SORBS1 gene, a new candidate for diabetic nephropathy: results from a multi-stage Genome Wide Association Study in type 1 diabetes patients

Abstract

Background

The genetic determinants of diabetic nephropathy remain poorly understood. We aimed to identify novel susceptibility genes for diabetic nephropathy.

Patients and methods

We performed a genome-wide association study using 1000 Genomes-based imputation in type 1 diabetic patients comparing diabetic nephropathy cases with proteinuria with or without renal failure to controls with diabetes for more than 15 years and no evidence of renal disease.

Results

None of the SNPs tested in a discovery cohort composed of 683 cases and 779 controls reached genome-wide statistical significance. The 46 top hits (p-value < 10⁻⁵) were then sought for first-stage analysis in US-GoKinD an independent population of 820 cases and 885 controls. Two SNPs, in strong linkage disequilibrium with each other, located in the SORBS1 gene, were consistently and significantly (p < 10⁻⁴) associated with diabetic nephropathy. The minor rs1326934-C allele was less frequent in cases than in controls (0.34 vs 0.43) and was associated with a decreased risk for diabetic nephropathy: OR = 0.70 [0.60 – 0.82]. However, this association was not observed in a second-stage with two additional diabetic nephropathy cohorts UK-ROI (p=0.15) and FINNDIANE (p = 0.44) totaling 2,142 cases and 2,494 controls. Altogether, the random-effect meta-analyzed rs1326934-C allele OR for diabetic nephropathy was 0.83 [0.72 – 0.96] (p = 0.009).

Conclusion

These data suggest that SORBS1 might be a gene involved in diabetic nephropathy.

Diabetic nephropathy is a frequent condition affecting up to 40% of diabetes patients, and is a leading cause of end-stage renal disease. The determinants of diabetic nephropathy are complex and include genetic factors, as supported by a strong familial aggregation of diabetic nephropathy [1].

The precise nature of the genetic burden involved in diabetic nephropathy remains poorly understood. An alternative approach to candidate gene studies is to perform unbiased genome-wide association studies (GWAS). To date, only a small number of GWAS for diabetic nephropathy have been performed. Recently the GENIE consortium identified two loci associated with a population attributable risk of 0.5 to 10.5%; therefore, additional studies are needed to discover novel loci associated with diabetic nephropathy [2].

The aim of our research was to conduct a GWAS in a large-scale case-control study including French and Danish patients, based on latest 1000 Genomes imputation techniques, and to replicate our findings in available cohorts of patients with type 1 diabetes from European ancestry.

Patients and methods

Studied participants were of White European origin [2–4]. Briefly, patients were classified as cases or controls at the time of recruitment in all but the FINNDIANE study, where they were selected among a population-based consecutive collection of people with type 1 diabetes, and nephropathy status of the patients was defined based on the latest available data. All cohorts were multi-centric except the single-center LEACE study. Local ethics committees approved the study protocols. All participants gave written informed consent.

Phenotypic determination

Type 1 diabetes

Type 1 diabetes was diagnosed using ADA criteria: rapid definitive insulin requirement (within one year of diagnosis) and age at diabetes onset below 31 or 36 years, differing according to each cohort (Supplementary Table 1).

Diabetic nephropathy status

Phenotype determination was based on the clinical criteria proposed by the US-GoKinD study: cases had proteinuria with or without renal failure. Proteinuria was uniformly defined on at least two of three sterile urine collections (details in Supplementary Table 1). Controls had long-term duration of diabetes (over 15 years), normo-albuminuria, without renal failure and were not prescribed drugs that blockade the renin-angiotensin system. Microalbuminuric patients were not considered for the current analyses.

Study organization

The general research strategy adopted in this project is summarized in Supplementary Figure 1. Brief descriptions of the studied cohorts and patients can be found in the Supplementary Tables 1 & 2.

We ultimately assessed expression of identified gene in a post-hoc analysis of micro-dissected glomerular and tubule samples (Supplementary Tables 4 & 5)[5].

Genotype, quality control and imputation

Genotyping platforms, quality control and imputation are detailed in ESM method.

Statistical analysis

A logistic regression analysis was conducted to evaluate the association of each imputed SNP with diabetic nephropathy under an additive genetic model (allele dosage used as covariate for characterizing the tested SNP). Analyses were adjusted for age, sex and the first four principal components derived from the genome-wide genotyped SNPs computed by the Eigenstrat program [6]. Association analyses were performed using the mach2dat (v 1.0.19) software.

