GENETIC AND EPIGENETIC MECHANISMS OF COMPLEX REPRODUCTIVE DISORDERS

Common, complex disorders are polygenic and multifactorial traits representing interactions between environmental, genetic and epigenetic risk factors. More often than not, contributions of these risk factors have been studied individually and this is especially true for complex reproductive traits where application of genomic technologies has been challenging and slow to progress. This thesis explores the potential of genetic and epigenetic components contributing to a better understanding of the biological pathways underlying disease risk in two specific female complex reproductive traits - polycystic ovary syndrome (PCOS) and preterm premature rupture of membranes (PPROM). The PCOS projects focus on characterization of a gene, DENND1A, whose association to PCOS has been established by Genome Wide Association Studies (GWAS) and is known to contribute to PCOS steroidogenic phenotype. In addition, differential microRNAs expression contributing to DENND1A expression regulation in PCOS theca cells was identified. The studies on PPROM utilize a Whole Exome Sequencing approach to identify rare variants in fetal genes contributing to extracellular matrix composition and synthesis contributing to PPROM risk. The results suggest that fetal contribution to PPROM is polygenic and is driven by a significant genetic burden of potentially damaging rare variants in genes contributing to fetal membrane strength and integrity. Tissue and location specific expression patterns of the Chromosome 21 miRNA cluster (miR-99a, miR-125b, let-7c) in fetal membranes from term pregnancies with spontaneous rupture were investigated. The results suggest that these miRNAs play potential roles in fetal membrane rupture and fetal membrane defects associated with T21

Mutations in fetal genes involved in innate immunity and host defense against microbes increase risk of preterm premature rupture of membranes (PPROM)

BackgroundTwin studies have revealed a significant contribution of the fetal genome to risk of preterm birth. Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm delivery. Infection and inflammation of the fetal membranes is commonly found associated with PPROM.MethodsWe carried out whole exome sequencing (WES) of genomic DNA from neonates born of Africanâ American mothers whose pregnancies were complicated by PPROM (76) or were normal term pregnancies (N = 43) to identify mutations in 35 candidate genes involved in innate immunity and host defenses against microbes. Targeted genotyping of mutations in the candidates discovered by WES was conducted on an additional 188 PPROM cases and 175 controls.ResultsWe identified rare heterozygous nonsense and frameshift mutations in several of the candidate genes, including CARD6, CARD8, DEFB1, FUT2, MBL2, NLP10, NLRP12, and NOD2. We discovered that some mutations (CARD6, DEFB1, FUT2, MBL2, NLRP10, NOD2) were present only in PPROM cases.ConclusionsWe conclude that rare damaging mutations in innate immunity and host defense genes, the majority being heterozygous, are more frequent in neonates born of pregnancies complicated by PPROM. These findings suggest that the risk of preterm birth in Africanâ Americans may be conferred by mutations in multiple genes encoding proteins involved in dampening the innate immune response or protecting the host against microbial infection and microbial products.Rare damaging mutations in fetal innate immunity and host defense genes, the majority being heterozygous, are more frequent in neonates born of pregnancies complicated by preterm premature rupture of membranes. An increased risk of preterm birth may be conferred by mutations in multiple genes encoding proteins involved in dampening the innate immune response or protecting the host against microbial infection and microbial products.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140041/1/mgg3330.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/140041/2/mgg3330_am.pd

Functional genomics of PCOS: from GWAS to molecular mechanisms

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by increased ovarian androgen biosynthesis, anovulation, and infertility. PCOS has a strong heritable component based on familial clustering and twin studies. Genome-wide association studies (GWAS) identified several PCOS candidate loci including, DENND1A, LHCGR, FSHR, ZNF217, YAP1, INSR, RAB5B, and C9orf3. Here, we review the functional roles of strong PCOS candidate loci focusing on FSHR, LHCGR, INSR and DENND1A. We propose that these candidates comprise a hierarchical signaling network by which DENND1A, LHCGR, INSR, RAB5B, adapter proteins, and associated downstream signaling cascades converge to regulate theca cell androgen biosynthesis. Future elucidation of the functional gene networks predicted by the PCOS GWAS will result in new diagnostic and therapeutic approaches for women with PCOS

Expression patterns of the chromosome 21 MicroRNA cluster (miR-99a, miR-125b and let-7c) in chorioamniotic membranes

