Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis

Abstract

Background: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute.

Methods and findings: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting.

Conclusions: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.

Figure 1. Gene structure and linkage disequilibrium plots for ABCA4 (left panel) and COL2A1 (right panel).

Upper diagrams show positions of SNPs genotyped in relative to intron/exon structure of the gene. Lower diagrams show the linkage disequilibrium (D′) plots generated in Haploview (http://www.hapmap.org/) using data for each gene from the EMSCOT or NCCCTS cohorts as indicated. Linkage disequilibrium values (×100) between markers are indicated at the intercept of the two markers on the matrix. Where there is no value, D′ = 1 (i.e. 100). Haplotype blocks within each gene are outlined within the black triangles. The black (high) through grey to white (low) shading indicates the degree of confidence in the estimate of linkage disequilibrium between the markers. For the EMSCOT cohort, stepwise logistic regression analysis of associations observed in the mothers (Online Supplementary Material, Text S1 plus Table S5) indicate that all of the association at ABCA4 is accounted for by rs2997633 and rs1761375, implying that a single etiological variant in strong linkage disequilibrium with these two markers may account for the association. At COL2A1, SNPs rs2276455, rs1635544 and rs3803183 all add significant main effects when compared to rs2070739, but rs2070739 does not add significant main effects to any one of these markers. SNPs rs2276455, rs1635544 and rs3803183 do not add significant main effects to each other. Once pairs of these markers are taken into the model, the third SNP does not add significant main effects. A single etiological variant in this haplotype block could account for the association with COL2A1. Neither SNP (rs2070739, rs3803183) that results in a non-synonymous amino acid substitution appears to be the primary functional variant.

Figure 2. Experimental evidence for monoallelic expression at ABCA4 and COL2A1.

(A) and (B) show transcripts of ABCA4 and COL2A1expressed in human embryonic stem cell lines (HSF6 shown here; H9, data not shown) before (lane 1) and after commitment to extra-embryonic (lane 2) or ectoderm/neural (lane 3) lineages, in human placenta (lane 4) but not uterus (lane 5). ABCA4 with exon 10 is expressed in Y79 (lane 6) and WERI RB1 (lane 8) eye cell lines, adult (lane 10) and fetal (lane 11) brain, and EBV lines (e.g. lane 13) used for sequencing (C), but not in HEK293 (lane 12). The isoform without exon 10 is seen in eye lines (lanes 6, 8). COL2A1 isoform IIA is expressed in Y79 (lane 6) and WERI RB1 (lane 8) cells. Neither isoform is expressed in adult (lane 10) or fetal (lane 11) brain. Both are expressed in HEK293 (lane 12), and in EBV lines (e.g. lane 13) used for sequencing (D). Water and –RT lanes are indicated. (C) and (D) show sequence analysis of genomic DNA (gDNA) and cDNA in EBV lines heterozygous for exonic SNPs. (C) EBV lines heterozygous for ABCA4 rs3112831 in gDNA; lines EBV1 to EBV4 homozygous (i.e. monoallelic) in cDNA specific for the exon 10-containing isoform. Line EBV5 is heterozygous, indicating that mono-allelic silencing is polymorphic. (D) EBV lines show monoallelic expression for COL2A1 SNP rs3737548 in PCR products specific for isoform IIB, but not IIA. Positions of SNPs (*) indicated by N where heterozygous, with the bp underlined for mono-allelic expression in cDNA.