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Stanchev PE, Dimitrova M, Makakova D, Tilov B. Exploring the Differential Diagnosis of Adrenal Adenoma in the Context of Situs Ambiguous: A Clinical Case Study. MEDICINA (KAUNAS, LITHUANIA) 2024; 60:2010. [PMID: 39768890 PMCID: PMC11727780 DOI: 10.3390/medicina60122010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2024] [Revised: 11/21/2024] [Accepted: 12/02/2024] [Indexed: 01/16/2025]
Abstract
Situs anomalies, including situs inversus and situs ambiguous (SAMB), are rare congenital conditions typically noted in pediatric populations, with SAMB being particularly uncommon in adults. This case study addresses the incidental discovery of situs ambiguous with polysplenia in a 65-year-old man evaluated for suspected adrenal adenoma. The patient's medical history included benign prostatic hyperplasia and tuberculous pleurisy. Methods included a thorough physical examination and laboratory tests, which showed normal cortisol levels and ACTH rhythm. Contrast-enhanced CT imaging revealed multiple spleens near the right adrenal region, altered liver positioning, a truncated pancreas, and a right-sided stomach, while the right adrenal gland was not visualized. Notably, the patient exhibited minimal symptoms despite these significant anatomical anomalies. The findings underscore the rarity of situs ambiguous in adults and its unexpected association with endocrine pathology. This case highlights the importance of comprehensive imaging and a multidisciplinary approach in managing patients with unusual anatomical presentations. It suggests that situs anomalies may be more prevalent in adult populations than previously recognized and emphasizes the need for increased clinical awareness and evaluation in similar cases.
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Affiliation(s)
- Pavel E. Stanchev
- Clinic of Endocrinology and Metabolic Diseases, St. George University Hospital, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria;
| | - Mariya Dimitrova
- Department of Prosthetic Dentistry, Faculty of Dental Medicine, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria;
| | - Desislava Makakova
- Department of Prosthetic Dentistry, Faculty of Dental Medicine, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria;
| | - Boris Tilov
- Medical College, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria;
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Shiryagin VV, Devyatkin AA, Fateev OD, Petriaikina ES, Bogdanov VP, Antysheva ZG, Volchkov PY, Yudin SM, Woroncow M, Skvortsova VI. Genomic complexity and clinical significance of the RCCX locus. PeerJ 2024; 12:e18243. [PMID: 39512309 PMCID: PMC11542561 DOI: 10.7717/peerj.18243] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2024] [Accepted: 09/15/2024] [Indexed: 11/15/2024] Open
Abstract
Nearly identical, repetitive elements in the genome contribute to the variability in genetic inheritance patterns, particularly in regions like the RCCX locus, where such repeats can lead to structural variations. In addition, during the formation of gametes as a result of meiosis, variants of loci with repetitive elements that do not code for the required proteins may occur. As a result, an individual with certain genetic rearrangements in this region may have an increased risk of developing a congenital disorder, particularly in cases where the non-functional allele is inherited dominantly. At the same time, there is still no routine or generally recognized diagnostic method to determine the sequence of the repetitive fragments. The functionally important RCCX locus consists of such repetitive fragments. The available knowledge about the genomic variants of the RCCX locus is fragmented, as there is no standardized method to determine its structure. It should be noted that in some structural variants of the RCCX locus, the sequence of protein-coding genes is disrupted, leading to the development of diseases such as congenital adrenal hyperplasia (CAH). Although genetic testing is generally accepted as a gold standard for CAH diagnosis, there are a myriad of strategies on which exact methods to use and in which order. The reason for this inconsistency lies in the complexity of the RCCX locus and the fact that each patient or carrier may have a highly individualized mutation or combination thereof. In this review, we have discussed all known methods that can be used to study the structure of the RCCX locus. As a result, optimal approaches are proposed for the diagnosis of the most common disease caused by lesions in the RCCX-CAH due to CYP21A2 deficiency.
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Affiliation(s)
- Vladimir V. Shiryagin
- Federal State Budgetary Institution “Centre for Strategic Planning and Management of Biomedical Health Risks” of the Federal Medical Biological Agency (Centre for Strategic Planning of FMBA of Russia), Moscow, Russia
| | - Andrey A. Devyatkin
- Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, Moscow, Russia
| | - Oleg D. Fateev
- Federal State Budgetary Institution “Centre for Strategic Planning and Management of Biomedical Health Risks” of the Federal Medical Biological Agency (Centre for Strategic Planning of FMBA of Russia), Moscow, Russia
| | - Ekaterina S. Petriaikina
- Federal State Budgetary Institution “Centre for Strategic Planning and Management of Biomedical Health Risks” of the Federal Medical Biological Agency (Centre for Strategic Planning of FMBA of Russia), Moscow, Russia
| | - Viktor P. Bogdanov
- Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, Moscow, Russia
| | - Zoia G. Antysheva
- Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, Moscow, Russia
| | - Pavel Yu Volchkov
- Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, Moscow, Russia
- Department of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russia
| | - Sergey M. Yudin
- Federal State Budgetary Institution “Centre for Strategic Planning and Management of Biomedical Health Risks” of the Federal Medical Biological Agency (Centre for Strategic Planning of FMBA of Russia), Moscow, Russia
| | - Mary Woroncow
- Department of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russia
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Lan T, Wang J, Chen K, Zhang J, Chen X, Yao H. Comparison of long-read sequencing and MLPA combined with long-PCR sequencing of CYP21A2 mutations in patients with 21-OHD. Front Genet 2024; 15:1472516. [PMID: 39553475 PMCID: PMC11563783 DOI: 10.3389/fgene.2024.1472516] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Accepted: 10/14/2024] [Indexed: 11/19/2024] Open
Abstract
Background 21-Hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. Due to the complex structure and the high genetic heterogeneity of the CYP21A2 gene, genetic testing for 21-OHD is currently facing challenges. Moreover, there are no comparative studies on detecting CYP21A2 mutations by both second-generation sequencing and long-read sequencing (LRS, also known as third-generation sequencing). Objective To detect CYP21A2 variations in 21-OHD patients using targeted capture with LRS method based on the PacBio (Pacific Biosciences) Sequel II platform. Methods A total of 67 patients with 21-OHD were admitted in Wuhan Children's Hospital. The full sequence of CYP21A2 gene was analyzed by targeted capture combined with LRS based on the PacBio Sequel II platform. The results were compared with those of long-polymerase chain reaction (Long-PCR) combined with multiplex ligation probe amplification (MLPA) detection. Based on the in vitro study of 21-hydroxylase activity of common mutations, the patient genotypes were divided into groups of Null, A, B, and C, from severe to mild. The correlation between different genotype groups and clinical typing was observed. Results The study analyzed a total of 67 patients. Among them, 44 (65.67%) were males and 23 (34.33%) were females, with a male-to-female ratio of approximately 1.9:1. A total of 27 pathogenic variants were identified in the 67 patients, of which micro-conversion accounted for 61.9%, new variants of CYP21A2 accounted for 8.2%; deletion accounted for 22.4% (CYP21A2 single deletion and chimeric TNXA/TNXB accounted for 12.7%, chimeric CYP21A1P/CYP21A2 accounted for 9.7%); and duplication accounted for 3.0% (CYP21A2 Gene Duplication). I2G was the most common variant (26.9%). Targeted capture LRS and MLPA combined with Long-PCR detection of CYP21A2 mutations showed 30 detection results with differences. The overall genotype-phenotype correlation was 82.1%. The positive predictive rate of the Null group for salt wasting (SW) type was 84.6%, the A group for SW type was 88.9%, the group B for simple virilization (SV) type was 82.4%, and the group C for SV type was 62.5%. The correlation coefficient rs between the severity of the phenotype and the genotype group was 0.682 (P < 0.05). Conclusion Targeted capture combined with LRS is an integrated approach for detecting CYP21A2 mutations, allowing precise determination of connected sites for multiple deletions/insertions and cis/trans configurations without analyzing parental genomic samples. The overall genotype-phenotype correlation for 21-OHD is generally strong, with higher associations observed between genotype and phenotype for group Null, A, and B mutations, and larger genotype-phenotype variation in group C mutations. Targeted capture with LRS sequencing offers a new method for genetic diagnosis in 21-OHD patients.
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Affiliation(s)
- Tian Lan
- Department of Genetics, Metabolism and Endocrinology, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jin Wang
- Department of Genetics, Metabolism and Endocrinology, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Kaibi Chen
- Department of Genetics, Metabolism and Endocrinology, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | | | - Xiaohong Chen
- Department of Genetics, Metabolism and Endocrinology, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Hui Yao
- Department of Genetics, Metabolism and Endocrinology, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Xia Y, Yu F, Bai Y, Jiang L, Shi P, Jiang Z, Kong X. Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency. Orphanet J Rare Dis 2024; 19:397. [PMID: 39468716 PMCID: PMC11514819 DOI: 10.1186/s13023-024-03414-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Accepted: 10/13/2024] [Indexed: 10/30/2024] Open
Abstract
BACKGROUND Molecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing. RESULTS Among the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD. CONCLUSION CNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.