AbstractTrisomy 21 (T21) is the most common chromosome abnormality in humans and is associated with a spectrum of phenotypes, including cognitive impairment, congenital heart defects and immune system defects. In addition, T21 is also associated with abnormalities of fetal membranes including chorioamniotic separation, delayed fusion of the chorioamniotic membranes, defects in syncytiotrophoblast formation, as well as amniocyte senescence. There is evidence indicating miRNAs encoded by sequences on chromosome 21 (Chr-21) are involved in several of the cognitive and neurological phenotypes of T21, but the role of Chr-21 derived miRNAs in fetal membrane abnormalities associated with T21 has not been investigated. In the current study, we determined the expression patterns of three miRNAs derived from a cluster on Chr-21 – hsa-miR-99a, hsa-miR-125b and hsa-let-7c in chorioamniotic membranes obtained from term pregnancies with spontaneous rupture (n = 20). Tissue and location specific expression patterns within the chorioamniotic membranes were identified. The rupture zone in the choriodecidua had distinct expression patterns compared to other fetal membrane locations. Despite the increased gene dosage associated with T21, the expression of all three miRNAs was reduced in cultured T21 amniocytes as compared to cultured euploid amniocytes. In silico analysis of experimentally validated targets of the three miRNAs suggest these Chr-21 derived miRNAs play a potential role in fetal membrane rupture and the fetal membrane defects associated with T21

Rare mutations and potentially damaging missense variants in genes encoding fibrillar collagens and proteins involved in their production are candidates for risk for preterm premature rupture of membranes

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest-fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans

Clinical IRAK4 deficiency caused by homozygosity for the novel <i>IRAK4</i> (c.1049delG, p.Gly350Glufs*15) variant

The innate immune system allows for rapid recognition of pathogens. Toll-like receptor (TLR) signaling is a key aspect of the innate immune response, and interleukin-1 receptor-associated kinase 4 (IRAK4) plays a vital role in the TLR signaling cascade. Each TLR recognizes a distinct set of pathogen-associated molecular patterns (PAMPs) that encompass conserved microbial components such as lipopolysaccharides and flagellin. Upon binding of PAMPs and TLR activation, TLR intracellular domains initiate the oligomerization of the myeloid differentiation primary response 88 (MyD88), IRAK1, IRAK2, and IRAK4 signaling platform known as the Myddosome complex while also triggering the Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathway. The Myddosome complex initiates signal transduction pathways enabling the activation of NF-κB and mitogen-activated protein kinase (MAPK) transcription factors and the subsequent production of inflammatory cytokines. Human IRAK4 deficiency is an autosomal recessive inborn error of immunity that classically presents with blunted or delayed inflammatory response to infection and susceptibility to a narrow spectrum of pyogenic bacteria, particularly Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. We describe a case of IRAK4 deficiency in an 11-mo-old boy with concurrent S. pneumoniae bacteremia and S. aureus cervical lymphadenitis with a blunted inflammatory response to invasive infection. Although initial clinical immune profiling was unremarkable, a high degree of suspicion for an innate immune defect prompted genetic sequencing. Genetic testing revealed a novel variant in the IRAK4 gene (c.1049delG, p.(Gly350Glufs*15)) predicted to be likely pathogenic. Functional testing showed a loss of IRAK4 protein expression and abolished TLR signaling, confirming the pathogenicity of this novel IRAK4 variant.</jats:p

miRNA Profiling Reveals miRNA-130b-3p Mediates DENND1A Variant 2 Expression and Androgen Biosynthesis

