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

作者：Coeli Lacchini Fernanda Borchers; Turatti Wendy; Lamparelli Elias Paula Conde; Kagohara Elias Lucila Leico; Martinelli Carlos Eduardo Jr; Moreira Ayrton Custodio; Antonini Sonir Roberto; de Castro Margaret^*

来源：Gene, 2013, 526(2): 239-245.

DOI：10.1016/j.gene.2013.03.082

• Congenital adrenal hyperplasia
• CYP21A2 gene
• 21-Hydroxylase deficiency
• MLPA
• CYP21A1P

Context: Molecular diagnosis of congenital adrenal hyperplasia (CAB) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.
Objective: To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAI-21OHD diagnosis.
Patients and methods: 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.
Results: 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 (63%). 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.
Conclusion: 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.

• 出版日期2013-9-10