A comprehensive genetic analysis of candidate genes regulating response to Trypanosoma congolense infection in mice

Abstract

Background: African trypanosomes are protozoan parasites that cause "sleeping sickness" in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9-12 cM.

Principal findings: Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus.

Conclusions: By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.

Figure 1. CNV plots from Agilent DNA Analytics software.

A: Reduced copy numbers in C57BL/6 of Itlnb and Cd244 near Tir3c relative to two susceptible breeds of mice (Chr 1: 172,831,532–173,931,532 bp). B: CNV data at the proximal end of Tir1 showing a deletion of Glo1 and Dnahc8 in C57BL/6 and BALB/c relative to A/J and 129P3. (Chr 17: 29,854,972–30,954,972). Probes are plotted at their genomic position relative to their respective log₂ fluorescence intensity ratios (Y-axis) along with genes on the x-axis (filled blue rectangles). Green dots are negative ratios and red dots positive ratios (threshold 0.5). Lines are a moving average over a 10 Kbp window for A/J (blue); 129P3 (red) and BALBc (yellow). Genomic positions are based on mouse build mm8 (NCBI36).

Figure 2. Expression of A/J OlaHsdnd (A/J), BALB/cJ OlaHsdce (BALB/c) and C57BL/6JOlaHSD (C57BL/6) mouse genes in the Tir3c locus at five time points in the course of infection (0 days; 3 days; 5days; 9 days; 17 days).

Graphs include a small x-axis offset to improve spatial clarity. A Cd244 in the spleen, B Cd48 in the liver, C Apcs in the liver D Ifi202b in liver and spleen. Cd244 expression was low in liver in all strains until Day 7 when it rose above background and C57BL/6 had slightly lower levels than A/J or BALB/c (data not shown).

Figure 3. Array-based sequence capture and next generation sequencing of a 6.2 Mbp region of Mmu17 in four breeds of mice: A/J; BALB/c; C3H/HeJ and 129/J (Mmu17:30,637,692–36,837,814 bp).

Plot is circular for ease of display . Tir1 is highlighted in black on the inside track. Genomic positions are in Mbp. The outer tracks (blue and brown) show genes and designed capture probes, respectively. The four, coloured, inner tracks show SNP called in each of the four sequencing experiments, with the black tick marks highlighting areas of common SNP. Haplotype blocks can clearly be seen as clustering of high- and low- density regions of SNP. A magnified region around Tir1 is displayed underneath the circular plot. Tracks are identically coloured and include a moving average (window 1 Kbp) of sequence read coverage across the region (top). Genes in the region are displayed for the forward strand (above) and reverse strand (below).

Figure 4. SNP plots of Tir1 between 31 and 31.65 Mbp.

The C57BL/6 row represents the reference allele for all loci that are polymorphic in either the Perlegen set or our 454 set. The SNP density is much greater in the 454 data set, in which haplotype blocks are clearly identifiable by eye. It appears that SNP are much better represented in the Perlegen data set in some regions than in others. Between 31.2 and 31.30 the two data sets are very similar with high density of SNP in BALB/c and 129 substrains in each dataset. However in the region between 31.1 and 31.20 the SNP in BALB/c and 129 are relatively much sparser in Perlegen than in the 454 data.