Phenotype-Genotype Correlation in 56 Women with Nonclassical Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

Complete analysis of the CYP21 gene was performed in 56 unrelated French women with symptomatic nonclassical congenital adrenal hyperplasia. The mutational spectrum and the phenotype-genotype correlation were examined. The overall predominant mutation was V281L, which was present on 51% of alleles and in 80% of women. Three novel mutations were found: L317M, R435C, and a 5′-end gene conversion. Sixty-three percent of the women were carrying a severe mutation of the CYP21 gene, and hence risk giving birth to children with a classical form of the disease. In such cases, screening for heterozygosity in the partner is crucial. Potential genotype/phenotype correlations were examined by classifying the patients into three groups according to the CYP21 allelic combinations: A (mild/mild), B (mild/severe), and C (severe/severe). Primary amenorrhea was more frequent, and mean basal and stimulated 17-hydroxyprogesterone levels were higher in compound heterozygotes for mild and severe mutations (group B) compared with women with two mild mutations (group A), but there was a considerable overlap for individual values. Surprisingly, in two women, a severe mutation was found on both alleles (group C). Therefore, the phenotype cannot be accurately predicted from the genotype. Variability in phenotypic expression may be conditioned by mechanisms other than genetic heterogeneity at the CYP21 locus.

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Congenital adrenal hyperplasia: molecular mechanisms resulting in 21-hydroxylase deficiency

Acta Endocrinologica ◽

10.1530/acta.0.112s315 ◽

1986 ◽

Vol 113 (4_Suppl) ◽

pp. S315-S320 ◽

Cited By ~ 7

Author(s):

Patricia A. Donohoue ◽

Cornelis Van Dop ◽

Nicholas Jospe ◽

Claude J. Migeon

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Molecular Mechanisms ◽

Sequence Data ◽

Phenotypic Expression ◽

Restriction Pattern ◽

Class Iii ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Wide Range ◽

21 Hydroxylase Deficiency

Abstract 21-Hydroxylase deficiency resulting in congenital adrenal hyperplasia (CAH) is a HLA-linked autosomal recessive disorder that has a wide range of phenotypic expression. Two homologous 21-hydroxylase genes (21-OHA and 21-OHB) occur within the Class III region of the major histocompatibility complex, but only one (21-OHB) appears to function in adrenal steroidogenesis. Our restriction maps, and initial sequence data from White et al. (Pediatr Res 20:274A (1986)) for the two human 21-OH genes reveal a high degree of homology between these genes and a reading frame shift mutation in the 21-OHA gene respectively. Among fourteen control subjects, the intragenic restriction patterns of the 21-OHA and 21-OHB genes are invariant. The few restriction fragment length polymorphisms (RFLPs) found in some controls result from polymorphic restriction sites outside the 21-OH genes. In patients with CAH, several different mechanisms for mutation of the 21-OHB gene have been described: 1) deletion of the unique sequences of the 21-OHB gene, 2) conversion of the unique sequences of the 21-OHB gene to those of 21-OHA, and 3) mutations of 21-OHB which do not result in a detectable alteration of restriction pattern (e.g., point mutations). Duplication of the 21-OHA gene has been found in some patients with attenuated CAH; however, the significance of this finding remains unclear.

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Mutation of IVS2 –12A/C>G in Combination with 707–714delGAGACTAC in the CYP21 Gene Is Caused by Deletion of the C4-CYP21 Repeat Module with Steroid 21-Hydroxylase Deficiency

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2003-030047 ◽

2003 ◽

Vol 88 (6) ◽

pp. 2726-2729 ◽

Cited By ~ 12

Author(s):

Hsien-Hsiung Lee ◽

Shwu-Fen Chang ◽

Fuu-Jen Tsai ◽

Li-Ping Tsai ◽

Ching-Yu Lin

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Donor Site ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Aberrant Splicing ◽

