Targeted next-generation sequencing identification of mutations in patients with disorders of sex development

Abstract

BackgroundThe identification of causative mutations is important for treatment decisions and genetic counseling of patients with disorders of sex development (DSD). Here, we designed a new assay based on targeted next-generation sequencing (NGS) to diagnose these genetically heterogeneous disorders.MethodsAll coding regions and flanking sequences of 219 genes implicated in DSD were designed to be included on a panel. A total of 45 samples were used for sex chromosome dosage validation by targeted sequencing using the NGS platform. Among these, 21 samples were processed to find the causative mutation.ResultsThe sex chromosome dosages of all 45 samples in this assay were concordant with their corresponding karyotyping results. Among the 21 DSD patients, a total of 11 mutations in SRY, NR0B1, AR, CYP17A1, GK, CHD7, and SRD5A2 were identified, including five single nucleotide variants, three InDels, one in-frame duplication, one SRY-positive 46,XX, and one gross duplication with an estimated size of more than 427,038 bp containing NR0B1 and GK. We also identified six novel mutations: c.230_231insA in SRY, c.7389delA in CHD7, c.273C>G in NR0B1, and c.2158G>A, c.1825A>G, and c.2057_2065dupTGTGTGCTG in AR.ConclusionsOur assay was able to make a genetic diagnosis for eight DSD patients (38.1 %), and identified variants of uncertain clinical significance in the other three cases (14.3 %). Targeted NGS is therefore a comprehensive and efficient method to diagnose DSD. This work also expands the pathogenic mutation spectrum of DSD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0286-2) contains supplementary material, which is available to authorized users.

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort

“…This suggests that if applied as a first-tier diagnostic test, we could expect our panel to provide an even greater diagnostic outcome. Our results support previous conclusions reached by others [7, 8, 10] indicating that diagnosis of 46,XY DSD can be significantly enhanced through use of MPS technologies, albeit on a much larger scale.…”

“…These were further filtered for frequency in our database, inheritance, and quality/depth (see “Methods”). Remaining variants were curated in accordance with previous publications using MPS analysis of DSD cohorts [8, 10] (see “Methods”), which were based on the American College of Medical Genetics and Genomics (ACMG) guidelines [15]. Rare variants in a clinically relevant DSD gene are reported here if our curation processes classified them as pathogenic, likely pathogenic, or variants of uncertain significance (VUS; not predicted to be damaging or the affected gene has not been previously reported with the described phenotype).…”

“…In particular, 46,XY DSD are not well diagnosed at the molecular level. However, MPS is now fast becoming a standard assay for molecular diagnosis of rare Mendelian disorders and has been successfully used on small cohorts of DSD patients [7, 8, 10]; in particular, a research study of 40 cases using WES provided a likely genetic diagnosis in 35% [8]. We present a MPS targeted DSD gene panel on one of the largest collections of 46,XY DSD reported to date (278 patients).…”

“…In this environment, an MPS targeted gene panel offers many advantages, such as relatively low cost, shorter turnaround time, and smaller overheads in data handling and analysis compared to WES or WGS. Indeed, numerous gene panels have been successfully employed in the genetic diagnosis of a variety of monogenic disorders [9], including small cohorts of patients with 46,XY DSD [7, 10]. Finally, no studies have reported the usefulness of MPS for patients with 46,XX DSD, nor have any large-scale studies looked at the contribution of known DSD genes to this heterogeneous condition.…”

Anomalies in human sex determination provide unique insights into the complex genetic interactions of early gonad development

“…Baxter et al found that in a WES screen, a genetic diagnosis was established in 14 of 40 (35%) cases of an unbiased group of 46,XY DSD patients that presented with a wide spectrum of phenotypes (63). In a second study by Dong et al (64), a genetic diagnosis for possible for 38% of DSD cases, and variants of uncertain clinical significance were found in a further 14%.…”