All SNPs with suggestive evidence of association with diabetic nephropathy (p<10⁻⁵) were moved forward in the first-stage study. Their center- and sex- adjusted association with diabetic nephropathy was tested by use of the SNPTEST program (https://mathgen.stats.ox.ac.uk/genetics_software/snptest/snptest.html#introduction).

In the second-stage samples, association of candidate SNP with diabetic nephropathy was tested using the Cochran-Armitage (CA) trend test. Odds ratios derived from the application of the CA test in the four case-controls samples were finally combined into a random-effect model based meta-analysis using the GWAMA program [7].

Results

Discovery analysis

None of the 11,133,962 tested SNPs achieved the statistical threshold of 5 × 10⁻⁸ for declaring genome-wide significance (Supplementary Figures 2 & 3). All SNPs with suggestive evidence for association at p < 10⁻⁵ (n = 46) were selected for further association testing in a first independent sample of 820 cases and 885 controls, the US-GoKinD (Supplementary Figure 1).

First-stage study

After Bonferroni correction, three SNPs demonstrated significant association with diabetic nephropathy: rs11188343 (p = 9.06 ×10⁻⁵), rs1326934 (p = 9.85 × 10⁻⁵) and rs4917695 (p = 1.27 × 10⁻⁴) (Supplementary Table 3). These SNPs mapped to SORBS1 gene and were in nearly complete linkage disequilibrium (r²~ 1).

In the discovery GWAS, the minor rs1326934-C allele was less frequent in cases than in controls (0.34 vs. 0.43) and was associated with a decreased risk for diabetic nephropathy of OR = 0.70 [0.60 – 0.82] (p = 7.87 × 10⁻⁶). In the first-stage population, consistent association was observed with the rs1326934-C allele at lower frequency in cases (combined statistical evidence for association of p = 3.52 × 10⁻⁹) (Table 1).

Table 1.

Association of SORBS1 rs1326934 with diabetic nephropathy in the four studied cohorts

	Cases				Controls				P value*
	TT	TC	CC	MAF	TT	TC	CC	MAF
Discovery cohort	291	314	78	0.344	247	398	134	0.427	2.94 10-6
1st Stage analysis	289	407	124	0.399	260	428	197	0.464	1.32 10-4
2nd Stage analysis
UK ROI**	263	426	134	0.422	269	462	170	0.445	0.155
FinnDiane	681	539	99	0.279	819	640	132	0.284	0.756

MAF, Minor Allele Frequency

^*Cochran-Armitage Trend Test for association

^**2 patients with low call rate for rs1326934 in controls from the UK ROI cohort

Second-stage study

To validate further the association of rs13626934 with diabetic nephropathy, we studied it in two additional case-control studies composed of 823 cases and 903 (UK-ROI) and 1,335 cases and 1,633 controls (FinnDiane) (Table 1). In UK-ROI, the same trend of association, though not significant (p = 0.15), was observed: the rs13626934-C allele was less frequent in cases than in controls (0.42 vs. 0.45). However, no association was observed in the FinnDiane population (p = 0.44) where the allele frequency of C allele was very similar in cases and controls (0.27 vs. 0.28).

Combining the results of the four studies into a fixed-effect meta-analysis leads to an overall OR for diabetic nephropathy of 0.84 [0.79–0.90] (p = 5.69 × 10⁻⁷). However, this association was statistically heterogeneous across the four samples (p = 3.94 × 10⁻³), and the random-effect meta-analyzed OR for diabetic nephropathy was then 0.83 [0.72 – 0.96] (p = 0.009). When excluding FINNDIANE patients, meta-analyzed OR was 0.795 [0.733 – 0.861], (p = 2.40 × 10⁻⁸).

Gene expression analysis

SORBS1 gene over-expression was observed in tubules of type 2 diabetes patients compared to controls (Figure 1a). We also observed significant inverse correlation between SORBS1 expression and estimated glomerular filtration rate (eGFR) (Figure 1b). Among the other genes mapping within 250kb of rs1326934, SORBS1 was the one demonstrated the strongest correlation with eGFR in control and diabetic nephropathy tubule samples (Figure 1c). SORBS1 was also found highly expressed in renal tubules and medium expressed in glomeruli at a protein level (www.proteinatlas.org).

Figure 1. Expression profile of SORBS1 in diabetic nephropathy.

The y-axis represents the relative expression of the SORBS1 transcript. Control samples: white bars, diabetic kidney disease (DKD): black bars. The tubular expression of SORBS1 is significantly up-regulated in DKD compared to control samples in tubules (P = 0.0006) (a).

