Genetic variants in major histocompatibility complex-linked genes associate with pediatric liver transplant rejection

Abstract

Background & aims: Limited access to large samples precludes genome-wide association studies of rare but complex traits. To localize candidate genes with family-based genome-wide association, a novel exploratory analysis was first tested on 1774 major histocompatibility complex single nucleotide polymorphisms (SNPs) in 240 DNA samples from 80 children with primary liver transplantation and their biologic parents.

Methods: Initially, 57 SNPs with large differences (P < .05) in minor allele frequencies were selected when parents of children with early rejection (rejectors) were compared with parents of nonrejectors.

Results: In hypothesis testing of selected SNPs, the gamete competition statistic identified the minor allele G of the SNP rs9296068, near HLA-DOA, as being significantly different (P = .018) between outcome groups in parent-to-child transmission. Subsequent simple association testing confirmed over- and undertransmission of rs9296068 based on the most significant differences between outcome groups, of 1774 SNPs tested (P = .002), and allele (G) frequencies that were greater among rejectors (51.4% vs 36.8%, respectively, P = .015) and lower among nonrejectors (26.8% vs 36.8%, respectively, P = .074) compared with 400 normal control Caucasian children. In early functional validation, rejectors demonstrated significant repression of the first HLA-DOA exon closest to rs9296068. Also, intragraft B lymphocytes, whose antigen-presenting function is selectively inhibited by HLA-DOA were 3-fold more numerous during rejection among rejectors with the risk allele, than those without.

Conclusions: The minor allele of the SNP rs9296068 is significantly associated with liver transplantation rejection and with enhanced B-lymphocyte participation in rejection, likely because of a dysfunctional HLA-DOA gene product.

Figure 1

Upper panel shows −log₁₀(p-values) for comparison of minor allele frequencies of 1,776 SNPs in parents of Rejectors, and parents of Non-Rejectors. Fifty seven SNPs show large differences (p<0.05, −log₁₀(p-value)>1.3) in allele frequencies in this comparison and are shown in the lower panel. Only one of these 57 SNPs (lower panel) also shows significant differences in parent-to-child transmission when Rejectors were compared with Non-Rejectors in the gamete competition statistic. This SNP rs9296068, marked by the red square is located in the HLA-DOA gene.

Figure 2

Splicing index values, defined as the fold change between mean gene-level normalized exon intensities in the Rejector and Non-Rejector groups and 95% confidence intervals for the 10 exons that map to the HLA-DOA gene. The physical position ranges of each exon are represented on the x-axis and any exon point with a significant p-value (p<.05) is shaded red. Significant negative fold changes represent exon skipping or repression, while significant positive fold changes represent exon enrichment for the gene.

Figure 3

a). Significant correlation exists between B-lymphocyte content in liver grafts with ACR, and numbers of risk allele G at rs9296068 (r²=0.194, p=0.021). Accompanying micrographs show ACR, with portal areas containing brown, horseradish-peroxidase-stained CD79a+ B-lymphocyte/plasmacytoid cells. B-lymphocyte content is lowest, when the risk allele is absent (panel b), higher in heterozygotes (panel c), and highest when the risk allele is homozygous (panel d).

Figure 4

The HLA-DOA gene is transcribed from the minus strand. The upstream 5′ flanking UTR region defined by five discriminatory SNPs in simple association testing of Rejectors vs Non-Rejectors lies between rs9296068 to the right and rs6457699 to the left, and shows marked linkage disequilibrium. Rs6457699 lies ≈4kb from the first HLA-DOA exon, on the left. Rs9296068 is associated with allele-dependent decrease in HLA-DOA expresion among CEU and YRI in public databases (Source: WGAViewer).