Genome-wide association study on coronary artery disease in type 1 diabetes suggests beta-defensin 127 as a risk locus

Aims Diabetes is a known risk factor for coronary artery disease (CAD). There is accumulating evidence that CAD pathogenesis differs for individuals with type 1 diabetes (T1D). However, the genetic background has not been extensively studied. We aimed to discover genetic loci increasing CAD susceptibility, especially in T1D, to examine the function of these discoveries and to study the role of the known risk loci in T1D. Methods and results We performed the largest genome-wide association study to date for CAD in T1D, comprising 4869 individuals with T1D (cases/controls: 941/3928). Two loci reached genome-wide significance, rs1970112 in CDKN2B-AS1 [odds ratio (OR) =1.32, P = 1.50 x 10(-8)], and rs6055069 on DEFB127 promoter (OR= 4.17, P= 2.35 x 10(-9)), with consistent results in survival analysis. The CDKN2B-AS1 variant replicated (P = 0.04) when adjusted for diabetic kidney disease in three additional T1D cohorts (cases/controls: 434/3123). Furthermore, we explored the function of the lead discoveries with a cardio-phenome-wide analysis. Among the eight suggestive loci (P <1 x 10(-6)), rs70962766 near B3GNT2 associated with central blood pressure, rs1344228 near CNTNAP5 with intima media thickness, and rs2112481 on GRAMD2B promoter with serum leucocyte concentration. Finally, we calculated genetic risk scores for individuals with T1D with the known susceptibility loci. General population risk variants were modestly but significantly associated with CAD also in T1D (P=4.21 x 10(-7)). Conclusion While general population CAD risk loci had limited effect on the risk in T1D, for the first time, variants at the CDKN2B-AS1 locus were robustly associated with CAD in individuals with T1D. The novel finding on beta-defensin DEFB127 promoter provides a link between diabetes, infection susceptibility, and CAD, although pending on future confirmation. [GRAPHICS] .Peer reviewe

Haptoglobin Genotype Does Not Confer a Risk of Stroke in Type 1 Diabetes

The exon copy number variant in the haptoglobin gene is associated with cardiovascular and kidney disease. For stroke, previous research is inconclusive. We aimed to study the relationship between haptoglobin Hp1/2 genotype and stroke in individuals with type 1 diabetes from the Finnish Diabetic Nephropathy Study. We included two partially overlapping cohorts: one with haptoglobin genotypes determined using genotyping for 179 stroke cases and 517 matched controls, and the other using haptoglobin genotype imputation for a larger cohort of 500 stroke cases and 3,806 controls. We observed no difference in the Hp1-1, Hp2-1, and Hp2-2 genotype frequencies between the stroke cases and controls, neither in the genotyping nor the imputation cohorts. Haptoglobin genotypes were also not associated with the ischemic or hemorrhagic stroke subtypes. In our imputed haptoglobin cohort, 61% of individuals with stroke died during follow-up. However, the risk of death was not related to the haptoglobin genotype. Diabetic kidney disease and cardiovascular events were common in the cohort, but the haptoglobin genotypes were not associated with stroke when stratified by these complications. To conclude, the Hp1/2 genotypes did not affect the risk of stroke or survival after stroke in our type 1 diabetes cohort.Peer reviewe

Whole-exome sequencing identifies novel protein-altering variants associated with serum apolipoprotein and lipid concentrations

Background: Dyslipidemia is a major risk factor for cardiovascular disease, and diabetes impacts the lipid metabolism through multiple pathways. In addition to the standard lipid measurements, apolipoprotein concentrations provide added awareness of the burden of circulating lipoproteins. While common genetic variants modestly affect the serum lipid concentrations, rare genetic mutations can cause monogenic forms of hypercholesterolemia and other genetic disorders of lipid metabolism. We aimed to identify low-frequency protein-altering variants (PAVs) affecting lipoprotein and lipid traits. Methods: We analyzed whole-exome (WES) and whole-genome sequencing (WGS) data of 481 and 474 individuals with type 1 diabetes, respectively. The phenotypic data consisted of 79 serum lipid and apolipoprotein phenotypes obtained with clinical laboratory measurements and nuclear magnetic resonance spectroscopy. Results: The single-variant analysis identified an association between the LIPC p.Thr405Met (rs113298164) and serum apolipoprotein A1 concentrations (p=7.8×10-8). The burden of PAVs was significantly associated with lipid phenotypes in LIPC, RBM47, TRMT5, GTF3C5, MARCHF10, and RYR3 (p170,000 individuals from multiple ancestries (p=0.0013). Two PAVs in GTF3C5 were highly enriched in the Finnish population and associated with cardiovascular phenotypes in the general population. In the previously known APOB gene, we identified novel associations at two protein-truncating variants resulting in lower serum non-HDL cholesterol (p=4.8×10-4), apolipoprotein B (p=5.6×10-4), and LDL cholesterol (p=9.5×10-4) concentrations. Conclusions: We identified lipid and apolipoprotein-associated variants in the previously known LIPC and APOB genes, as well as PAVs in GTF3C5 associated with LDLC, and in RBM47 associated with apolipoprotein C-III concentrations, implicated as an independent CVD risk factor. Identification of rare loss-of-function variants has previously revealed genes that can be targeted to prevent CVD, such as the LDL cholesterol-lowering loss-of-function variants in the PCSK9 gene. Thus, this study suggests novel putative therapeutic targets for the prevention of CVD. Keywords: APOB; Apolipoprotein A1; Apolipoprotein C-III; GTF3C5; LIPC; Lipidomics; MARCHF10; RBM47; RYR3; Whole-exome sequencing.Peer reviewe

