A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

• Published in: Gene 2013-09, Vol.526 (2), p.239-245
• Main Authors: Coeli-Lacchini, Fernanda Borchers, Turatti, Wendy, Elias, Paula Conde Lamparelli, Elias, Lucila Leico Kagohara, Martinelli, Carlos Eduardo, Moreira, Ayrton Custodio, Antonini, Sonir Roberto, de Castro, Margaret
• Format: Article
• Language: English
• Subjects: 21-Hydroxylase deficiency; Adolescent; Adrenal Hyperplasia, Congenital - diagnosis; Adrenal Hyperplasia, Congenital - genetics; algorithms; Alleles; Child; Child, Preschool; Congenital adrenal hyperplasia; cost effectiveness; CYP21A1P; CYP21A2 gene; dissociation; Exons; Genotype; Humans; hyperplasia; Infant; Infant, Newborn; Introns; MLPA; patients; Phenotype; Point Mutation; Polymerase Chain Reaction; promoter regions; Promoter Regions, Genetic; Steroid 21-Hydroxylase - genetics; Young Adult
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2013.03.082

Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward. To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis. We 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. ASO-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. Using 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. •The algorithm using ASO-PCR/MLPA/direct sequencing elucidated all CYP21A2 alleles.•Algorithm and paternal segregation clarified some genotype/phenotype dissociation.•Mutations/SNPs in the probe-binding regions cause false-positive results in MLPA.•Association of ASO-PCR/paternal segregation elucidates false-positive MLPA results.•MLPA is a cost-effective method for the molecular diagnosis of CAH-21OHD.