Experiment
Blood brain barrier (BBB) disruption is an integral feature of numerous immune-mediated neurological disorders. Comprehensive genetic analysis was performed in this
study to identify QTL linked to functional motor deficits and CNS vascular permeability,
which are both devastating characteristics associated with CD8 T cell-initiated BBB disruption in the peptide-induced fatal syndrome (PIFS) model.
C57BL/6J (B6) mice are highly susceptible to PIFS, exhibiting functional motor deficits, increased astrocyte activation, and severe CNS vascular permeability, while 129S1/SvImJ (129S1) mice are resistant.
(B6 x 129S1)F1 hybrids were brother-sister mated to produce 273 (B6 x 129S1)F2 progeny.
All mice were intracranially infected with 2 10 6 PFU Daniel's strain of TMEV. Seven days post-TMEV infection, during the peak of CD8 T cell expansion, mice were intravenously administered 0.1 mg VP2 121-130 (FHAGSLLVFM) peptide. In order to evaluate functional motor deficits, mice were placed on the Rotamex-5 rotarod apparatus (Columbus Instruments) increasing from 5-40 RPMs over 7 minutes. Mice were then injected with VP2 121-130
peptide to induce CNS vascular permeability and their motor performance was assessed again 24 hours later. Final scores were depicted as percent initial motor ability. Mice were given an intravenous injection of 10 mg FITC-albumin (Sigma #A9771) 23 hours post-VP2
121-130 peptide administration to induce PIFS. rotein concentration was assessed using the BCA protein assay (Pierce #23223). Samples were then normalized for protein content and
read on a fluorescent plate reader at 488 nm excitation and 525 nm emission to detect FITC-albumin leakage into the brain.
DNA from 273 F2 progeny (124 males; 149 females) were analyzed on 2 SNP chips using Illumina Chip Technologies. Two B6 and 2 129S1 mice were included as controls. 874 SNPs
were found to be unique between the two strains, 671 had a known cM value and thus were analyzed for the relationship between quantifiable traits and genotype. Functional motor deficits measured by rotarod, CNS vascular permeability measured by FITC-albumin leakage, and astrocyte activation measured by GFAP expression as quantifiable traits predictive of susceptibility to PIFS.
Interval mapping was used to identify significant linkage using a critical value of p = 0.05 and a 95% confidence interval. A chi-square test statistic for each SNP marker showing linkage within a QTL interval was obtained using 2 3 contingency tables to demonstrate the distribution of parental strain alleles within the F2 population.
Since F2 mice displayed variable susceptibility, they were divided into two groups, low F2s and high F2s, based on whether or not they exhibited functional motor deficits or CNS vascular permeability. For functional motor deficits, low F2s were defined as those mice
displaying <50% initial ability, while high F2s were defined as mice displaying >50% initial ability. For CNS vascular permeability, mice with FITC scores <500 were
placed in the low F2 group while mice with FITC scores >500 were placed in the high F2 group for analysis.
QTL on Chromosomes 12 and 17 were significantly linked with susceptibility to PIFS.
QTL Pifs1 (peptide-induced fatal syndrome 1), linked with functional motor deficits, mapped to Chr 12 with a LOD score of 3.3 at marker rs3655057, peaking at 6.0 cM. The 95% CI mapped between 4.7 and 10.1 cM. The 129S1 strain contributed to low rotarod performance on Chr 12 markers rs6292954, rs13481303, rs3655057 and rs13481324 within the Pifs1 interval while the B6 strain contributed to high rotarod performance on these markers. [Table 1].
QTL Pifs2 (peptide-induced fatal syndrome 2), linked with CNS vascular permeability, mapped to Chr 17 with a LOD score of 3.7 at marker rs6196216, peaking at 10.8 cM. The 95% CI mapped between 5.7 and 12.5 cM. B6 alleles contributed to less CNS vascular permeability on Chr 17 markers rs6196216 and rs3672065 within the Pifs2 interval while 129S1 alleles contributed more vascular permeability on the same markers. [Table 1.]
Using the Mouse Genomen Informatics Database resources a search was performed to locate genes in proximity with the significant loci found for each QTL. Then candidate genes were proritized based on known or presumed biological functions and previous studies.
Six genes of interest were identified for Pifs1: Vsnl1, Ddx1, Trib2, Lpin1, Kcnf1 and Rock2. [Table 3.]
Eight genes of interest were identified for Pifs2: Pde10a, Qk, Parcg, Park2, Plg, Igf2r, Mas1 and Mllt4. [Table 3].
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