Identification of a novel QTL (Spir2) on chromosome 4 contributing to both survival time and bacteraemia

Selective genotyping of CCBAF₂ mice, with phenotypes representing the extremes of the response after infection challenge, identified a region of significant linkage on chromosome 4 with the traits of survival time and bacteraemia (Figure 1). The peak of linkage was located at SNP 4_80 (rs4224562) at 82.5 Mb. The QTL location on chromosome 4, estimated by the one LOD support interval, was from SNP 4_47 (rs13477699) at 50.7 Mb to SNP 4_87 (rs3022987) at 88.2 Mb and was ~ 38 Mb in size. The LOD score for the peak of linkage exceeded the genome wide significance threshold of 3.10 with a LOD score of 4.56 for survival and 4.25 for bacteraemia. The QTL identified on chromosome 4 was estimated to account for 8% of the phenotypic variance. Detection of the Spir1 locus on chromosome 7 was replicated in this study, also accounting for ~ 8% of the phenotypic variance (data not shown).

Effect of genotype at Spir2on phenotype

In order to investigate the effect of genotype at SNP 4_80 on the time to become moribund, a Kaplan-Meier survival analysis was performed (Figure 2A). CCBAF₂ mice heterozygous for SNP 4_80 showed a significant difference in the time before exhibiting disease signs when compared to mice homozygous for BALB/c (p = 0.01) or CBA/Ca (p = 0.001). The mean and median survival times were calculated for each CCBAF₂ genotype group. The median survival time, at which 50% of the group became severely lethargic, was 44 hours for both BALB/c and CBA/Ca genotypes. There was no median survival time calculated for the heterozygous genotype, as more than 50% of the group survived to the end of the experiment. The numbers of bacteria in the blood at 24 hours after intranasal infection were also significantly lower in CCBAF₂ mice, which were heterozygous for the peak of linkage, when compared to those which were homozygous for BALB/c or CBA/Ca at the same SNP (Figure 2B). These data suggest that being heterozygous at this location on chromosome 4 is advantageous.

Congenic breeding confirms the contribution of the Spir2locus to susceptibility to pneumococcal infection

The strategy of congenic breeding was used to replicate detection of the chromosome 4 QTL and to assess its contribution to the infection phenotype. Incipient congenic strains consisting of different portions of the chromosome 4 QTL from BALB/cByJ on a CBA/CaH background, and vice versa, were produced using a marker-assisted breeding scheme.

The percentages of heterozygosity observed in the N2 and N3 males used for breeding further generations were between 37 and 42% for the N2 generation and 7 to 10% for the N3 generation. Mice with the CBA/CaH Spir2 locus on a BALB/cByJ background were bred to the N6 generation before intercrossing. These incipient congenics were named BYJCBAN6-4. Mice with the BALB/cByJ Spir2 locus on a CBA/CaH background were bred to the N7 generation and were named CBABYJN7-4. The estimated percentage recipient genome in the final incipient congenic mice tested for infection susceptibility was 99.36 to 99.78%. Therefore these mice were similar to N9 mice produced by conventional congenic breeding.

BALB/cByJ Spir2locus on CBA/CaH background (CBABYJN7-4)

A total of 75 CBABYJN7-4 intercross mice were tested for susceptibility to S. pneumoniae infection. Of these 75 mice 18 (24%) were resistant to pneumococcal infection and the remaining 57 (76%) were susceptible. Six of the susceptible mice survived 50 hours or longer, while the rest only survived to between 28 and 49 hours.

A survival analysis was performed on the CBABYJN7-4 mice for each SNP on Chr4 and it was found that animals that were heterozygous at SNP 4_80 survived longer by comparison with those homozygous for the CBA/Ca allele. Similar results were found at SNP 4_103 (Additional file 1: Table S2). These results do not achieve statistical significance (SNP 4_80 p = 0.064 and SNP 4_103 p = 0.087), yet are noteworthy because they show the same trend as the Spir2 QTL detected in the CCBAF₂. (Figure 3 and Additional file 2: Figure S1). There was no difference in time to morbidity between mice homozygous for BALB/cByJ and those homozygous for CBA/CaH at any Chr4 marker. There was also no significant difference in the numbers of bacteria in the blood at 24 hours post-infection in CBABYJN7-4 mice homozygous for BALB/cByJ or CBA/CaH or heterozygous for any of the chromosome 4 SNP markers (data not shown).

A total of 64 mice could be divided into 11 haplotypes based on their combination of genotypes at SNPs 4_38 to 4_103. These groups were called CBABYJN7-4-A to K (Figure 4). There was no significant difference in numbers of bacteria in the blood at 24 hours post-infection between any of the CBABYJN7-4 haplotypes (data not shown). However, there was a significant difference between the survival curves of CBABYJN7-4-B and G (p = 0.014) with a lower risk of disease in the CBABYJN7-4-G mice compared to the CBABYJN7-4-B mice (Figure 5).