Cyp21 Gene ◽

Pcr Product ◽

Intergenic Recombination ◽

21 Hydroxylase Deficiency

More than 90% of the cases of congenital adrenal hyperplasia 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. Among them, mutation of the aberrant splicing donor site of IVS2 –12A/C>G at nucleotide (nt) 655 is believed to be a result derived from this mechanism and is the most prevalent case among all ethnic groups. However, mutation of 707–714delGAGACTAC rarely exists alone, although this locus is a distance of 53 nt away from IVS2 –12A/C>G. From the molecular characterization of the mutation of IVS2 –12A/C>G combined with 707–714delGAGACTAC in patients with congenital adrenal hyperplasia, we found that it appeared to be in a 3.2-rather than a 3.7-kb fragment generated by Taq I digestion in a PCR product of the CYP21 gene. Interestingly, the 5′ end region of such a CYP21 haplotype had CYP21P-specific sequences. Our results indicate that the coexistence of these two mutations is caused by deletion of the CYP21P, XA, RP2, and C4B genes and intergenic recombination in the C4-CYP21 repeat module. Surprisingly, this kind of the haplotype of the mutated CYP21 gene has not been reported as a gene deletion.

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Study of a Kindred with Classic Congenital Adrenal Hyperplasia: Diagnostic Challenge due to Phenotypic Variance1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.6.4887 ◽

1998 ◽

Vol 83 (6) ◽

pp. 1940-1945

Author(s):

Daisy Chin ◽

Phyllis W. Speiser ◽

Julianne Imperato-McGinley ◽

Naznin Dixit ◽

Naveen Uli ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Newborn Screening ◽

Point Mutation ◽

Phenotypic Expression ◽

Variable Degree ◽

Adrenal Hyperplasia ◽

Cyp21 Gene ◽

Classic Congenital Adrenal Hyperplasia ◽

Genital Ambiguity ◽

21 Hydroxylase Deficiency

We sought to determine the concordance of the phenotype and genotype in a kindred with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency. The variation in phenotypic expression within this family underscores the difficulty of establishing the diagnosis in the absence of newborn screening, even with a heightened index of suspicion. Steroidogenic profiles were obtained for the three affected siblings. The available clinical history of the two affected aunts was retrieved. Genotyping was performed on several members of the kindred. Detailed sequencing of the entire CYP21 gene of two clinically dissimilar subjects in this family was undertaken to explore the possibility of other mutations or polymorphisms. PCR with ligase detection reaction analysis of CYP21 revealed that the affected family members III-2, III-3, III-4, II-3, and II-4, all were compound heterozygotes carrying the intron 2 point mutation known to interfere with splicing (nucleotide 656 A to G) and the exon 4 point mutation causing a nonconservative substitution of asparagine for isoleucine at codon 172 (I172N). Detailed sequencing of the gene was performed for the two most phenotypically dissimilar subjects. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4, but not in patient III-4, and no additional mutations were found. Classic congenital adrenal hyperplasia remains a difficult diagnosis to make in the absence of newborn screening because of the variability of phenotypic expression. Likewise, the variable degree of genital ambiguity in affected females in this family serves to question universal advocacy of prenatal steroid treatment in pregnancies at risk for congenital adrenal hyperplasia. Extensive molecular exploration did not provide an explanation of the phenotypic heterogeneity and supports the possibility of influences other than the CYP21 gene for the observed divergence.

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Analysis of the Chimeric CYP21P/CYP21 Gene in Steroid 21-Hydroxylase Deficiency

Clinical Chemistry ◽

10.1093/clinchem/46.5.606 ◽

2000 ◽

Vol 46 (5) ◽

pp. 606-611 ◽

Cited By ~ 16

Author(s):

Hsien-Hsiung Lee ◽

Jan-Gowth Chang ◽

Chang-Hai Tsai ◽

Fuu-Jen Tsai ◽

Hsiang-Tai Chao ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Direct Method ◽

Pcr Amplification ◽

Chimeric Gene ◽

Wild Type ◽

Adrenal Hyperplasia ◽

Specific Primer ◽

Hydroxylase Deficiency ◽

Cyp21 Gene ◽

21 Hydroxylase Deficiency

Abstract Background: A single nonfunctional chimeric gene with its 5′ and 3′ ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance. Methods: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3′-specific primer for the CYP21 gene and two different 5′ primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel. Results: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method. Conclusions: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.

