case_images: PMC9294228_01
case_images: Histologic examination of the proband's testicular biopsies. (A) Left testes: a low magnification (x20) from the surgical specimen of the left "testis" where epididymal tissue (E) is seen along with adjacent fatty tissue that is rich in blood vessels (BV). Note the total absence of any testicular tissue. (B) Right testis: an intermediate magnification (x100) image from the right testis showing seminiferous tubules (ST) that lack germ cells (lined by Sertoli cells only). The interstitium between the tubules shows clusters of hyperplastic Leydig cells (lLC) and fibrosis (F) secondary to tubular atrophy. TA, tunica albuginea. Both sections were stained using hematoxylin and eosin staining.
case_images: fgene-13-885589-g001.jpg
case_images: PMC9294228_01_fgene-13-885589-g001.jpg
case_images: F1
case_images: The histopathological examination using hematoxylin and eosin staining showed a left undescended testis, atrophied with vasa efferentia, and epididymal tissue, negative for neoplasm at x20 magnification (Figure 1A). The right testis showed total loss of germ cells in the seminiferous tubules, which were lined by mature Sertoli cells and negative for neoplasm or intratubular germ cell neoplasm at x100 magnification (Figure 1B).
case_images: PMC9294228_02
case_images: Molecular characterization of the identified c.2T>C missense variant in the NR5A1 gene. (A) Chromatogram of Sanger sequencing segregation analysis of the wild-type control and affected proband. Red arrow represents the identified variant. (B) Annotation of the identified missense variant in the NR5A1 gene (c.2T>C, p.Met1Thr) is located on chromosome 9p33.3. The variant falls in the first transcribed exon (exon 2) of the N-terminal region and interrupts the starting codon (ATG>ACG). The variant located in some of the ENCODE functional data tracks in the USCS genome browser (http://genome.ucsc.edu/). The variant interrupt DNaseI hypersensitivity and some predicted transcription factor binding sites (TFBSs) such as EZH2, NR3C1, and SUZ12.
case_images: fgene-13-885589-g002.jpg
case_images: PMC9294228_02_fgene-13-885589-g002.jpg
case_images: F2
case_images: ENCODE annotation on the UCSC genome browser revealed that the variant falls in a functional region of NR5A1 and thus predicted to be pathogenic (Figure 2B). The variant was predicted to fall in several transcriptional factor binding motifs such as EZH2, NR3C1, and SUZ12 (Figure 2B).
case_images: Predicted effect of Met1Thr mutation on NR5A1 function. (A) Schematic diagram of the identified variant at amino acid 1 is located on the DBD (green) in the N-terminus of NR5A1. The diagram was generated using PROSITE (https://prosite.expasy.org/cgi-bin/prosite). (B) Alignment of the sequences of H. Sapiens NR5A1 (UniProt: Q13285) with the Mouse NR5A1 (UniProt: P33242), Pig NR5A1 (UniProt: P79387), Rat NR5A1 (UniProt: P50569), Bovin NR5A1 (Uniprot: Q04752), and Horse NR5A1 (UniProt: Q9GKL2). The alignment was generated using UniProt Align (http://www.uniprot.org/align/). Start codon (Met) are represented by green rectangles. The alignment file was generated using UniProt Align (http://www.uniprot.org/align/), and final processing was performed in ESPript (http://espript.ibcp.fr). (C) Schematic representation of the NR5A1 interactions with other proteins using GeneMANIA (http://genemania.org/): physical interaction with other proteins (pink lines), genetics interaction (green lines), co-localization (blue lines), and predicted interaction (orange lines).
case_images: fgene-13-885589-g003.jpg
case_images: PMC9294228_02_fgene-13-885589-g003.jpg
case_images: F3
case_images: The variant falls in a functional region of the N-terminal region of NR5A1 and disrupts the start codon in the first coding exon, exon 2 (Figure 3A). Sapiens NR5A1 (UniProt: Q13285) with the Mouse NR5A1 (UniProt: P33242), Pig NR5A1 (UniProt: P79387), Rat NR5A1 (UniProt: P50569), Horse NR5A1 (UniProt: Q9GKL2) and Bovine NR5A1 (UniProt: Q04752) was generated (Figure 3B). The p.Met1Thr variant is located in a highly conserved position across mammals and higher eukaryotes, and thus, this strengthens the pathogenicity of the identified variant (Figure 3B). The STRING protein interaction shows that there is a strong interaction between NR5A1 and several other DSD proteins such as SOX9 (Figure 3C).