The y-axis shows the relative normalised tubular expressions of SORBS1 (sorbin and SH3 domain containing 1), while the x-axis represents eGFR (estimated glomerular filtration rate, ml/min/1.73m²) for each sample. Each dot represents transcript levels and eGFR values from a single kidney sample. The line represents the fitted linear correlation values (b).

The x-axis represents the genomic position of each gene on chromosome 10 (q24.1) in the 500 kb vicinity of rs1326934 and rs11188343 loci (triangle). The y-axis represents the negative logarithm of the p-value (significance) between the expression of each gene and eGFR (estimated glomerular filtration rate, ml.min⁻¹ per 1.73m²). Not only the SORBS1 transcript, but also other transcripts correlate with renal function in the vicinity of rs1326934 and rs11188343 loci. Gene symbols are official symbols approved by HGNC (HUGO Gene Nomenclature Committee): PDLIM1 (PDZ and LIM domain 1), ALDH18A1 (aldehyde dehydrogenase 18 family, member A1), TCTN3 (tectonic family member 3) and ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) (c).

Discussion

In this report, a multi-stage based GWAS looked for novel susceptibility genes associated with diabetic nephropathy in patients with type 1 diabetes. We did not detect any new loci passing genome-wide significance but observed promising evidence for association of SORBS1 rs1326934 (or any SNP in complete LD with it) with diabetic nephropathy, in three of four of the studied populations. Given the frequency of the risk allele observed in our control populations (i.e. ~0.55) and the associated OR for diabetic nephropathy (~1.25), the population attributable risk of the identified polymorphisms would be 12%.

No association was observed in the Finish population, which exhibited a marked difference in allele frequency at the candidate SNP compared to the three other studied populations. We further interrogated databases with genome-wide genotype and gene expression but did not observe any association in monocytes, macrophages, hepatocytes, adipocytes [8] nor endothelial cells [9] with the SORBS1 gene expression. Nevertheless, the SORBS1 rs1188343 (in complete LD with the rs1326934) was predicted in the RegulomeDB database (http://regulome.stanford.edu) to be located at a potential binding site either for RFX3 or for histone interaction.

The sorbin protein, coded by SORBS1, was found to be differentially up-regulated in glomeruli of rats with DN compared to rats without diabetic nephropathy [10]. High tubular and moderate glomerular expression of sorbin protein was observed in human kidney samples. These findings from type 2 diabetes patients provide additional support for the association of SORBS1 with diabetic nephropathy. Gene expression changes of the SORBS1 were easier to detect in tubules, as SORBS1 has a higher tubular expression. Although SORBS1 expression was significantly up-regulated only in tubules, we cannot exclude the importance of glomerular SORBS1. Diabetic nephropathy not only involves glomeruli but also tubules [11], and genes identified through GWAS are likely to impact both renal structures.

The function of sorbin is not fully established but we speculate it plays a key role in several processes involved in diabetic nephropathy including insulin resistance and cytoskeleton architecture. Sorbin acts in the genesis of stress fibers and might then be involved in podocyte alterations of the slit diaphragm barrier.

The lack of homogenous replicated associations in our work is unlikely due to clinical heterogeneity of the studied populations as the same definitions for type 1 diabetes and diabetic nephropathy were used. Conversely, the different patterns of allelic heterogeneity and LD across European populations may explain the lack of replication of the SORBS1 signal in the Finnish population. Characterizing the exact variability at the SORBS1 locus is needed to validate SORBS1 as a new susceptibility gene for diabetic nephropathy and identify the disease-associated functional variant(s).

The main limitation of the present study is its design. We adopted a multi-stage strategy using all available GWAS resources imputed for 1000G reference dataset at the time this work was launched. A more powerful approach would have been to conduct a comprehensive meta-analysis of the four populations. An international initiative has been set up to overcome this limitation. Another caveat is that the diabetic nephropathy phenotype was defined by the presence of proteinuria, regardless renal function, and genetic susceptibility to proteinuria might differ from genetic predisposition to renal failure. However, the diabetic nephropathy cohorts we used all had this combined diabetic nephropathy phenotype permitting international collaboration.

In conclusion, our study provides preliminary support for SORBS1 gene acting as a new susceptibility gene for diabetic nephropathy.

Abreviations

1000G

1000 genomes

ADA

American Diabetes Association

ESM

electronic supplement material

eGFR

estimated Glomerular Filtration Rate

GWAS

Genome wide association study

OR

Odds Ratio

SNP

single nucleotide polymorphism