Hazel Wilkie

Suggestive evidence for association of two potassium channel genes with different idiopathic generalised epilepsy syndromes

Several potassium channel genes have been implicated in epilepsy. We have investigated three such genes, KCNJ3, KCNJ6 and KCNQ2, by association studies using a broad sample of idiopathic generalised epilepsy (IGE) unselected by syndrome. One of the two single nucleotide polymorphisms (SNPs) examined in one of the inward rectifying potassium channel genes, KCNJ3, was associated with IGE by genotype (P=0.0097), while its association by allele was of borderline significance (P=0.051). Analysis of the different clinical subgroups within the IGE sample showed more significant association with the presence of absence seizures (P=0.0041) and which is still significant after correction for multiple testing. Neither SNP in the other rectifying potassium channel gene, KCNJ6, was associated with IGE or any subgroup. None of the three SNPs in the voltage-gated potassium channel gene, KCNQ2, was associated with IGE. However, one SNP was associated with epilepsy with generalised tonic clonic seizures only (P=0.016), as was an SNP approximately 56 kb distant in the closely linked nicotinic acetylcholine gene CHRNA4 (P=0.014). These two SNPs were not in linkage disequilibrium with each other, suggesting that if they are not true associations they have independently occurred by chance. Neither association remains significant after correcting for multiple testing.

Haplotype and linkage disequilibrium analysis to characterise a region in the calcium channel gene CACNA1A associated with idiopathic generalised epilepsy

Idiopathic generalised epilepsy (IGE) is a common form of epilepsy, including several defined and overlapping syndromes, and likely to be due to the combined actions of mutations in several genes. In a recent study we investigated the calcium channel gene CACNA1A for involvement in IGE, unselected for syndrome, by means of association studies using several polymorphisms within the gene. We reported a highly significant case/control association with a silent single nucleotide polymorphism (SNP) in exon 8 that we confirmed by within-family analyses. In this present study we screened the gene for novel SNPs within 25 kb of exon 8, which have enabled us to define the critical region of CACNA1A in predisposing to IGE. Several intronic SNPs were identified and three, within 1.5 kb of exon 8 and in strong linkage disequilibrium with each other and with the original SNP, were significantly associated with IGE (P=0.00029, P=0.0015 and P=0.010). The associations were not limited to an IGE syndrome or other subgroup. Another SNP, 25 kb away, in intron 6 was also significantly associated with IGE (P=0.0057) but is not in linkage disequilibrium with the SNPs around exon 8. Haplotype predictions revealed even more significant associations (3-marker haplotype: P<10−6). Logistic regression showed that all the data can be explained by two of the SNPs, which is consistent with two functionally significant variants being responsible for all five associations, although a single variant cannot be excluded. The functionally significant variant(s) are unlikely to be exonic and suggests an effect on expression or alternative splicing.

Association of μ-opioid receptor subunit gene and idiopathic generalized epilepsy

Objective: To replicate and extend the previously reported association between the opioid receptor μ subunit gene (OPRM1) and idiopathic absence epilepsy (IAE), using a sample of 230 probands with idiopathic generalized epilepsy (IGE). Background: In humans and in animal models, several lines of evidence implicate opioid receptors with seizures. The G118 allele of OPRM1 was associated with IAE (p = 0.019). Methods: Three single nucleotide polymorphisms (SNP) of OPRM1 were investigated by association studies with IGE using a case/control design, one of which also used a within-family design. Results: Association was found for G118 with IGE (p = 0.00027, odds ratio [OR] = 1.86), replicating the previous association. Within-family tests of linkage and association (haplotype-based haplotype relative risk and transmission disequilibrium test) confirmed this result. Further evidence for involvement of OPRM1 in IGE was provided by an association with G-172T, located in the 5′ untranslated region (p = 0.0015, OR = 2.36). Haplotypes of the two SNPs were associated with IGE with a greater level of significance (p = 0.000087) suggesting that both SNPs might be in linkage disequilibrium with a single functional variant. Analysis of the results by subgroups of IGE showed association with all subgroups tested. Conclusions: These results confirm the previous association and support the hypothesis of a role for OPRM1 in IGE, including absence syndromes. However, the authors found no evidence for a specific association between OPRM1 and idiopathic absence epilepsy. The data suggest that the functional variant predisposing to IGE is located within 60kb of exon 1.

