Insights into the genetics of menopausal vasomotor symptoms: genome-wide analyses of routinely-collected primary care health records.

BACKGROUND: Vasomotor symptoms (VMS) can often significantly impact women's quality of life at menopause. In vivo studies have shown that increased neurokinin B (NKB) / neurokinin 3 receptor (NK3R) signalling contributes to VMS, with previous genetic studies implicating the TACR3 gene locus that encodes NK3R. Large-scale genomic analyses offer the possibility of biological insights but few such studies have collected data on VMS, while proxy phenotypes such as hormone replacement therapy (HRT) use are likely to be affected by changes in clinical practice. We investigated the genetic basis of VMS by analysing routinely-collected health records. METHODS: We performed a GWAS of VMS derived from linked primary-care records and cross-sectional self-reported HRT use in up to 153,152 women from UK Biobank, a population-based cohort. In a subset of this cohort (n = 39,356), we analysed exome-sequencing data to test the association with VMS of rare deleterious genetic variants. Finally, we used Mendelian randomisation analysis to investigate the reasons for HRT use over time. RESULTS: Our GWAS of health-records derived VMS identified a genetic signal near TACR3 associated with a lower risk of VMS (OR=0.76 (95% CI 0.72,0.80) per A allele, P=3.7x10-27), which was consistent with previous studies, validating this approach. Conditional analyses demonstrated independence of genetic signals for puberty timing and VMS at the TACR3 locus, including a rare variant predicted to reduce functional NK3R levels that was associated with later menarche (P = 5 × 10-9) but showed no association with VMS (P = 0.6). Younger menopause age was causally-associated with greater HRT use before 2002 but not after. CONCLUSIONS: We provide support for TACR3 in the genetic basis of VMS but unexpectedly find that rare genomic variants predicted to lower NK3R levels did not modify VMS, despite the proven efficacy of NK3R antagonists. Using genomics we demonstrate changes in genetic associations with HRT use over time, arising from a change in clinical practice since the early 2000s, which is likely to reflect a switch from preventing post-menopausal complications in women with earlier menopause to primarily treating VMS. Our study demonstrates that integrating routinely-collected primary care health records and genomic data offers great potential for exploring the genetic basis of symptoms.

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage.

AIMS/HYPOTHESIS: Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes. METHODS: We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes. RESULTS: We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.

Loss of MANF Causes Childhood-Onset Syndromic Diabetes Due to Increased Endoplasmic Reticulum Stress.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse ß-cells, its precise role in human ß-cell development and function is unknown. In this study, we show that lack of MANF in humans results in diabetes due to increased ER stress, leading to impaired ß-cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human ß-cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin-processing capacity of ß-cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in recipients with diabetes. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human ß-cell function and demonstrate the crucial role of MANF in this process.

Development and Standardization of an Improved Type 1 Diabetes Genetic Risk Score for Use in Newborn Screening and Incident Diagnosis.

OBJECTIVE: Previously generated genetic risk scores (GRSs) for type 1 diabetes (T1D) have not captured all known information at non-HLA loci or, particularly, at HLA risk loci. We aimed to more completely incorporate HLA alleles, their interactions, and recently discovered non-HLA loci into an improved T1D GRS (termed the "T1D GRS2") to better discriminate diabetes subtypes and to predict T1D in newborn screening studies. RESEARCH DESIGN AND METHODS: In 6,481 case and 9,247 control subjects from the Type 1 Diabetes Genetics Consortium, we analyzed variants associated with T1D both in the HLA region and across the genome. We modeled interactions between variants marking strongly associated HLA haplotypes and generated odds ratios to create the improved GRS, the T1D GRS2. We validated our findings in UK Biobank. We assessed the impact of the T1D GRS2 in newborn screening and diabetes classification and sought to provide a framework for comparison with previous scores. RESULTS: The T1D GRS2 used 67 single nucleotide polymorphisms (SNPs) and accounted for interactions between 18 HLA DR-DQ haplotype combinations. The T1D GRS2 was highly discriminative for all T1D (area under the curve [AUC] 0.92; P < 0.0001 vs. older scores) and even more discriminative for early-onset T1D (AUC 0.96). In simulated newborn screening, the T1D GRS2 was nearly twice as efficient as HLA genotyping alone and 50% better than current genetic scores in general population T1D prediction. CONCLUSIONS: An improved T1D GRS, the T1D GRS2, is highly useful for classifying adult incident diabetes type and improving newborn screening. Given the cost-effectiveness of SNP genotyping, this approach has great clinical and research potential in T1D.

