Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common endocrine diseases, yet genetic diagnosis is among the most complicated of all monogenic disorders. It has an overall incidence of 1:10000-1:20000, it is inherited in autosomal recessive pattern and caused by mutations affecting CYP21A2 gene. Based on the phenotypic expression, this disease is categorized into severe, classical form revealed at birth and mild, non-classical form. Although diagnosis could be established based on biochemical tests and distinctive clinical features, molecular genetic testing is crucial for diagnosis confirmation, detection of carriers and asymptomatic patients, disease prognosis, as well as for providing proper genetic counselling and prenatal diagnosis. Based on CYP21A2 mutational spectrum and frequencies in Serbia, in this paper we propose an optimal molecular genetic diagnostic algorithm for CAH and discuss genetic mechanisms underlying the disease. The complete diagnostic procedure combines multiplex minisequencing technique (SNaPshot PCR) as a method for rapid detection of common point mutations, direct sequencing of whole CYP21A2 gene and PCR with sequence specific primers (PCR-SSP) for large gene rearrangements detection (CYP21A1P/CYP21A2 chimeras). While SNaPshot PCR assay analyses ten common mutations (c.290-13A/C>G, p.P30L, p.R356W, p.G110fs, p.V281L, p.Q318X, p.L307fs, p.I172N, Cluster p.[I236N;V237E;M239K] and p.P453S) which account for over 80% of all CYP21A2 mutations in Serbian population, direct sequencing of CYP21A2 gene is needed to identify potential rare or novel mutations present in Serbian population with frequency of 1.8%. Additionally, large gene rearrangements which are present with frequency of 16.7% make PCR-SSP analysis an unavoidable part of molecular characterization of CAH in Serbia. Described molecular genetic strategy is intended to facilitate correct diagnosis assessment in CAH affected individuals and their families in Serbia but it will also contribute to molecular genetic testing of CAH patients across Europe.

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Molecular Analysis of CYP21A2 Gene Mutations among Iraqi Patients with Congenital Adrenal Hyperplasia

Enzyme Research ◽

10.1155/2016/9040616 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 6

Author(s):

Ruqayah G. Y. Al-Obaidi ◽

Bassam M. S. Al-Musawi ◽

Munib Ahmed K. Al-Zubaidi ◽

Christian Oberkanins ◽

Stefan Németh ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Molecular Analysis ◽

Point Mutations ◽

Gene Mutations ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Cyp21a2 Gene ◽

Gene Deletions ◽

Large Gene ◽

21 Hydroxylase Deficiency

Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

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Low incidence of KIT gene mutations and no PDGFRA gene mutations in primary cutaneous melanoma: an immunohistochemical and molecular genetic study of Japanese cases

International Journal of Clinical Oncology ◽

10.1007/s10147-010-0087-0 ◽

2010 ◽

Vol 15 (5) ◽

pp. 453-456 ◽

Cited By ~ 23

Author(s):

Tadashi Terada

Keyword(s):

Genetic Study ◽

Cutaneous Melanoma ◽

Molecular Genetic ◽

Gene Mutations ◽

Molecular Genetic Study ◽

Primary Cutaneous Melanoma ◽

Pdgfra Gene ◽

Kit Gene ◽

Low Incidence

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Comprehensive Genetic Testing of CYP21A2: A Retrospective Analysis in Patients with Suspected Congenital Adrenal Hyperplasia

Journal of Clinical Medicine ◽

10.3390/jcm10061183 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1183

Author(s):

Madalina Nicoleta Nan ◽

Rosa Roig ◽

Susana Martínez ◽

Jose Rives ◽

Eulàlia Urgell ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Point Mutations ◽

Gene Mutations ◽

Pathogenic Variant ◽

Adrenal Hyperplasia ◽

Cyp21a2 Gene ◽

Pathogenic Variants ◽

New Variant ◽

Allelic Distribution ◽

Large Rearrangements

The most common form of congenital adrenal hyperplasia (CAH) results from a deficiency of the 21-hydroxylase enzyme (21-OHD), presenting with a broad spectrum of clinical phenotypes according to the CYP21A2 gene mutations. Of the 59 patients with suspected CAH, 62.7% presented a positive genetic result. Of them, 78.4% and 18.9% presented with non-classical and classical forms, respectively. An overall phenotype-genotype correlation of 88.9% was observed. Biochemically, 17-hydroxiprogesterone concentrations were significantly higher in genetically confirmed patients. Genetically, 36 patients presented with previously reported pathogenic variants, and one presented a new variant in homozygosis. Among the 74 alleles tested, point mutations were found in 89.2% and large rearrangements were found in the rest. The most prevalent pathogenic variant was p.(Val282Leu). The inclusion of relatives revealed one further case. Interestingly, 87.5% of relatives were carriers of a pathogenic variant, including two siblings initially classified as genetically positive. In addition, the study of male partners with gestational desire identified several carriers of mild mutations. Studying the allelic distribution of the variants also allowed for reclassifying one patient. In conclusion, a genetic approach including Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, and allelic distribution of the pathogenic variants represents a beneficial tool for better classifying patients with 21-OHD.