Abstract Polycystic ovary syndrome (PCOS) is a common endocrine disorder of reproductive-age women involving overproduction of ovarian androgens and, in some cases, from the adrenal cortex. Family studies have established that PCOS is a complex heritable disorder with genetic and epigenetic components. Several small, noncoding RNAs (miRNAs) have been shown to be differentially expressed in ovarian cells and follicular fluid and in the circulation of women with PCOS. However, there are no reports of global miRNA expression and target gene analyses in ovarian theca cells isolated from normal cycling women and women with PCOS, which are key to the elucidation of the basis for the hyperandrogenemia characteristic of PCOS. With the use of small RNA deep sequencing (miR-seq), we identified 18 differentially expressed miRNAs in PCOS theca cells; of these, miR-130b-3p was predicted to target one of the PCOS genome-wide association study candidates, differentially expressed in neoplastic vs normal cells domain containing 1A (DENND1A). We previously reported that DENND1A variant 2 (DENND1A.V2), a truncated isoform of DENND1A, is upregulated in PCOS theca cells and mediates augmented androgen biosynthesis in PCOS theca cells. The comparison of miR-130b-3p in normal and PCOS theca cells demonstrated decreased miR-130b-3p expression in PCOS theca cells, which was correlated with increased DENND1A.V2, cytochrome P450 17α-hydroxylase (CYP17A1) mRNA and androgen biosynthesis. miR-130b-3p mimic studies established that increased miR130b-3p is correlated with decreased DENND1A.V2 and CYP17A1 expression. Thus, in addition to genetic factors, post-transcriptional regulatory mechanisms via miR-130b-3p underly androgen excess in PCOS. Ingenuity® Pathway Analysis Core Pathway and Network Analyses suggest a network by which miR-130b-3p, DENND1A, the luteinizing hormone/choriogonadotropin receptor, Ras-related protein 5B, and signaling pathways that they potentially target may mediate hyperandrogenism in PCOS.</jats:p

Inborn Errors of Immunity Associated With Type 2 Inflammation in the USIDNET Registry.

BackgroundMonogenic conditions that disrupt proper development and/or function of the immune system are termed inborn errors of immunity (IEIs), also known as primary immunodeficiencies. Patients with IEIs often suffer from other manifestations in addition to infection, and allergic inflammation is an increasingly recognized feature of these conditions.MethodsWe performed a retrospective analysis of IEIs presenting with allergic inflammation as reported in the USIDNET registry. Our inclusion criteria comprised of patients with a reported monogenic cause for IEI where reported lab eosinophil and/or IgE values were available for the patient prior to them receiving potentially curative therapy. Patients were excluded if we were unable to determine the defective gene underlying their IEI. Patients were classified as having eosinophilia or elevated IgE when their record included at least 1 eosinophil count or IgE value that was greater than the age stratified upper limit of normal. We compared the proportion of patients with eosinophilia or elevated IgE with the proportion of samples in a reference population that fall above the upper limit of normal (2.5%).ResultsThe query submitted to the USIDNET registry identified 1409 patients meeting inclusion criteria with a monogenic cause for their IEI diagnosis, of which 975 had eosinophil counts and 645 had IgE levels obtained prior to transplantation or gene therapy that were available for analysis. Overall, 18.8% (183/975) of the patients evaluated from the USIDNET registry had eosinophilia and 20.9% (135/645) had an elevated IgE. IEIs caused by defects in 32 genes were found to be significantly associated with eosinophilia and/or an elevated IgE level, spanning 7 of the 10 IEI categories according to the International Union of Immunological Societies classification.ConclusionType 2 inflammation manifesting as eosinophilia or elevated IgE is found in a broad range of IEIs in the USIDNET registry. Our findings suggest that allergic immune dysregulation may be more widespread in IEIs than previously reported

Human DENND1A.V2 Drives Cyp17a1 Expression and Androgen Production in Mouse Ovaries and Adrenals

The DENND1A locus is associated with polycystic ovary syndrome (PCOS), a disorder characterized by androgen excess. Theca cells from ovaries of PCOS women have elevated levels of a DENND1A splice variant (DENND1A.V2). Forced expression of this variant in normal theca cells increases androgen biosynthesis and CYP17A1 expression, whereas knockdown of the transcript in PCOS theca cells reduced androgen production and CYP17A1 mRNA. We attempted to create a murine model of PCOS by expressing hDENND1A.V2 using standard transgenic approaches. There is no DENND1A.V2 protein equivalent in mice, and the murine Dennd1a gene is essential for viability since Dennd1a knockout mice are embryonically lethal, suggesting that Dennd1a is developmentally critical. Three different hDENND1A.V2 transgenic mice lines were created using CMV, Lhcgr, and TetOn promoters. The hDENND1A.V2 mice expressed hDENND1A.V2 transcripts. While hDENND1A.V2 protein was not detectable by Western blot analyses, appropriate hDENND1A.V2 immunohistochemical staining was observed. Corresponding Cyp17a1 mRNA levels were elevated in ovaries and adrenals of CMV transgenic mice, as were plasma steroid production by theca interstitial cells isolated from transgenic ovaries. Even though the impact of robust hDENND1A.V2 expression could not be characterized, our findings are consistent with the notion that elevated hDENND1A.V2 has a role in the hyperandrogenemia of PCOS.Other UBCNon UBCReviewedFacult