{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T18:44:24Z","timestamp":1787165064570,"version":"build-2736575974"},"reference-count":37,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2024,5,16]],"date-time":"2024-05-16T00:00:00Z","timestamp":1715817600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic defects in cortisol synthesis and shows elevated ACTH concentrations, which in turn has downstream effects. The most common variant of CAH, 21-hydroxylase deficiency (21OHD), is the result of pathogenic variants in the CYP21A2 gene and is one of the most common monogenic disorders. However, the genetics of 21OHD is complex and challenging. The CYP21A2 gene is located in the RCCX copy number variation (CNV), a complex, multiallelic, and tandem CNV in the major histocompatibility complex (MHC) class III region on chromosome 6 (band 6p21.3). Here, CYP21A2 and its pseudogene CYP21A1P are located 30 kb apart and share a high nucleotide homology of approximately 98% and 96% in exons and introns, respectively. This high-sequence homology facilitates large structural rearrangements, copy number changes, and gene conversion through intergenic recombination. There is a good genotype\u2013phenotype correlation in 21OHD, and genotyping can be performed to confirm the clinical diagnosis, predict long-term outcomes, and determine genetic counseling. Thus, genotyping in CAH is clinically relevant but the interpretations can be challenging for non-initiated clinicians. Here, there are some concrete examples of how molecular diagnosis can sometimes require the use of multiple molecular strategies.<\/jats:p>","DOI":"10.3390\/cimb46050291","type":"journal-article","created":{"date-parts":[[2024,5,16]],"date-time":"2024-05-16T11:26:17Z","timestamp":1715858777000},"page":"4832-4844","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Challenging Molecular Diagnosis of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency: Case Series and Novel Variants of CYP21A2 Gene"],"prefix":"10.3390","volume":"46","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0523-5744","authenticated-orcid":false,"given":"Paola","family":"Concolino","sequence":"first","affiliation":[{"name":"Dipartimento di Scienze di Laboratorio ed Ematologiche, UOC Chimica, Biochimica e Biologia Molecolare Clinica, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,16]]},"reference":[{"key":"ref_1","first-page":"245","article-title":"Congenital adrenal hyperplasia due to 21-hydroxylase deficiency","volume":"21","author":"White","year":"2000","journal-title":"Endocr. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.jsbmb.2016.06.015","article-title":"Steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia","volume":"165","author":"Parsa","year":"2017","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/S0140-6736(22)01330-7","article-title":"Congenital adrenal hyperplasia","volume":"401","author":"Auer","year":"2023","journal-title":"Lancet"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2194","DOI":"10.1016\/S0140-6736(17)31431-9","article-title":"Congenital adrenal hyperplasia","volume":"390","author":"Arlt","year":"2017","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4043","DOI":"10.1210\/jc.2018-01865","article-title":"Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: An Endocrine Society Clinical Practice Guideline","volume":"103","author":"Speiser","year":"2018","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1276","DOI":"10.1038\/gim.2017.46","article-title":"Revisiting the prevalence of nonclassic congenital adrenal hyperplasia in US Ashkenazi Jews and Caucasians","volume":"19","author":"Morissette","year":"2017","journal-title":"Genet. Med."},{"key":"ref_7","first-page":"447","article-title":"Distribution of the V281L mutation of the CYP21 gene in Israeli congenital adrenal hyperplasia patients and its association with HLA-B14","volume":"3","author":"Israel","year":"2006","journal-title":"Pediatr. Endocrinol. Rev."},{"key":"ref_8","first-page":"650","article-title":"High frequency of nonclassical steroid 21-hydroxylase deficiency","volume":"37","author":"Speiser","year":"1985","journal-title":"Am. J. Hum. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"709758","DOI":"10.3389\/fendo.2021.709758","article-title":"Genes and Pseudogenes: Complexity of the RCCX Locus and Disease","volume":"12","author":"Carrozza","year":"2021","journal-title":"Front. Endocrinol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1073\/pnas.82.4.1089","article-title":"Two Genes Encoding Steroid 21-Hydroxylase are Located Near the Genes Encoding the Fourth Component of Complement in Man","volume":"82","author":"White","year":"1985","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5111","DOI":"10.1073\/pnas.83.14.5111","article-title":"Structure of Human Steroid 21-Hydroxylase Genes","volume":"83","author":"White","year":"1986","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","first-page":"17","article-title":"Evidence for Frequent Gene Conversion in the Steroid 21-Hydroxylase P-450(C21) Gene: Implications for Steroid 21-Hydroxylase Deficiency","volume":"42","author":"Higashi","year":"1988","journal-title":"Am. J. Hum. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"10796","DOI":"10.1073\/pnas.92.23.10796","article-title":"Gene Conversions and Unequal Crossovers Between CYP21 (Steroid 21-Hydroxylase Gene) and CYP21P Involve Different Mechanisms","volume":"92","author":"White","year":"1995","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"432","DOI":"10.3389\/fendo.2019.00432","article-title":"The Complexities in Genotyping of Congenital Adrenal Hyperplasia: 21-Hydroxylase Deficiency","volume":"10","author":"Pignatelli","year":"2019","journal-title":"Front. Endocrinol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1007\/s40291-018-0319-y","article-title":"Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency: A Comprehensive Focus on 233 Pathogenic Variants of CYP21A2 Gene","volume":"22","author":"Concolino","year":"2018","journal-title":"Mol. Diagn. Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1007\/s12098-023-04951-9","article-title":"Navigating the Complex Landscape of CYP21A2 Variants","volume":"91","author":"Dubey","year":"2024","journal-title":"Indian J. Pediatr."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1002\/humu.23351","article-title":"CYP21A2 Mutation Update: Comprehensive Analysis of Databases and Published Genetic Variants","volume":"39","author":"Simonetti","year":"2018","journal-title":"Hum. Mutat."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1373\/clinchem.2011.174037","article-title":"Junction Site Analysis of Chimeric CYP21A1P\/CYP21A2 Genes in 21-Hydroxylase Deficiency","volume":"58","author":"Chen","year":"2012","journal-title":"Clin. Chem."},{"key":"ref_19","first-page":"123","article-title":"Detection of a Frequent Duplicated CYP21A2 Gene Carrying a Q318X Mutation in a General Population with Quantitative PCR Methods. Diagn","volume":"20","author":"Kharrat","year":"2011","journal-title":"Mol. Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.cca.2013.05.019","article-title":"Genes, Pseudogenes and Like Genes: The Case of 21-Hydroxylase in Italian Population","volume":"424","author":"Concolino","year":"2013","journal-title":"Clin. Chim. Acta."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"803226","DOI":"10.3389\/fendo.2022.803226","article-title":"Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome","volume":"13","author":"Marino","year":"2022","journal-title":"Front. Endocrinol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1038\/s41431-021-00870-5","article-title":"Molecular genetic testing of congenital adrenal hyperplasia due to 21-hydroxylase deficiency should include CAH-X chimeras","volume":"29","author":"Lao","year":"2021","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1007\/s40291-023-00666-x","article-title":"Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency","volume":"27","author":"Concolino","year":"2023","journal-title":"Mol. Diagn. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"788812","DOI":"10.3389\/fendo.2021.788812","article-title":"Characteristics of In2G Variant in Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency","volume":"12","author":"Kocova","year":"2022","journal-title":"Front. Endocrinol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1016\/j.ygeno.2003.11.006","article-title":"PCR-based detection of the CYP21 deletion and TNXA\/TNXB hybrid in the RCCX module","volume":"83","author":"Lee","year":"2004","journal-title":"Genomics"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.ab.2011.02.016","article-title":"Analysis of the CYP21A1P pseudogene: Indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes","volume":"413","author":"Tsai","year":"2011","journal-title":"Anal. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2611","DOI":"10.1073\/pnas.1300057110","article-title":"Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency","volume":"110","author":"New","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1007\/s40291-019-00415-z","article-title":"Issues with the Detection of Large Genomic Rearrangements in Molecular Diagnosis of 21-Hydroxylase Deficiency","volume":"23","author":"Concolino","year":"2019","journal-title":"Mol. Diagn. Ther."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1373\/clinchem.2011.162230","article-title":"Multiplex ligation-dependent probe amplification analysis is useful for diagnosing congenital adrenal hyperplasia but requires a deep knowledge of CYP21A2 genetics","volume":"57","author":"Concolino","year":"2011","journal-title":"Clin. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"6789","DOI":"10.12998\/wjcc.v9.i23.6789","article-title":"2+0 CYP21A2 deletion carrier\u2014A limitation of the genetic testing and counseling: A case report","volume":"9","author":"Xi","year":"2021","journal-title":"World J. Clin. Cases."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Parajes, S., Quinteiro, C., Dom\u00ednguez, F., and Loidi, L. (2008). High frequency of copy number variations and sequence variants at CYP21A2 locus: Implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002138"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1007\/s00439-002-0810-7","article-title":"Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: Characteristics of three unusual haplotypes","volume":"111","author":"Koppens","year":"2002","journal-title":"Hum. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1341","DOI":"10.1038\/s41431-020-0653-5","article-title":"EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency","volume":"28","author":"Hoeppner","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"834549","DOI":"10.3389\/fendo.2022.834549","article-title":"Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency: A Practical Approach","volume":"13","author":"Arriba","year":"2022","journal-title":"Front. Endocrinol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1002\/pd.1584","article-title":"Gene duplications in 21-hydroxylase deficiency: The importance of accurate molecular diagnosis in carrier detection and prenatal diagnosis","volume":"26","author":"Ezquieta","year":"2006","journal-title":"Prenat. Diagn."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1038\/jp.2012.5","article-title":"Erroneous prenatal diagnosis of congenital adrenal hyperplasia owing to a duplication of the CYP21A2 gene","volume":"33","author":"Lekarev","year":"2013","journal-title":"J. Perinatol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1978","DOI":"10.1093\/bioinformatics\/bty897","article-title":"VarSome: The human genomic variant search engine","volume":"35","author":"Kopanos","year":"2019","journal-title":"Bioinformatics"}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/46\/5\/291\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:43:15Z","timestamp":1760107395000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/46\/5\/291"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,16]]},"references-count":37,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5]]}},"alternative-id":["cimb46050291"],"URL":"https:\/\/doi.org\/10.3390\/cimb46050291","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,16]]}}}