CBA/CaH Spir2locus on BALB/cByJ background (BYJCBAN6-4)

A total of 26 BYJCBAN6-4 intercross mice were tested for susceptibility to infection with S. pneumoniae. Ten (38%) of these mice were susceptible and 16 (62%) were resistant to pneumococcal infection. An analysis of the survival of these mice, based on their genotype at markers on Chr4, revealed significant differences for SNPs 4_38, 4_80 and 4_103 (Figure 6A and Additional file 3: Figure S2). Mice homozygous for the BALB/cByJ allele of any of these three SNPS were at significantly less risk of disease than those homozygous for CBA/CaH at the same SNP. Survival curves for mice that were heterozygous for the SNP 4_38, were significantly different from those for CBA/CaH homozygotes (p = 0.007), with more heterozygotes surviving. There were no significant differences in the survival curves for animals of any of the genotypes for SNP 4_47, however this marker did follow the same trend as for SNPs 4_38, 4_80 and 4_103 (data not shown).

Numbers of bacteria in the blood of BYJCBAN6-4 mice were assessed at 24 hours post-infection. There were significantly more bacteria present in mice homozygous for the CBA/CaH allele at SNP marker 4_38 or 4_47 when compared with mice either heterozygous or homozygous for BALB/cByJ at the same SNP (p = 0.022 and 0.009 respectively). Although not significant, the trend was the same for SNP markers 4_80 and 4_103 (Figure 6B).

Analysis of the combination of alleles at SNPs 4_38 to 4_103 was performed on the BYJCBAN6-4 mice. Of the 26 mice, 21 could be placed into one of five different haplotypes, named BYJCBAN6-4-A to E (Figure 7). Kaplan-Meier analysis of the five haplotype groups revealed a significant difference in the survival curve of BYJCBAN6-4-A mice, which were homozygous for BALB/cByJ at all four SNPs when compared with BYJCBAN6-4-E mice, which were homozygous for CBA/CaH at these SNPs (p = 0.017) (Figure 8A). Analysis of the bacteraemia results at 24 hours post-infection also revealed a significant difference between BYJCBAN6-4-A and E mice (p = 0.005). BYJCBAN6-4-E mice had significantly higher numbers of bacteria present in the blood when compared with BYJCBAN6-4-A mice, which had no bacteraemia at 24 hours (Figure 8B).

Ethics statement

This study was performed in strict accordance with U.K. Home Office guidelines. Both the U.K. Home Office and the University of Leicester ethics committee approved the protocol. Every effort was made to minimize suffering and in bacterial infection experiments mice were humanely culled if they became lethargic. All animal experiments were carried out at the University of Leicester.

Mice

BALB/cByJ and CBA/CaH were obtained from the MRC Mary Lyon Centre (MLC) in Harwell and were used for the congenic breeding scheme. The congenic breeding was performed at the MLC and infection studies were done at the University of Leicester.

Congenic breeding scheme

A semi-speed congenic breeding scheme was implemented in this study. An alternating set of 73 SNP markers, spanning the genome, with an average spacing of 28 Mb, was used in order to reduce cost and time and ensure good coverage of the genome [32]. During the first two backcross generations (N2 and N3) the alternate genome scan was performed where at least 20 male progeny for each generation, which were heterozygous for the QTL, were typed genome wide in order to select the best male for breeding [32, 33]. The next generations (from N4 to N6 or N7) were only genotyped for the chromosome 4 QTL markers. Once the mice reached the N6 or N7 generation, female mice heterozygous for the region of interest were crossed to males heterozygous for the same SNP markers to produce offspring for infection testing. Whole litters were tested for susceptibility to S. pneumoniae.

Bacterial culture and infections

Incipient congenic mice were infected intranasally with S. pneumoniae D39 as described previously [12, 13]. Blood was taken at 24 hours post-infection from the tail vein (for bacterial culture) and survival times of the mice were recorded. Mice that survived to the end point of the experiment (more than 168 hours) were considered resistant. All mice that succumbed to infection (the endpoint was severely lethargic) and had survival times less than 168 hours were considered susceptible.

DNA extractions

For QTL mapping, DNA from the (BALB/cOlaHsd × CBA/CaOlaHsd)F₂ (CCBAF₂) intercross, reported in Denny et al., [13], was used. DNA samples from CCBAF₂ mice from the phenotypic extremes were selected for QTL mapping. Thirty eight of the most susceptible mice (survival < 46 hours and high levels of bacteria in blood) and thirty eight of the most resistant mice (survival ≥ 168 hours and no bacteria in blood) were genotyped for linkage analysis. DNA was diluted to 5 ng/μl in double-distilled H₂O.

In congenic breeding, DNA was extracted from ear clips using the Viagen DirectPCR ear lysis reagent (Viagen Biotech cat 402-E). 195 μl Direct PCR lysis reagent and 5 μl proteinase K (10 mg/ml) was added to each earclip and incubated overnight at 55°C. After digestion, the samples were heated to 85°C for 45 min and centrifuged for 10 seconds. 1 μl of lysate was used in each PCR reaction.

