Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development

Abstract

Disorders of sex development (DSD) are rare disorders in which there is discordance between chromosomal, gonadal, and phenotypic sex. Only a minority of patients clinically diagnosed with DSD obtains a molecular diagnosis, leaving a large gap in our understanding of the prevalence, management, and outcomes in affected patients. We created a novel DSD-genetic diagnostic tool, in which sex development genes are captured using RNA probes and undergo massively parallel sequencing. In the pilot group of 14 patients, we determined sex chromosome dosage, copy number variation, and gene mutations. In the patients with a known genetic diagnosis (obtained either on a clinical or research basis), this test identified the molecular cause in 100% (7/7) of patients. In patients in whom no molecular diagnosis had been made, this tool identified a genetic diagnosis in two of seven patients. Targeted sequencing of genes representing a specific spectrum of disorders can result in a higher rate of genetic diagnoses than current diagnostic approaches. Our DSD diagnostic tool provides for first time, in a single blood test, a comprehensive genetic diagnosis in patients presenting with a wide range of urogenital anomalies.

Fig. 1

Sex chromosome complement. Depth of coverage (DOC) along the X- and Y-chromosomes was normalized to the autosomal DOC and plotted to determine sex chromosome complement. All XY samples (upper left) clustered together and had normalized coverage ranging from 0.43 to 0.63 for the X-chromosome and 0.58 to 0.72 for the Y-chromosome. All XX samples (lower right) clustered together, had null Y-chromosome coverage, and had close to 1 for normalized X-chromosome coverage, indicating an absence of the Y-chromosome and two copies of the X-chromosome. An X marks the mean coverage for the 46, XY cluster or the 46, XX cluster. The p-values for separating the two clusters were <0.001 for both directions, and DSDpt3 that had the highest DOC (0.63) along the X-chromosome was separated from the 46, XX cluster with a p-value of <0.001.

Fig. 2

Copy number variant analysis. To determine CNV status for NROB1 (DAX1 ), coverage for each gene was normalized to autosomal coverage of each sample and all samples plotted based on normalized coverage. Since NROB1 is an X-chromosome gene, normalized coverage was plotted separately based on the X-chromosome karyotype. DSDpt2, with 46, XY GD, had significantly higher coverage than other XY individuals, indicating a duplication of the gene (p = 0.002). An X indicates the mean coverage for the gene.

Fig. 3

Proposed integration of targeted sequencing approach to clinical management of suspected DSD. Current clinical management begins with the identification of an abnormal phenotype and is followed by multiple metabolic and endocrine tests, genetic tests, and imaging studies in order to identify the mostly likely candidate for sequencing. Targeted sequencing approach would prioritize a genetic diagnosis, which would be functionally assayed and confirmed with endocrine and imaging studies of the patient.