ESPE Yearbook of Paediatric Endocrinology

Sex Dev. 2021;15(5-6):392-410. PMID: 34634785, doi: 10.1159/000519238.

Brief Summary: This review summarizes how non-coding genomic variants are identified and validated, and why non-coding defects found in DSD patients have to be considered when investigating the genetic etiology of DSD.

Accurate genetic diagnosis of 46,XY DSD patients remains crucial for early treatment and prediction of associated risks of malignant tumors. Genetic studies that aim to investigate the etiology of DSD have focussed mainly on the coding genome. Despite the availability of techniques, such as targeted gene panel sequencing, whole-exome sequencing, and microarray analysis, the current DSD diagnostic rate for 46,XY DSD is limited to ~40%, and the rest of these patients yet lack a specific genetic diagnosis. On the other hand, a wide variety of non-coding defects are found in DSD patients, suggesting that aberrations in non-coding genomic regions could account for many of the remaining cases.

Therefore, at a genomic level, it is necessary to define regulatory regions of gonadal genes that are disrupted in DSD patients, identify active enhancers within these regions and detect their target promoters. Validation experiments both in vitro and in vivo will provide evidence of the functionality of these enhancers. The tools and approach algorithm used for the identification of variants in non-coding genomic regions associated to DSD etiology and in vivo and in vitro validation methods of these variants and enhancers were proposed in Figure 1.

Using these tools several CNVs disrupting the regulatory regions of gonad development genes including SOX9, SOX3, SOX8, NR0B1, GATA4, DMRT1, WT1, NR5A1, AR, and SRY have been discovered in DSD patients, with the SOX9 regulatory region being the most extensively studied. Several duplications and deletions far upstream of SOX9 were identified using array comparative genomic hybridization (CGH) on genomic DNA from 46,XX and 46,XY DSD patients, respectively. The review provides an excellent clinical update on these functional regulatory elements of the non-coding genome described in the pathogenesis of DSD so far.

Figure 1. An approach to variant discovery in the non-coding genome for DSD patients. ATAC-seq, assay for transposase-accessible chromatin with high-throughput sequencing; CGH, comparative genomic hybridization; ChIP-seq, chromatin immunoprecipitation sequencing; CRISPR, clustered regulatory interspaced short palindromic repeat; dREG, discriminative regulatory element detection from GRO-seq; DSD, disorders of sex development; ENCODE, encyclopedia of DNA elements; FANTOM5, functional annotation of the mammalian genome project; GWAS, genome-wide association study; Hi-C, high throughput chromosome conformation capture; MLPA, multiplex ligation dependent probe amplification; TFBS, transcription factor binding site; UCSC, University of California Santa Cruz; WGS, whole genome sequencing.