CACNB2 Is a Novel Susceptibility Gene for Diabetic Retinopathy in Type 1 Diabetes

Diabetic retinopathy is a common diabetes complication that threatens the eyesight and may eventually lead to acquired visual impairment or blindness. While a substantial heritability has been reported for proliferative diabetic retinopathy (PDR), only a few genetic risk factors have been identified. Using genome-wide sib pair linkage analysis including 361 individuals with type 1 diabetes, we found suggestive evidence of linkage with PDR at chromosome 10p12 overlapping the CACNB2 gene (logarithm of odds = 2.73). Evidence of association between variants in CACNB2 and PDR was also found in association analysis of 4,005 individuals with type 1 diabetes with an odds ratio of 0.83 and P value of 8.6 x 10(-4) for rs11014284. Sequencing of CACNB2 revealed two coding variants, R476C/rs202152674 and S502L/rs137886839. CACNB2 is abundantly expressed in retinal cells and encodes the beta 2 subunit of the L-type calcium channel. Blocking vascular endothelial growth factor (VEGF) by intravitreous anti-VEGF injections is a promising clinical therapy to treat PDR. Our data show that L-type calcium channels regulate VEGF expression and secretion from retinal pigment epithelial cells (ARPE19) and support the role of CACNB2 via regulation of VEGF in the pathogenesis of PDR. However, further genetic and functional studies are necessary to consolidate the findings.Peer reviewe

Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy

We formed the GEnetics of Nephropathy–an International Effort (GENIE) consortium to examine previously reported genetic associations with diabetic nephropathy (DN) in type 1 diabetes. GENIE consists of 6,366 similarly ascertained participants of European ancestry with type 1 diabetes, with and without DN, from the All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Republic of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic Nephropathy Study (FinnDiane), combined with reanalyzed data from the Genetics of Kidneys in Diabetes U.S. Study (U.S. GoKinD). We found little evidence for the association of the EPO promoter polymorphism, rs161740, with the combined phenotype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, P = 0.60). However, a fixed-effects meta-analysis that included the previously reported cohorts retained a genome-wide significant association with that phenotype (OR 1.31, P = 2 × 10−9). An expanded investigation of the ELMO1 locus and genetic regions reported to be associated with DN in the U.S. GoKinD yielded only nominal statistical significance for these loci. Finally, top candidates identified in a recent meta-analysis failed to reach genome-wide significance. In conclusion, we were unable to replicate most of the previously reported genetic associations for DN, and significance for the EPO promoter association was attenuated

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

New susceptibility loci associated with kidney disease in type 1 diabetes

WOS:000309817900008Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.Peer reviewe