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Affiliation(s)
- Yanjie Xia
- Genetics and Prenatal Diagnosis Center, Henan Engineering Research Center for Gene Editing of Human Genetic Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Feng Yu
- Genesky Diagnostics (Suzhou) Inc, Rm# 5F, Suite# C13, 218 Xinghu Street, SIP, Suzhou, Jiangsu, China
| | - Ying Bai
- Genetics and Prenatal Diagnosis Center, Henan Engineering Research Center for Gene Editing of Human Genetic Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Lili Jiang
- Genesky Diagnostics (Suzhou) Inc, Rm# 5F, Suite# C13, 218 Xinghu Street, SIP, Suzhou, Jiangsu, China
| | - Panlai Shi
- Genetics and Prenatal Diagnosis Center, Henan Engineering Research Center for Gene Editing of Human Genetic Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Zhengwen Jiang
- Genesky Diagnostics (Suzhou) Inc, Rm# 5F, Suite# C13, 218 Xinghu Street, SIP, Suzhou, Jiangsu, China.
| | - Xiangdong Kong
- Genetics and Prenatal Diagnosis Center, Henan Engineering Research Center for Gene Editing of Human Genetic Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
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Fylaktou I, Mertzanian A, Farakla I, Gryparis A, Vasilakis IA, Binou M, Charmandari E, Kanaka-Gantenbein C, Sertedaki A. Genetics of 21-OH Deficiency and Genotype-Phenotype Correlation: Experience of the Hellenic National Referral Center. Curr Issues Mol Biol 2024; 46:10696-10713. [PMID: 39451515 PMCID: PMC11506624 DOI: 10.3390/cimb46100635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Revised: 09/16/2024] [Accepted: 09/20/2024] [Indexed: 10/26/2024] Open
Abstract
21-hydroxylase deficiency (21-OHD) represents the most common form of congenital adrenal hyperplasia (CAH) due to CYP21A2 gene pathogenic variants. Τhe aim of this study was the identification of CYP21A2 variants in 500 subjects of Greek origin with a suspicion of 21-OHD and, by using the existing hormonal assessment and genotypes of the 500 subjects tested, to identify a biomarker that could differentiate between the heterozygotes and the cases with no pathogenic variants identified. Five hundred subjects with clinical suspicion of 21-OHD underwent CYP21A2 gene sequencing and Multiplex Ligation Dependent Probe Amplification (MLPA). Genetic diagnosis was achieved in 27.4% of the subjects tested, most of which presented with the non-classic form (NC) of 21-OHD. Heterozygotes accounted for 42.6% of cases, whereas no pathogenic variants were identified in 27% of cases. De novo aberrations, duplications, and five novel variants were also identified. Statistical analysis revealed that the difference between the basal and 60' post-ACTH stimulation 17-hydroxyprogesterone concentrations (Δ17-OHP60-0) could be a potential biomarker (p < 0.05) distinguishing the heterozygotes from the cases with no pathogenic variants identified, although no clear cut-off value could be set. Further analysis revealed overlapping clinical manifestations among all the subjects tested. The presented phenotypic traits of the subjects tested and the inability to identify a discriminative biochemical marker highlight the importance of comprehensive CYP21A2 genotyping to ascertain the correct genetic diagnosis and proper genetic counselling.
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Affiliation(s)
- Irene Fylaktou
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Anny Mertzanian
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Ioanna Farakla
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Alexandros Gryparis
- Department of Speech and Language Therapy, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece;
| | - Ioannis Anargyros Vasilakis
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Maria Binou
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Evangelia Charmandari
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Christina Kanaka-Gantenbein
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
| | - Amalia Sertedaki
- Division of Endocrinology, Diabetes and Metabolism ‘Aghia Sophia’ Children’s Hospital ENDO-ERN Center for Rare Paediatric Endocrine Diseases, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, 11527 Athens, Greece; (A.M.); (I.F.); (I.A.V.); (M.B.); (E.C.); (C.K.-G.); (A.S.)
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Concolino P. Challenging Molecular Diagnosis of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency: Case Series and Novel Variants of CYP21A2 Gene. Curr Issues Mol Biol 2024; 46:4832-4844. [PMID: 38785559 PMCID: PMC11119849 DOI: 10.3390/cimb46050291] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2024] [Revised: 05/07/2024] [Accepted: 05/10/2024] [Indexed: 05/25/2024] Open
Abstract
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-phenotype 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.
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Affiliation(s)
- Paola Concolino
- 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
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Carsote M, Gheorghe AM, Nistor C, Trandafir AI, Sima OC, Cucu AP, Ciuche A, Petrova E, Ghemigian A. Landscape of Adrenal Tumours in Patients with Congenital Adrenal Hyperplasia. Biomedicines 2023; 11:3081. [PMID: 38002081 PMCID: PMC10669095 DOI: 10.3390/biomedicines11113081] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2023] [Revised: 11/01/2023] [Accepted: 11/15/2023] [Indexed: 11/26/2023] Open
Abstract
Our aim is to update the topic of adrenal tumours (ATs) in congenital adrenal hyperplasia (CAH) based on a multidisciplinary, clinical perspective via an endocrine approach. This narrative review is based on a PubMed search of full-length, English articles between January 2014 and July 2023. We included 52 original papers: 9 studies, 8 case series, and 35 single case reports. Firstly, we introduce a case-based analysis of 59 CAH-ATs cases with four types of enzymatic defects (CYP21A2, CYP17A1, CYP17B1, and HSD3B2). Secondarily, we analysed prevalence studies; their sample size varied from 53 to 26,000 individuals. AT prevalence among CAH was of 13.3-20%. CAH prevalence among individuals with previous imaging diagnosis of AT was of 0.3-3.6%. Overall, this 10-year, sample-based analysis represents one of the most complex studies in the area of CAH-ATs so far. These masses should be taken into consideration. They may reach impressive sizes of up to 30-40 cm, with compressive effects. Adrenalectomy was chosen based on an individual multidisciplinary decision. Many tumours are detected in subjects with a poor disease control, or they represent the first step toward CAH identification. We noted a left lateralization with a less clear pathogenic explanation. The most frequent tumour remains myelolipoma. The risk of adrenocortical carcinoma should not be overlooked. Noting the increasing prevalence of adrenal incidentalomas, CAH testing might be indicated to identify non-classical forms of CAH.
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Affiliation(s)
- Mara Carsote
- Department of Endocrinology, Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania;
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
| | - Ana-Maria Gheorghe
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
- Ph.D. Doctoral School of Carol Davila, University of Medicine and Pharmacy, 020021 Bucharest, Romania;
| | - Claudiu Nistor
- Department 4—Cardio-Thoracic Pathology, Thoracic Surgery II Discipline, Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania;
- Thoracic Surgery Department, “Dr. Carol Davila” Central Emergency University Military Hospital, 020021 Bucharest, Romania
| | - Alexandra-Ioana Trandafir
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
- Ph.D. Doctoral School of Carol Davila, University of Medicine and Pharmacy, 020021 Bucharest, Romania;
| | - Oana-Claudia Sima
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
- Ph.D. Doctoral School of Carol Davila, University of Medicine and Pharmacy, 020021 Bucharest, Romania;
| | - Anca-Pati Cucu
- Ph.D. Doctoral School of Carol Davila, University of Medicine and Pharmacy, 020021 Bucharest, Romania;
- Thoracic Surgery Department, “Dr. Carol Davila” Central Emergency University Military Hospital, 020021 Bucharest, Romania
| | - Adrian Ciuche
- Department 4—Cardio-Thoracic Pathology, Thoracic Surgery II Discipline, Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania;
- Thoracic Surgery Department, “Dr. Carol Davila” Central Emergency University Military Hospital, 020021 Bucharest, Romania
| | - Eugenia Petrova
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
- Department of Endocrinology, Faculty of Midwifery and Nursing, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania
| | - Adina Ghemigian
- Clinical Endocrinology Department, C.I. Parhon National Institute of Endocrinology, 020021 Bucharest, Romania; (A.-I.T.); (O.-C.S.); (E.P.); (A.G.)
- Department of Endocrinology, Faculty of Midwifery and Nursing, Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania
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Fanis P, Skordis N, Toumba M, Picolos M, Tanteles GA, Neocleous V, Phylactou LA. The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes. Front Endocrinol (Lausanne) 2023; 14:1156616. [PMID: 37324257 PMCID: PMC10266209 DOI: 10.3389/fendo.2023.1156616] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Accepted: 05/04/2023] [Indexed: 06/17/2023] Open
Abstract
Objective The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). Methods 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Results Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). Conclusion The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.
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Affiliation(s)
- Pavlos Fanis
- Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Nicos Skordis
- Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- Division of Paediatric Endocrinology, Paedi Center for specialized Paediatrics, Nicosia, Cyprus
- School of Medicine, University of Nicosia, Nicosia, Cyprus
| | - Meropi Toumba
- Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
- Department of Paediatrics, Paediatric Endocrinology Clinic, Aretaeio Hospital, Nicosia, Cyprus
| | - Michalis Picolos
- Department of Endocrinology, Alithias Endocrinology Center, Nicosia, Cyprus
| | - George A. Tanteles
- Department of Clinical Genetics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Vassos Neocleous
- Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Leonidas A. Phylactou
- Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
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9
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Arriba M, Ezquieta B. Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency: A Practical Approach. Front Endocrinol (Lausanne) 2022; 13:834549. [PMID: 35422767 PMCID: PMC9001848 DOI: 10.3389/fendo.2022.834549] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Accepted: 02/28/2022] [Indexed: 11/18/2022] Open
Abstract
Adrenal insufficiency in paediatric patients is mostly due to congenital adrenal hyperplasia (CAH), a severe monogenic disease caused by steroid 21-hydroxylase deficiency (21-OHD, encoded by the CYP21A2 gene) in 95% of cases. CYP21A2 genotyping requires careful analyses that guaranty gene-specific PCR, accurate definition of pseudogene-gene chimeras, gene duplications and allele dropout avoidance. A small panel of well-established disease-causing alterations enables a high diagnostic yield in confirming/discarding the disorder not only in symptomatic patients but also in those asymptomatic with borderline/positive results of 17-hydroxyprogesterone. Unfortunately, the complexity of this locus makes it today reluctant to high throughput techniques of massive sequencing. The strong relationship existing between the molecular alterations and the degree of enzymatic deficiency has allowed genetic studies to demonstrate its usefulness in predicting/classifying the clinical form of the disease. Other aspects of interest regarding molecular studies include its independence of physiological variations and analytical interferences, its usefulness in the diagnosis of simple virilizing forms in males and its inherent contribution to the genetic counseling, an aspect of great importance taking into account the high carrier frequency of CAH in the general population. Genetic testing of CYP21A2 constitutes an irreplaceable tool to detect severe alleles not just in family members of classical forms but also in mild late-onset forms of the disease and couples. It is also helpful in areas such as assisted reproduction and preimplantation diagnosis. Molecular diagnosis of 21-OHD under expert knowledge definitely contributes to a better management of the disease in every step of the clinical course.
