New technologies to uncover the molecular basis of disorders of sex development

Abstract

The elegant developmental biology experiments conducted in the 1940s by French physiologist Alfred Jost demonstrated that the sexual phenotype of a mammalian embryo depended whether the embryonic gonad develops into a testis or not. In humans, anomalies in the processes that regulate development of chromosomal, gonadal or anatomic sex result in a spectrum of conditions termed Disorders/Differences of Sex Development (DSD). Each of these conditions is rare, and understanding of their genetic etiology is still incomplete. Historically, DSD diagnoses have been difficult to establish due to the lack of standardization of anatomical and endocrine phenotyping procedures as well as genetic testing. Yet, a definitive diagnosis is critical for optimal management of the medical and psychosocial challenges associated with DSD conditions. The advent in the clinical realm of next-generation sequencing methods, with constantly decreasing price and turnaround time, has revolutionized the diagnostic process. Here we review the successes and limitations of the genetic methods currently available for DSD diagnosis, including Sanger sequencing, karyotyping, exome sequencing and chromosomal microarrays. While exome sequencing provides higher diagnostic rates, many patients still remain undiagnosed. Newer approaches, such as whole-genome sequencing and whole-genome mapping, along with gene expression studies, have the potential to identify novel DSD-causing genes and significantly increase total diagnostic yield, hopefully shortening the patient's journey to an accurate diagnosis and enhancing health-related quality-of-life outcomes for patients and families.

Keywords: DSD; Exome; Genome mapping; Testis development; Whole genome sequencing.

Fig. 1. DNA labeling for next-generation genome mapping (NGM)

The DNA-labeling workflow is divided into four consecutive steps. First, the high-molecular-weight DNA is nicked with an endonuclease of choice (Nt.BspQI or Nb.BssSI, New England BioLabs/Bionano Genomics), which introduces single-strand nicks throughout the genome. Second, Taq polymerase (NEB) recognizes these sites and replaces several nucleotides with fluorescently tagged nucleotides added to the solution. Third, the two ends of the DNA are ligated together using Taq DNA ligase (NEB). Fourth, the DNA backbone is stained with DNA Stain (Bionano Genomics). Figure originally published in Barseghyan et al. 2017, Genome Medicine 9:90. DOI 10.1186/s13073-017-0479-0. Reproduced here courtesy of publisher BioMed Central’s policy of free sharing of its open access articles.

Fig. 2. Chip nanochannel structure and DNA loading for next-generation genome mapping (NGM)

The labeled double-stranded DNA is loaded into two flow cells (Irys or Saphyr, Bionano Genomics). The applied voltage concentrates the coiled DNA at the lip (left). Later, DNA is pushed through pillars (middle) to uncoil/straighten, then into nanochannels (right). DNA is stopped and imaged in the nanochannels. Blue - staining of DNA backbone. Green - fluorescently labeled nicked sites. Figure originally published in Barseghyan et al. 2017, Genome Medicine 9:90. DOI 10.1186/s13073-017-0479-0. Reproduced here courtesy of publisher BioMed Central’s policy of free sharing of its open access articles.

Fig. 3. Next-generation genome mapping (NGM) identifies two small deletions in intron 5 of the WWOX gene in a 46,XY DSD patient

Top panel: assembly of the proband’s contigs (yellow) generated using nicking endonuclease Nb.BssSI (sites are shown as small black vertical bars), aligned to the reference genome (blue). The individual DNA molecules used to assemble the bottom allele are shown as grey lines and black dots corresponding to linearized DNA and nicking sites respectively. The genomic locations of the region displayed and of the long non-coding RNA RP11-190D6.2 (in the GRCh38/hg38 human genome assembly reference map) are shown in the top purple bar representing the WWOX gene. The middle and bottom panels show the maps of the father and mother respectively, which helped to identify parent of origin for the proband’s alleles. Similar results were obtained with a different endonuclease, Nt.BspQI (not shown). The 2.3 kbp and 13.2 kbp deletions were present in respectively 1.3% and 15% of the reference maps in the Bionano database of 150 healthy controls.