Next-Generation Sequencing Reveals Novel Genetic Variants (SRY, DMRT1, NR5A1, DHH, DHX37) in Adults With 46,XY DSD

Abstract

Context: The genetic basis of human sex development is slowly being elucidated, and >40 different genetic causes of differences (or disorders) of sex development (DSDs) have now been reported. However, reaching a specific diagnosis using traditional approaches can be difficult, especially in adults where limited biochemical data may be available.

Objective: We used a targeted next-generation sequencing approach to analyze known and candidate genes for DSDs in individuals with no specific molecular diagnosis.

Participants and design: We studied 52 adult 46,XY women attending a single-center adult service, who were part of a larger cohort of 400 individuals. Classic conditions such as17β-hydroxysteroid dehydrogenase deficiency type 3, 5α-reductase deficiency type 2, and androgen insensitivity syndrome were excluded. The study cohort had broad working diagnoses of complete gonadal dysgenesis (CGD) (n = 27) and partially virilized 46,XY DSD (pvDSD) (n = 25), a group that included partial gonadal dysgenesis and those with a broad "partial androgen insensitivity syndrome" label. Targeted sequencing of 180 genes was undertaken.

Results: Overall, a likely genetic cause was found in 16 of 52 (30.8%) individuals (22.2% CGD, 40.0% pvDSD). Pathogenic variants were found in sex-determining region Y (SRY; n = 3), doublesex and mab-3-related transcription factor 1 (DMRT1; n = 1), NR5A1/steroidogenic factor-1 (SF-1) (n = 1), and desert hedgehog (DHH; n = 1) in the CGD group, and in NR5A1 (n = 5), DHH (n = 1), and DEAH-box helicase 37 (DHX37; n = 4) in the pvDSD group.

Conclusions: Reaching a specific diagnosis can have clinical implications and provides insight into the role of these proteins in sex development. Next-generation sequencing approaches are invaluable, especially in adult populations or where diagnostic biochemistry is not possible.

Keywords: DHX37; DSD; SRY; desert hedgehog; sex determination; steroidogenic factor-1.

Figure 1.

Overview of the study cohort and summary of findings.

Figure 2.

Overview of genetic diagnoses reached.

Figure 3.

(A) Depiction of SRY demonstrating the mutations identified, with amino acid conservancy shown below. The position of variants is indicated by red arrowheads. (B) Aligned sequence and chromatogram showing the complex indel variant. The position of the typical stop codon at 205 is shown with a blue arrow. The red asterisk indicates where the frameshift ends. HMG, high-mobility group.

Figure 4.

(A) Depiction of DMRT1 demonstrating the p.R80S mutation identified, with amino acid conservancy shown below. The position of p.R80 is indicated by a red arrowhead. (B) Schematic representation of the DMRT1–DNA complex (PDB ID 4YJ0). The DMRT1 protein is shown in white and the R80 residues are depicted as spheres; zinc atoms are shown in magenta. (C) Snapshot of the R80 or S80 vicinity (upper) and electrostatic potential of surface of the respective DMRT1-DNA complexes (lower). (D) Cα-root-mean-squared (RMSD) deviation and distances between Arg or Ser 80 and the phosphate backbone of the DNA along the molecular dynamics simulation.

Figure 5.

(A) Depiction of NR5A1 (SF-1) demonstrating the mutations identified, with amino acid conservancy shown below. The positions of variants are indicated by red arrowheads. (B) Transient transfection assays showing activation of a Cyp11a1 promoter by WT SF-1 and impaired transcriptional activity by the p.G22D and p.L420P mutants. Results are shown as a percentage of WT SF-1 activity (relative light units). Data are shown as mean ±SEM of three experiments performed in triplicate. Zn, Zinc finger.

Figure 6.

(A) Depiction of DHH demonstrating the mutations identified, with amino acid conservancy shown below. The positions of variants are indicated by red arrowheads. (B) Immunohistochemistry of human fetal testis at 9 wk postconception showing expression of DHH (green) predominantly in interstitial Leydig cells but also in Sertoli cells. Anti-Müllerian hormone (AMH; red) is expressed strongly in Sertoli cells of primitive seminiferous tubules. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). No staining is seen in the peripheral capsular region at the bottom right of the image. Scale bar, 100 μm.

Figure 7.

Depiction of DHX37 demonstrating the mutations identified, with amino acid conservancy shown below. The positions of variants are indicated by red arrowheads. CTD, C-terminal domain; HA2, helicase-associated 2 domain; NTD, N-terminal domain; OB, oligonucleotide/oligosaccharide-like domain; RecA1, ATP binding DEAH box helicase; RecA2, C-terminal helicase.