TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function.

Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. Here, we show that the canonical transient receptor potential 6 (TRPC6) ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. We identified five families with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function.

Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons.

BACKGROUND: Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. RESULTS: Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. CONCLUSIONS: Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

Tagging single-nucleotide polymorphisms in antioxidant defense enzymes and susceptibility to breast cancer.

It is generally believed that the initiation of breast cancer is a consequence of cumulative genetic damage leading to genetic alterations and provoking uncontrolled cellular proliferation and/or aberrant programmed cell death, or apoptosis. Reactive oxygen species have been related to the etiology of cancer as they are known to be mitogenic and therefore capable of tumor promotion. The aim of this study was to assess the role of common variation in 10 polymorphic genes coding for antioxidant defense enzymes in modulating individual susceptibility to breast cancer using a case-control study (N cases = 4,474 and N controls = 4,580). Both cases and controls were from the East Anglian region of the United Kingdom. We have identified a set of 54 single nucleotide polymorphisms (SNPs) that efficiently tag all the known SNPs in the 10 genes and are also expected to tag any unknown SNPs in each gene. We found no evidence for association of common variants in SOD1, SOD2, GPX1, GPX4, GSR, TXNRD1, and TXN2. There was borderline evidence for association of variants in CAT g27168a {P [2 degrees of freedom (df)] = 0.05}, TXN t2715c [P (2 df) = 0.007], and TXNRD2 A66S and TXNRD2 g23524a (P(trend) = 0.074 and 0.046, respectively). For TXNRD2 A66S [AS versus AA: odds ratio (OR), 1.05; 95% confidence intervals (95% CI), 0.96-1.15; SS versus AA: OR, 1.12; 95% CI, 0.98-1.29], there are bioinformatics data to suggest that it is functional but confirmation in independent data sets is required before they can be regarded as definitive breast cancer susceptibility alleles. Even if confirmed, these four alleles would account for just 0.32% of the excess familial risk of breast cancer.

Investigating diagnostic bias in autism spectrum conditions: An item response theory analysis of sex bias in the AQ-10.

Diagnostic bias is a concern in autism spectrum conditions (ASC) where prevalence and presentation differ by sex. To ensure that females with ASC are not under-identified, it is important that ASC screening tools do not systematically underestimate autistic traits in females relative to males. We evaluated whether the AQ-10, a brief screen for ASC recommended by the National Institute of Clinical Excellence in cases of suspected ASC, exhibits such a bias. Using an item response theory approach, we evaluated differential item functioning and differential test functioning. We found that although individual items showed some sex bias, these biases at times favored males and at other times favored females. Thus, at the level of test scores the item-level biases cancelled out to give an unbiased overall score. Results support the continued use of the AQ-10 sum score in its current form; however, suggest that caution should be exercised when interpreting responses to individual items. The nature of the item level biases could serve as a guide for future research into how ASC affects males and females differently. Autism Res 2017, 10: 790-800. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Association of the progesterone receptor gene with breast cancer risk: a single-nucleotide polymorphism tagging approach.

Association studies on susceptibility to breast cancer using single nucleotide polymorphisms (SNP) in the progesterone receptor (PGR) gene have been previously published, but the results have been inconclusive. We used a comprehensive SNP-tagging approach to search for low-penetrance susceptibility alleles in a study of up to 4,647 cases and 4,564 controls, in a two-stage study design. We identified seven tagging SNPs using genotype data from the National Institute of Environmental Health Sciences (NIEHS) Environmental Genome Project and typed these, and an additional three SNPs, in 2,345 breast cancer cases and 2,284 controls (set 1). Three SNPs showed no evidence for association and were not studied further, whereas seven SNPs (rs11571171, rs7116336, rs660149, rs10895068, rs500760, rs566351, and rs1042838) exhibited significant associations at P < 0.1 using either a heterogeneity or trend test and progressed to be genotyped in set 2. After both stages, only one SNP was significantly associated with an increased risk of breast cancer - the PGR-12 (rs1042638) V660L valine to leucine polymorphism [VL heterozygotes (odds ratio, 1.13; 95% confidence interval, 1.03-1.24) and the LL homozygotes (odds ratio, 1.30; 95% confidence interval, 0.98-1.73), P(het) = 0.008, P(trend) = 0.002]. Similar estimates were obtained in a combined analysis of our data with those from three other published studies. We conclude that the 660L allele may be associated with a moderately increased risk of breast cancer, but that other common SNPs in the PGR gene are unlikely to be associated with a substantial risk of breast cancer.