Genetic aspects of type 1 diabetes and its complications

Type 1 diabetes (T1D) is an autoimmune form of diabetes with a high incidence in Finland. The typical onset age of T1D is in childhood, but the disease may also manifest in adults. The late complications of diabetes in the kidneys (diabetic nephropathy) and in the eyes (diabetic retinopathy), however, appear only after living for years with diabetes. High blood glucose levels are a major risk factor for these microvascular complications. Glycated hemoglobin (HbA1c), a measure of long-term hyperglycemia, constantly forms when free glucose attaches to hemoglobin in the red blood cells. Earlier genetic and heritability analyses have shown that all these phenotypes, T1D, HbA1c, and complications of diabetes are affected by the genome. The studies included in this thesis had four specific aims: 1) to examine the relationship between leukocyte telomere length and diabetic nephropathy, 2) to study genetic variability in three histone methyltransferases and the risk of diabetes complications, 3) to find genetic factors associated with HbA1c in type 1 diabetes in a genome-wide association study, and 4) to look for genetic variants and genes associated with age at diagnosis of type 1 diabetes (Study IV). The Finnish Diabetic Nephropathy (FinnDiane) Study was the primary study cohort. Several other diabetes cohorts served as replication cohorts (Studies II and III) or were included together with the FinnDiane in a genome-wide association study meta-analysis as in Study IV. As commonly shown, telomere length shortened with age in our cohort. Most importantly, short telomere length and a higher proportion of short telomeres predicted diabetic nephropathy progression. In the candidate gene study, an exonic SNP in SUV39H2 was associated with diabetic retinopathy. Study III showed that HbA1c is partly an inherited trait in diabetes. A locus on chromosome 13 closest to the gene RXFP2 was associated with HbA1c with genome-wide significance. Additionally, many variants known to be associated with HbA1c in the general population had a similar direction in our diabetes cohort. In Study IV, our top associations for T1D diagnosis age were in known T1D risk loci in the HLA region on chromosome 6 and chromosome 17q12, a known risk locus for childhood-onset asthma as well. However, most of the suggestive associations were at genomic regions not previously implicated in T1D. A transcriptome-wide association study highlighted genes such as IKZF3, GSDMB, ORMDL3, and ZBPB2 in the chr17q12 locus. To conclude, the four studies included in this thesis utilized various genetic study approaches and found significant associations between genetic variants and age of diagnosis of T1D, HbA1c, and diabetes complications. More importantly, the HbA1c values are surprisingly stable over time and are affected by genetic variants. An analysis integrating the information from genetic variants and gene expression suggested genes that have potential age-related effects in the pathogenesis of T1D.Tyypin 1 diabetes on autoimmuunitauti, jonka ilmaantuvuus on Suomessa korkea. Tyypin 1 diabetes diagnosoidaan yleensä lapsuusiällä, mutta sairaus voi myös alkaa aikuisena. Diabeteksen aiheuttamat myöhäiskomplikaatiot munuaisiin (diabeettinen munuaistauti) ja silmiin (diabeettinen retinopatia) alkavat kuitenkin vasta kun diabeteksen kanssa on eletty jo vuosia. Korkea verensokeri on suurin yksittäinen mikrovaskulaaristen myöhäis-komplikaatioiden riskitekijä. Pitkäaikaisen sokerialtistuksen mittarina käytetään sokerihemoglobiinia (HbA1c), jota muodostuu jatkuvasti, kun glukoosimolekyylit tarttuvat hemoglobiin veren punasoluissa. Aikaisemmat tutkimukset ovat osoittaneet, että perimällä on vaikutusta sekä diabetesriskiin, sokerihemoglobiinitasoihin, että diabeteksen myöhäis-komplikaatioihin riskiin. Väitöskirjaan kuuluvien neljän osatutkimuksen tavoitteet olivat: 1) tutkia, onko valkosolujen telomeerien lyhyys diabeettisen munuaistaudin riskitekijä, 2) selvittää, assosioiko geneettinen monimuotoisuus kolmessa histoni metyylitransferaasi -geenissä diabeteksen myohäiskomplikaatioiden riskiin, 3) löytää tyypin 1 diabeetikon HbA1c-tasoihin vaikuttavia geneettisiä variantteja genominlaajuisella assosiaatiotutkimuksella, 4) sekä tunnistaa geneettisiä variantteja ja geenejä, jotka assosioivat tyypin 1 diabeteksen alkamisikään. Pääasiallinen tutkimuskohorttimme oli FinnDiane (The Finnish Diabetic Nephropathy Study), mutta muilla diabetes-kohorteilla oli tärkeä rooli löydösten toistamisessa (Tutkimukset II ja III), tai ne olivat samanarvoisena osana genominlaajuisessa assosiaatiotutkimuksen meta-analyysissa (Tutkimus IV). Kuten yleensä huomataan, telomeerien pituus korreloi käänteisesti ikään myös meidän aineistossamme. Telomeerin lyhyt keskipituus ja suuri lyhyiden telomeerien osuus ennusti diabeettisen munuaistaudin etenemistä. Ehdokasgeenitutkimuksessa eräs SUV39H2 geenin eksonissa sijaitseva SNP yhdistyi diabeettiseen retinopatiaan. Tutkimus III osoitti HbA1c:n olevan osin perinnöllinen ominaisuus myös tyypin 1 diabeetikoilla. Tutkimuksessa löytyi kromosomista 13 HbA1c tasoihin merkitsevästi assosioiva lokus, jonka lähin geeni on RXFP2. Lisäksi muutaman muun tunnetun HbA1c tasoihin vaikuttavan SNP:n assosiaatio oli samansuuntainen diabetes-kohortissamme. Tutkimuksessa 4 tyypin 1 diabeteksen alkamisikään assosioi erityisesti kaksi tunnettua diabeteksen riskialuetta genomissa: HLA-alue kromosomissa 6 ja kromosomi 17q12, joka on myös tunnettu lapsuusiän astman riskilokus. Useimmat variantit, jotka eivät aivan yltäneet genominlaajuiseen merkitsevyyteen olivat kuitenkin genomissa alueilla, joita ei ole aiemmin yhdistetty diabetesriskiin. Kromosomin 17q12 lokuksen geeneistä IKZF3, GSDMB, ORMDL3 ja ZBPB2 nousivat esiin geenien ennustettua ilmenemistasoa hyödyntävässä genominlaajuisessa assosiaatiotutkimuksessa. Yhteenvetona, väitöskirjan neljä osatutkimusta hyödynsivät monimuotoisesti geneettisiä tutkimusmenetelmiä ja löysivät merkitseviä yhteyksiä perimän vaihtelun ja sokerihemoglobiinin, sekä tyypin 1 diabeteksen ja sen komplikaatioiden välillä. HbA1c-tasot ovat yllättävän pysyviä tyypin 1 diabeetikoilla ja perimä vaikuttaa niihin. Analyysimme, joka yhdisti tietoa geneettisistä varianteista ja geenien ilmenemisestä osoitti joukon mahdollisia geenejä, joiden vaikutus tyypin 1 diabeksen syntyyn on riippuvainen iästä