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Affiliation(s)
- María Arriba
- Molecular Diagnostics Laboratory, Department of Laboratory Medicine, Hospital General Universitario Gregorio Marañón, Madrid, Spain
- Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain
| | - Begoña Ezquieta
- Molecular Diagnostics Laboratory, Department of Laboratory Medicine, Hospital General Universitario Gregorio Marañón, Madrid, Spain
- Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain
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10
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Claahsen - van der Grinten HL, Speiser PW, Ahmed SF, Arlt W, Auchus RJ, Falhammar H, Flück CE, Guasti L, Huebner A, Kortmann BBM, Krone N, Merke DP, Miller WL, Nordenström A, Reisch N, Sandberg DE, Stikkelbroeck NMML, Touraine P, Utari A, Wudy SA, White PC. Congenital Adrenal Hyperplasia-Current Insights in Pathophysiology, Diagnostics, and Management. Endocr Rev 2022; 43:91-159. [PMID: 33961029 PMCID: PMC8755999 DOI: 10.1210/endrev/bnab016] [Citation(s) in RCA: 258] [Impact Index Per Article: 86.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Indexed: 11/19/2022]
Abstract
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders affecting cortisol biosynthesis. Reduced activity of an enzyme required for cortisol production leads to chronic overstimulation of the adrenal cortex and accumulation of precursors proximal to the blocked enzymatic step. The most common form of CAH is caused by steroid 21-hydroxylase deficiency due to mutations in CYP21A2. Since the last publication summarizing CAH in Endocrine Reviews in 2000, there have been numerous new developments. These include more detailed understanding of steroidogenic pathways, refinements in neonatal screening, improved diagnostic measurements utilizing chromatography and mass spectrometry coupled with steroid profiling, and improved genotyping methods. Clinical trials of alternative medications and modes of delivery have been recently completed or are under way. Genetic and cell-based treatments are being explored. A large body of data concerning long-term outcomes in patients affected by CAH, including psychosexual well-being, has been enhanced by the establishment of disease registries. This review provides the reader with current insights in CAH with special attention to these new developments.
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Affiliation(s)
| | - Phyllis W Speiser
- Cohen Children’s Medical Center of NY, Feinstein Institute, Northwell Health, Zucker School of Medicine, New Hyde Park, NY 11040, USA
| | - S Faisal Ahmed
- Developmental Endocrinology Research Group, School of Medicine Dentistry & Nursing, University of Glasgow, Glasgow, UK
| | - Wiebke Arlt
- Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
- Department of Endocrinology, Queen Elizabeth Hospital, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
| | - Richard J Auchus
- Division of Metabolism, Endocrinology, and Diabetes, Departments of Internal Medicine and Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Henrik Falhammar
- Department of Molecular Medicine and Surgery, Karolinska Intitutet, Stockholm, Sweden
- Department of Endocrinology, Karolinska University Hospital, Stockholm, Sweden
| | - Christa E Flück
- Pediatric Endocrinology, Diabetology and Metabolism, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland
| | - Leonardo Guasti
- Centre for Endocrinology, William Harvey Research Institute, Bart’s and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK
| | - Angela Huebner
- Division of Paediatric Endocrinology and Diabetology, Department of Paediatrics, Universitätsklinikum Dresden, Technische Universität Dresden, Dresden, Germany
| | - Barbara B M Kortmann
- Radboud University Medical Centre, Amalia Childrens Hospital, Department of Pediatric Urology, Nijmegen, The Netherlands
| | - Nils Krone
- Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Deborah P Merke
- National Institutes of Health Clinical Center and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
| | - Walter L Miller
- Department of Pediatrics, Center for Reproductive Sciences, and Institute for Human Genetics, University of California, San Francisco, CA 94143, USA
| | - Anna Nordenström
- Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden
- Pediatric Endocrinology, Karolinska University Hospital, Stockholm, Sweden
| | - Nicole Reisch
- Medizinische Klinik IV, Klinikum der Universität München, Munich, Germany
| | - David E Sandberg
- Department of Pediatrics, Susan B. Meister Child Health Evaluation and Research Center, University of Michigan, Ann Arbor, MI 48109, USA
| | | | - Philippe Touraine
- Department of Endocrinology and Reproductive Medicine, Center for Rare Endocrine Diseases of Growth and Development, Center for Rare Gynecological Diseases, Hôpital Pitié Salpêtrière, Sorbonne University Medicine, Paris, France
| | - Agustini Utari
- Division of Pediatric Endocrinology, Department of Pediatrics, Faculty of Medicine, Diponegoro University, Semarang, Indonesia
| | - Stefan A Wudy
- Steroid Research & Mass Spectrometry Unit, Laboratory of Translational Hormone Analytics, Division of Paediatric Endocrinology & Diabetology, Justus Liebig University, Giessen, Germany
| | - Perrin C White
- Division of Pediatric Endocrinology, UT Southwestern Medical Center, Dallas TX 75390, USA
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11
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12
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Xi N, Song X, Wang XY, Qin SF, He GN, Sun LL, Chen XM. 2+0 CYP21A2 deletion carrier — a limitation of the genetic testing and counseling: A case report. World J Clin Cases 2021; 9:6789-6797. [PMID: 34447826 PMCID: PMC8362542 DOI: 10.12998/wjcc.v9.i23.6789] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Revised: 03/04/2021] [Accepted: 06/28/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND CYP21A2 gene mutations may all cause reduction or loss of 21-hydroxylase activity, leading to development of congenital adrenal hyperplasia (CAH) with different clinical phenotypes. For families with CAH children, genetic testing of the parents and genetic counseling are recommended to assess the risk of recurrence.
CASE SUMMARY We report a case of CAH with a high suspicion before delivery. The risk of the child suffering from CAH during the pregnancy had been underestimated due to the deviation of genetic counseling and genetic testing results. Our report confirmed a CYP21A2 homozygous deletion in this case, CYP21A2 heterozygous deletion in the mother, and a rare 2+0 CYP21A2 deletion in the father.
CONCLUSION It is important to analyze the distribution of CYP21A2 gene in the two alleles of parents of children with CAH.
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Affiliation(s)
- Na Xi
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Xiao Song
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Xue-Yan Wang
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Sheng-Fang Qin
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Guan-Nan He
- Department of Ultrasound, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Ling-Ling Sun
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
| | - Xi-Min Chen
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Hospital for Women and Children, Chengdu 610045, Sichuan Province, China
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Comprehensive Genetic Testing of CYP21A2: A Retrospective Analysis in Patients with Suspected Congenital Adrenal Hyperplasia. J Clin Med 2021; 10:jcm10061183. [PMID: 33809035 PMCID: PMC8001222 DOI: 10.3390/jcm10061183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 03/01/2021] [Accepted: 03/09/2021] [Indexed: 11/17/2022] Open
Abstract
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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Carrozza C, Foca L, De Paolis E, Concolino P. Genes and Pseudogenes: Complexity of the RCCX Locus and Disease. Front Endocrinol (Lausanne) 2021; 12:709758. [PMID: 34394006 PMCID: PMC8362596 DOI: 10.3389/fendo.2021.709758] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2021] [Accepted: 07/19/2021] [Indexed: 11/13/2022] Open
Abstract
Copy Number Variations (CNVs) account for a large proportion of human genome and are a primary contributor to human phenotypic variation, in addition to being the molecular basis of a wide spectrum of disease. Multiallelic CNVs represent a considerable fraction of large CNVs and are strictly related to segmental duplications according to their prevalent duplicate alleles. RCCX CNV is a complex, multiallelic and tandem CNV located in the major histocompatibility complex (MHC) class III region. RCCX structure is typically defined by the copy number of a DNA segment containing a series of genes - the serine/threonine kinase 19 (STK19), the complement 4 (C4), the steroid 21-hydroxylase (CYP21), and the tenascin-X (TNX) - lie close to each other. In the Caucasian population, the most common RCCX haplotype (69%) consists of two segments containing the genes STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB, with a telomere-to-centromere orientation. Nonallelic homologous recombination (NAHR) plays a key role into the RCCX genetic diversity: unequal crossover facilitates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers. The results of these events increased the RCCX genetic diversity and are responsible of specific human diseases. This review provides an overview on RCCX complexity pointing out the molecular bases of Congenital Adrenal Hyperplasia (CAH) due to CYP21A2 deficiency, CAH-X Syndrome and disorders related to CNV of complement component C4.
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Affiliation(s)
- Cinzia Carrozza
- Dipartimento di Scienze di Laboratorio e Infettivologiche, UOC Chimica, Biochimica e Biologia Molecolare Clinica, Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Roma, Italy
- Dipartimento di Scienze Biotecnologiche di base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Roma, Italy
| | - Laura Foca
- Dipartimento di Scienze di Laboratorio e Infettivologiche, UOC Chimica, Biochimica e Biologia Molecolare Clinica, Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Roma, Italy
| | - Elisa De Paolis
- Dipartimento di Scienze di Laboratorio e Infettivologiche, UOC Chimica, Biochimica e Biologia Molecolare Clinica, Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Roma, Italy
| | - Paola Concolino
- Dipartimento di Scienze di Laboratorio e Infettivologiche, UOC Chimica, Biochimica e Biologia Molecolare Clinica, Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Roma, Italy
- *Correspondence: Paola Concolino,
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15
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EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency. Eur J Hum Genet 2020; 28:1341-1367. [PMID: 32616876 PMCID: PMC7609334 DOI: 10.1038/s41431-020-0653-5] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 05/05/2020] [Accepted: 05/13/2020] [Indexed: 11/25/2022] Open
Abstract
Molecular genetic testing for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is offered worldwide and is of importance for differential diagnosis, carrier detection and adequate genetic counseling, particularly for family planning. In 2008 the European Molecular Genetics Quality Network (EMQN) for the first time offered a European-wide external quality assessment scheme for CAH (due to 21-OH deficiency). The interest was great and over the last years at about 60 laboratories from Europe, USA and Australia regularly participated in that scheme. These best practice guidelines were drafted on the basis of the extensive knowledge and experience got from those annually organized CAH-schemes. In order to obtain the widest possible consultation with practicing laboratories the draft was therefore circulated twice by EMQN to all laboratories participating in the EQA-scheme for CAH genotyping and was updated by that input. The present guidelines address quality requirements for diagnostic molecular genetic laboratories, as well as criteria for CYP21A2 genotyping (including carrier-testing and prenatal diagnosis). A key aspect of that article is the use of appropriate methodologies (e.g., sequencing methods, MLPA (multiplex ligation dependent probe amplification), mutation specific assays) and respective limitations and analytical accuracy. Moreover, these guidelines focus on classification of variants, and the interpretation and standardization of the reporting of CYP21A2 genotyping results. In addition, the article provides a comprehensive list of common as well as so far unreported CYP21A2-variants.
