Endocrine Abstracts

JOINT328

Introduction: Short Read Next Generation Sequencing (SR-NGS) has been widely applied in the genetic diagnosis of several mendelian disorders. Nevertheless, one of the major disadvantages remains the inability to fully discriminate genes from their pseudogenes/homologous genes. To date, SR-NGS is not widely used in the diagnostic genotyping of the CYP21A2 gene due to its highly homologous pseudogene, thus rendering the PCR-based sequencing and MLPA analysis the recommended CYP21A2 genotyping methodologies. The aim of this study is to assess one of the first SR-NGS assays developed for the genotyping of the CYP21A2 gene and to present the results of 151 samples referred to the Laboratory of Molecular Endocrinology for CYP21A2 genotyping employing the above-mentioned technique.

Patients and methods: A total of 172 subjects were studied. Subjects were categorized in 2 groups; the Pilot and the Study Group. The Pilot Group, employed for the assessment of the SR-NGS assay, consisted of 21 samples, previously analyzed using PCR-based sequencing and MLPA analysis. The Study Group consisted of 151 subjects referred for CYP21A2 genotyping. Both Groups underwent Long Range PCR followed by SR-NGS. Two different bioinformatic pipelines for variant calling (varscan mpileup2cns and GATK HaplotypeCaller functions) were employed in the Study Group. Filtration of the data was performed using VarAFT v2.17. In cases with suspicion of CYP21A2 gene duplication/deletion MPLA analysis was also employed.

Results: The pilot study assessment of the SR-NGS assay resulted in a sensitivity and precision of 100%. Minor differences were observed in the use of the two different bioinformatics pipelines. No alteration was observed in the frequencies of cases harboring duplication of the gene when compared to cases carrying two copies of the CYP21A2 gene. Advantage of this methodology is the identification of variants in regions with heterozygous deletions/insertions that could not be covered by PCR-based sequencing. In the Study Group, pathogenic variants were identified in 54.3% of cases while duplications in 4.6%.

Discussion: This study verifies that the application of the SR-NGS assay for CYP21A2 genotyping presents high sensitivity and precision, short turnaround time and cost effectiveness compared to the PCR based sequencing. Both bioinformatic pipelines can be employed for the SR-NGS data analysis although the GATK algorithm exhibits higher accuracy. MLPA analysis is still required for the identification of deletions/duplications of the CYP21A2 gene. Hence, the presented SR-NGS assay and MLPA can be employed for the CYP21A2 diagnostic genotyping.