The Common HNF1A Variant I27L Is a Modifier of Age at Diabetes Diagnosis in Individuals With HNF1A-MODY.

There is wide variation in the age at diagnosis of diabetes in individuals with maturity-onset diabetes of the young (MODY) due to a mutation in the HNF1A gene. We hypothesized that common variants at the HNF1A locus (rs1169288 [I27L], rs1800574 [A98V]), which are associated with type 2 diabetes susceptibility, may modify age at diabetes diagnosis in individuals with HNF1A-MODY. Meta-analysis of two independent cohorts, comprising 781 individuals with HNF1A-MODY, found no significant associations between genotype and age at diagnosis. However after stratifying according to type of mutation (protein-truncating variant [PTV] or missense), we found each 27L allele to be associated with a 1.6-year decrease (95% CI -2.6, -0.7) in age at diagnosis, specifically in the subset (n = 444) of individuals with a PTV. The effect size was similar and significant across the two independent cohorts of individuals with HNF1A-MODY. We report a robust genetic modifier of HNF1A-MODY age at diagnosis that further illustrates the strong effect of genetic variation within HNF1A upon diabetes phenotype.

Functional characterisation of ADIPOQ variants using individuals recruited by genotype.

Four non-coding GWAS variants in or near the ADIPOQ gene (rs17300539, rs17366653, rs3821799 and rs56354395) together explain 4% of the variation in circulating adiponectin. The functional basis for this is unknown. We tested the effect of these variants on ADIPOQ transcription, splicing and stability respectively in adipose tissue samples from participants recruited by rs17366653 genotype. Transcripts carrying rs17300539 demonstrated a 17% increase in expression (p = 0.001). Variant rs17366653 was associated with disruption of ADIPOQ splicing leading to a 7 fold increase in levels of a non-functional transcript (p = 0.002). Transcripts carrying rs56354395 demonstrated a 59% decrease in expression (p = <0.0001). No effects of rs3821799 genotype on expression was observed. Association between variation in the ADIPOQ gene and serum adiponectin may arise from effects on mRNA transcription, splicing or stability. These studies illustrate the utility of recruit-by-genotype studies in relevant human tissues in functional interpretation of GWAS signals.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1.

AIMS/HYPOTHESIS: Intronic single nucleotide polymorphisms (SNPs) in the CDKAL1 gene are associated with risk of developing type 2 diabetes. A strong correlation between risk alleles and lower levels of the non-coding RNA, CDKAL1-v1, has recently been reported in whole blood extracted from Japanese individuals. We sought to replicate this association in two independent cohorts: one using whole blood from white UK-resident individuals, and one using a collection of human pancreatic islets, a more relevant tissue type to study with respect to the aetiology of diabetes. METHODS: Levels of CDKAL1-v1 were measured by real-time PCR using RNA extracted from human whole blood (n = 70) and human pancreatic islets (n = 48). Expression with respect to genotype was then determined. RESULTS: In a simple linear regression model, expression of CDKAL1-v1 was associated with the lead type 2 diabetes-associated SNP, rs7756992, in whole blood and islets. However, these associations were abolished or substantially reduced in multiple regression models taking into account rs9366357 genotype: a moderately linked SNP explaining a much larger amount of the variation in CDKAL1-v1 levels, but not strongly associated with risk of type 2 diabetes. CONCLUSIONS/INTERPRETATION: Contrary to previous findings, we provide evidence against a role for dysregulated expression of CDKAL1-v1 in mediating the association between intronic SNPs in CDKAL1 and susceptibility to type 2 diabetes. The results of this study illustrate how caution should be exercised when inferring causality from an association between disease-risk genotype and non-coding RNA expression.

Targeted allelic expression profiling in human islets identifies cis-regulatory effects for multiple variants identified by type 2 diabetes genome-wide association studies.

