Testicular dysgenesis/regression without campomelic dysplasia in patients carrying missense mutations and upstream deletion of SOX9

Abstract

SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1-3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C-terminal domain but not in other known 46,XY DSD causative genes. These substitutions were absent from ~120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited ~491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short-nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C-terminal missense substitutions which lead to target-gene-specific protein dysfunction, and enhancer-containing upstream microdeletions mediated by nonhomologous end-joining.

Keywords: Campomelic dysplasia; deletion; enhancer; mutation; testis.

Figure 1

SOX9 abnormalities in patients 1–3. (A) Genomic and protein structures of SOX9/SOX9. The positions of the c.1180C>G (p.Arg394Gly) and c.1309C>T (p.Arg437Cys) mutations are indicated by arrows. White and black boxes in the upper panel indicate the untranslated and coding regions, respectively. Colored boxes in the lower panel indicate dimerization (codon 60–101) (Bernard et al. 2003), high‐mobility group (HMG: codon 101–184), proline/glutamine/alanine (PQA: codon 339–379), and proline/glutamine/serine‐rich (PQS‐rich: codon 386–509) domains (McDowall et al. 1999). (B) Nucleotide substitutions detected in patients 1 and 2. Left panel: electro chromatograms of the mutations. The mutated nucleotides are indicated by arrows. Right panel: in silico functional prediction of mutant proteins. (C) In vitro assays using reporters containing the Col2a1 enhancer or Amh promoter. The transactivating activity of p.Arg394Gly and p.Arg437Cys mutants was compared to that of the known ACD‐associated SOX9 mutant p.Pro176Leu (Michel‐Calemard et al. 2004). The results are expressed as the mean ± one standard deviation. Relative transactivating activities of the SOX9 mutants against the wild‐type are shown. Empty: empty expression vector; ns: not significant. (D) SOX9 upstream deletion in patient 3. Upper panel: array‐based comparative genomic hybridization analysis. The black, red, and green dots denote signals indicative of the normal, increased (> +0.5) and decreased (< −1.0) copy‐numbers, respectively. The blue and red boxes represent previously reported XY sex reversal region (XYSR) (Kim et al. 2015) and SOX9 exons, respectively. Genomic positions refer to the UCSC database (http://genome.ucsc.edu/; GRCh37/hg19). Lower panel: sequence of the fusion junction. The junction is accompanied by a short‐nucleotide insertion of unknown origin (the red‐shaded area).

Figure 2

Schematic representation of the SOX9 upstream region. Upper panel represents positions of SOX9 and repeat sequences (UCSC database, http://genome.ucsc.edu/; GRCh37/hg19). The numbers indicate the distance from SOX9. Lower panel represents genomic rearrangements in the present and previous cases. Blue and black arrows indicate chromosomal translocations and deletions, respectively. Broken arrows indicate breakpoint regions of multiple patients. The red‐shaded area represents the XYSR reported by Kim et al. (2015). XYSR, XY sex reversal region; CD, campomelic dysplasia; ACD, acampomelic CD; DSD, disorders of sex development.