Phenotype specific association of the TGFBR3 locus with nonsyndromic cryptorchidism

Abstract

Purpose: Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal.

Materials and methods: We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum.

Results: We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum.

Conclusions: These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFβ signaling.

Keywords: cryptorchidism; genetic association studies; phenotype; testis.

Figure 1

SNAP plots showing the association analysis results of imputed data from (A) Group 1 cases and controls, (B) Group 2 cases and controls and (C) Meta-analysis of all cases and controls.

Figure 2

SNAP plots of meta-analysis results of imputed data at the TGFBR3 locus. (A) Cryptorchidism cases with testes located proximal to the external inguinal ring and all controls and (B) Cryptorchidism cases with testes located distal to the external inguinal ring.

Figure 3

Linkage disequilibrium (LD) plot of markers identified in the primary group and subgroup analyses that are available in HaploView (http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview). Lower portion shows an overview of LD blocks within the imputed TGFBR3 locus, with the black rectangle centered on the 3′ end of the TGFBR3 gene.

Figure 4

Tgfbr3 mRNA expression in gubernaculum from wild type (wt; blue bars) and cryptorchid orl (green bars) rat fetuses. Baseline expression is shown for freshly isolated embryonic (E) day 17, 19 and 21 gubernacula and for E17 wt gubernacular explants established in culture followed by exposure to insulin-like 3 (INSL3; 0, 10 or 100 nM) or dihydrotestosterone (DHT; 0, 10 or 30 nM) for 24 hours. *p<0.05 and **p<0.01 for wt vs orl.

Figure 5

A TGFBR3-A (green; 1:50), myosin (red) and nuclei (DAPI; blue) in embryonic (E) day 21 gubernaculum shows an example of prominent soluble betaglycan collections that we observe in some fetuses (10X, scale bar, 200μm). B TGFBR3/betaglycan expression in E17, E19 and E21 gubernacula from wild type (wt) and cryptorchid orl fetuses (green). Staining for myosin (differentiated muscle; red) and TGFBR3-A (extracellular domain, top 2 rows) or TGFBR3-B (cystoplasmic domain, bottom 2 rows), both green (20X, scale bar, 100 μm).