Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes

Koppens, Paul F. J.; Hoogenboezem, Theo; Degenhart, H. J.

doi:10.1007/s00439-002-0810-7

Cited by 69 publications

(38 citation statements)

References 24 publications

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“…The CYP21A2 large-scale conversions into CYP21A1P generating the TNXB/TNXA hybrid gene represent the majority part of these (3.1% of the mutant alleles). Koppens et al described that CYP21A2 large-scale conversions generating the TNXA/TNXB hybrid gene are associated with a presence of 2 CYP21A1P pseudogenes on the involved chromosome (13). The presence of 2 copies of the CYP21A1P pseudogenes on 1 chromosome was identified in all the …”

Section: Resultsmentioning

confidence: 99%

“…These mutations are generated by the unequal crossingover during meiosis. However, the mechanism of small-scale gene conversions, that result in short-range mutations in CYP21A2 derived from CYP21A1P, is not yet well understood (12,13). Unequal crossing-over can cause a 30 kb deletion, involving the 3' end of CYP21A1P, all of TNXA, RP2, C4B, and the 5' end of CYP21A2, which produces a non-functional chimeric gene with 5' and 3' ends corresponding to CYP21A1P and CYP21A2, respectively (14,15).…”

Section: Introductionmentioning

confidence: 99%

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Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency

Vrzalová

Hrubá²,

Hrabincová³

et al. 2010

Int J Mol Med

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Abstract. Congenital adrenal hyperplasia (CAH) is comprised of a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in most of the severe cases, also the biosynthesis of aldosterone. Approximately 90-95% of all the CAH cases are due to mutations in the steroid 21-hydroxylase gene (CYP21A2). In this study, the molecular genetic analysis of CYP21A2 was performed in 267 Czech probands suspected of 21-hydroxylase deficiency (21OHD). 21OHD was confirmed in 241 probands (2 mutations were detected). In 26 probands, a mutation was found only in 1 CYP21A2 allele. A set of 30 different mutant alleles was determined. We describe i) mutated CYP21A2 alleles carrying novel point mutations (p.Thr168Asn, p.Ser169X and p.Pro386Arg), ii) mutated CYP21A2 alleles carrying the novel chimeric gene designated as CH-7, which was detected in 21.4% of the mutant alleles, iii) an unusual genotype with a combination of the CYP21A2 duplication, 2 point mutations and the CYP21A2 large-scale gene conversion on the second allele, and (iv) a detailed analysis of the chimeric CYP21A1P/CYP21A2 genes. In conclusion, our genotyping approach allowed for the accurate identification of the CYP21A2 gene mutations in 21OHD patients and their families and provided some useful information on diagnosis and genetic counselling.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency

Vrzalová

Hrubá²,

Hrabincová³

et al. 2010

Int J Mol Med

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“…Minisequencing results for wildtype amplicon 4, if present, produce an identical pattern to that of amplicon 1. Although the presence of amplicon 4 may be considered an unexpected result, it has been observed on multiple occasions and likely corresponds to cis-gene duplications detected by other groups, 33,34 gene conversion at the 3Ј end of the gene, or polymorphism under the primer binding site, none of which cause coding sequence changes. Regardless of the mechanism that creates these fragments, downstream minisequencing (or complete sequencing) will inform the user whether or not these amplicons 4 contain any genedeactivating mutation.…”

Section: Assay Designmentioning

confidence: 87%

“…When used in combination with family studies to determine phase of mutations, this assay provides informative haplotype data as is advocated by Koppens and colleagues. 33 The minisequencing portion of this assay is modified from the principles described by Krone and colleagues. 31 These reactions are multiplexed, semiautomated, and efficient, providing simultaneous detection of 11 point mutations and a small 8-bp deletion within a single tube.…”

Section: Discussionmentioning

confidence: 99%

Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency)

Keen-Kim

Redman

Alanes

et al. 2005

The Journal of Molecular Diagnostics

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Congenital adrenal hyperplasia is an autosomal recessive disorder caused by defective adrenal steroid biosynthesis, resulting in reduced glucocorticoid and increased androgen production. The majority of cases are due to inactivation of the 21-hydroxylase gene (CYP21A2), most commonly caused by genomic rearrangements with the adjacent, highly homologous pseudogene CYP21A. The most common deletions and gene conversion events have been defined and are typically detected by Southern hybridization detection of CYP21 rearrangements and/or polymerase chain reaction (PCR). However, Southern hybridization is laborious, and allele-specific PCR results may be difficult to interpret. We have therefore developed a locus-specific, PCR-based, minisequencing method for detecting the 12 most common CYP21A2 mutations. We validated the assay using a panel of 20 previously genotyped samples obtained from individuals who collectively have a broad spectrum of mutations causing 21-hydroxylase deficiency. We also used 19 control samples having no CYP21 mutations. All validation samples were correctly typed, and we identified haplotypes that may be useful for clinical diagnosis. Results from 102 clinical samples demonstrate that this assay is a rapid, reliable, and robust method for locus-specific identification of mutations and is suitable for routine clinical use and prenatal diagnosis. (J Mol Diagn 2005, 7:236 -246)

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“…Most chromosomes bear 1 CYP21A1P pseudogene and 1 CYP21A2 gene, although deletions and duplications have been described (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Intergenic recombinations are relatively frequent in this region (14 ), and are responsible for 95% of the mutations associated with 21OHD; the remain-ing 5% of mutations are apparently not the result of gene conversion events (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Among the intergenic recombinations, approximately 75% are deleterious or partially inactivating mutations, which are typically present in the CYP21A1P pseudogene and appear in the CYP21A2 gene as a result of gene conversion events (14 ).…”

mentioning

confidence: 99%

A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency

et al. 2007

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Background: Correct diagnosis of 21-hydroxylase deficiency (21OHD) requires the identification of CYP21A2 gene deletions and CYP21A1P/CYP21A2 chimeric genes, which are disease-causing alleles, and gene duplications, which can lead to false-positive 21OHD allele results. Because lack of suitable CYP21A2 dosage assessment methods hampers correct 21OHD diagnosis, we developed a new assay based on the relative quantification of the CYP21A2 gene using the DSP gene as a reference. Methods: The assay to determine CYP21A2 copy number is based on real-time PCR. The method also detects the presence of the CYP21A1P/CYP21A2 chimeric gene. We used a duplex PCR to coamplify the DSP gene, included as an internal control, along with CYP21A2. The difference in threshold cycles between CYP21A2 and DSP genes (⌬Ct) was used to assess CYP21A2 copy number. Results:The ⌬Ct values obtained from 24 samples used to set up the method clearly differentiated 3 nonoverlapping intervals, which corresponded to the number of CYP21A2 copies: ؊1.35 to ؊0.25 defined 2 gene copies, ؉0.20 to ؉2.00 defined 1 copy, and ؊2.50 to ؊1.50 defined 3 copies. With these intervals we were able to assess the gene copy number in 24 additional samples. Conclusions: This new method for gene copy assessment detects homozygous and heterozygous CYP21A2 gene deletions, CYP21A1P/CYP21A2 chimeric genes, and gene duplications. Moreover, the method is robust, fast, and easy to use in a molecular diagnosis laboratory.

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Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes

Cited by 69 publications

References 24 publications

Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency

Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency

Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency)

A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency

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