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AimsIndividuals with type 1 diabetes (T1D) carry a markedly increased risk of stroke, with distinct clinical and neuroimaging characteristics as compared to those without diabetes. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we aimed to find rare and low-frequency genomic variants associated with stroke in T1D. The lead findings were followed up in various datasets to replicate the findings and to assess their specificity to diabetes.Methods and ResultsWe studied stroke genetics in 1,051 individuals with T1D using WGS or WES. We analysed the genome with single-variant analyses, gene aggregate analyses, and aggregate analyses on genomic windows, enhancers and promoters. Furthermore, we attempted replication in T1D using a genome-wide association study (N=3,945) and direct genotyping (N=3,600), and in the general population from the FinnGen project and UK Biobank summary statistics. We identified a rare missense mutation onSREBF1associated with hemorrhagic stroke (rs114001633, p.Pro227Leu,p-value=8.96×10-9), which further replicated in T1D. Using gene aggregate analysis with protein altering or protein truncating variants, we identified exome-wide significant genes:ANK1andLRRN1displayed replication evidence in T1D, whileLRRN1,HAS1andUACAreplicated in the general population (UK Biobank). Furthermore, we performed sliding-window analyses and identified 14 genome-wide significant windows for stroke on 4q33-34.1, of which two replicated in T1D, and a suggestive genomic window onLINC01500, which replicated in T1D. Finally, with the regulatory region aggregate analyses, we identified a stroke-associatedTRPM2-ASpromoter (p-value=5.78×10-6), which we validated with an in vitro cell-based assay.TRPM2has been previously linked to ischemic stroke.ConclusionsHere, we report the first genome-wide analysis on stroke in individuals with diabetes. We identified multiple stroke risk loci with evidence of replication: 4q33-34.1,SREBF1, andANK1for stroke in T1D; andHAS1,UACA,LRRN1,LINC01500, andTRPM2-ASpromoter for stroke potentially generalizable to the non-diabetic population.

The early prediction of health outcomes for people with diabetes mellitus is desirable, as are adjunct therapies to reduce the related chronic complications and risk of premature death. The length of telomeres, protective caps on chromosome ends, is influenced by genetic and acquired factors, and shorter telomeres have been associated with and predictive of adverse cardiometabolic outcomes. Many studies have shown associations between telomere length in white blood cells (WBC) and diabetes per se and its chronic complications, and some studies show that telomeres do not always progressively shorten in people with diabetes. With the pandemic of diabetes and taking into consideration the calculations of residual risk using existent risk equations, additional tests to stratify subject risk are desirable. In this evolving era of precision medicine for people with diabetes, this 'global biomarker' of WBC telomere length may be useful to help predict health outcomes, to monitor health status, and may be a therapeutic target. We comment on the field of telomere investigations in diabetes, including recommending areas for further clinical research.

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.

Background Type 1 diabetes (T1D) is an autoimmune disease affecting individuals in the early years of life. Although previous studies have identified genetic loci influencing T1D diagnosis age, these studies did not investigate the genome with high resolution. Objective and methods We performed a genome-wide meta-analysis for age at diagnosis with cohorts from Finland (Finnish Diabetic Nephropathy Study), the United Kingdom (UK Genetic Resource Investigating Diabetes) and Sardinia. Through SNP associations, transcriptome-wide association analysis linked T1D diagnosis age and gene expression. Results We identified two chromosomal regions associated with T1D diagnosis age: multiple independent variants in the HLA region on chromosome 6 and a locus on chromosome 17q12. We performed gene-level association tests with transcriptome prediction models from two whole blood datasets, lymphocyte cell line, spleen, pancreas and small intestine tissues. Of the non-HLA genes, lower PNMT expression in whole blood, and higher IKZF3 and ZPBP2, and lower ORMDL3 and GSDMB transcription levels in multiple tissues were associated with lower T1D diagnosis age (FDR = 0.05). These genes lie on chr17q12 which is associated with T1D, other autoimmune diseases, and childhood asthma. Additionally, higher expression of PHF20L1, a gene not previously implicated in T1D, was associated with lower diagnosis age in lymphocytes, pancreas, and spleen. Altogether, the non-HLA associations were enriched in open chromatin in various blood cells, blood vessel tissues and foetal thymus tissue. Conclusion Multiple genes on chr17q12 and PHF20L1 on chr8 were associated with T1D diagnosis age and only further studies may elucidate the role of these genes for immunity and T1D onset.

AIMS: Diabetes is a known risk factor for coronary artery disease. There is accumulating evidence that coronary artery disease pathogenesis differs for individuals with type 1 diabetes. However, the genetic background has not been extensively studied. We aimed to discover genetic loci increasing coronary artery disease susceptibility especially in type 1 diabetes, to examine the function of these discoveries and to study the role of the known risk loci in type 1 diabetes. METHODS AND RESULTS: We performed the largest genome-wide association study to date for coronary artery disease in type 1 diabetes, comprising 4869 individuals with type 1 diabetes (cases/controls: 941/3928). Two loci reached genome-wide significance, rs1970112 in CDKN2B-AS1 (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 type 1 diabetes 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

Additional file 1: Supplementary material. A combined document including all supplementary tables (Tables S1-S11), figures (Figs. S1-S7), and code (Text S1).

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 the 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 individuals with stroke and 517 matched control subjects, and the other using haptoglobin genotype imputation for a larger cohort of 500 individuals with stroke and 3,806 individuals without stroke. We observed no difference in the Hp1-1, Hp2-1, and Hp2-2 genotype frequencies between individuals with or without stroke, 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 cohort with type 1 diabetes.

