BASIC RESEARCH www.jasn.org Whole-Genome Sequencing of Finnish Type 1 Diabetic Siblings Discordant for Kidney Disease Reveals DNA Variants associated with Diabetic Nephropathy Jing Guo ,1,2 Owen J. L. Rackham ,2 Niina Sandholm ,3,4,5 Bing He ,1 Anne-May Österholm,1,2 Erkka Valo ,3,4,5 Valma Harjutsalo ,3,4,5,6 Carol Forsblom,3,4,5 Iiro Toppila,3,4,5 Maija Parkkonen,3,4,5 Qibin Li,7 Wenjuan Zhu,7 Nathan Harmston ,2,8 Sonia Chothani,2 Miina K. Öhman ,2 Eudora Eng,2 Yang Sun,2 Enrico Petretto ,2,9 Per-Henrik Groop,3,4,5,10 and Karl Tryggvason1,2,11 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Several genetic susceptibility loci associated with diabetic nephropathy have been documen- ted, but no causative variants implying novel pathogenetic mechanisms have been elucidated. Methods We carried out whole-genome sequencing of a discovery cohort of Finnish siblings with type 1 diabetes who were discordant for the presence (case) or absence (control) of diabetic nephropathy. Con- trols had diabetes without complications for 15–37 years. We analyzed and annotated variants at genome, gene, and single-nucleotide variant levels. We then replicated the associated variants, genes, and regions in a replication cohort from the Finnish Diabetic Nephropathy study that included 3531 unrelated Finns with type 1 diabetes. Results We observed protein-altering variants and an enrichment of variants in regions associated with the presence or absence of diabetic nephropathy. The replication cohort confirmed variants in both regulatory and protein-coding regions. We also observed that diabetic nephropathy–associated variants, when clus- tered at the gene level, are enriched in a core protein-interaction network representing proteins essential for podocyte function. These genes include protein kinases (protein kinase C isoforms « and i) and protein tyrosine kinase 2. Conclusions Our comprehensive analysis of a diabetic nephropathy cohort of siblings with type 1 di- abetes who were discordant for kidney disease points to variants and genes that are potentially caus- ative or protective for diabetic nephropathy. This includes variants in two isoforms of the protein kinase C family not previously linked to diabetic nephropathy, adding support to previous hypotheses that the protein kinase C family members play a role in diabetic nephropathy and might be attractive therapeutic targets. JASN 31: 309–323, 2020. doi: https://doi.org/10.1681/ASN.2019030289 With the increase in the incidence of diabetes worldwide, complications such as diabetic nephrop- Received March 22, 2019. Accepted October 19, 2019. athy (DN), retinopathy, neuropathy, skin ulcers, and Published online ahead of print. Publication date available at amputations have become a major global health and www.jasn.org. socioeconomic threat. In addition to intensive blood Correspondence: Prof. Karl Tryggvason or Prof. Enrico Petretto, 1 glucose control, the only drugs providing a signif- Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical icant delay in progression of DN are angiotensin- School, 8 College Road Singapore 169857. Email: karl.tryggvason@ converting enzyme inhibitors or angiotensin receptor duke-nus.edu.sg or [email protected] blockers, which reduce intraglomerular pressure and Copyright © 2020 by the American Society of Nephrology JASN 31: 309–323, 2020 ISSN : 1046-6673/3102-309 309 BASIC RESEARCH www.jasn.org efferent arteriolar vasoconstriction.2 The molecular pathogene- Significance Statement sis of DN is still poorly understood. Hyperglycemia, a major risk factor for complications, causes accumulation of toxic glucose Although diabetic nephropathy is partly genetic in nature, the un- derivatives, such as methylglyoxal, that bind covalently to the side derlying pathogenetic mechanisms are obscure. The authors as- chains of amino acids, particularly arginine and lysine, and also sembled from the homogeneous Finnish population a cohort of 76 sibling pairs with type 1 diabetes who were discordant for diabetic 3,4 fi methionine and cysteine. Hyperglycemia alone is not suf cient nephropathy. Using whole-genome sequencing and multiple to trigger the development of complications, as only 30%–40% of analytic approaches, they identified DNA variants associated with individuals with type 1 diabetes (T1D) develop diabetic micro- nephropathy or its absence and validated their findings in a 3531- angiopathy.1,5,6 Independent familial studies have shown a trend member cohort of unrelated Finns with type 1 diabetes. The genes of family aggregation of DN in different populations,7,8 most strongly associated with diabetic nephropathy encode two protein kinase C isoforms (isoforms « and i) not previously impli- suggesting a genetic predisposition