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16
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Fernández CS, Taboas M, Bruque CD, Benavides-Mori B, Belli S, Stivel M, Oneto A, Pasqualini T, Delea M, Espeche LD, Kolomenski JE, Alba L, Buzzalino N, Dain L. Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency. Clin Endocrinol (Oxf) 2020; 93:19-27. [PMID: 32289882 DOI: 10.1111/cen.14190] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 03/31/2020] [Accepted: 04/07/2020] [Indexed: 01/25/2023]
Abstract
CONTEXT 21-hydroxylase deficiency is the most common cause of Congenital Adrenal Hyperplasia. It presents as severe or classical forms-salt wasting and simple virilizing-and a mild or nonclassical (NC). Several studies have reported the frequency of pathogenic variants in different populations, although few of them included a large number of NC patients. OBJECTIVE To analyse the CYP21A2 gene defects in a large cohort of Argentine patients. DESIGN Molecular characterization of 628 patients (168 classical, 460 nonclassical, representing 1203 nonrelated alleles), 398 relatives, 126 partners. METHODS Genetic variants were assessed by allele-specific PCR, PCR-RFLP or direct sequencing. Deletions, duplications and large gene conversions (LGC) were studied by Southern blot/MLPA or long-range PCR. Biological implications of novel variants were analysed by structure-based in silico studies. RESULTS The most frequent pathogenic variants were p.V282L (58%) in NC alleles and c.293-13C>G (31.8%) and p.I173N (21.1%) in classical. Deletions and LGC were found at low frequency (6.2%), 57 alleles had rare pathogenic variants, and 3 had novel variants: p.(S166F); p.(P189R), p.(R436L). Genotype-phenotype correlation was observed in 98.6% of the cases, 11 asymptomatic first-degree relatives had pathogenic variants in both alleles, and 21/126 partners were carriers. CONCLUSIONS We conducted a comprehensive genetic characterization of the largest cohort of 21-hydroxylase patients from the region. In particular, we add to the molecular characterization of a large number of NC patients and to the estimation of the disease carrier's frequency in our population.
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Affiliation(s)
- Cecilia S Fernández
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
- Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Melisa Taboas
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Carlos D Bruque
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
- Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Belén Benavides-Mori
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Susana Belli
- División Endocrinología, Hospital Durand, Buenos Aires, Argentina
| | - Mirta Stivel
- División Endocrinología, Hospital Durand, Buenos Aires, Argentina
| | - Adriana Oneto
- División Endocrinología, Hospital Durand, Buenos Aires, Argentina
| | - Titania Pasqualini
- Sección Endocrinología, Crecimiento y Desarrollo, Departamento de Pediatría, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina
| | - Marisol Delea
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Lucía D Espeche
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Jorge E Kolomenski
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina
| | - Liliana Alba
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Noemí Buzzalino
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
| | - Liliana Dain
- Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) 'Dr. Carlos G. Malbrán', Buenos Aires, Argentina
- Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina
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A rare CYP21A2 haplotype clarifies the phenotype-genotype discrepancy in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). Mol Biol Rep 2020; 47:3049-3052. [PMID: 32185686 DOI: 10.1007/s11033-020-05379-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Accepted: 03/12/2020] [Indexed: 10/24/2022]
Abstract
RCCX haplotypes with two copies of the CYP21A2 gene and one copy of the CYP21A1P pseudogene have been widely described in different populations. In most cases, the CYP21A2-like gene downstream of the TNXA gene showed a wild-type sequence or the c.293-13A/C > G variant while the CYP21A2 gene next to TNXB carried the p.(Gln319Ter) variant. Here is the discovery of a novel rare CYP21A2 haplotypes detected in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). The molecular family study was performed clarifying the previously found phenotype-genotype discrepancy.
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18
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Carvalho B, Marques CJ, Santos-Silva R, Fontoura M, Carvalho D, Carvalho F. Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency: An Update on Genetic Analysis of CYP21A2 Gene. Exp Clin Endocrinol Diabetes 2020; 129:477-481. [PMID: 32131114 DOI: 10.1055/a-1108-1419] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Congenital Adrenal Hyperplasia is a group of genetic autosomal recessive disorders that affects adrenal steroidogenesis in the adrenal cortex. One of the most common defects associated with Congenital Adrenal Hyperplasia is the deficiency of 21-hydroxylase enzyme, responsible for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. The impairment of cortisol and aldosterone production is directly related to the clinical form of the disease that ranges from classic or severe to non-classic or mild late onset. The deficiency of 21-hydroxylase enzyme results from pathogenic variants on CYP21A2 gene that, in the majority of the cases, compromise enzymatic activity and are strongly correlated with the clinical severity of the disease. Due to the exceptionally high homology and proximity between the gene and the pseudogene, more than 90% of pathogenic variants result from intergenic recombination. Around 75% are deleterious variants transferred from the pseudogene by gene conversion, during mitosis. About 20% are due to unequal crossing over during meiosis and lead to duplications or deletions on CYP21A2 gene. Molecular genetic analysis of CYP21A2 variants is of major importance for confirmation of clinical diagnosis, predicting prognosis and for an appropriate genetic counselling. In this review we will present an update on the genetic analysis of CYP21A2 gene variants in CAH patients performed in our department.
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Affiliation(s)
- Berta Carvalho
- Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.,i3S, Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal
| | - C Joana Marques
- Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.,i3S, Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal
| | - Rita Santos-Silva
- Department of Pediatrics, Centro Hospitalar Universitário S. João, Porto, Portugal
| | - Manuel Fontoura
- Department of Pediatrics, Centro Hospitalar Universitário S. João, Porto, Portugal
| | - Davide Carvalho
- i3S, Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal.,Department of Endocrinology, Diabetes e Metabolism, Centro Hospitalar Universitário S. João, Faculty of Medicine, Porto, Portugal
| | - Filipa Carvalho
- Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.,i3S, Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal
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19
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Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression. Hum Genet 2019; 139:137-149. [PMID: 31786673 DOI: 10.1007/s00439-019-02093-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Accepted: 11/23/2019] [Indexed: 12/13/2022]
Abstract
Structural variants including copy number variations (CNV) have gained widespread attention, especially in pharmacogenomics but for several genes functional relevance and clinical evidence are still lacking. Detection of CNVs in next-generation sequencing data is challenging but offers widespread applications. We developed a cohort-based CNV detection workflow to extract CNVs from read counts of targeted NGS of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. We applied our method to 150 human liver tissue samples and correlated identified CNVs to mRNA expression levels. In total, we identified 445 deletions (73%) and 167 duplications (27%) in 36 pharmacogenes including all well-known CNVs of CYPs, GSTs, SULTs, UGTs, numerous described rare CNVs of CYP2E1, SLC16A3 or UGT2B15 as well as novel observations, e.g., for SLC22A12, SLC22A17 and GPS2 (G Protein Pathway Suppressor 2). We were able to fine-map complex CNVs of CYP2A6 and CYP2D6 with exon resolution. Correlation analysis confirmed known expression patterns for common CNVs and suggested an influence on expression variability for some rare CNVs. Our straightforward CNV detection workflow can be easily applied to any NGS coverage data and helped to analyze CNVs in an ADME-NGS panel of 340 pharmacogenes to improve genotype-phenotype correlations.
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Neves Cruz J, da Costa KS, de Carvalho TAA, de Alencar NAN. Measuring the structural impact of mutations on cytochrome P450 21A2, the major steroid 21-hydroxylase related to congenital adrenal hyperplasia. J Biomol Struct Dyn 2019; 38:1425-1434. [PMID: 30982438 DOI: 10.1080/07391102.2019.1607560] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Congenital adrenal hyperplasia is an inherited autosomal recessive disorder related to deficient cortisol synthesis. The deficiency of steroid 21-hydroxylase (cytochrome P450 21A2), an enzyme involved in cortisol synthesis, is responsible for ∼95% of cases of congenital adrenal hyperplasia. This metabolic disease exhibits three clinical forms: salt-wasting, simple virilizing, and non-classical form, which are divided according to the degree of severity. In the present study, structural and mutational analyses were performed in order to identify the structural impact of mutations on cytochrome P450 21A2 and correlate them with patient clinical severity. The following mutations were selected: arginine-356 to tryptophan (R356W), proline-30 to leucine (P30L), isoleucine-172 to asparagine (I172N), valine-281 to leucine (V281L), and the null mutation glutamine-318 (Q318X). Our computational approach mapped the location of residues on P450 and identified their implications on enzyme electrostatic potential mapping to progesterone and heme binding pockets. Using molecular dynamics simulations, we analyzed the structural stability of ligand binding and protein structure, as well as possible conformational changes at the catalytic pocket that leads to impairment of enzymatic activity. Our study sheds light on the impact structural mutations have over steroid 21-hydroxylase structure-function in the cell.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Jorddy Neves Cruz
- Faculty of Pharmaceutical Sciences, University of the Amazon, Belém, Brazil.,Institute of Natural Sciences, Federal University of Pará, Belém, Brazil
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21
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Pignatelli D, Carvalho BL, Palmeiro A, Barros A, Guerreiro SG, Macut D. The Complexities in Genotyping of Congenital Adrenal Hyperplasia: 21-Hydroxylase Deficiency. Front Endocrinol (Lausanne) 2019; 10:432. [PMID: 31333583 PMCID: PMC6620563 DOI: 10.3389/fendo.2019.00432] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Accepted: 06/17/2019] [Indexed: 12/13/2022] Open
Abstract
The deficiency of 21-hydroxylase due to CYP21A2 pathogenic variants is a rather frequent disease with serious consequences, going from a real mortality risk to infertility and to milder symptoms, nevertheless important for affecting the patients' self-esteem. In the most severe cases life-threatening adrenal salt wasting crises may occur. Significant morbidity including the possibility of mistaken gender determination, precocious puberty, infertility and growth arrest with consequent short stature may also affect these patients. In the less severe cases milder symptoms like hirsutism will likely affect the image of the self with strong psychological consequences. Its diagnosis is confirmed by 17OH-progesterone dosages exceeding the cut-off value of 10/15 ng/ml but genotyping is progressively assuming an essential role in the study of these patients particularly in confirming difficult cases, determining some aspects of the prognosis and allowing a correct genetic counseling. Genotyping is a difficult process due to the occurrence of both a gene and a highly homologous pseudo gene. However, new tools are opening new possibilities to this analysis and improving the chances of a correct diagnosis and better understanding of the underlying mechanisms of the disease. Beyond the 10 classic pathogenic variants usually searched for in most laboratories, a correct analysis of 21OH-deficiency cases implies completely sequencing of the entire gene and the determination of gene duplications. These are now recognized to occur frequently and can be responsible for some false positive cases. And finally, because gene conversions can include several pathogenic variants one cannot be certain of identifying that both alleles are affected without studying parental DNA samples. A complete genotype characterization should be considered essential in the preparation for pregnancy, even in the case of parents with milder forms of the disease, or even just carriers, since it has been reported that giving birth to progeny with the severe classic forms occurs with a much higher frequency than expected.
