Genome-Wide Association Study in Acute Tubulointerstitial Nephritis

Abstract

Significance statement: Polymorphisms of HLA genes may confer susceptibility to acute tubulointerstitial nephritis (ATIN), but small sample sizes and candidate gene design have hindered their investigation. The first genome-wide association study of ATIN identified two significant loci, risk haplotype DRB1*14-DQA1*0101-DQB1*0503 (DR14 serotype) and protective haplotype DRB1*1501-DQA1*0102-DQB1*0602 (DR15 serotype), with amino acid position 60 in the peptide-binding groove P10 of HLA-DR β 1 key. Risk alleles were shared among different causes of ATIN and HLA genotypes associated with kidney injury and immune therapy response. HLA alleles showed the strongest association. The findings suggest that a genetically conferred risk of immune dysregulation is part of the pathogenesis of ATIN.

Background: Acute tubulointerstitial nephritis (ATIN) is a rare immune-related disease, accounting for approximately 10% of patients with unexplained AKI. Previous elucidation of the relationship between genetic factors that contribute to its pathogenesis was hampered because of small sample sizes and candidate gene design.

Methods: We undertook the first two-stage genome-wide association study and meta-analysis involving 544 kidney biopsy-defined patients with ATIN and 2346 controls of Chinese ancestry. We conducted statistical fine-mapping analysis, provided functional annotations of significant variants, estimated single nucleotide polymorphism (SNP)-based heritability, and checked genotype and subphenotype correlations.

Results: Two genome-wide significant loci, rs35087390 of HLA-DQA1 ( P =3.01×10 -39 ) on 6p21.32 and rs2417771 of PLEKHA5 on 12p12.3 ( P =2.14×10 -8 ), emerged from the analysis. HLA imputation using two reference panels suggested that HLA-DRB1*14 mainly drives the HLA risk association . HLA-DRB1 residue 60 belonging to pocket P10 was the key amino acid position. The SNP-based heritability estimates with and without the HLA locus were 20.43% and 10.35%, respectively. Different clinical subphenotypes (drug-related or tubulointerstitial nephritis and uveitis syndrome) seemed to share the same risk alleles. However, the HLA risk genotype was associated with disease severity and response rate to immunosuppressive therapy.

Conclusions: We identified two candidate genome regions associated with susceptibility to ATIN. The findings suggest that a genetically conferred risk of immune dysregulation is involved in the pathogenesis of ATIN.

Graphical abstract

Figure 1

A flow diagram of building the study cohort. Five hundred seventy-five individuals were previously recruited with a clinical diagnosis of ATIN. Three of the largest renal pathology centers in China (North of China, Peking University First hospital; South of China, Ruijin Hospital and Huashan Hospital) provided diagnostic pathology. To assess patient eligibility for entry to this study, in each center, at least two nephrologists and at least two pathologists reviewed each patient. After quality controls, 544 patients were included in the final GWAS analysis.

Figure 2

The genome-wide association results in ATIN. (A) Manhattan plot. Observed −log₁₀P values versus chromosomal location. The y-axis represents the −log₁₀P value for association of SNPs with ATIN. The threshold for genome-wide significance (P<5×10⁻⁸) is represented by a horizontal red line. Genome-wide significant variants in the two genome-wide significant loci are in blue. (B) Quantile-quantile plot. Quantile-quantile plot of the association before (red, λ=1.028) and after (blue, λ=1.021) removing HLA (–chr 6 –from-kb 25,000 –to-kb 34,000). Figure 2 can be viewed in color online at www.jasn.org.

Figure 3

Results of meta-analyses of HLA signals and location of associated amino acid positions in ATIN. (A) Association results of the unconditional analysis (upper) of SNPs (gray), HLA classical alleles (orange), and amino acid residues (AA, red) and results of the same variants after conditioning (below) on lead SNP of rs35087390. Each point corresponds to the P_Meta value in each type of variant. The figures presented were drawn by HLA-TAPAS (https://github.com/immunogenomics/HLA-TAPAS). (B) The key amino acid positions identified by association analysis are highlighted in three-dimensional ribbon models for the HLA-DR proteins. All protein structures are positioned to accommodate the view of the peptide binding groove and the associated amino acid residues using UCSF Chimera. The amino acids at positions 60 and 57 are located in the peptide-binding groove of HLA-DRB1. Figure 3 can be viewed in color online at www.jasn.org.

Figure 4

Representation of molecular surface change for amino acid at position 60 of HLA-DRB1. The molecular graphics program PyMOL was used to analyze the changes caused by the variant and the molecular surface charge distribution and to display the results (with PDB code 3pdo). Right upper for the structure for Tyr60 and lower for the His60 variant. The electrostatic surface was shown in color varying from blue (positive) to red (negative). Protein structural elements involved were shown as ribbons and Tyr60 and His60 as sticks with the rest of the side chains as lines. The substitution of Tyr60 to His60 strengthened the positive charge distribution on the molecular surface. The shorter His side chain created an empty volume causing conformational changes of the molecular surface. PDB, protein data bank. Figure 4 can be viewed in color online at www.jasn.org.

Figure 5

Clinical correlations stratified by different genotypes of HLA variant rs35087390. (A–F) Comparisons were on the basis of baseline data collection in pooling. (G–I) Comparisons were on the basis of available therapy and prognosis data in the discovery cohort. Detailed statistics could be referred to in Supplemental Table 13. *Significant P<0.05. AKD, acute kidney disease. Figure 5 can be viewed in color online at www.jasn.org.