes and the close proximity of the CYP21A pseudogene, which has several potentially deleterious mutations.     

p.H282N and p.Y191H: 2 novel CYP21A2 mutations in Italian congenital adrenal hyperplasia patients

More than 90% of all cases of congenital adrenal hyperplasia (CAH) result from steroid 21-hydroxylase gene (CYP21A2) mutations. The CYP21A2 gene is located in the human leukocyte antigen (HLA) class III region on the short arm of chromosome 6p21.3, along with an inactive pseudogene, CYP21A1P, that is 98% homologous in its coding sequence with CYP21A2. Most CYP21A2 mutations result from intergenic recombinations between CYP21A2 and the closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for only 5% to 10% of 21-hydroxylase deficiency alleles. However, detection of these rare and spontaneous mutations has continued to expand worldwide. We identified 2 novel CYP21A2 missense mutations (p.H282N and p.Y191H) in 2 Italian patients with simple-virilizing and nonclassic CAH forms. Functional analysis of these CYP21A2 mutations was performed. Functional in vitro assay for mutagenized CYP21A2 enzymes was performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained allowed us to classify the p.H282N and p.Y191H variants as simple-virilizing and nonclassic CAH associated mutations, respectively. These results correlate with the rate of severity of the patients' disease. This finding provides a further contribution for assisting in the diagnosis of CAH patients.