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Affiliation(s)
- Duarte Pignatelli
- Hospital S. João, Porto, Portugal
- Department of Biomedicine, Faculty of Medicine of Porto, Porto, Portugal
- IPATIMUP/I3S Research Institute, University of Porto, Porto, Portugal
- *Correspondence: Duarte Pignatelli
| | - Berta L. Carvalho
- Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal
- I3S Research Institute, University of Porto, Porto, Portugal
| | | | - Alberto Barros
- Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal
- I3S Research Institute, University of Porto, Porto, Portugal
| | - Susana G. Guerreiro
- Department of Biomedicine, Faculty of Medicine of Porto, Porto, Portugal
- IPATIMUP/I3S Research Institute, University of Porto, Porto, Portugal
| | - Djuro Macut
- Clinic of Endocrinology, Diabetes and Metabolic Diseases, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
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Kang Y, Nam SH, Park KS, Kim Y, Kim JW, Lee E, Ko JM, Lee KA, Park I. DeviCNV: detection and visualization of exon-level copy number variants in targeted next-generation sequencing data. BMC Bioinformatics 2018; 19:381. [PMID: 30326846 PMCID: PMC6192323 DOI: 10.1186/s12859-018-2409-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2018] [Accepted: 10/04/2018] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Targeted next-generation sequencing (NGS) is increasingly being adopted in clinical laboratories for genomic diagnostic tests. RESULTS We developed a new computational method, DeviCNV, intended for the detection of exon-level copy number variants (CNVs) in targeted NGS data. DeviCNV builds linear regression models with bootstrapping for every probe to capture the relationship between read depth of an individual probe and the median of read depth values of all probes in the sample. From the regression models, it estimates the read depth ratio of the observed and predicted read depth with confidence interval for each probe which is applied to a circular binary segmentation (CBS) algorithm to obtain CNV candidates. Then, it assigns confidence scores to those candidates based on the reliability and strength of the CNV signals inferred from the read depth ratios of the probes within them. Finally, it also provides gene-centric plots with confidence levels of CNV candidates for visual inspection. We applied DeviCNV to targeted NGS data generated for newborn screening and demonstrated its ability to detect novel pathogenic CNVs from clinical samples. CONCLUSIONS We propose a new pragmatic method for detecting CNVs in targeted NGS data with an intuitive visualization and a systematic method to assign confidence scores for candidate CNVs. Since DeviCNV was developed for use in clinical diagnosis, sensitivity is increased by the detection of exon-level CNVs.
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Affiliation(s)
- Yeeok Kang
- SD Genomics Co., Ltd., 11F, Seoul Gangnam Post Office, 619 Gaepo-ro, Gangnam-gu, Seoul, 06336 Republic of Korea
- Department of Bio and Brain Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Seong-Hyeuk Nam
- SD Genomics Co., Ltd., 11F, Seoul Gangnam Post Office, 619 Gaepo-ro, Gangnam-gu, Seoul, 06336 Republic of Korea
| | - Kyung Sun Park
- SD Genomics Co., Ltd., 11F, Seoul Gangnam Post Office, 619 Gaepo-ro, Gangnam-gu, Seoul, 06336 Republic of Korea
| | - Yoonjung Kim
- Department of Laboratory Medicine, Yonsei University College of Medicine, 211 Eonjuro, Gangnam-gu, Seoul, 06273 Republic of Korea
| | - Jong-Won Kim
- Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
| | - Eunjung Lee
- Division of Genetics and Genomics, Boston Children’s Hospital and Harvard Medical School, Boston, USA
| | - Jung Min Ko
- Department of Pediatrics, Seoul National University Children’s Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Kyung-A Lee
- Department of Laboratory Medicine, Yonsei University College of Medicine, 211 Eonjuro, Gangnam-gu, Seoul, 06273 Republic of Korea
| | - Inho Park
- SD Genomics Co., Ltd., 11F, Seoul Gangnam Post Office, 619 Gaepo-ro, Gangnam-gu, Seoul, 06336 Republic of Korea
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23
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Su L, Yin X, Cheng J, Cai Y, Wu D, Feng Z, Liu L. Clinical presentation and mutational spectrum in a series of 166 patients with classical 21-hydroxylase deficiency from South China. Clin Chim Acta 2018; 486:142-150. [PMID: 30048636 DOI: 10.1016/j.cca.2018.07.039] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Revised: 07/09/2018] [Accepted: 07/22/2018] [Indexed: 10/28/2022]
Abstract
Classical 21-hydroxylase deficiency (21-OHD) due to mutations in the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene is the most common type of congenital adrenal hyperplasia (CAH). In this study, we analyzed clinical and molecular data of 166 patients with classical CAH in South China. Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) method were used to detect mutations in these 99 salt wasting (SW) patients and 67 simple virilizing (SV) patients. Micro-conversion mutation IVS2-13A/C > G (I2G) was the most frequent mutation in both SW form (42.9%) and SV form (41.8%) in our large cohort, and large gene deletion or large gene conversion also commonly resulted in classical CAH. Rare mutations only account for 8.4% of all alleles, among them four novel variants p.S126X, p.C429X, c.1209_1210insT and c.840delG were responsible for the clinical presentations. CYP21A2 gene duplications linked to the mutation Q319X were found in our cohort, though these cases were rather rare. In this study, we provided detailed clinical data and mutation spectrum to confirm the common mutations in Chinese populations, especially in South China,which will contribute to further genetic consultation and prenatal diagnosis. Sanger sequencing combined with MLPA method could detect most mutation types in the CYP21A2 gene effectively.
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Affiliation(s)
- Ling Su
- Southern Medical University, Guangzhou 510515, China; Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
| | - Xi Yin
- Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
| | - Jing Cheng
- Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
| | - Yanna Cai
- Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
| | - Dongyan Wu
- Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
| | - Zhichun Feng
- Southern Medical University, Guangzhou 510515, China; Department of Neonatology, Bayi Children's Hospital Affiliated to PLA Army General Hospital, Beijing 100700, China..
| | - Li Liu
- Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China.
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24
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Dubey S, Tardy V, Chowdhury MR, Gupta N, Jain V, Deka D, Sharma P, Morel Y, Kabra M. Prenatal diagnosis of steroid 21-hydroxylase-deficient congenital adrenal hyperplasia: Experience from a tertiary care centre in India. Indian J Med Res 2018. [PMID: 28639595 PMCID: PMC5501051 DOI: 10.4103/ijmr.ijmr_329_16] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
Background & objectives: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder with a wide range of clinical manifestations. The disease is attributed to mutations in CYP21A2 gene encoding 21-hydroxylase enzyme. In view of severe phenotype in salt-losing cases, issues related to genital ambiguity in girls and precocity in boys, most families opt for prenatal testing and termination of affected foetus. CAH can be diagnosed in utero through direct molecular analysis of CYP21A2 gene, using DNA extracted from foetal tissues or cells obtained from chorionic villus sampling or amniocentesis. The objective of this study was to evaluate the feasibility and accuracy of prenatal diagnosis (PND) using sequencing and multiplex ligation probe amplification (MLPA) methods in families at risk for CAH. Methods: Fifteen pregnant women at risk of having an affected offspring with CAH were included in this study. Ten families had previous affected children with salt-wasting/simple virilising form of CAH and five families did not have live children but had a high index of suspicion for CAH in previous children based on history or records. Mutation analysis was carried out by Sanger sequencing and MLPA method. Results: Seven different mutations were identified in 15 families. Deletions and I2g mutation were the most common. Of the 15 foetuses analyzed, nine were unaffected while six were affected. Unaffected foetuses were delivered, they were clinically normal and their genotype was found to be concordant to the prenatal report. All except two families reported in the second trimester. None of the couples opted for prenatal treatment. Interpretation & conclusions: Our preliminary findings show that PND by direct mutation analysis along with MLPA is a feasible strategy that can be offered to families at risk.