SNP panel

The 76 CCBAF₂ mice from the phenotypic extremes were genotyped by Pyrosequencing (as described below) across the whole genome using a panel of 73 SNPs with an average spacing of 28 Mb. Chromosome 4 was genotyped with an additional set of SNP markers to narrow down areas of suggestive linkage. A total of nine SNPs for chromosome 4 were typed by Pyrosequencing. Genotyping data from chromosome 4 microsatellite markers used in Denny et al., [13] were incorporated into the analysis. There were a total of 5 microsatellite markers that had been typed on 38 of the mice. Details of the SNP primers can be found in Additional file 4: Table S1.

Primer design

SNPs were selected using the Mouse Phenome Database (http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home) and the SNP sequences were exported from the NCBI Entrez SNP database (http://www.ncbi.nlm.nih.gov/sites/entrez). Primers for Pyrosequencing were designed using the PSQ Assay Design software from Biotage AB. Primer sets of three primers for each SNP were designed, one pair of primers for the PCR (one of which was biotinylated) and a sequencing primer for the Pyrosequencing reaction. The minimum and maximum T_m for the PCR primers were 64 to 66°C. The sequencing primers were designed with a maximum distance of three bases from the SNP. Primers were manufactured by either Biomers.net or MWG-Biotech.

PCR

10 μl PCR reactions were set up using 5 μl Qiagen Taq PCR master mix (cat. 201445), 0.2 μl forward primer and 0.2 μl reverse primer (at 10 pmol/μl), 2.6 - 3.6 μl nuclease free water and 1–2 μl DNA(~5 ng/μl). PCR reactions were run using the following PCR program: 95°C for 5 min, followed by 45 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 15 sec. The final extension step was for 5 min at 72°C.

Pyrosequencing

10 μl PCR product, 2 μl streptavidin-Sepharose beads (GE Healthcare 17-5113-01), 38 μl binding buffer (Biotage AB 40–0033) and 30 μl H₂O were combined in a 96-well plate and mixed vigorously on a plate shaker for 5 min so that the biotin-labelled PCR product bound to the streptavidin coated beads. The PCR products were then prepared using a vacuum work table (Biotage AB). The biotinylated PCR products, attached to the filter probes of the vacuum tool, were immersed in 70% (v/v) ethanol for 5 seconds, denatured in PyroMark Denaturation solution (Biotage AB 40–0034) for 5 sec (allowing only the biotin labelled strand of the PCR product to stay attached to the filter probes) and immersed in 1X PyroMark Wash buffer (Biotage AB 40–0035) for 5 sec. The single-stranded PCR products were then re-suspended in a PSQ HS 96-well plate containing 0.5 μl sequencing primer (at 10 pmol/μl) and 11.5 μl annealing buffer (Biotage AB 40–0036) per well.

The plate was incubated at 80°C for 2 minutes to allow the sequencing primer to anneal to the single-stranded PCR product. The PSQ 96-well plate and a PSQ HS 96 capillary dispensing tip holder (Biotage AB 60–0211) containing enzyme, substrate and dNTPs (PyroGold reagent kit Biotage AB 40–0047), were placed into a PSQ HS 96 Pyrosequencer (Biotage AB). The assays were performed and the data were analysed using the SNP software (Biotage AB).

Linkage analysis

SNP genotype data from the 76 CCBAF₂ mice and phenotype data from 168 non-genotyped CCBAF₂ mice were incorporated into the linkage analysis. Data were analysed using R-QTL for non parametric analysis [34]. One thousand permutation tests were performed in order to establish genome-wide LOD significance thresholds at the 90% and 95% confidence level. Loci with LOD scores exceeding the 90% confidence level were identified as suggestive and those exceeding the 95% confidence level were identified as significant regions of linkage. The confidence interval for each QTL was determined by a one LOD support interval (−1 LOD drop from the peak of linkage). For the marker at the peak of each QTL, data were analysed using a Chi-squared (χ²) test. Numbers of mice resistant and susceptible to pneumococcal infection were grouped by their genotype at the appropriate SNP marker. Observed numbers of mice were compared to the expected numbers (1:2:1 ratio) if genotype had no effect (χ²). To investigate the effect the genotype, at the peak of linkage, on time to reach a moribund state, Kaplan Meier survival analysis was performed using the statistics package SPSS (version 17). The difference between the survival curves was analysed using the Log Rank test. p values of less than 0.05 were considered significant. The heritability equation (1-10^-2LOD/n) was used to estimate the percentage of phenotypic variance accounted for by the QTL.

Congenic breeding data analysis

Survival data and bacteraemia data were firstly analysed on a single SNP basis for chromosome 4. Mice were then grouped into shared haplotypes based on the combination of SNPs for chromosome 4 from SNP 4_38 to SNP 4_103. Survival data were analysed using Kaplan-Meier survival analysis with Log Rank pair wise comparison for statistical significance using SPSS (version 17). Bacteraemia data for the three genotypes (BALB/cByJ homozygous, CBA/CaH homozygous or heterozygous) were compared for each SNP using a two-tailed unpaired Student t-test. Numbers of bacteria in the blood in the haplotypes were compared in the same way. Results with p values less than 0.05 were considered significant.