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.

Dyslipidemia is a major risk factor for cardiovascular disease. While common genetic variants are known to modestly affect the serum lipid concentrations, rare genetic mutations can cause monogenic forms of hypercholesteremia and other genetic disorders of lipid metabolism. Aiming to identify low-frequency protein-altering variants (PAVs) affecting lipoprotein and lipid traits, we analyzed whole-exome and whole-genome sequencing data of 481 and 573 individuals with type 1 diabetes, respectively. The phenotypic data consisted of 97 serum lipid, apolipoprotein, or other metabolic phenotypes obtained with clinical laboratory measurements and nuclear magnetic resonance (NMR) technology. Single variant analysis identified a novel association between LIPC p.Thr405Met (rs113298164) and serum apolipoprotein-A1 levels (p=7.8×10−8). In the APOB gene, we identified novel associations at two protein-truncating variants (PTVs) resulting in lower serum apolipoprotein B levels (p=5.6×10−4). The burden of PAVs was significantly associated with lipid phenotypes in LIPC, RBM47, TRMT5, and GTF3C5 (p−6). The RBM47 gene is required for apolipoprotein-B post-translational modifications, and in our data, the association between RBM47 and apolipoprotein C-III levels was led by a rare 21 base pair Ala496-Ala502 deletion; as replication, the burden of rare deleterious variants in RBM47 was associated with TG-to-HDLC ratio in WES of 20,917 individuals (p=0.0093). Two PAVs in GTF3C5 were highly Finnish-enriched and associated with cardiovascular phenotypes in external data, whereby the TRMT5 p.Ser185Cys lead variant was associated with stroke phenotypes. Altogether, we identified both novel variant associations in known lipid genes, as well as novel genes implicated in lipoprotein metabolism.

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.

Glycated hemoglobin (HbA(1c)) is an important measure of glycemia in diabetes. HbA(1c) is influenced by environmental and genetic factors both in people with and in people without diabetes. We performed a genome-wide association study (GWAS) for HbA(1c) in a Finnish type 1 diabetes (T1D) cohort, FinnDiane. Top results were examined for replication in T1D cohorts DCCT/EDIC, WESDR, CACTI, EDC, and RASS, and a meta-analysis was performed. Three SNPs in high linkage disequilibrium on chromosome 13 near relaxin family peptide receptor 2 (RXFP2) were associated with HbA(1c) in FinnDiane at genome-wide significance (P

Aims We aimed to determine how white blood cell (WBC) telomeres and telomere length change over time are associated with health status in type 1 diabetes. Methods Relative telomere length (rTL) was measured in WBC DNA from two time-points (median 6.8 years apart) in 618 individuals from the Finnish Diabetic Nephropathy Study by quantitative PCR, with interassay CV ≤ 4%. Results Baseline rTL correlated inversely with age and was shorter in men. Individuals in the shortest vs. longest rTL tertile had adverse cardiometabolic profiles, worse renal function, and were prescribed more antihypertensive and lipid-lowering drugs. While overall rTL tended to decrease during the median 6.8-years of follow-up, telomeres shortened in 55.3% of subjects, lengthened in 40.0%, and did not change in 4.7%. Baseline rTL correlated inversely with rTL change. Telomere lengthening was associated with higher HDL-Cholesterol (HDL-C), HDL-C/ApoA1, and with antihypertensive drug and (inversely) with lipid-lowering drug commencement during follow-up. Correlates of rTL percentage change per-annum (adjusted model) were baseline BMI, eGFR, previous retinal laser treatment, HDL-C, and HDL-C/ApoA1. Conclusions Telomere length measurements may facilitate the treatment and monitoring of the health status of individuals with type 1 diabetes.

Background A subset of individuals with type 1 diabetes mellitus (T1DM) are predisposed to developing diabetic kidney disease (DKD), the most common cause globally of end-stage kidney disease (ESKD). Emerging evidence suggests epigenetic changes in DNA methylation may have a causal role in both T1DM and DKD. The aim of this exploratory investigation was to assess differences in blood-derived DNA methylation patterns between individuals with T1DM-ESKD and individuals with long-duration T1DM but no evidence of kidney disease upon repeated testing to identify potential blood-based biomarkers. Blood-derived DNA from individuals (107 cases, 253 controls and 14 experimental controls) were bisulphite treated before DNA methylation patterns from both groups were generated and analysed using Illumina’s Infinium MethylationEPIC BeadChip arrays (n = 862,927 sites). Differentially methylated CpG sites (dmCpGs) were identified (false discovery rate adjusted p ≤ × 10–8 and fold change ± 2) by comparing methylation levels between ESKD cases and T1DM controls at single site resolution. Gene annotation and functionality was investigated to enrich and rank methylated regions associated with ESKD in T1DM. Results Top-ranked genes within which several dmCpGs were located and supported by functional data with methylation look-ups in other cohorts include: AFF3, ARID5B, CUX1, ELMO1, FKBP5, HDAC4, ITGAL, LY9, PIM1, RUNX3, SEPTIN9 and UPF3A. Top-ranked enrichment pathways included pathways in cancer, TGF-β signalling and Th17 cell differentiation. Conclusions Epigenetic alterations provide a dynamic link between an individual’s genetic background and their environmental exposures. This robust evaluation of DNA methylation in carefully phenotyped individuals has identified biomarkers associated with ESKD, revealing several genes and implicated key pathways associated with ESKD in individuals with T1DM.