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Affiliation(s)
- Sudhisha Dubey
- Department of Pediatrics, Division of Genetics, All India Institute of Medical Sciences, New Delhi, India
| | - Veronique Tardy
- Department of Molecular Endocrinology and Rare Diseases, Center for Biology and Eastern Pathology, Civil Hospitals of Lyon, Bron Cedex, France
| | - Madhumita Roy Chowdhury
- Department of Pediatrics, Division of Genetics, All India Institute of Medical Sciences, New Delhi, India
| | - Neerja Gupta
- Department of Pediatrics, Division of Genetics, All India Institute of Medical Sciences, New Delhi, India
| | - Vandana Jain
- Division of Pediatric Endocrinology, All India Institute of Medical Sciences, New Delhi, India
| | - Deepika Deka
- Department of Obstetrics & Gynecology, All India Institute of Medical Sciences, New Delhi, India
| | - Pankaj Sharma
- Department of Pediatrics, Division of Genetics, All India Institute of Medical Sciences, New Delhi, India
| | - Yves Morel
- Department of Molecular Endocrinology and Rare Diseases, Center for Biology and Eastern Pathology, Civil Hospitals of Lyon, Bron Cedex, France
| | - Madhulika Kabra
- Department of Pediatrics, Division of Genetics, All India Institute of Medical Sciences, New Delhi, India
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25
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Congenital Adrenal Hyperplasia (CAH) due to 21-Hydroxylase Deficiency: A Comprehensive Focus on 233 Pathogenic Variants of CYP21A2 Gene. Mol Diagn Ther 2018; 22:261-280. [DOI: 10.1007/s40291-018-0319-y] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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26
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Abstract
The congenital adrenal hyperplasias comprise a family of autosomal recessive disorders that disrupt adrenal steroidogenesis. The most common form is due to 21-hydroxylase deficiency associated with mutations in the 21-hydroxylase gene, which is located at chromosome 6p21. The clinical features associated with each disorder of adrenal steroidogenesis represent a clinical spectrum that reflect the consequences of the specific mutations. Treatment goals include normal linear growth velocity and "on-time" puberty in affected children. For adolescent and adult women, treatment goals include regularization of menses, prevention of progression of hirsutism, and preservation of fertility. For adolescent and adult men, prevention and early treatment of testicular adrenal rest tumors is beneficial. In this article key aspects regarding pathophysiology, diagnosis, and treatment of congenital adrenal hyperplasia are reviewed.
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Affiliation(s)
- Selma Feldman Witchel
- Division of Pediatric Endocrinology, Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, Pennsylvania.
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27
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Carmina E, Dewailly D, Escobar-Morreale HF, Kelestimur F, Moran C, Oberfield S, Witchel SF, Azziz R. Non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency revisited: an update with a special focus on adolescent and adult women. Hum Reprod Update 2017; 23:580-599. [DOI: 10.1093/humupd/dmx014] [Citation(s) in RCA: 86] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2016] [Accepted: 05/12/2017] [Indexed: 01/29/2023] Open
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28
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A unique haplotype of RCCX copy number variation: from the clinics of congenital adrenal hyperplasia to evolutionary genetics. Eur J Hum Genet 2017; 25:702-710. [PMID: 28401898 DOI: 10.1038/ejhg.2017.38] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2016] [Revised: 02/08/2017] [Accepted: 02/14/2017] [Indexed: 01/26/2023] Open
Abstract
There is a difficulty in the molecular diagnosis of congenital adrenal hyperplasia (CAH) due to the c.955C>T (p.(Q319*), formerly Q318X, rs7755898) variant of the CYP21A2 gene. Therefore, a systematic assessment of the genetic and evolutionary relationships between c.955C>T, CYP21A2 haplotypes and the RCCX copy number variation (CNV) structures, which harbor CYP21A2, was performed. In total, 389 unrelated Hungarian individuals with European ancestry (164 healthy subjects, 125 patients with non-functioning adrenal incidentaloma and 100 patients with classical CAH) as well as 34 adrenocortical tumor specimens were studied using a set of experimental and bioinformatic methods. A unique, moderately frequent (2%) haplotypic RCCX CNV structure with three repeated segments, abbreviated to LBSASB, harboring a CYP21A2 with a c.955C>T variant in the 3'-segment, and a second CYP21A2 with a specific c.*12C>T (rs150697472) variant in the middle segment occurred in all c.955C>T carriers with normal steroid levels. The second CYP21A2 was free of CAH-causing mutations and produced mRNA in the adrenal gland, confirming its functionality and ability to rescue the carriers from CAH. Neither LBSASB nor c.*12C>T occurred in classical CAH patients. However, CAH-causing CYP21A2 haplotypes with c.955C>T could be derived from the 3'-segment of LBSASB after the loss of functional CYP21A2 from the middle segment. The c.*12C>T indicated a functional CYP21A2 and could distinguish between non-pathogenic and pathogenic genomic contexts of the c.955C>T variant in the studied European population. Therefore, c.*12C>T may be suitable as a marker to avoid this genetic confound and improve the diagnosis of CAH.
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29
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Abulí A, Boada M, Rodríguez-Santiago B, Coroleu B, Veiga A, Armengol L, Barri PN, Pérez-Jurado LA, Estivill X. NGS-Based Assay for the Identification of Individuals Carrying Recessive Genetic Mutations in Reproductive Medicine. Hum Mutat 2016; 37:516-23. [PMID: 26990548 DOI: 10.1002/humu.22989] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Accepted: 03/09/2016] [Indexed: 12/25/2022]
Abstract
Next-generation sequencing (NGS) has the capacity of carrier screening in gamete donation (GD) programs. We have developed and validated an NGS carrier-screening test (qCarrier test) that includes 200 genes associated with 368 disorders (277 autosomal recessive and 37 X-linked). Carrier screening is performed on oocyte donation candidates and the male partner of oocyte recipient. Carriers of X-linked conditions are excluded from the GD program, whereas donors are chosen who do not carry mutations for the same gene/disease as the recipients. The validation phase showed a high sensitivity (>99% sensitivity) detecting all single-nucleotide variants, 13 indels, and 25 copy-number variants included in the validation set. A total of 1,301 individuals were analysed with the qCarrier test, including 483 candidate oocyte donors and 635 receptor couples, 105 females receiving sperm donation, and 39 couples seeking pregnancy. We identified 56% of individuals who are carriers for at least one genetic condition and 1.7% of female donors who were excluded from the program due to a carrier state of X-linked conditions. Globally, 3% of a priori assigned donations had a high reproductive risk that could be minimized after testing. Genetic counselling at different stages is essential for helping to facilitate a successful and healthy pregnancy.
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Affiliation(s)
- Anna Abulí
- Unit of Medical Genomics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain
| | - Montserrat Boada
- Service of Reproduction Medicine, Obstetrics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain
| | | | - Buenaventura Coroleu
- Service of Reproduction Medicine, Obstetrics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain
| | - Anna Veiga
- Service of Reproduction Medicine, Obstetrics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain
| | - Lluís Armengol
- Research and Development Department, qGenomics Laboratory, Barcelona, Spain
| | - Pedro N Barri
- Service of Reproduction Medicine, Obstetrics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain
| | - Luis A Pérez-Jurado
- Genetics Unit, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), Barcelona, Spain.,Hospital del Mar Research Institute (IMIM), Barcelona, Spain.,CIBER in Rare Disorders (CIBERER), Barcelona, Spain
| | - Xavier Estivill
- Unit of Medical Genomics, Department of Obstetrics, Gynaecology and Reproduction, Dexeus Women's Health, Barcelona, Spain.,Genetics Unit, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), Barcelona, Spain.,Hospital del Mar Research Institute (IMIM), Barcelona, Spain.,Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.,CIBER in Epidemiology and Public Health (CIBERESP), Barcelona, Spain.,Experimental Genetics Division, Sidra Medical and Research Centre, Doha, Qatar
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30
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Abstract
Prenatal treatment of congenital adrenal hyperplasia by administering dexamethasone to a woman presumed to be carrying an at-risk fetus remains a controversial experimental treatment. Review of data from animal experimentation and human trials indicates that dexamethasone cannot be considered safe for the fetus. In animals, prenatal dexamethasone decreases birth weight, affects renal, pancreatic beta cell and brain development, increases anxiety and predisposes to adult hypertension and hyperglycemia. In human studies, prenatal dexamethasone is associated with orofacial clefts, decreased birth weight, poorer verbal working memory, and poorer self-perception of scholastic and social competence. Numerous medical societies have cautioned that prenatal treatment of adrenal hyperplasia with dexamethasone is not appropriate for routine clinical practice and should only be done in Institutional Review Board approved, prospective clinical research settings with written informed consent. The data indicate that this treatment is inconsistent with the classic medical ethical maxim to 'first do no harm'.
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Affiliation(s)
- Walter L Miller
- Department of Pediatrics and Center for Reproductive Sciences, University of California, San Francisco, San Francisco CA 94143-0556, USA.
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31
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Lack of genotypephenotype correlation in congenital adrenal hyperplasia due to a CYP21A2-like gene. Clin Chim Acta 2014; 437:48-51. [PMID: 25025300 DOI: 10.1016/j.cca.2014.07.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2014] [Revised: 07/07/2014] [Accepted: 07/07/2014] [Indexed: 11/22/2022]
Abstract
CONTEXT Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. OBJECTIVES We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype-phenotype correlation in individuals of two different families. DESIGN AND METHODS To verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. RESULTS The amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. CONCLUSIONS The strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.
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32
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Lu X, Ye K, Zou K, Chen J. Identification of copy number variation-driven genes for liver cancer via bioinformatics analysis. Oncol Rep 2014; 32:1845-52. [PMID: 25174835 DOI: 10.3892/or.2014.3425] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2014] [Accepted: 06/10/2014] [Indexed: 11/05/2022] Open
Abstract
To screen out copy number variation (CNV)-driven differentially expressed genes (DEGs) in liver cancer and advance our understanding of the pathogenesis, an integrated analysis of liver cancer-related CNV data from The Cancer Genome Atlas (TCGA) and gene expression data from EBI Array Express database were performed. The DEGs were identified by package limma based on the cut-off of |log2 (fold-change)|>0.585 and adjusted p-value<0.05. Using hg19 annotation information provided by UCSC, liver cancer-related CNVs were then screened out. TF-target gene interactions were also predicted with information from UCSC using DAVID online tools. As a result, 25 CNV-driven genes were obtained, including tripartite motif containing 28 (TRIM28) and RanBP-type and C3HC4-type zinc finger containing 1 (RBCK1). In the transcriptional regulatory network, 8 known cancer-related transcription factors (TFs) interacted with 21 CNV-driven genes, suggesting that the other 8 TFs may be involved in liver cancer. These genes may be potential biomarkers for early detection and prevention of liver cancer. These findings may improve our knowledge of the pathogenesis of liver cancer. Nevertheless, further experiments are still needed to confirm our findings.