The AIB1 polyglutamine repeat does not modify breast cancer risk in BRCA1 and BRCA2 mutation carriers.

This is by far the largest study of its kind to date, and further suggests that AIB1 does not play a substantial role in modifying the phenotype of BRCA1 and BRCA2 carriers. The AIB1 gene encodes the AIB1/SRC-3 steroid hormone receptor coactivator, and amplification of the gene and/or protein occurs in breast and ovarian tumors. A CAG/CAA repeat length polymorphism encodes a stretch of 17 to 29 glutamines in the HR-interacting carboxyl-terminal region of the protein which is somatically unstable in tumor tissues and cell lines. There is conflicting evidence regarding the role of this polymorphism as a modifier of breast cancer risk in BRCA1 and BRCA2 carriers. To further evaluate the evidence for an association between AIB1 glutamine repeat length and breast cancer risk in BRCA1 and BRCA2 mutation carriers, we have genotyped this polymorphism in 1,090 BRCA1 and 661 BRCA2 mutation carriers from Australia, Europe, and North America. There was no evidence for an increased risk associated with AIB1 glutamine repeat length. Given the large sample size, with more than adequate power to detect previously reported effects, we conclude that the AIB1 glutamine repeat does not substantially modify risk of breast cancer in BRCA1 and BRCA2 mutation carriers.

Polymorphisms in the initiators of RET (rearranged during transfection) signaling pathway and susceptibility to sporadic medullary thyroid carcinoma.

CONTEXT: Medullary thyroid carcinoma (MTC) is a characteristic tumor occurring in individuals with multiple endocrine neoplasia type 2 who carry germ-line mutations in RET (rearranged during transfection). However, most MTC occur in individuals without a family history. OBJECTIVES: The objective of this study was to explore the possibility that susceptibility in these cases results from low penetrance alleles of RET, its coreceptors, and ligands. DESIGN: We carried out an association study in 135 sporadic MTC (sMTC) patients and 533 controls from the United Kingdom population. RESULTS AND CONCLUSIONS: We analyzed 33 polymorphisms in all nine genes involved in the glial cell line-derived neurotropic factor receptor-alpha (GFRalpha)-RET complex. This is the first association study in which all genes involved in this complex have been investigated for susceptibility to sMTC. We did not find any association between single nucleotide polymorphisms in the exonic regions of the GFRalpha2, GFRalpha3, GFRalpha4, glial cell line-derived neurotropic factor, neurturin, or persephin genes and risk of developing sMTC. We found a strong association between the disease and specific haplotypes of RET. We not only confirmed the previously described association with G691S and S904S (for heterozygotes: odds ratio, 1.85; range, 1.22-2.82; P = 0.004), but we found a novel protective effect associated with a specific haplotype (odds ratio, 0.39; range, 0.21-0.72; P = 0.005) revealing the existence of different genetic variants in the RET oncogene that either increase or decrease risk of sMTC.

The androgen receptor CAG repeat polymorphism and modification of breast cancer risk in BRCA1 and BRCA2 mutation carriers.

