Abstract 109: Long-range PCRs and MLPA for molecular analysis of large rearrangements in 21 hydroxylase deficiency

Background: Genetics of 21 hydroxylase deficiency is complex with pseudogene derived point mutations and rearrangements in the CYP21A2 gene. 20-30% of these mutations are due to large 30kb deletion forming chimeric genes and large gene conversion. In general, MLPA is the choice of technique in molecular analysis of large deletions and duplications. Aim and Objectives: To evaluate long-range PCRs, restriction digestion, ASPCR and MLPA to identify various chimeric forms of CYP21A2 gene. Results: In this study, 24.2% (n=16/66) were identified with large rearrangements based on long-range PCR and restriction digestion results. Thirteen subjects were positive for chimeric genes described in 21-hydroxylase deficiency. Junction site analysis with both MLPA and ASPCR results revealed that ASPCR could detect the actual extent of rearrangement in all the chimeric genes while MLPA could miss two of the chimeric genes in four subjects. Conclusion: In this study, MLPA as a direct stand-alone technique has shown to be inadequate in identifying the extent of some rearrangements in the CYP21A2 gene. Since, it is crucial to differentiate various chimeric forms of this gene; we recommend a multistep approach including long-range PCR, restriction digestion and ASPCR/MLPA to interpret and understand the associated phenotypic severity in 21-hydroxylase deficiency.