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Affiliation(s)
- Xiaojie Lu
- Department of Gastroenterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Kun Ye
- Department of Gastroenterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Kailin Zou
- Department of Gastroenterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Jinlian Chen
- Department of Gastroenterology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
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33
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Novel method to characterize CYP21A2 in Florida patients with congenital adrenal hyperplasia and commercially available cell lines. Mol Genet Metab Rep 2014; 1:312-323. [PMID: 27896104 PMCID: PMC5121304 DOI: 10.1016/j.ymgmr.2014.07.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2014] [Revised: 07/08/2014] [Accepted: 07/08/2014] [Indexed: 11/22/2022] Open
Abstract
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder and affects approximately 1 in 15,000 births in the United States. CAH is one of the disorders included on the Newborn Screening (NBS) Recommended Uniform Screening Panel. The commonly used immunological NBS test is associated with a high false positive rate and there is interest in developing second-tier assays to increase screening specificity. Approximately 90% of the classic forms of CAH, salt-wasting and simple virilizing, are due to mutations in the CYP21A2 gene. These include single nucleotide changes, insertions, deletions, as well as chimeric genes involving CYP21A2 and its highly homologous pseudogene CYP21A1P. A novel loci-specific PCR approach was developed to individually amplify the CYP21A2 gene, the nearby CYP21A1P pseudogene, as well as any 30 kb deletion and gene conversion mutations, if present, as single separate amplicons. Using commercially available CAH positive specimens and 14 families with an affected CAH proband, the single long-range amplicon approach demonstrated higher specificity as compared to previously published methods.
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34
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Ma D, Chen Y, Sun Y, Yang B, Cheng J, Huang M, Zhang J, Zhang J, Hu P, Lin Y, Jiang T, Xu Z. Molecular analysis of the CYP21A2 gene in Chinese patients with steroid 21-hydroxylase deficiency. Clin Biochem 2014; 47:455-63. [PMID: 24503005 DOI: 10.1016/j.clinbiochem.2014.01.019] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2013] [Revised: 01/16/2014] [Accepted: 01/20/2014] [Indexed: 11/26/2022]
Abstract
OBJECTIVE 21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired cortisol synthesis. This study aimed to design a reliable and rational approach for identifying mutations in the CYP21A2 gene and to characterize the molecular basis of 21-OHD in 30 Chinese patients. DESIGN AND METHODS Copy number variations were investigated by multiplex ligation-dependent probe amplification (MLPA). Locus-specific polymerase chain reaction (PCR)/restriction endonuclease analysis was then used to verify CYP21A2 rearrangement products and prevent allele dropout. Direct sequencing of rearrangement products was performed to further refine recombination breakpoint locations. Direct sequencing of the entire CYP21A2 gene was used to detect microconversions. RESULTS We successfully characterized 60 CYP21A2 alleles from 30 patients with genetic defects. The most common one was intron 2 splice mutation (38.3%). Eighteen alleles with large gene deletions/conversions were identified, which accounted for nearly one-third (30.0%) of the genetic defects. Among these, three types of CYP21A1P/CYP21A2 chimeric genes (CH-1, CH-2, and CH-4) were characterized. Two novel CYP21A2 rearrangement genes were revealed and further demonstrated to be located downstream of the TNXB gene. CONCLUSIONS Our results indicate that the stepwise diagnostic procedure involving MLPA analysis, locus-specific PCR/restriction endonuclease analysis, and direct DNA sequencing can provide detailed genetic information about Chinese 21-OHD patients, which is helpful for characterizing structural rearrangements of CYP21A2.
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Affiliation(s)
- Dingyuan Ma
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Yulin Chen
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Yun Sun
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Bing Yang
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Jian Cheng
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Meilian Huang
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Jin Zhang
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Jingjing Zhang
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Ping Hu
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Ying Lin
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China
| | - Tao Jiang
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China.
| | - Zhengfeng Xu
- State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China.
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Cavarzere P, Vincenzi M, Teofoli F, Gaudino R, Lauriola S, Maines E, Camilot M, Antoniazzi F. Genotype in the diagnosis of 21-hydroxylase deficiency: who should undergo CYP21A2 analysis? J Endocrinol Invest 2013; 36:1083-9. [PMID: 24081139 DOI: 10.3275/9096] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
AIMS to confirm the diagnosis of 21-hydroxylase deficiency (21-OHD) by the analysis of CYP21A2 gene in infants with clinical and/or biochemical features of 21-OHD in order to clarify which patients to submit to genetic analysis; to analyze the genotype-phenotype concordance in these infants. SUBJECTS AND METHODS We studied 25 children with clinical and/or biochemical features of 21-OHD. All of them and their parents were submitted to genetic analysis of CYP21A2. Patients were classified in 3 groups according to mutations' severity: severe (group A), moderate (group B) or mild (group C). RESULTS CYP21A2 gene mutations were found in 17 children. Whereas all infants of groups A and B presented a classical form of 21- OHD, children of group C had a non-classical form of 21-OHD. Four infants resulted heterozygotes and 4 children were wildtype. A girl clinically presenting a non-classical form of 21-OHD resulted compound heterozygote with one of the mutations not described in literature (R25W) and whose residual enzymatic activity is not already known. All affected children presented a 17-OHP level after ACTH stimulation greater than 100 nmol/l. We found an optimal concordance between 17-OHP levels after ACTH test and genotype. CONCLUSIONS CYP21A2 analysis permitted to confirm the diagnosis of 21-OHD in 68% of our children. To improve this percentage we suggest to perform the CYP21A2 analysis only when 17-OHP after ACTH test is greater than 100 nmol/l. Moreover, we found an optimal genotype-phenotype concordance in the 21-OHD patients.
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Affiliation(s)
- P Cavarzere
- Pediatric Clinic, Department of Life and Reproduction Sciences, University of Verona, Piazzale Scuro 10, 37134 Verona, Italy.
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Comprehensive mutation analysis of the CYP21A2 gene: an efficient multistep approach to the molecular diagnosis of congenital adrenal hyperplasia. J Mol Diagn 2013; 15:745-53. [PMID: 24071710 DOI: 10.1016/j.jmoldx.2013.06.001] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Revised: 05/13/2013] [Accepted: 06/11/2013] [Indexed: 11/21/2022] Open
Abstract
Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of adrenal steroidogenesis caused by mutations in the CYP21A2 gene. Direct comparison of established and novel methodologies of CYP21A2 genetic analysis in a large cohort representing a wide range of genotypes has not been previously reported. We genotyped a cohort of 129 unrelated patients with 21-OHD, along with 145 available parents, using Southern blot (SB) analysis, multiplex ligation-dependent probe amplification (MLPA), PCR-based restriction fragment length polymorphism (RFLP) analysis, multiplex minisequencing and conversion-specific PCR, duplication-specific amplification, and DNA sequencing. CYP21A2 genotyping identified four duplicated CYP21A2 genes (1.53%) and 79 chimeric CYP21A1P/CYP21A2 genes (30.15%). Parental SB data were essential for determining the CYP21 haplotype in three cases, whereas PCR-based RFLP analysis was necessary for MLPA results to be accurately interpreted in the majority of cases. The comparison of different methods in detecting deletion and duplication showed that MLPA with PCR-based RFLP was comparable with SB analysis, with parental data of 100% sensitivity and specificity. DNA sequencing was required for the identification of 16 (6.1%) rare point mutations and determination of clinically significant chimera junction sites. MLPA with PCR-based RFLP analysis is an excellent substitute for SB analysis in detecting CYP21A2 deletion and duplication and a combination of MLPA, PCR-based RFLP, duplication-specific amplification, and DNA sequencing is a convenient and comprehensive strategy for mutation analysis of the CYP21A2 gene in patients with 21-OHD.
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Concolino P, Mello E, Minucci A, Giardina B, Capoluongo E. Genes, pseudogenes and like genes: The case of 21-hydroxylase in Italian population. Clin Chim Acta 2013; 424:85-9. [DOI: 10.1016/j.cca.2013.05.019] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2013] [Revised: 05/15/2013] [Accepted: 05/15/2013] [Indexed: 10/26/2022]
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Yoo Y, Chang MS, Lee J, Cho SY, Park SW, Jin DK, Park HD. Genotype-phenotype correlation in 27 pediatric patients in congenital adrenal hyperplasia due to 21-hydroxylase deficiency in a single center. Ann Pediatr Endocrinol Metab 2013; 18:128-34. [PMID: 24904866 PMCID: PMC4027076 DOI: 10.6065/apem.2013.18.3.128] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Revised: 09/16/2013] [Accepted: 09/30/2013] [Indexed: 11/20/2022] Open
Abstract
PURPOSE The purpose of the study was to evaluate endocrine patterns of patients with congenital adrenal hyperplasia and each gene mutation and to analyze the correlation between each phenotype and genotype. METHODS This was a retrospective study of the patients with congenital adrenal hyperplasia in the pediatric outpatient clinic at the Samsung Medical Center from November 1994 to December 2012. We analyzed the medical records of 27 patients (male, 19; female, 8) with congenital adrenal hyperplasia who had been diagnosed by genetic testing to have 21-hydroxylase deficiency. RESULTS In genetic analysis of 54 alleles from 27 patients, 13 types of mutations were identified. The distribution of 21-hydroxylase deficiency gene mutations revealed that intron 2 splice site (c.293-13A/C>G) mutations and large deletions were the most common, at 31.5% and 22.2% respectively, followed by p.I173N, p.R356W, and p.I172N mutations at 11.1%, 9.3%, and 9.3%, respectively. Other mutations were observed at 1.9-3.7%. No novel mutations were detected. CONCLUSION The analysis of 54 alleles revealed 13 types of mutation. The salt wasting form showed a good correlation between genotype and phenotype, but the simple virilizing and nonclassic forms showed inconsistencies between genotype and phenotype. The distribution of CYP21A2 mutations was evaluated for 21-hydroxylase deficiency patients from a single center. This study provides limited data on mutation spectrum and genotype-phenotype correlation of 21-hydroxylase deficiency in Korea.
