BACKGROUND. Proteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding. METHODS. We used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods. RESULTS. We identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts. CONCLUSION. Collectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.

Aims/hypothesisIdentifying rare coding variants associated with albuminuria may open new avenues for preventing chronic kidney disease and end-stage renal disease, which are highly prevalent in individuals with diabetes. Efforts to identify genetic susceptibility variants for albuminuria have so far been limited, with the majority of studies focusing on common variants.MethodsWe performed an exome-wide association study to identify coding variants in a two-stage (discovery and replication) approach. Data from 33,985 individuals of European ancestry (15,872 with and 18,113 without diabetes) and 2605 Greenlanders were included.ResultsWe identified a rare (minor allele frequency [MAF]: 0.8%) missense (A1690V) variant in CUBN (rs141640975, β = 0.27, p = 1.3 × 10−11) associated with albuminuria as a continuous measure in the combined European meta-analysis. The presence of each rare allele of the variant was associated with a 6.4% increase in albuminuria. The rare CUBN variant had an effect that was three times stronger in individuals with type 2 diabetes compared with those without (pinteraction = 7.0 × 10−4, β with diabetes = 0.69, β without diabetes = 0.20) in the discovery meta-analysis. Gene-aggregate tests based on rare and common variants identified three additional genes associated with albuminuria (HES1, CDC73 and GRM5) after multiple testing correction (pBonferroni < 2.7 × 10−6).Conclusions/interpretationThe current study identifies a rare coding variant in the CUBN locus and other potential genes associated with albuminuria in individuals with and without diabetes. These genes have been implicated in renal and cardiovascular dysfunction. The findings provide new insights into the genetic architecture of albuminuria and highlight target genes and pathways for the prevention of diabetes-related kidney disease.Electronic supplementary materialThe online version of this article (10.1007/s00125-018-4783-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

No abstract

Pa g e 2 Significance statement (120 words summary)Increased albuminuria is a key manifestation of major health burdens, including chronic kidney disease and/or cardiovascular disease. Although being partially heritable, there is a lack of knowledge on rare genetic variants that contribute to albuminuria. The current study describes the discovery and validation, of a new rare gene mutation (~1%) in the CUBN gene which associates with increased albuminuria. Its effect multiplies 3 folds among diabetes cases compared to nondiabetic individuals. The study further uncovers 3 additional genes modulating albuminuria levels in humans. Thus the current study findings provide new insights into the genetic architecture of albuminuria and highlight novel genes/pathways for prevention of diabetes related kidney disease. P a g e 3 AbstractIdentifying rare coding variants associated with albuminuria may open new avenues for preventing chronic kidney disease (CKD) and end-stage renal disease which are highly prevalent in patients with diabetes. Efforts to identify genetic susceptibility variants for albuminuria have so far been limited with the majority of studies focusing on common variants.We performed an exome-wide association study to identify coding variants in a two phase (discovery and replication) approach, totaling to 33,985 individuals of European ancestry (15,872 with and 18,113 without diabetes) and further testing in Greenlanders (n = 2,605). We identify a P a g e 4 P a g e 6 P a g e 7 run (wherever required). Replication meta-analyses with p replication <0.05, were considered significant. Combined (EUR/EUR-GL) Meta analysesThe combined meta-analyses was firstly performed with all individuals of European ancestry (Combined EUR) followed by pooling of Greenlandic data (Combined EUR-GL).Any SNP with a) p replication < 0.05 and b) p meta_EUR/EUR-GL < 5.0 × 10 -8 was considered overall significant while those with 5.0 × 10 -5 > p meta_EUR/EUR-GL > 5.0 × 10 -8 were considered suggestive. Conditional analysesConditional analyses for novel SNPs identified in known loci (and/or in low LD, LD r2 <0.01) were performed to determine if the signal was independent (in discovery set). This was performed using the following linear model:trait ~ top identified SNP + secondary known SNP + other covariates. If the top SNP retained the association estimates and p value it was considered an independent signal. Gene Aggregate TestsGene-based multi-marker association testing for rare and common (MAF > 0.0001) variants was performed using the Meta Analyses for SNP-Set (Sequence) Kernel Association Test (MetaSKAT) R package (https://cran.r-project.org/web/packages/MetaSKAT/index.html). At the study-specific level, the gene-based analyses were performed against the null model accounting for gender and ten principal components, (using the previously described SKAT-O method) 14 generating SKAT objects individually for each cohort with complete ExomeWAS data (discovery studies + MDCS study, n studies = 6)) which were then meta-analyzed.The ...