br Discussion Standard protocols of the assessment of neurop

Discussion Standard protocols of the assessment of neuropathic pain have been widely agreed for specialist settings and primary care (Haanpää et al., 2011; Jones & Backonja, 2013). However, there is no common approach or consensus reached by clinicians or researchers to define neuropathic pain in population-based settings or in general cohorts. As GoDARTS participants were recruited through community-based clinics and general hospitals, there is no formal record of neuropathic pain status made by specialists. We acknowledge that expert clinical examination would have increased the robustness of the case definition in this cohort. However, without clinical examination evidence, it protein phosphatase is reasonable to use an alternative, acceptable definition to represent neuropathic pain cases. We adopted a pragmatic approach to define cases using a multiple prescription history of the five main drugs used exclusively or mainly to treat neuropathic pain (rather than other disorders) in a diabetic population. A combination of diagnostic codes for type 2 diabetes and prescription of neuropathic pain drugs has been used in previous epidemiological studies to identify patients with painful diabetic neuropathy (Hall et al., 2013; Dieleman et al., 2008). Members of our population-based cohort were already identified as having type 2 diabetes, and so our method of identifying neuropathic pain makes this study reasonably consistent with these previous studies. While amitriptyline, carbamazepine, and nortriptyline are also frequently used in neuropathic pain, we considered that these are relatively likely to be used for indications other than neuropathic pain and we did not include individuals who had been prescribed these drugs as either cases or controls. To have a more homogeneous population, we removed individuals with only a single prescription of the five neuropathic pain drugs from both cases and controls. It has previously been highlighted that patients in primary care with neuropathic pain are often not prescribed any of the specific medications for its treatment (Hall et al., 2008; Torrance et al., 2007, 2013). As there is no pain status recorded in the GoDARTS, no direct assessment of the presence of (neuropathic) pain can be made among cases or controls. Furthermore, we did not assess whether cases or controls had received any other prescriptions for pain, such as opioid medications, and it is possible that some with neuropathic pain were treated with drugs that are not specifically indicated for this. Therefore the definition in our study is possible to have classified some who have neuropathic pain as controls but few controls as cases. The subsequent P values and ORs may be underestimated, though we cannot measure the extent of this. The most significant SNP cluster in the overall dataset was found in Chr1p35.1 with a lowest P value of 3.84×10 at rs35260355, spanning ZSCAN20-TLR12P area. The function of ZSCAN20 (zinc finger and SCAN domain containing 20) gene is not known yet and it has not been noted to be associated with any disorders. One of the proteins it codes contain typical C2H2 zinc finger domain, which enables zinc finger protein to bind other molecules such as RNA and DNA and affect transcription and translation (Krishna et al., 2003). There have been attempts to use zinc finger proteins to treat neuropathic pain since the receptor specific transcription factors of zinc-finger proteins have been developed to target gene repression in cell line models and in vitro (Tan et al., 2005). It is worth noting that the top SNP from the female only dataset rs71647933 is suggested to be a transcription factor binding site of the zinc interaction domain (SNPnexus). Toll-like receptors (TLRs) are a class of proteins which exist in various cell types in the central nervous system, including neuronal and non-neuronal cells (Liu et al., 2012). TLRs share structural and functional similarities. Specifically, the deletion or inhibition of TLR2 and TLR4 in animal models will impair nerve injury-induced neuropathic pain (Kim et al., 2007; Tanga et al., 2005). When using a TLR4 antagonist to treat both wild type mice and TLR4 knockout mice suffering neuropathic pain, pain relief can be achieved in the wild type mice but not in the TLR4 knockout mice (Bettoni et al., 2008). TLR12P is a unitary pseudogene with a transcript but there is no protein product of this gene in the human. The function of its homolog in mice is unclear although it is suggested it may be involved in the immune system against pathogens (Koblansky et al., 2012). There is emerging evidence showing that TLRs are involved in the control of (neuropathic) pain while the mechanisms are still far from being elucidated (Liu et al., 2012). In the females only dataset (1730 individuals), the P value of the SNPs in the cluster were lower than in the overall dataset, indicating that the male samples were not contributing so much to the associations in this cluster, and that the identified ZSCAN20-TLR12P locus has a gender specific influence on diabetic neuropathic pain. This is consistent with the findings of other TLR genes. Studies have found that variants in TLR genes are gender-specifically linked with multiple situations (Roberts et al., 2012). The mechanism of sex-specific phenomena is not clear and the evidence for hormone involvement is insufficient and controversial (Roberts et al., 2012; Berghöfer et al., 2006).