Development of CYP21A2 Genotyping Assay for the Diagnosis of Congenital Adrenal Hyperplasia

Background Steroid 21-hydroxylase deficiency due to CYP21A2 gene mutations represents more than 90% of all congenital adrenal hyperplasia cases. This deficiency is screened by measuring levels of 17-hydroxyprogesterone, which may vary, causing false positive or false negative results. In order to as...

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Bibliographic Details
Published in:Molecular diagnosis & therapy 2017-12, Vol.21 (6), p.663-675
Main Authors: Prado, Mayara Jorgens, de Castro, Simone Martins, Kopacek, Cristiane, de Mello, Maricilda Palandi, Rispoli, Thaiane, Grandi, Tarciana, da Silva, Cláudia Maria Dornelles, Rossetti, Maria Lucia Rosa
Format: Article
Language:English
Subjects:
Adrenal glands
Assaying
Biomedical and Life Sciences
Biomedicine
Biosynthesis
Cancer Research
Diagnosis
Enzymes
Gene conversion
Genotype & phenotype
Genotyping
Hormones
Human Genetics
Hydroxylase
Hyperplasia
Laboratory Medicine
Medical screening
Molecular Medicine
Mutation
Original Research Article
Pharmacotherapy
Point mutation
Population studies
Steroid 21-hydroxylase
Studies
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Summary:Background Steroid 21-hydroxylase deficiency due to CYP21A2 gene mutations represents more than 90% of all congenital adrenal hyperplasia cases. This deficiency is screened by measuring levels of 17-hydroxyprogesterone, which may vary, causing false positive or false negative results. In order to assist the diagnosis, molecular methodologies have been employed. This work aimed to perform genotyping assays to detect mutations in the CYP21A2 gene and compare the findings with other population studies. Methods The SNaPshot assay was developed to simultaneously detect 12 frequent point mutations in the CYP21A2 gene (p.Arg409Cys, p.Gln319Ter, p.Arg357Trp, p.Leu308PhefsTer6, p.Val237Glu, IVS2-13A/C > G, p.Ile173Asn, p.Pro31Leu, p.Pro454Ser, p.Val282Leu, p.Gly111ValfsTer21 and p.His63Leu). The direct sequencing and multiplex ligation-dependent probe amplification assays were used to confirm point mutations present in the developed method. The latter was also used to search large deletions and gene conversion, complementing the investigation. A total of 166 cases were studied. Results The SNaPshot assay was successfully developed to detect the 12 mutations. The results of mutation analysis indicated 84 pathogenic alleles in 48 cases, with p.Val282Leu (27.1%) and IVS2-13A/C > G (20.8%) being the most frequently found mutations. Between the findings of this study and those of other South American studies, there were significant differences in frequency for p.Pro31Leu and p.Val282Leu ( p   G was identified in two patients via the SNaPshot assay. Conclusion The molecular strategy developed for CYP21A2 gene mutation screening allowed us to detect the principle mutations described around the world. Furthermore, the first Southern Brazilian mutation frequencies concerning the CYP21A2 gene were obtained.
ISSN:1177-1062
1179-2000
DOI:10.1007/s40291-017-0296-6