Whole-exome and whole-genome sequencing of 1064 individuals with type 1 diabetes reveals novel genes for diabetic kidney disease.

AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is a severe diabetic complication that affects one third of individuals with type 1 diabetes. Although several genes and common variants have been shown to be associated with DKD, much of the predicted inheritance remains unexplained. Here, we performed next-generation sequencing to assess whether low-frequency variants, extending to a minor allele frequency (MAF) ≤10% (single or aggregated) contribute to the missing heritability in DKD. METHODS: We performed whole-exome sequencing (WES) of 498 individuals and whole-genome sequencing (WGS) of 599 individuals with type 1 diabetes. After quality control, next-generation sequencing data were available for a total of 1064 individuals, of whom 541 had developed either severe albuminuria or end-stage kidney disease, and 523 had retained normal albumin excretion despite a long duration of type 1 diabetes. Single-variant and gene-aggregate tests for protein-altering variants (PAV) and protein-truncating variants (PTV) were performed separately for WES and WGS data and combined in a meta-analysis. We also performed genome-wide aggregate analyses on genomic windows (sliding window), promoters and enhancers using the WGS dataset. RESULTS: In the single-variant meta-analysis, no variant reached genome-wide significance, but a suggestively associated common THAP7 rs369250 variant (p=1.50 × 10-5, MAF=49%) was replicated in the FinnGen general population genome-wide association study (GWAS) data for chronic kidney disease and DKD phenotypes. The gene-aggregate meta-analysis provided suggestive evidence (p<4.0 × 10-4) at four genes for DKD, of which NAT16 (MAFPAV≤10%) and LTA (also known as TNFß, MAFPAV≤5%) are replicated in the FinnGen general population GWAS data. The LTA rs2229092 C allele was associated with significantly lower TNFR1, TNFR2 and TNFR3 serum levels in a subset of FinnDiane participants. Of the intergenic regions suggestively associated with DKD, the enhancer on chromosome 18q12.3 (p=3.94 × 10-5, MAFvariants≤5%) showed interaction with the METTL4 gene; the lead variant was replicated, and predicted to alter binding of the MafB transcription factor. CONCLUSIONS/INTERPRETATION: Our sequencing-based meta-analysis revealed multiple genes, variants and regulatory regions that were suggestively associated with DKD. However, as no variant or gene reached genome-wide significance, further studies are needed to validate the findings.

Whole-genome sequencing identifies variants in ANK1, LRRN1, HAS1, and other genes and regulatory regions for stroke in type 1 diabetes.

Individuals 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-exome or whole-genome sequencing of 1,051 individuals with T1D, we aimed to find rare and low-frequency genomic variants associated with stroke in T1D. We analysed the genome comprehensively with single-variant analyses, gene aggregate analyses, and aggregate analyses on genomic windows, enhancers and promoters. In addition, we attempted replication in T1D using a genome-wide association study (N = 3,945) and direct genotyping (N = 3,263), and in the general population from the large-scale population-wide FinnGen project and UK Biobank summary statistics. We identified a rare missense variant on SREBF1 exome-wide significantly associated with stroke (rs114001633, p.Pro227Leu, p-value = 7.30 × 10-8), which replicated for hemorrhagic stroke in T1D. Using gene aggregate analysis, we identified exome-wide significant genes: ANK1 and LRRN1 displayed replication evidence in T1D, and LRRN1, HAS1 and UACA 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 on LINC01500, which replicated in T1D. Finally, we identified a suggestively stroke-associated TRPM2-AS promoter (p-value = 5.78 × 10-6) with borderline significant replication in T1D, which we validated with an in vitro cell-based assay. Due to the rarity of the identified genetic variants, future replication of the genomic regions represented here is required with sequencing of individuals with T1D. Nevertheless, we here report the first genome-wide analysis on stroke in individuals with diabetes.

Residual insulin secretion in individuals with type 1 diabetes in Finland: longitudinal and cross-sectional analyses.

