Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat.
OBJECTIVE
Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis.


RESULTS
Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively.


BACKGROUND
Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility.


METHODS
The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines.


RESULTS
LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis.


CONCLUSIONS
The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype.


CONCLUSIONS
Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism.


METHODS
Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/).


BACKGROUND
This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare.
Pediatric Urology Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA julia.barthold@nemours.org.
Julia Spencer
JS
Barthold
Pediatric Urology Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Joan
J
Pugarelli
Center for Bioinformatics and Computational Biology, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Madolyn L
ML
MacDonald
Center for Bioinformatics and Computational Biology, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Jia
J
Ren
Center for Bioinformatics and Computational Biology, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Modupeore O
MO
Adetunji
Center for Bioinformatics and Computational Biology, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Shawn W
SW
Polson
Pediatric Urology Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Abigail
A
Mateson
Pediatric Urology Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Yanping
Y
Wang
Biomolecular Core Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Katia
K
Sol-Church
Bioinformatics Core, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Suzanne M
SM
McCahan
Tissue Engineering and Regenerative Medicine Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Robert E
RE
Akins
Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Biostatistics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Molecular Medicine, Sapienza University, Rome, Italy.
Marcella
M
Devoto
Pediatric Urology Research Laboratory, Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
Alan K
AK
Robbins
D000483:Alleles; D000728:Androgens / Q000502:physiology; D000818:Animals; D003456:Cryptorchidism / Q000235:genetics / Q000662:veterinary*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000502:physiology; D005298:Fertility / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D056726:Genetic Association Studies; D008040:Genetic Linkage; D020022:Genetic Predisposition to Disease; D007248:Infertility, Male / Q000235:genetics / Q000662:veterinary; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D020412:Multifactorial Inheritance; D056918:Nuclear Receptor Coactivators / Q000235:genetics / Q000502:physiology; D051381:Rats; D011921:Rats, Inbred WKY; D020318:Rats, Long-Evans / Q000235:genetics*; D011922:Rats, Mutant Strains; D060888:Real-Time Polymerase Chain Reaction; D012376:Rodent Diseases / Q000235:genetics*; D013737:Testis / Q000196:embryology
D000728:Androgens; D003598:Cytoskeletal Proteins; C000611215:Ncoa4 protein, rat; D056918:Nuclear Receptor Coactivators; C000611214:SYNE2 protein, rat
cryptorchidism; genetics; gubernaculum; linkage analysis; rat strains; whole genome sequencing