Identification of novel candidate genes for 46,XY disorders of sex development (DSD) using a C57BL/6J-Y POS mouse model - PubMed (original) (raw)

doi: 10.1186/s13293-018-0167-9.

Aleisha Symon 3, Mariam Zadikyan 2, Miguel Almalvez 1 2, Eva E Segura 2, Ascia Eskin 2, Matthew S Bramble 1 2, Valerie A Arboleda 2, Ruth Baxter 2, Stanley F Nelson 2, Emmanuèle C Délot 1 2 4, Vincent Harley 3, Eric Vilain 5 6 7

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Identification of novel candidate genes for 46,XY disorders of sex development (DSD) using a C57BL/6J-Y POS mouse model

Hayk Barseghyan et al. Biol Sex Differ. 2018.

Abstract

Background: Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes.

Methods: To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (Y POS ) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-Y B6 and undervirilized B6-Y POS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-Y POS males).

Results: We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-Y POS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9.

Conclusion: The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation.

Keywords: 46,XY DSD; C57BL/6J mouse; Disorders of sex development; Exome; Gonadal dysgenesis; RNA-Seq; Undervirilization.

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Conflict of interest statement

Research involving human subjects, human material, and human data have been performed in accordance with protocols (IRB# 11-001491; IRB# 11-001775) approved by the UCLA Institutional Review Board.

Experimental studies involving animals, animal tissues, and data have been performed in accordance with approved protocol (ARC# 2000-088-51A) by UCLA Chancellor’s Animal Research Committee as well as Animal Ethics Committee of Southern Health (Clayton, Australia, ethics number MMCB/2009/30).

Participants have consented by approved IRB protocol to share their de-identified data.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig 1

Fig 1

C57BL/6J-Y poschiavinus mice — a model for 46,XY DSD with gonadal dysgenesis. a The morphology of gonadal development in mice shown at embryonic day E11.5 (when it is still capable of giving rise to both testes and ovaries) and at E15.5. Top panel: testicular development in wild-type B6-Y_B6_ male and ovarian development in WT B6-XX female. Bottom panel: B6-Y_POS_ males show development of ovotestis (left) or ovary (right). b Pie chart representing the number of differentially expressed genes between WT B6-Y_B6_ and undervirilized B6-Y_POS_ male gonads at E11.5, as detected by RNASeq. Variants were found in exomes of the cohort of patients with 46,XY DSD in 189 of the 308 underexpressed and 116 of the 207 overexpressed genes. c Expression (shown as fragments per kilobase of transcript per million reads, FPKM) of the two major sex-determining genes Sry and Sox9. Sry expression was present in both B6-Y_B6_ and B6-Y_POS_ males. However, expression of Sox9 was dramatically lower in B6-Y_POS_ males (as expected [41]); as a consequence, expression of some of the candidate genes for 46,XY DSD may be Sox9_-dependent. d Expression values (shown as fold change differences between B6-Y_B6 and B6-Y_POS_ males) for the 9 genes present in the primary gene list used for exome variant filtration that are also downregulated in B6-Y_POS_ males. The red line represents a 2-fold cutoff

Fig. 2

Fig. 2

Gonadal expression of 15 novel candidate genes in the B6-Y_POS_ mouse model. a Gene expression differences in candidate genes between B6-Y_B6_ (blue) males, B6-Y_POS_ (red) males, and WT B6 females (green). The expression values, as measured by RNASeq, are shown in FPKM values (fragments per kilobase of transcript per million reads). Data generated from gonads dissected at E11.5. b Expression values are shown as fold change differences between B6-Y_B6_ and B6-Y_POS_ males using RNA-Seq data (blue) and qPCR data (red). Both methods show similar direction of gene expression in B6-Y_B6_ and B6-Y_POS_ males

Fig. 3

Fig. 3

Expression of the novel candidate genes in AmhCre Sox9floxflox XY gonads. a Immunofluorescence of the wild-type and Sox9 knockout gonad at E13.5. Sox9 protein is lost from the testicular cords (white arrows) in the Amh-Cre Sox9floxflox mice yet the testicular cords remain intact, as shown by the laminin stain. Sox9/Laminin is shown in green, and nuclei stained with DAPI are shown in blue. b Expression levels of candidate genes in AmhCre Sox9floxflox XY gonads (red) and WT B6 XY gonads (blue). Expression in WT B6 female gonads is also shown (XX in green). The expression values are shown in TMM values (trimmed mean of M values). RNA-seq was done n = 3 with six pooled E13.5 gonads in each sample. Error bars represent standard error of the mean. Asterisks indicate significantly differentially expressed genes based on an adjusted P value < 0.05

Fig. 4

Fig. 4

Profiles of candidate gene expression in the gonad at different sex developmental stages. Candidate gene profile graphs were generated from the microarray performed by Jameson et al. [42] where gene expression was profiled in each cell population of the gonad at E11.5, E12.5, and E13.5. Similar to the Sox9 target genes Amh and Ptgds (only Amh is shown—outlined in green), the new candidate genes show strong expression in the male supporting lineage (solid blue line) compared to the female (dotted blue line). There was no information available in the microarray data for Tox2

Fig. 5

Fig. 5

Fbln2 protein expression in WT B6 females and males at E12.5 by immunohistochemistry. Section of WT B6 female and male embryos at E12.5 stained for Fbln2 (red) and cell nuclei (purple). Fbln2 is expressed in a sexually dimorphic pattern as no expression is present in WT B6 female (left), whereas the expression in WT B6 male is high. The gonads are encircled by yellow dashed lines

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