BACKGROUND: Contrary to the presumption that type 1 diabetes leads to an absolute insulin deficiency, many individuals with type 1 diabetes have circulating C-peptide years after the diagnosis. We studied factors affecting random serum C-peptide concentration in individuals with type 1 diabetes and the association with diabetic complications. METHODS: Our longitudinal analysis included individuals newly diagnosed with type 1 diabetes from Helsinki University Hospital (Helsinki, Finland) with repeated random serum C-peptide and concomitant glucose measurements from within 3 months of diagnosis and at least once later. The long-term cross-sectional analysis included data from participants from 57 centres in Finland who had type 1 diabetes diagnosed after 5 years of age, initiation of insulin treatment within 1 year from diagnosis, and a C-peptide concentration of less than 1·0 nmol/L (FinnDiane study) and patients with type 1 diabetes from the DIREVA study. We tested the association of random serum C-peptide concentrations and polygenic risk scores with one-way ANOVA, and association of random serum C-peptide concentrations, polygenic risk scores, and clinical factors with logistic regression. FINDINGS: The longitudinal analysis included 847 participants younger than 16 years and 110 aged 16 years or older. In the longitudinal analysis, age at diagnosis strongly correlated with the decline in C-peptide secretion. The cross-sectional analysis included 3984 participants from FinnDiane and 645 from DIREVA. In the cross-sectional analysis, at a median duration of 21·6 years (IQR 12·5-31·2), 776 (19·4%) of 3984 FinnDiane participants had residual random serum C-peptide secretion (>0·02 nmol/L), which was associated with lower type 1 diabetes polygenic risk compared with participants without random serum C-peptide (p<0·0001). Random serum C-peptide was inversely associated with hypertension, HbA1c, and cholesterol, but also independently with microvascular complications (adjusted OR 0·61 [95% CI 0·38-0·96], p=0·033, for nephropathy; 0·55 [0·34-0·89], p=0·014, for retinopathy). INTERPRETATION: Although children with multiple autoantibodies and HLA risk genotypes progressed to absolute insulin deficiency rapidly, many adolescents and adults had residual random serum C-peptide decades after the diagnosis. Polygenic risk of type 1 and type 2 diabetes affected residual random serum C-peptide. Even low residual random serum C-peptide concentrations seemed to be associated with a beneficial complications profile. FUNDING: Folkhälsan Research Foundation; Academy of Finland; University of Helsinki and Helsinki University Hospital; Medical Society of Finland; the Sigrid Juselius Foundation; the "Liv and Hälsa" Society; Novo Nordisk Foundation; and State Research Funding via the Helsinki University Hospital, the Vasa Hospital District, Turku University Hospital, Vasa Central Hospital, Jakobstadsnejdens Heart Foundation, and the Medical Foundation of Vaasa.

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 (p<2.9×10-6). The RBM47 gene is required for apolipoprotein B post-translational modifications, and in our data, the association between RBM47 and apolipoprotein C-III concentrations was due to a rare 21 base pair p.Ala496-Ala502 deletion; in replication, the burden of rare deleterious variants in RBM47 was associated with lower triglyceride concentrations in WES of >170,000 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.

Association of Coding Variants in Hydroxysteroid 17-beta Dehydrogenase 14 (HSD17B14) with Reduced Progression to End Stage Kidney Disease in Type 1 Diabetes.

BACKGROUND: Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. METHODS: Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (n=2372/1115 events all cohorts) and replicating in two retrospective case-control studies (n=1072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. RESULTS: Protein coding variants in the hydroxysteroid 17-ß dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n=4196; P value=3.3 × 10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. CONCLUSIONS: HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

Novel Linkage Peaks Discovered for Diabetic Nephropathy in Individuals With Type 1 Diabetes.