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Affiliation(s)
- Yangho Yoo
- Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Mi Sun Chang
- Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Jieun Lee
- Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Sung Yoon Cho
- Department of Pediatrics, Hanyang University Guri Hopistal, Hanyang University College of Medicine, Guri, Korea
| | - Sung Won Park
- Department of Pediatrics, Cheil General Hospital & Woman's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea
| | - Dong-Kyu Jin
- Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Hyung-Doo Park
- Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
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Lee HH. Variants of the CYP21A2 and CYP21A1P genes in congenital adrenal hyperplasia. Clin Chim Acta 2013; 418:37-44. [DOI: 10.1016/j.cca.2012.12.030] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2012] [Revised: 12/30/2012] [Accepted: 12/31/2012] [Indexed: 10/27/2022]
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40
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Toraman B, Ökten A, Kalay E, Karagüzel G, Dinçer T, Açıkgöz EG, Karagüzel A. Investigation of CYP21A2 mutations in Turkish patients with 21-hydroxylase deficiency and a novel founder mutation. Gene 2013; 513:202-8. [DOI: 10.1016/j.gene.2012.10.059] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2012] [Accepted: 10/07/2012] [Indexed: 10/27/2022]
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Erroneous prenatal diagnosis of congenital adrenal hyperplasia owing to a duplication of the CYP21A2 gene. J Perinatol 2013; 33:76-8. [PMID: 23269230 DOI: 10.1038/jp.2012.5] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder where steroidogenesis in the adrenal cortex is impaired. The most common form is caused by 21-hydroxylase deficiency (21OHD). Classical 21OHD is characterized by glucocorticoid and mineralocorticoid deficiency and by overproduction of adrenal androgens. The diagnosis rests on biochemical and genetic analyses. In families with history of CAH, prenatal genetic diagnosis is offered. We herein present a case of an infant whose parents were identified to carry mutations on the CYP21A2 gene. The fetal DNA analysis demonstrated that the fetus carried a paternal exon 8 (Q318X) mutation and a maternal exon 8 (R356X) mutation. The fetus was presumed to be affected with CAH, yet his clinical presentation at birth was not consistent with the diagnosis. Repeated genetic analysis identified a paternal CYP21A2 gene duplication with Q318X mutation on one copy of CYP21A2. We conclude that a duplication of the CYP21A2 gene should be suspected when clinical and hormonal findings do not support the genetic diagnosis. Furthermore, because individuals with Q318X mutation frequently have a duplication of the CYP21A2 gene, when Q318X is detected, it is important to distinguish the severe point mutation in single gene copy alleles from the non-deficient variant in gene-duplicated alleles.
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Chen W, Xu Z, Nishitani M, Van Ryzin C, McDonnell NB, Merke DP. Complement component 4 copy number variation and CYP21A2 genotype associations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hum Genet 2012; 131:1889-94. [PMID: 22886582 DOI: 10.1007/s00439-012-1217-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2012] [Accepted: 08/01/2012] [Indexed: 11/30/2022]
Abstract
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.
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Affiliation(s)
- Wuyan Chen
- Laboratory of Clinical Investigation, National Institute on Aging, Baltimore, MD, USA.
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Malikova J, Votava F, Vrzalova Z, Lebl J, Cinek O. Genetic analysis of the CYP21A2 gene in neonatal dried blood spots from children with transiently elevated 17-hydroxyprogesterone. Clin Endocrinol (Oxf) 2012; 77:187-94. [PMID: 22313422 DOI: 10.1111/j.1365-2265.2012.04358.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Neonatal screening for congenital adrenal hyperplasia (CAH) identifies a certain proportion of newborns with transient moderate elevation of 17-hydroxyprogesterone (17-OHP). These children require regular follow-up until normalization of their 17-OHP levels. We investigated the possibility of reducing the individuals' recall rates by using genetic methods on their original neonatal dried blood spots. PATIENTS AND METHODS We analysed neonatal dried blood spots from 753 subjects with transiently elevated levels of 17-OHP. The CYP21A2 gene was sequenced to detect point mutations, and the presence of CYP21A2 was further confirmed by two methods utilizing the difference between CYP21A2 and its CYP21A1P pseudogene in the sequence of exon 3 (8-bp deletion). The accuracy of the methods was verified using samples from 70 subjects with known CYP21A2 mutations and 181 healthy children. RESULT Among the 701 successfully sequenced samples from subjects with transiently elevated 17-OHP, 670 (95%) had no point mutations or novel variants in the CYP21A2 gene. We found no individuals carrying genotypes consistent with the diagnosis of CAH (i.e. homozygotes or compound heterozygotes for point mutations, large deletions or rearrangements). However, 21 heterozygous carriers of known point mutations that cause the classic and nonclassic forms of CAH were identified. Additionally, we detected eight heterozygous and two homozygous point variants with unknown functional significance. CONCLUSION Although CAH caused by 21-hydroxylase deficiency could be genetically excluded with a reasonable degree of confidence in 95% of the genotyped subjects that had transiently elevated 17-OHP, the performance of the tests was suboptimal when performed using dried blood spots and time-consuming in comparison with the current practice of repeated measurements of 17-OHP. The introduction of this method into clinical practice seems to be impractical at this stage.
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Affiliation(s)
- Jana Malikova
- Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
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Tsai LP, Lee HH. Analysis of CYP21A1P and the duplicated CYP21A2 genes. Gene 2012; 506:261-2. [PMID: 22771554 DOI: 10.1016/j.gene.2012.06.045] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2012] [Accepted: 06/18/2012] [Indexed: 11/19/2022]
Abstract
The RCCX module on chromosome 6p21.3 has 3 possible forms: monomodular, bimodular, and trimodular. Chromosomes with 4 RCCX modules are very rare. In the monomodule, most of the CYP21A1P genes do not exist. However, haplotypes of the RCCX module with more than one CYP21A2 gene were observed. Obviously, the gene located downstream of the XA gene can possibly include the CYP21A2 as well as the CYP21A1P gene.
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Witchel SF, Miller WL. Prenatal Treatment of Congenital Adrenal Hyperplasia—Not Standard of Care. J Genet Couns 2012; 21:615-24. [DOI: 10.1007/s10897-012-9508-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2011] [Accepted: 05/01/2012] [Indexed: 10/28/2022]
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Ji W, Li J, Liu J. A Method for Measuring Gene Copy Number in Biological Samples without Using Control Samples of Known Copies. J Mol Diagn 2012; 14:280-5. [DOI: 10.1016/j.jmoldx.2012.01.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2011] [Revised: 12/18/2011] [Accepted: 01/18/2012] [Indexed: 10/28/2022] Open
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Huidobro Fernández B, Roldán Martín M, Rodríguez Arnao M, Ezquieta Zubicaray B. Consejo genético en la hiperplasia suprarrenal congénita por déficit de 21-hidroxilasa. An Pediatr (Barc) 2012; 76:51-2. [DOI: 10.1016/j.anpedi.2011.08.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2011] [Revised: 07/18/2011] [Accepted: 08/07/2011] [Indexed: 11/25/2022] Open
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Rabbani B, Mahdieh N, Ashtiani MTH, Larijani B, Akbari MT, New M, Parsa A, Schouten JP, Rabbani A. Mutation analysis of the CYP21A2 gene in the Iranian population. Genet Test Mol Biomarkers 2011; 16:82-90. [PMID: 22017335 DOI: 10.1089/gtmb.2011.0099] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Defects in the CYP21A2 gene cause steroid 21-hydroxylase deficiency, which is the most frequent cause of congenital adrenal hyperplasia. Forty four affected families were investigated to identify the mutation spectrum of the CYP21A2 gene. METHODS Families were subjected to clinical, biochemical, and molecular analyses. Allele-specific polymerase chain reaction amplification was used for eight common mutations followed by dosage analysis to exclude CYP21A2 deletions. RESULTS The most frequent mutations detected were gene deletions and chimera (31.8%). Other mutation frequencies were as follows: Q318X, 15.9%; I2G, 14.8%; I172N, 5.8%; gene duplication, 5.7%; R356W, 8%; and E6 cluster mutations, 2.3%. Direct sequencing of the CYP21A2 gene revealed R316X, P453S, c.484insT, and a change at the start codon. Different modules carried by patients were classified into five different haplotypes. The genotype phenotype correlation (positive predictive value) for group null, A, B, and C were 92.3%, 85.7%, 100%, and 0, respectively. CONCLUSIONS Methods used will be helpful for carrier detection and antenatal diagnosis, especially with inclusion of the multiplex ligation probe dependent amplification technique, which is easier for routine tests in comparison with other methods. Mutation frequencies indicate that Iranians are possible descendants of Asians and Europeans.
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Affiliation(s)
- Bahareh Rabbani
- Growth and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
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Finkielstain GP, Chen W, Mehta SP, Fujimura FK, Hanna RM, Van Ryzin C, McDonnell NB, Merke DP. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2011; 96:E161-72. [PMID: 20926536 PMCID: PMC3038490 DOI: 10.1210/jc.2010-0319] [Citation(s) in RCA: 135] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
BACKGROUND Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. STUDY OBJECTIVE The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. METHODS Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. RESULTS In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. CONCLUSIONS Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.
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Affiliation(s)
- Gabriela P Finkielstain
- Program in Developmental Endocrinology and Genetics, National Institutes of Health, Bethesda, Maryland 20892-1932, USA
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Cantürk C, Baade U, Salazar R, Storm N, Pörtner R, Höppner W. Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia. Clin Chem 2010; 57:511-7. [PMID: 21148302 DOI: 10.1373/clinchem.2010.156893] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND The high homology between the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genes is the major obstacle to risk-free genetic diagnosis of congenital adrenal hyperplasia, especially regarding the quantification of gene dosage. Because of the lack of a comprehensive study providing useful information about the detailed genetic structure of CYP21A1P, we used a large data set to analyze and characterize this pseudogene. METHODS We amplified and directly sequenced the CYP21A1P and CYP21A2 genes of 200 unrelated individuals. The resulting sequence data were aligned against the manually curated transcript ENST0000448314 from Havana/Vega matching to the genebuild ENSG00000198457; all differences were documented. Copy number was measured by multiplex ligation-dependent probe amplification when necessary. RESULTS We found that 40 potentially variable positions in CYP21A2 were conserved in CYP21A1P in all study participants. In addition, we detected 14 CYP21A1P variants that were not previously reported in either CYP21A2 or CYP21A1P. Unlike CYP21A2, CYP21A1P possessed certain mutation haplotypes. CONCLUSIONS The genetic structure of CYP21A1P and the potential risks of false conclusions it may introduce are essential considerations in designing a PCR-based diagnosis procedure for congenital adrenal hyperplasia.
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