Genome-wide association studies (GWAS) have identified variation at >65 genomic loci associated with susceptibility to type 2 diabetes, but little progress has been made in elucidating the molecular mechanisms behind most of these associations. Using samples heterozygous for transcribed single nucleotide polymorphisms (SNPs), allelic expression profiling is a powerful technique for identifying cis-regulatory variants controlling gene expression. In this study, exonic SNPs, suitable for measuring mature mRNA levels and in high linkage disequilibrium with 65 lead type 2 diabetes GWAS SNPs, were identified and allelic expression determined by real-time PCR using RNA and DNA isolated from islets of 36 white nondiabetic donors. A significant allelic expression imbalance (AEI) was identified for 7/14 (50%) genes tested (ANPEP, CAMK2B, HMG20A, KCNJ11, NOTCH2, SLC30A8, and WFS1), with significant AEI confirmed for five of these genes using other linked exonic SNPs. Lastly, results of a targeted islet expression quantitative trait loci experiment support the AEI findings for ANPEP, further implicating ANPEP as the causative gene at its locus. The results of this study support the hypothesis that changes to cis-regulation of gene expression are involved in a large proportion of SNP associations with type 2 diabetes susceptibility.

A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells.

Changes in the levels of specific microRNAs (miRNAs) can reduce glucose-stimulated insulin secretion and increase beta-cell apoptosis, two causes of islet dysfunction and progression to type 2 diabetes. Studies have shown that single nucleotide polymorphisms (SNPs) within miRNA genes can affect their expression. We sought to determine whether miRNAs, with a known role in beta-cell function, possess SNPs within the pre-miRNA structure which can affect their expression. Using published literature and dbSNP, we aimed to identify miRNAs with a role in beta-cell function that also possess SNPs within the region encoding its pre-miRNA. Following transfection of plasmids, encoding the pre-miRNA and each allele of the SNP, miRNA expression was measured. Two rare SNPs located within the pre-miRNA structure of two miRNA genes important to beta-cell function (miR-34a and miR-96) were identified. Transfection of INS-1 and MIN6 cells with plasmids encoding pre-miR-34a and the minor allele of rs72631823 resulted in significantly (p < 0.05) higher miR-34a expression, compared to cells transfected with plasmids encoding the corresponding major allele. Similarly, higher levels were also observed upon transfection of HeLa cells. Transfection of MIN6 cells with plasmids encoding pre-miR-96 and each allele of rs41274239 resulted in no significant differences in miR-96 expression. A rare SNP in pre-miR-34a is associated with increased levels of mature miR-34a. Given that small changes in miR-34a levels have been shown to cause increased levels of beta-cell apoptosis this finding may be of interest to studies looking at determining the effect of rare variants on type 2 diabetes susceptibility.

The splice site variant rs11078928 may be associated with a genotype-dependent alteration in expression of GSDMB transcripts.

BACKGROUND: Many genetic variants have been associated with susceptibility to complex traits by genome wide association studies (GWAS), but for most, causal genes and mechanisms of action have yet to be elucidated. Using bioinformatics, we identified index and proxy variants associated with autoimmune disease susceptibility, with the potential to affect splicing of candidate genes. PCR and sequence analysis of whole blood RNA samples from population controls was then carried out for the 8 most promising variants to determine the effect of genetic variation on splicing of target genes. RESULTS: We identified 31 splice site SNPs with the potential to affect splicing, and prioritised 8 to determine the effect of genotype on candidate gene splicing. We identified that variants rs11078928 and rs2014886 were associated with altered splicing of the GSDMB and TSFM genes respectively. rs11078928, present in the asthma and autoimmune disease susceptibility locus on chromosome 17q12-21, was associated with the production of a novel Δ exon5-8 transcript of the GSDMB gene, and a separate decrease in the percentage of transcripts with inclusion of exon 6, whereas the multiple sclerosis susceptibility variant rs2014886, was associated with an alternative TFSM transcript encompassing a short cryptic exon within intron 2. CONCLUSIONS: Our findings demonstrate the utility of a bioinformatic approach in identification and prioritisation of genetic variants effecting splicing of their host genes, and suggest that rs11078928 and rs2014886 may affect the splicing of the GSDMB and TSFM genes respectively.

MicroRNA expression profiling of human islets from individuals with and without type 2 diabetes: promises and pitfalls.

Recent studies in mouse, involving the ß-cell-specific deletion of Dicer1, have highlighted the crucial role of miRNAs (microRNAs) in regulating insulin secretion and consequently Type 2 diabetes. Identifying the individual miRNAs involved in human islet dysfunction may be of diagnostic and therapeutic interest. miRNA expression profiling of human islets isolated from donors with and without Type 2 diabetes may represent one of the first steps in the discovery of these specific miRNAs. The present review discusses some of the potential pitfalls and promises of such an approach.