Genome-wide association studies (GWAS) and linkage studies have had limited success in identifying genome-wide significantly linked regions or risk loci for diabetic nephropathy (DN) in individuals with type 1 diabetes (T1D). As GWAS cohorts have grown, they have also included more documented and undocumented familial relationships. Here we computationally inferred and manually curated pedigrees in a study cohort of >6,000 individuals with T1D and their relatives without diabetes. We performed a linkage study for 177 pedigrees consisting of 452 individuals with T1D and their relatives using a genome-wide genotyping array with >300,000 single nucleotide polymorphisms and PSEUDOMARKER software. Analysis resulted in genome-wide significant linkage peaks on eight chromosomal regions from five chromosomes (logarithm of odds score >3.3). The highest peak was localized at the HLA region on chromosome 6p, but whether the peak originated from T1D or DN remained ambiguous. Of other significant peaks, the chromosome 4p22 region was localized on top of ARHGAP24, a gene associated with focal segmental glomerulosclerosis, suggesting this gene may play a role in DN as well. Furthermore, rare variants have been associated with DN and chronic kidney disease near the 4q25 peak, localized on top of CCSER1.

Differential metabolomic signatures of declining renal function in Types 1 and 2 diabetes.

BACKGROUND: Chronic kidney disease (CKD) shows different clinical features in Types1 (T1D) and 2 diabetes (T2D). Metabolomics have recently provided useful contribution to the identification of biomarkers of CKD progression in either form of the disease. However, no studies have so far compared plasma metabolomics between T1D and T2D in order to identify differential signatures of progression of estimated glomerular filtration rate (eGFR) decline. METHODS: We used two large cohorts of T1D (from Finland) and T2D (from Italy) patients followed up to 7 and 3 years, respectively. In both groups, progression was defined as the top quartile of yearly decline in eGFR. Pooled data from the two groups were analysed by univariate and bivariate random forest (RF), and confirmed by bivariate partial least squares (PLS) analysis, the response variables being type of diabetes and eGFR progression. RESULTS: In progressors, yearly eGFR loss was significantly larger in T2D [-5.3 (3.0), median (interquartile range)mL/min/1.73 m2/year] than T1D [-3.7 (3.1) mL/min/1.73 m2/year ; P = 0.018]. Out of several hundreds, bivariate RF extracted 22 metabolites associated with diabetes type (all higher in T1D than T2D except for 5-methylthioadenosine, pyruvate and ß-hydroxypyruvate) and 13 molecules associated with eGFR progression (all higher in progressors than non-progressors except for sphyngomyelin). Three of the selected metabolites (histidylphenylalanine, leucylphenylalanine, tryptophylasparagine) showed a significant interaction between disease type and progression. Only eight metabolites were common to both bivariate RF and PLS. CONCLUSIONS: Identification of metabolomic signatures of CKD progression is partially dependent on the statistical model. Dual analysis identified molecules specifically associated with progressive renal impairment in both T1D and T2D.

Liver nucleotide biosynthesis is linked to protection from vascular complications in individuals with long-term type 1 diabetes.

Identification of biomarkers associated with protection from developing diabetic complications is a prerequisite for an effective prevention and treatment. The aim of the present study was to identify clinical and plasma metabolite markers associated with freedom from vascular complications in people with very long duration of type 1 diabetes (T1D). Individuals with T1D, who despite having longer than 30 years of diabetes duration never developed major macro- or microvascular complications (non-progressors; NP) were compared with those who developed vascular complications within 25 years from diabetes onset (rapid progressors; RP) in the Scandinavian PROLONG (n = 385) and DIALONG (n = 71) cohorts. The DIALONG study also included 75 healthy controls. Plasma metabolites were measured using gas and/or liquid chromatography coupled to mass spectrometry. Lower hepatic fatty liver indices were significant common feature characterized NPs in both studies. Higher insulin sensitivity and residual ß-cell function (C-peptide) were also associated with NPs in PROLONG. Protection from diabetic complications was associated with lower levels of the glycolytic metabolite pyruvate and APOCIII in PROLONG, and with lower levels of thiamine monophosphate and erythritol, a cofactor and intermediate product in the pentose phosphate pathway as well as higher phenylalanine, glycine and serine in DIALONG. Furthermore, T1D individuals showed elevated levels of picolinic acid as compared to the healthy individuals. The present findings suggest a potential beneficial shunting of glycolytic substrates towards the pentose phosphate and one carbon metabolism pathways to promote nucleotide biosynthesis in the liver. These processes might be linked to higher insulin sensitivity and lower liver fat content, and might represent a mechanism for protection from vascular complications in individuals with long-term T1D.