Variations in T Cell Transcription Factor Sequence and Expression Associated with Resistance to the Sheep Nematode Teladorsagia circumcincta

This study used selected lambs that varied in their resistance to the gastrointestinal parasite Teladorsagia circumcincta. Infection over 12 weeks identified susceptible (high adult worm count, AWC; high fecal egg count, FEC; low body weight, BW; low IgA) and resistant sheep (no/low AWC and FEC, high BW and high IgA). Resistance is mediated largely by a Th2 response and IgA and IgE antibodies, and is a heritable characteristic. The polarization of T cells and the development of appropriate immune responses is controlled by the master regulators, T-bet (TBX21), GATA-3 (GATA3), RORγt (RORC2) and RORα (RORA); and several inflammatory diseases of humans and mice are associated with allelic or transcript variants of these transcription factors. This study tested the hypothesis that resistance of sheep to T. circumcincta is associated with variations in the structure, sequence or expression levels of individual master regulator transcripts. We have identified and sequenced one variant of sheep TBX21, two variants of GATA3 and RORC2 and five variants of RORA from lymph node mRNA. Relative RT-qPCR analysis showed that TBX21, GATA3 and RORC2 were not significantly differentially-expressed between the nine most resistant (AWC, 0; FEC, 0) and the nine most susceptible sheep (AWC, mean 6078; FEC, mean 350). Absolute RT-qPCR on all 45 animals identified RORVv5 as being significantly differentially-expressed (p = 0.038) between resistant, intermediate and susceptible groups; RORCv2 was not differentially-expressed (p = 0.77). Spearman’s rank analysis showed that RORAv5 transcript copy number was significantly negatively correlated with parameters of susceptibility, AWC and FEC; and was positively correlated with BW. RORCv2 was not correlated with AWC, FEC or BW but was significantly negatively correlated with IgA antibody levels. This study identifies the full length RORA variant (RORAv5) as important in controlling the protective immune response to T. circumcincta infection in sheep.

Mucosal Expression of T Cell Gene Variants Is Associated with Differential Resistance to Teladorsagia circumcincta.

Resistance of sheep to the gastrointestinal nematode Teladorsagia circumcincta is a heritable characteristic. Control of parasite colonization and egg production is strongly linked to IgA antibody levels regulated by Th2 T cell activation within lymphoid tissue; and persistently-infected susceptible animals develop an inflammatory Th1/Th17 response within the abomasum that fails to control infection. Differential T cell polarization therefore is associated with parasite resistance and/or susceptibility and is controlled by a specific set of transcription factors and cytokine receptors. Transcript variants of these genes have been characterized in sheep, while in humans and mice different variants of the genes are associated with inflammatory diseases. RT-qPCR was used to quantify mucosal expression of the transcript variants of the sheep genes in trickle-infected animals with defined phenotypic traits. Genes that encode full-length GATA3 and IL17RB were shown to be significantly increased in resistant sheep that had controlled parasite infection. Expression levels of both were significantly negatively correlated with abomasal worm count (a parameter of susceptibility) and positively correlated with body weight (a parameter of resistance). These data show that polarized Th2 T cells within the abomasal mucosa play an important role in the maintenance of resistance.

Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta

Graphical abstract

A candidate gene approach to study nematode resistance traits in naturally infected sheep

Highlights • SNPs within genes associated with the development of nematode resistance were sequenced.• The SNPs were segregating in two unrelated, domestic and feral, sheep breeds.• More samples are required to determine significant associations between SNPs and traits.

Variations in T cell transcription factor gene structure and expression associated with the two disease forms of sheep paratuberculosis

Two different forms of clinical paratuberculosis in sheep are recognised, related to the level of bacterial colonization. Paucibacillary lesions are largely composed of lymphocytes with few bacteria, and multibacillary pathology is characterized by heavily-infected macrophages. Analysis of cytokine transcripts has shown that inflammatory Th1/Th17 T cells are associated with development of paucibacillary pathology and Th2 cytokines are correlated with multibacillary disease. The master regulator T cell transcription factors TBX21, GATA3, RORC2 and RORA are critical for the development of these T cell subsets. Sequence variations of the transcription factors have also been implicated in the distinct disease forms of human mycobacterial and gastrointestinal inflammatory diseases. Relative RT-qPCR was used to compare expression levels of each transcript variant of the master regulators in the ileo-caecal lymph nodes of uninfected controls and sheep with defined paucibacillary and multibacillary pathology. Low levels of GATA3 in multibacillary sheep failed to confirm that multibacillary paratuberculosis is caused simply by a Th2 immune response. However, high levels of TBX21, RORC2 and RORC2v1 highlights the role of Th1 and Th17 activation in paucibacillary disease. Increased RORAv1 levels in paucibacillary tissue suggests a role for RORα in Th17 development in sheep; while elevated levels of RORAv4 hints that this variant might inhibit RORα function and depress Th17 development in multibacillary sheep.