A rational,non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency |
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Authors: | Fernanda Borchers Coeli-Lacchini Wendy Turatti Paula Conde Lamparelli Elias Lucila Leico Kagohara Elias Carlos Eduardo Martinelli Jr. Ayrton Custodio Moreira Sonir Roberto Antonini Margaret de Castro |
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Affiliation: | 1. Department of Internal Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil;2. Department of Pediatrics, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil;3. Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil |
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Abstract: | ContextMolecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.ObjectiveTo conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.Patients and methodsWe studied 99 patients from 90 families with salt-wasting (SW; n = 32), simple-virilizing (SV; n = 29), and non-classical (NC; n = 29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.ResultsASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3′-end of CYP21A1P, C4B, and the 5′-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.ConclusionUsing the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD. |
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Keywords: | CAH, congenital adrenal hyperplasia 21OHD, 21-hydroxylase deficiency RCCX module, RP, C4, CYP21 and TNX genes SW, salt-wasting SV, simple-virilizing NC, non-classical ACTH, Adrenocorticotropic Hormone 17-OHP, 17-hydroxyprogesterone DHEAS, Dehydroepiandrosterone Sulfate ASO-PCR, allele specific oligonucleotide polymerase chain reaction MLPA, Multiplex Ligation dependent Probe Amplification RFLP, restriction fragment length polymorphism 5&prime UTR, 5&prime untranslated 3&prime UTR, 3&prime untranslated 30-kb deletion, deletion of 30-kb including 3&prime -end CYP21A1P, C4B, and 5&prime -end CYP21A2 |
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