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Metabolomic Profile Predicts Development of Microalbuminuria in Individuals with Type 1 Diabetes.

Elevated urinary albumin excretion (microalbuminuria) is an early marker of diabetic nephropathy, but there is an unmet need for better biomarkers that capture the individuals at risk with higher accuracy and earlier than the current markers do. We performed an untargeted metabolomic study to assess baseline differences between individuals with type 1 diabetes who either developed microalbuminuria or remained normoalbuminuric. A total of 102 individuals progressed to microalbuminuria during a median follow-up of 3.2 years, whereas 98 sex-, age- and body mass index (BMI) matched non-progressors remained normoalbuminuric during a median follow-up of 7.1 years. Metabolomic screening identified 1,242 metabolites, out of which 111 differed significantly between progressors and non-progressors after adjustment for age of diabetes onset, baseline glycosylated hemoglobin A1c (HbA1c), and albumin excretion rate (AER). The metabolites that predicted development of microalbumiuria included several uremic toxins and carnitine metabolism related molecules. Iterative variable selection indicated erythritol, 3-phenylpropionate, and N-trimethyl-5-aminovalerate as the best set of variables to predict development of microalbuminuria. A metabolomic index based on these metabolites improved the prediction of incident microalbuminuria on top of the clinical variables age of diabetes onset, baseline HbA1c and AER (ROCAUC = 0.842 vs 0.797), highlighting their ability to predict early-phase diabetic nephropathy.

Confirmation of GLRA3 as a susceptibility locus for albuminuria in Finnish patients with type 1 diabetes.

Urinary albumin excretion is an early sign of diabetic kidney disease, affecting every third individual with diabetes. Despite substantial estimated heritability, only variants in the GLRA3 gene have been genome-wide significantly associated (p-value < 5 × 10-8) with diabetic albuminuria, in Finnish individuals with type 1 diabetes; However, replication attempt in non-Finnish Europeans with type 1 diabetes showed nominally significant association in the opposite direction, suggesting a population-specific effect, but simultaneously leaving the finding controversial. In this study, the association between the common rs10011025 variant in the GLRA3 locus, and albuminuria, was confirmed in 1259 independent Finnish individuals with type 1 diabetes (p = 0.0013), and meta-analysis of all Finnish individuals yielded a genome-wide significant association. The association was particularly pronounced in subjects not reaching the treatment target for blood glucose levels (HbA1c > 7%; N = 2560, p = 1.7 × 10-9). Even though further studies are needed to pinpoint the causal variants, dissecting the association at the GLRA3 locus may uncover novel molecular mechanisms for diabetic albuminuria irrespective of population background.

Evolution of the metazoan mitochondrial replicase.

The large number of complete mitochondrial DNA (mtDNA) sequences available for metazoan species makes it a good system for studying genome diversity, although little is known about the mechanisms that promote and/or are correlated with the evolution of this organellar genome. By investigating the molecular evolutionary history of the catalytic and accessory subunits of the mtDNA polymerase, pol γ, we sought to develop mechanistic insight into its function that might impact genome structure by exploring the relationships between DNA replication and animal mitochondrial genome diversity. We identified three evolutionary patterns among metazoan pol γs. First, a trend toward stabilization of both sequence and structure occurred in vertebrates, with both subunits evolving distinctly from those of other animal groups, and acquiring at least four novel structural elements, the most important of which is the HLH-3ß (helix-loop-helix, 3 ß-sheets) domain that allows the accessory subunit to homodimerize. Second, both subunits of arthropods and tunicates have become shorter and evolved approximately twice as rapidly as their vertebrate homologs. And third, nematodes have lost the gene for the accessory subunit, which was accompanied by the loss of its interacting domain in the catalytic subunit of pol γ, and they show the highest rate of molecular evolution among all animal taxa. These findings correlate well with the mtDNA genomic features of each group described above, and with their modes of DNA replication, although a substantive amount of biochemical work is needed to draw conclusive links regarding the latter. Describing the parallels between evolution of pol γ and metazoan mtDNA architecture may also help in understanding the processes that lead to mitochondrial dysfunction and to human disease-related phenotypes.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.