to DN. At least four metabolic cated in the condition. Besides providing a resource for studies on pathways have been implicated in the development of complica- diabetic complications, these findings support previous hypotheses tions: polyol flux, increased the formation of advanced glycation that the protein kinase C family plays a role in diabetic nephropathy end products, hyperactivity of the hexosamine pathway, and and suggest potential targets for treatment. activation of protein kinase C (PKC) isoforms.4,9,10 Genome-wide association studies (GWAS) and candidate Study Participants fi gene approaches have identi ed several potential genomic loci The discovery cohort consisted of sibling pairs and small fami- 11 for DN susceptibility, but no variants with a major effect on lies, whereas the replication cohort consisted of unrelated indi- the risk of complications have been found, suggesting that DN viduals, all having T1D. The renal status was on the basis of the is modulated by a number of variants in genes that cooperate albumin excretion rate (AER) in a 24-hour urine collection or the within complex pathways. It is intriguing, however, that sev- albumin-to-creatinine ratio (ACR) in a random spot-urine col- eral independent, genome-wide linkage analysis studies car- lection. The presence of ESKD was defined according to whether ried out in white Americans, Pima Indians, black Americans, patients were undergoing dialysis or had received a kidney trans- fi and Finns have identi ed the same DN susceptibility locus plant. DN was defined by (1) persistent macroalbuminuria 12–15 on chromosome 3q. The complex interaction between (AER$300 mg/24 h or ACR.30 mg/mmol) in two of three con- genetics, risk factors such as hyperglycemia and environmen- secutive measurements or the presence of ESKD; and (2)the fi fi tal components makes it more challenging to nd speci c absence of clinical or laboratory evidence of nondiabetic renal genes for DN using genetic association studies. To that end, or urinary tract disease. Control status was defined by normoal- it could be advantageous to search for DN susceptibility genes buminuria (AER,30 mg/24 h or ACR,3 mg/mmol) despite in populations such as Finns, a uniquely homogeneous European duration of diabetes for at least 15 years (range, 15–37 years). In 16 ’ 17,18 population with the world s highest incidence of T1D. the discovery cohort, all study participants had been diagnosed With a combination of founder effects and genetic isolation, with T1D for at least 15 years, with the age at onset ,30 years; the population has accumulated rare genetic traits referred to in the replication cohort, age at diabetes onset was #40 years, “ ”19 as the Finnish Disease Heritage. In addition, Finland has a with insulin dependence within 1 year after the diagnosis of di- good public health care system, including nationwide disease abetes (or age at diabetes onset #15 years). Controls in the fi and treatment registries, which facilitates identi cation of replication cohort had minimum diabetes duration of 15 years. patients and follow-up of their clinical records. The replication cohort included 2187 controls with normal AER and 1344 cases with macroalbuminuria and ESKD. METHODS Ethical Permits All patients with diabetes gave written, informed consent Experimental Design to participate in the study and the Ethical Committee of the To search for DN susceptibility genes, we have assembled a Finnish National Public Institute, the Ethical Committee of cohort of Finnish T1D siblings with extreme phenotypes re- the Helsinki and Uusimaa Health District, and Karolinska In- garding the presence (case) or absence (control) of DN. This stitutet approved the protocol for the study. The transgene discovery cohort contained 76 T1D sibling pairs discordant manipulation in zebrafish was approved by the local ethical for DN, and three T1D families with three siblings (total of committee (the North Stockholm District Court). 80 cases and 81 controls). The samples came from two sources: the Finnish National Institutes of Health and Welfare diabetes Whole-Genome Sequencing collections, as described elsewhere15; and the Finnish Diabetic Whole-genome sequencing (WGS) was carried out on the dis- Nephropathy (FinnDiane) study.20 Furthermore, 3531 unre- covery cohort using both Illumina HiSeq 2000 and Complete lated individuals with T1D (1344 cases and 2187 controls) Genomics platforms. To evaluate the quality of the two different (Figure 1A) from FinnDiane were used for replication of findings sequencing methods, we sequenced four discordant sibling made in the discovery cohort. The main clinical characteristics of pairs with both platforms and compared the difference of the patients