Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection

Abstract

Background: In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection.

Methods: We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses.

Results: In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P = 9.8×10^-5). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor-recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P = 6.5×10^-5). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P = 4.7×10^-8). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses.

Conclusions: We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3. (Funded by the Columbia University Transplant Center and others.).

Figure 1. Discovery Phase.

Panel A shows our strategy for selecting high-priority deletions for tagging and typing in the discovery cohort. A total of 44 of 50 deletions were successfully tagged and genotyped in the discovery cohort; 6 of 50 deletion-tagging single-nucleotide polymorphisms (SNPs) were either monomorphic or failed our genotype quality-control analysis. Annotations were based on the human reference genome hg18 (accessed in July 2010). Copy-number polymorphisms (CNPs) were common copy-number variants (CNVs with an allele frequency of >1%). MAF denotes minor allele frequency. Panel B shows the probability–probability plot for the genetic screen for rejection in the discovery cohort of 705 recipients under a recessive model. The blue dots represent P values for 44 successfully typed common deletions; the red dotted lines represent significance thresholds of 0.05 (unadjusted analysis) and 0.0011 (Bonferroni-corrected for 44 independent tests). The blue dotted line indicates the expectation under the null hypothesis, and the shaded area corresponds to a 95% confidence interval for the null hypothesis of no association. The top SNP (rs893403) represents a near-perfect tag (r² = 0.98) for a common 1.5-kb deletion (CNVR915.1) on chromosome 2q12.3. Panel C shows the genomic characteristics of the 44 CNP-tagging SNPs that were tested in the discovery phase. Plus–minus values are means ±SD. Details are provided in Table S3 in the Supplementary Appendix.

Figure 2. Effects of rs893403 on Rejection-free Allograft Survival in Study Cohorts.

Panel A shows the results in the discovery phase (involving 705 kidney transplant recipients [the Columbia cohort] who had either a non-risk genotype [blue] or a risk genotype [red]). Tick marks indicate censored data. Panel B shows the results in the replication phase, which involved a stratified analysis of three other cohorts (Belfast, TransplantLines, and Torino) that included a total of 2004 donor–recipient pairs. The P values correspond to the minimally adjusted model, with adjustment for cohort only (if applicable). Panel C shows the results in all the cohorts combined, which involved a stratified analysis of the four cohorts (i.e., 2709 kidney transplants [in 705 recipients from the discovery cohort and 2004 donor–recipient pairs from the replication cohorts]). Panel D shows the estimated hazard ratios (with 95% confidence intervals) of rejection in each of the four cohorts individually, in all the replication cohorts, and in all the cohorts combined. The effects were estimated before (blue [recipient only]) and after (red [donor–recipient pairs]) accounting for donor compatibility in order to show that the inclusion of genetic information from the donors resulted in consistently improved hazard ratio estimates.

Figure 3. Detection of Anti-LIMS1 Antibodies in Kidney Transplant Recipients at Genetic Risk for Rejection.

Panel A shows the change in intensity (x axis) as compared with the −log P value (y axis) for the top-ranking proteins on the basis of the mean signal intensity in a protein array; the change is calculated as a ratio of the mean normalized intensity in the high-risk rejection group to the mean normalized intensity of all other groups (termed “fold change”). The findings suggest the presence of anti-LIMS1 reactivity in high-risk recipients with rejection. Panel B shows the normalized intensity levels for LIMS1 on the protein array for the comparison between the high-risk rejection group and all other groups (P = 0.002); the horizontal lines represent the group means. Panel C shows the results of anti-LIMS1 total IgG seroreactivity studies with the use of an enzyme-linked immunosorbent assay that were performed in 318 persons across seven genotype- and phenotype-discordant groups. The results are shown as the change in the optical density (OD), defined as a ratio of the measured OD for each sample to the mean OD of the same 5 normalization controls (serum samples obtained from healthy persons) that were used on each plate. These studies included 52 controls who had not undergone transplantation (Control), 37 recipients who were homozygous for the risk allele and did not have rejection (Risk-NR), 31 recipients who were homozygous for the risk allele and had rejection (Risk-R; in red), 50 recipients who were heterozygous for the risk allele and did not have rejection (Het-NR), 50 recipients who were heterozygous for the risk allele and had rejection (Het-R), 63 recipients who were homozygous for the non-risk–associated allele and did not have rejection (Hom-NR), and 35 recipients who were homozygous for the non-risk–associated allele and had rejection (Hom-R). Total IgG seroreactivity was detected only in recipients with a high-risk genotype who had rejection. Horizontal lines represent group means, and the dotted line represents 3 SD above the mean for the control group. Panels D through G show the anti-LIMS1 reactivity of IgG1, IgG2, IgG3, and IgG4 subclasses, respectively; the results show predominant IgG2 and IgG3 responses. An asterisk indicates a P value of less than 0.001 and a dagger a P value of less than 0.01 for the comparisons of the group of recipients with a high-risk genotype who had rejection as compared with all other groups.