I investigated two conditions affecting female reproductive health, dizygotic (DZ) twinning or double ovulation as a complex trait influencing fertility, and endometriosis with common symptoms of pain and infertility. Research questions and hypotheses are addressed in two sections with large datasets and various experimental and bioinformatic methods.
Section I focuses on the investigation of variation in functional candidate genes in the TGF-β pathway with potential effects on DZ twinning. BMPR1B, BMPR2, and TGFBR1 are strong candidate genes involved in the regulation of follicular growth and ovulation rate in female reproduction. By screening these three genes using direct sequencing and High Resolution Melt (HRM) followed by sequencing, one novel exonic and two novel intronic variants in BMPR1B and BMPR2 were identified in individual mothers of DZ twins. The exonic novel variant (p.Q249E) in BMPR1B was predicted to affect protein function in silico. Moreover, this variant is located at the same position as the mutation (p.Q249R) in BMPR1B increasing ovulation rate in a number of sheep breeds. The identification of a mutation in the same codon suggests a possible role of this gene in DZ twinning and further validation studies are required.
The second study focussed on the investigations of genetic variation affecting endometriosis risk. Three studies were conducted including one candidate gene study and two fine mapping studies of two regions identified in genome-wide association (GWA) studies with strongest evidence for association with endometriosis. The first study of the candidate gene KRAS arose from a publication by Grechukhina and others. The authors reported that the SNP rs61764370 in KRAS at a binding site for the let-7 micRNA, was found at higher frequency in women with endometriosis than in the general population and made strong recommendations for a role of testing this SNP in diagnosis of endometriosis. By re-examining our large GWA data set and directly genotyping this SNP and other specific SNPs in a large sample of familial cases and controls, we failed to replicate results from their study. We found no evidence for association of common variants including rs61764370 in KRAS and endometriosis risk in our samples. Our findings emphasise the important roles of matched control selection, replication, and appropriate reporting of results for association studies.
Additionally, two robust regions of association with endometiosis identified in GWA studies with strong candidate genes WNT4 and CDC42 at 1p36 locus and VEZT and FGD6 at 12q22 locus were fine mapped in two studies using a variety of genetic approaches. Genetic architecture in two regions is slightly different. One rare novel exonic and one in-del variant in the 3’UTR were found 1 in WNT4, but they were restricted to members of the families where the variants were first identified. Some other rare coding variants in the WNT4 region were found only in cases, but not in controls. One novel variant was identified in the 5’UTR in VEZT, but this was not associated with the risk of endometriosis.
Further coding SNPs in genes underlying these GWA loci were selected from public databases and tested for association with endometriosis risk. Many reported coding variants were not polymorphic in our sample. Of the polymorphic SNPs, none of the low frequency or rare variants showed evidence of disease association that warranted further follow up. One common missense variant rs201317857 in VEZT showed suggestive evidence for a protective role in endometriosis risk in our genotype data.
Association signals for non-coding imputed SNPs in each region were evaluated by genotyping in cases and controls. Bioinformatic analyses were performed using integrative tools to annotate functional roles of non-coding SNPs at the two regions. Some non-coding SNPs were located in DNA sequences with potential functional roles in multiple ENCODE cell types. Non-coding SNPs rs12404660, rs3820282, and rs55938609 at 1p36 locus and rs6538617, rs6538618, and rs6538617 at 12q22 locus are located at positions overlapping regions of open chromatin, histone modification, regulatory motif alteration, and binding of transcription factors FOXA1, FOXA2, ESR1, and ESR2 which are important in cell cycle and target tissues of the human female reproductive tract. These findings suggested functional roles for non-coding SNPs in these two GWA regions.
A small functional experiment was conducted to initially evaluate gene expression of VEZT and FGD6 in endometriosis and endometrial normal tissues. Both VEZT and FDG6 were expressed in endometrial samples from cases and controls. The VEZT assays showed differences in the levels of expression in carriers of the minor allele of the top imputed SNP rs4762347 and the key GWA SNP rs10859871. The FGD6 transcripts were expressed at significantly different levels in all samples. VEZT and FGD6 mRNA expression decreased in carriers of minor C allele of rs4762347 compared to non-carriers, but the differences were not statistically significant after corrections for multiple testing.
Our results provide insights into the genetic architecture of these regions. Potential functional SNPs in candidate genes and non-coding regions suggest further investigations to address the role of causal variation in DZ twinning and endometriosis risk.