Genome-wide homozygosity analysis reveals HADH mutations as a common cause of diazoxide-responsive hyperinsulinemic-hypoglycemia in consanguineous pedigrees.

CONTEXT AND OBJECTIVE: Recessive mutations in the hydroxyacyl-CoA dehydrogenase (HADH) gene encoding the enzyme 3-hydroxyacyl-CoA dehydrogenase are a rare cause of diazoxide-responsive hyperinsulinemic hypoglycemia (HH) with just five probands reported to date. HADH deficiency in the first three identified patients was associated with detectable urinary 3-hydroxyglutarate and raised plasma 3-hydroxybutyryl-carnitine levels, but two recent cases did not have abnormal urine organic acids or acylcarnitines. RESEARCH DESIGN AND METHODS: We studied 115 patients with diazoxide-responsive HH in whom the common genetic causes of HH had been excluded. No patients were reported to have abnormal acylcarnitines or urinary organic acids. Homozygosity mapping was undertaken in probands from 13 consanguineous pedigrees to search for regions harboring mutations that are identical by descent. RESULTS: HADH sequencing was performed after genome-wide single nucleotide polymorphism analysis revealed a large shared region of homozygosity spanning the HADH locus in six unrelated probands. Homozygous mutations were identified in three of these patients and in a further two probands from consanguineous families. HADH analysis in the remainder of the cohort identified mutations in a further six probands for whom consanguinity was not reported, but who originated from countries with high rates of consanguinity. Six different HADH mutations were identified in 11/115 (10%) patients tested. CONCLUSION: HADH mutations are a relatively common cause of diazoxide-responsive HH with a frequency similar to that of GLUD1 and HNF4A mutations. We recommend that HADH sequence analysis is considered in all patients with diazoxide-responsive HH when recessive inheritance is suspected.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis.

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs. -2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

The diabetic phenotype in HNF4A mutation carriers is moderated by the expression of HNF4A isoforms from the P1 promoter during fetal development.

OBJECTIVE: Mutations in the alternatively spliced HNF4A gene cause maturity-onset diabetes of the young (MODY). We characterized the spatial and developmental expression patterns of HNF4A transcripts in human tissues and investigated their role as potential moderators of the MODY phenotype. RESEARCH DESIGN AND METHODS: We measured the expression of HNF4A isoforms in human adult tissues and gestationally staged fetal pancreas by isoform-specific real-time PCR. The correlation between mutation position and age of diagnosis or age-related penetrance was assessed in a cohort of 190 patients with HNF4A mutations. RESULTS: HNF4A was expressed exclusively from the P2 promoter in adult pancreas, but from 9 weeks until at least 26 weeks after conception, up to 23% of expression in fetal pancreas was of P1 origin. HNF4A4-6 transcripts were not detected in any tissue. In whole pancreas, HNF4A9 expression was greater than in islets isolated from the endocrine pancreas (relative level 22 vs. 7%). Patients with mutations in exons 9 and 10 (absent from HNF4A3, HNF4A6, and HNF4A9 isoforms) developed diabetes later than those with mutations in exons 2-8, where all isoforms were affected (40 vs. 24 years; P = 0.029). Exon 9/10 mutations were also associated with a reduced age-related penetrance (53 vs. 10% without diabetes at age 55 years; P < 0.00001). CONCLUSIONS: We conclude that isoforms derived from the HNF4A P1 promoter are expressed in human fetal, but not adult, pancreas, and that their presence during pancreatic development may moderate the diabetic phenotype in individuals with mutations in the HNF4A gene.

RNA processing and mRNA surveillance in monogenic diabetes.

In the eukaryotic cell a number of molecular mechanisms exist to regulate the nature and quantity of transcripts intended for translation. For monogenic diabetes an understanding of these processes is aiding scientists and clinicians in studying and managing this disease. Knowledge of RNA processing and mRNA surveillance pathways is helping to explain disease mechanisms, form genotype-phenotype relationships, and identifying new regions within genes to screen for mutations. Furthermore, recent insights into the regulatory role of micro RNAs (miRNAs) and RNA editing in the pancreas suggests that these mechanisms may also be important in the progression to the diabetic state.