Endocrine Abstracts

Introduction: Defects in the pseudogene, CYP21A1P, can be transferred to the functional CYP21A2 gene by recombination and account for approximately 95% of CYP21A2 mutations, leading to CAH-21OHD. We conducted a comprehensive genetic analysis to assess whether Multiplex Ligation dependent Probes Amplification (MLPA) could substitute southern blotting with radioactive probes without compromising reliability of the diagnosis.

Patients and Methods: We studied 90 families/99 patients presenting salt-wasting (SW; n=32), simple-virilizing (SV; n=29), and non-classical (NC; n=29) phenotypes. Molecular analysis was sequentially performed by detecting the 8 most frequent point mutations by allele specific oligonucleotide polymerase chain reaction (ASO-PCR), large deletions and conversions by MLPA (KIT-P050-B2-CAH), and rare mutations by direct sequencing. Parental segregation was evaluated.

Results: Mutated alleles were elucidated by ASO-PCR in 92.2%, by MLPA in 5.0% and by direct sequencing in 2.8%. In SW group, the most frequent mutations detected by ASO-PCR were IVS2-13A/C>G (43.7%), p.R356W (12.5%), and p.Q318X (11.0%), large conversions in 12.5% by MLPA, and the 1762_1763InsT (1.6%) by direct sequencing. In SV patients, the most frequent mutations detected by ASO-PCR were p.I172N (55.2%), p.R356W (10.3%) and IVS2-13A/C>G (6.9%), large conversion in one single allele (1.7%) by MLPA, and the p.G424S mutation was found in 1.7% by direct sequencing. In NC patients, the most frequent mutations detected by ASO-PCR were p.V281L (72.4%), p.I172N (5.2%), IVS2-13A/C>G (5.2%). Direct sequencing revealed rare mutations (p.P453S, p.R408C, and p.A265V) in one allele each (5.2%). No large conversions were found in this group.

Conclusion: Although MLPA false positive results could arise due to mutations/polymorphisms close to the probe binding regions and due to probe hybridization and ligation, these problems can be overcome by the association of MLPA with ASO-PCR and parental segregation. Using these approaches, we can successfully substitute southern blotting in a cost-effective laboratory routine.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.