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Molecular genetic analysis of CYP21A2 gene in patients with congenital adrenal hyperplasia

Indian Journal of Endocrinology and Metabolism ◽

10.4103/2230-8210.95679 ◽

2012 ◽

Vol 16 (3) ◽

pp. 384 ◽

Cited By ~ 11

Author(s):

AriacheryC Ammini ◽

Arundhati Sharma ◽

Rajesh Khadgawat ◽

Eunice Marumudi ◽

Bindu Kulshreshtha ◽

...

Keyword(s):

Genetic Analysis ◽

Congenital Adrenal Hyperplasia ◽

Molecular Genetic ◽

Molecular Genetic Analysis ◽

Adrenal Hyperplasia ◽

Cyp21a2 Gene

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ANALYSIS CYP21A2 GENE MUTATIONS TECHNIQUE IN PATIENTS WITH CONGENITAL ADRENAL HYPERPLASIA

Biotechnologia Acta ◽

10.15407/biotech7.01.075 ◽

2014 ◽

Vol 7 (1) ◽

pp. 75-79 ◽

Cited By ~ 1

Author(s):

S. Yu. Chernushyn ◽

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Gene Mutations ◽

Adrenal Hyperplasia ◽

Cyp21a2 Gene

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Characterization of CYP21A2 Gene Mutations in Dominican Subjects with Congenital Adrenal Hyperplasia.

10.1210/endo-meetings.2010.part1.p7.p1-314 ◽

2010 ◽

pp. P1-314-P1-314

Author(s):

C Tan ◽

JL Imperato-McGinley ◽

I Labour ◽

JJ Cordero ◽

C Montero ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Gene Mutations ◽

Adrenal Hyperplasia ◽

Cyp21a2 Gene

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A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations

Molecular Neurobiology ◽

10.1007/s12035-017-0535-1 ◽

2017 ◽

Vol 55 (4) ◽

pp. 3477-3489 ◽

Cited By ~ 30

Author(s):

Shaghayegh Taghavi ◽

Rita Chaouni ◽

Abbas Tafakhori ◽

Luis J. Azcona ◽

Saghar Ghasemi Firouzabadi ◽

...

Keyword(s):

Genetic Study ◽

Autosomal Recessive ◽

Molecular Genetic ◽

Gene Mutations ◽

Molecular Genetic Study ◽

Novel Gene

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Current Aspects of Genetic Diagnosis of 21-Hydroxylase Insufficiency

Doctor Ru ◽

10.31550/1727-2378-2021-20-6-73-79 ◽

2021 ◽

Vol 20 (6) ◽

pp. 73-79

Author(s):

N.S. Osinovskaya ◽

◽

Yu.A. Nasykhova ◽

M.I. Yarmolinskaya ◽

O.B. Glavnova ◽

...

Keyword(s):

Genetic Testing ◽

Congenital Adrenal Hyperplasia ◽

Genetic Counselling ◽

Molecular Genetic ◽

Genetic Diagnosis ◽

Clinical Manifestations ◽

Adrenal Hyperplasia ◽

Cyp21a2 Gene ◽

Gene Sequence Analysis ◽

Dideoxynucleotide Chain Termination Method

Objective of the Review: To discuss the current peculiarities of 21-hydroxylase insufficiency diagnostics. Key Points. Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive pathologies, associated with a defective enzyme or transport protein participating in cortisol biosynthesis. Currently, there is information on CAH genetics including information on 21-hydroxylase insufficiency. Neonatal screening, antenatal and postnatal methods to diagnose 21-hydroxylase deficit have been developed. Timely diagnosis and correct therapy have been found to facilitate normal physical and mental development of patients. Molecular genetic testing for CAH associated with 21-hydroxylase deficit is widely used both in Russia and globally and is of importance for differential diagnosis, identification of pathogenic CYP21A2 gene carriers and adequate genetic counselling. The best strategy to diagnose 21-hydroxylase insufficiency is 2-stage CYP21A2 gene analysis. The test should include both gene sequence analysis and identification of point replacements, minor deletions and duplication (e.g., dideoxynucleotide chain-termination method or Next Generation Sequencing), and identification of extended deletions and duplication (multiplex ligation dependent probe amplification, real-time polymerase chain reaction). Such a comprehensive approach can help finding a majority of types of possible changes. Conclusion. Correct genetic testing methods ensure detection of pathogen variants in CYP21A2 gene; evaluation of the possible rate of clinical manifestations of the disease, both during antenatal testing and if an unknown clinical CAH form is encountered; prescribing an adequate therapy; and ensuring genetic counselling in order to develop a management strategy, including when planning for pregnancy in a woman with confirmed CAH. Keywords: congenital adrenal hyperplasia, CYP21A2 gene, genetic counselling, 21-hydroxylase

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