Amplicon-based targeted next-generation sequencing using dried blood spots for 46,XY differences/disorders of sex development: Aiming for diagnosis by minimally invasive testing

Rapid genetic diagnosis of differences/disorders of sex development (DSD) through minimally invasive testing is desirable. In this study, we performed PCR amplicon-based next-generation sequencing (NGS) targeting <i>AR</i> and <i>SRD5A2</i> using dried blood spots from 22 patients with 46,XY DSD. We compared the results with those of an outsourced capture-based NGS using venous blood-derived DNA. We successfully extracted DNA from the dried blood spots and obtained analysis results for 19 of the 22 cases within a minimum of seven days. The DNA quantity required was significantly lower for amplicon-based NGS using dried blood spots than for capture-based NGS using venous blood (median 8.7 ng vs. 1434.8 ng). We identified four single-nucleotide substitutions in <i>SRD5A2</i> in 16 of the 19 cases. The results were consistent between the two NGS analyses and Sanger sequencing using venous blood, except for case 1. In this case, amplicon-based NGS using dried blood spots incorrectly identified a heterozygous <i>SRD5A2</i> variant as homozygous, presumably due to allelic dropout. In conclusion, we demonstrated that amplicon-based NGS using dried blood spots allowed for rapid and minimally invasive genetic testing in patients with 46,XY DSD. However, optimizing DNA extraction from dried blood spots and validating detected variants using Sanger sequencing are necessary.