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Rapid Second-Tier Molecular Genetic Analysis for Congenital Adrenal Hyperplasia Attributable to Steroid 21-Hydroxylase Deficiency

Clinical Chemistry ◽

10.1373/clinchem.2004.042416 ◽

2005 ◽

Vol 51 (2) ◽

pp. 298-304 ◽

Cited By ~ 37

Author(s):

Siegfried Kösel ◽

Siegfried Burggraf ◽

Ralph Fingerhut ◽

Helmut G Dörr ◽

Adelbert A Roscher ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Molecular Genetic ◽

Molecular Genetic Analysis ◽

False Positives ◽

High Rate ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Cyp21 Gene ◽

Second Tier ◽

21 Hydroxylase Deficiency

Abstract Background: Neonatal screening for steroid 21-hydroxylase (CYP21) deficiency is performed to identify congenital adrenal hyperplasia (CAH). The immunologic assay for 17α-hydroxyprogesterone (17-OHP) has a high rate of false positives. We assessed the potential for increasing the specificity for CAH by use of a second step involving analysis of the CYP21 gene. Methods: Between January 1999 and December 2003, a total of 810 000 newborns were screened. Of these, 7920 had to be retested because their 17-OHP values were above the cutoff of the assay. Sixty-one had positive 17-OHP values in their recall samples and were diagnosed as having CAH. We used a rapid assay for common mutations of the CYP21 gene to analyze these 61 samples. In a prospective study, 198 consecutive samples that had increased 17-OHP and 100 samples that had normal 17-OHP concentrations were genotyped. Results: Fifty-nine of 61 cases diagnosed as having CAH were confirmed genetically as CYP21 deficiencies. One patient had a 3β-hydroxysteroid dehydrogenase deficiency, and one patient carried no CYP21 mutations. The 198 increased 17-OHP results were designated as false positives after immunologic testing of recall samples. None of these samples exhibited the genetic pattern consistent with CYP21 deficiency. Conclusions: If samples with increased 17-OHP values were screened genetically, the number of retests would decrease by ∼90%, but the overall sensitivity of CAH screening would remain the same. Adding a second-tier genetic step would require a modest increase in costs, but is counterbalanced by fewer recalls, less clinical follow-up, and a reduction in unnecessary worry for families.

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Carrier Analysis and Prenatal Diagnosis of Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency in Chinese1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.85.2.6367 ◽

2000 ◽

Vol 85 (2) ◽

pp. 597-600

Author(s):

Hsien-Hsiung Lee ◽

Jing-Mei Kuo ◽

Hsiang-Tai Chao ◽

Yann-Jinn Lee ◽

Jan-Gowth Chang ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Congenital Adrenal Hyperplasia ◽

Autosomal Recessive Disorder ◽

Single Strand Conformation Polymorphism ◽

Severe Form ◽

Heterozygous Mutation ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Cyp21 Gene ◽

21 Hydroxylase Deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We screened 1,000 healthy people, using a previously developed differential PCR method combined with single-strand conformation polymorphism and amplification-created restriction site methods for the carrier detection of the CYP21 gene deficiency. Our results indicated that the rate of occurrence of the heterozygous CAH carrier was about 12 in 1,000, with a gene frequency of 0.0060 and an incidence frequency of 1 in 28,000 in the Chinese population. In addition, 9 cases of CAH families were performed with prenatal diagnosis. Among them, 3 cases were diagnosed as the severe form, 4 cases carried the heterozygous mutation, and 2 were normal. This is the first report of carrier frequency analysis and prenatal diagnosis of 21-hydroxylase deficiency in Chinese.

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