Allelic variation in the Tyk2 and EGF genes as potential genetic determinants of CNS repair

Abstract

The potential for endogenous remyelination and axonal protection can be an important factor in determining disease outcome in demyelinating diseases like multiple sclerosis. In many multiple sclerosis (MS) patients CNS repair fails or is incomplete whereas in others the disease is accompanied by extensive repair of demyelinated lesions. We have described significant differences in the ability of two strains of mice to repair CNS damage following Theiler's virus-induced demyelination: FVB/NJ (FVB) mice repair damaged myelin spontaneously and completely, whereas B10.D1-H2(q)/SgJ (B10.Q) mice are deficient in the repair process. A QTL analysis was performed to identify genetic loci that differentially regulate CNS repair following chronic demyelination in these strains and two QTL were detected: one on chromosome 3 with a LOD score of 9.3 and a second on chromosome 9 with a LOD score of 14.0. The mouse genes for epidermal growth factor (EGF) and Tyk2 are encoded within the QTL on chromosomes 3 and 9, respectively. Sequence polymorphisms between the FVB and B10.Q strains at both the EGF and Tyk2 loci define functional variations consistent with roles for these genes in regulating myelin repair. EGF is a key regulator of cell growth and development and we show a sevenfold increase in EGF expression in FVB compared to B10.Q mice. Tyk2 is a Janus kinase that plays a central role in controlling the T(H)1 immune response and we show that attenuation of Tyk2 function correlates with enhanced CNS repair.

Fig. 1.

Genetics of CNS repair. (A) Crosses used to generate F₁ and N₂ mice for the analysis of the genetics of CNS repair. FVB/NJ and B10.D1-H2^q/SgJ (B10.Q) were the parental strains. The intercross/backcross strategy was designed to identify dominant traits from the FVB strain and the genotype beneath each strain designation indicates the potential dominant (R) or recessive (r) nature of the genotype at any genomic locus with regard to remyelination. In the N₂ generation there are only two possible genotypes: FVB/B10.Q (R/r) or B10.Q/B10.Q (r/r). (B) Extent of remyelination in the FVB and B10.Q parental strains, and in F₁ and N₂ hybrid mice, as measured by quadrant analysis at 300 days postinfection. Mean repair values are indicated by the horizontal lines. (C) Extent of demyelination and remyelination in the 109 (FVB/B10.Q × B10.Q)N₂ mice used for the QTL analysis. Mean repair values are indicated by the horizontal lines. (D) Examples of the high and low remyelination phenotypes in (FVB/B10.Q × B10.Q)N₂ mice at 300 days postinfection. N, normal myelin; Ro, remyelination.

Fig. 2.

QTL for CNS repair. (A) Genomewide one-dimensional QTL scan. The horizontal lines across the plot indicate four confidence thresholds calculated at 1% (top, highly significant), 5%, 10%, and 63% (bottom, suggestive). Highly significant QTL were identified on chromosomes 3 and 9. (B and C) Confidence interval plots for chromosomes 3 and 9. The small horizontal bars at the base of the plots indicate the genomic segment that corresponds to the 95% confidence interval for each QTL. (D and E) Effect plots for QTL on chromosomes 3 and 9. Plots are based on the genotypes detected at a representative SNP located at the peak of LOD score (SNP rs3676039 for chromosome 3 and rs8270115 for chromosome 9). Effect plot for chromosome 3 shows that the FVB allele increases remyelination whereas on chromosome 9 it decreases remyelination. Genotypes: BB, B10.Q/B10.Q; FB, FVB/B10.Q. (F–H) Phenotypic values for remyelination were plotted following stratification of the animals based on their genotypes over the 95% confidence intervals for each QTL. F plots animals with the genotypes that should give the best or the poorest repair. G and H plot animals on the basis of their chromosome 3 or 9 genotypes.

Fig. 3.

Identification of candidate genes. (A) Structure of the EGF precursor protein showing the relative sizes of the extracellular, transmembrane, and cytoplasmic domains and the positions of the polymorphisms that differentiate the FVB and B10.Q alleles. The sequence below the structure shows the four nonsynonymous single-nucleotide polymorphisms and the resulting amino acid substitutions. (B) Western blot for EGF on protein from salivary gland from FVB and B10.Q mice. The arrow indicates the position of mature EGF growth factor that was run as a standard. (C) Structure of the Tyk2 protein. The sequence below the structure indicates the single nonsynonymous single-nucleotide polymorphism that differentiates the FVB and B10.Q alleles and the resulting amino acid substitution. (D) Western blot for Tyk2 on protein from the CNS. The positions of three molecular weight standards are indicated. The predominant Tyk2 band runs at ∼130 kDa. (E) Western blots for STAT4 and pSTAT4 from ConA-activated splenocytes from FVB and B10.Q animals after stimulation for varying times with IL-12.