Leveraging Long-Read Sequencing to Bridge the Diagnostic and Equity Gaps in Differences of Sex Development (DSD)

Congenital Adrenal Hyperplasia (CAH) can result from variants in several genes but is most frequently caused by deletions and gene conversions in the segmentally duplicated RCCX module, which contains the CYP21A2 gene and its pseudogene. Current genetic tests vary greatly by laboratory, method, and consequently diagnostic ability. Less severe forms of CAH are frequently under- or mis-diagnosed, and phenotype/genotype correlations remain incomplete, hampering genetic counseling and prediction of long-term outcomes. Emerging technologies using long-read sequencing (LRS) have transformed molecular testing for CAH, accurately identifying pathogenic single-nucleotide variants, full gene deletions, gene conversions, fusions creating non-functional hybrids between the gene and pseudogene ("30-kb deletion"), and phasing variants, even in the absence of parental samples. This has led to updated reports of previously unrecognized complex alleles in large cohorts. This is a transformative breakthrough, which offers the promise of consolidated testing for all forms of CAH and other conditions under the Differences of Sex Development (DSD) umbrella. DSD are notoriously difficult to disambiguate, resulting in a significant diagnostic gap. The large array of known genetic etiology, ranging from single nucleotide variants in coding or non-coding regions in dozens of genes to sex chromosome aneuploidies, associated with overlapping phenotypes, make precise DSD diagnosis one of the most difficult in medicine. We report on our experience with LRS tools for disambiguation of CAH alleles discuss some of the technical aspects to be considered prior to the deployment of LRS to ensure accurate and equitable results for the diagnosis of rare conditions such as DSD.