Telomeres in clinical diabetes research – Moving towards precision medicine in diabetes care?

Peer reviewe

Poster abstract: Epigenetic analyses of diabetic kidney disease attributed to type 1 diabetes in European populations

Objective: This research was conducted to identify blood-derived DNA methylation in individuals with type 1 diabetes (T1D) who progressed to diabetic kidney disease (T1DKD, n=651) compared to persons with T1D and no evidence of kidney disease (n=651). The study included participants with at least 10-year duration of T1D from the United Kingdom (UK), Republic of Ireland (ROI) and Finland.Design: Case-control analysis of differentially methylated CpG sites (dmCpGs) was undertaken. The Infinium HD Methylation Assay, MethylationEPIC BeadChips (Illumina) were used to evaluate the methylation status of 862,927 CpG sites. The distribution of single-site methylation levels between individuals with T1DKD and those with T1D was assessed adjusting for covariates including age, sex, white cell counts (WCCs) and smoking using RnBeads and Bioconductor R packages. Individuals from the UK-ROI and Finnish populations were analysed separately before results were compared. Quality control included the removal of cross-reactive probes, those located near common SNPs, or on sex chromosomes. Raw intensities were normalised using the bmiq method. Setting: Wet-lab work was completed in research laboratories within the Belfast HSC Trust regional genetics diagnostic centre. Harmonised data analysis was conducted using RnBeads and Bioconductor R packages for each population. Patients: DNA was extracted from peripheral blood from individuals with T1D and known renal status recruited to the UK-ROI (n=502) and Finnish Diabetic Nephropathy (FinnDiane, n=800) studies. Case individuals had T1DKD defined as persistent macroalbuminuria (≥500 mg/24hr), eGFR &lt;60 mL/min/m2, hypertension (≥135/85 mmHg) and diabetic retinopathy. Control individuals had T1D and no evidence of kidney disease on repeat testing.Main Outcome Measurements: For all CpG sites, β values were generated, M values derived, and p values computed. Comparisons of dmCpGs between cases and controls were performed. Results: When comparing independent outcomes from UK-ROI and FinnDiane, adjusting for the minimal model of age, sex and WCCs, comparisons of dmCpGs identified 11 dmCpGs common to both cohorts (FDRadjp≤x10-5). Of these, cg17944885, located close to ZNF44 and cg25544931 were more significant in both populations (FDRadjp≤x10-8). Both were located on chr19 within CpG shelves and shores. Further adjustment for smoking status reduced the number of common dmCpGs to two in both populations; cg17944885 (FDRadjp&lt;x10-8) and cg05710777 located within LOC101927438 (FDRadjp≤x10-5).Conclusions: Investigation of additional potential confounding factors such as duration of diabetes, BMI, HbA1c and lipid measures are ongoing. These analyses provided the opportunity to assess previously unexplored regions of the methylome for T1DKD using two independent populations with harmonised inclusion criteria. Blood-derived methylation signatures may have utility as minimally invasive biomarkers for DKD attributed to T1D