INTRODUCTION: The androgen receptor (AR) gene exon 1 CAG repeat polymorphism encodes a string of 9-32 glutamines. Women with germline BRCA1 mutations who carry at least one AR allele with 28 or more repeats have been reported to have an earlier age at onset of breast cancer. METHODS: A total of 604 living female Australian and British BRCA1 and/or BRCA2 mutation carriers from 376 families were genotyped for the AR CAG repeat polymorphism. The association between AR genotype and disease risk was assessed using Cox regression. AR genotype was analyzed as a dichotomous covariate using cut-points previously reported to be associated with increased risk among BRCA1 mutation carriers, and as a continuous variable considering smaller allele, larger allele and average allele size. RESULTS: There was no evidence that the AR CAG repeat polymorphism modified disease risk in the 376 BRCA1 or 219 BRCA2 mutation carriers screened successfully. The rate ratio associated with possession of at least one allele with 28 or more CAG repeats was 0.74 (95% confidence interval 0.42-1.29; P = 0.3) for BRCA1 carriers, and 1.12 (95% confidence interval 0.55-2.25; P = 0.8) for BRCA2 carriers. CONCLUSION: The AR exon 1 CAG repeat polymorphism does not appear to have an effect on breast cancer risk in BRCA1 or BRCA2 mutation carriers.

Fast and slow voltage sensor movements in HERG potassium channels.

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH(2)-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.

Peripheral mechanisms for the initiation of pain following trigeminal nerve injury.

Injury to a branch of the trigeminal nerve may lead to the development of chronic pain in the affected area. The etiology of this condition is not clear, but there is strong evidence to suggest that spontaneous and mechanically induced neural discharge from the injury site plays a crucial role. In laboratory studies, we have characterized this discharge following injury to the inferior alveolar or lingual nerves and have shown a temporal association with the accumulation of neuropeptides in the damaged axons. Substance P, calcitonin gene-related peptide, and vasoactive intestinal polypeptide were all found to be capable of increasing the discharge when applied systemically, and enkephalin caused a decrease. There were also changes in the expression of specific sodium channels and nitric oxide synthase, both at the injury site and in the trigeminal ganglion. Studies on lingual nerve neuromas taken from patients undergoing nerve repair also revealed accumulation of peptides, as well as inflammatory and structural changes, but the presence of these features did not correlate directly with the reported symptoms. The application of corticosteroids to an experimental injury site decreased the mechanically induced discharge, and the anticonvulsant carbamazepine reduced the spontaneous discharge in some axons. Some of the responses that result from damage to a branch of the trigeminal nerve appear to differ from those that follow damage to other peripheral nerves. These differences will need to be taken into account when developing new therapeutic approaches for the management of injury-induced trigeminal pain.

The heritability of mammographic breast density and circulating sex-hormone levels: two independent breast cancer risk factors.

BACKGROUND: Mammographic breast density and endogenous sex-hormone levels are both strong risk factors for breast cancer. This study investigated whether there is evidence for a shared genetic basis between these risk factors. METHODS: Using data on 1,286 women from 617 families, we estimated the heritabilities of serum estradiol, testosterone, and sex-hormone binding globulin (SHBG) levels and of three measures of breast density (dense area, nondense area, and percentage density). We tested for associations between hormone levels and density measures and estimated the genetic and environmental correlations between pairs of traits using variance and covariance components models and pedigree-based maximum likelihood methods. RESULTS: We found no significant associations between estradiol, testosterone, or SHBG levels and any of the three density measures, after adjusting for body mass index (BMI). The estimated heritabilities were 63%, 66%, and 65% for square root-transformed adjusted percentage density, dense area, and nondense area, respectively, and 40%, 25%, and 58% for log-transformed-adjusted estradiol, testosterone, and SHBG. We found no evidence of a shared genetic basis between any hormone levels and any measure of density, after adjusting for BMI. The negative genetic correlation between dense and nondense areas remained significant even after adjustment for BMI and other covariates (ρ = -0.34; SE = 0.08; P = 0.0005). CONCLUSIONS: Breast density and sex hormones can be considered as independent sets of traits. IMPACT: Breast density and sex hormones can be used as intermediate phenotypes in the search for breast cancer susceptibility loci.

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Bisandrographolide from Andrographis paniculata activates TRPV4 channels.

Many transient receptor potential (TRP) channels are activated or blocked by various compounds found in plants; two prominent examples include the activation of TRPV1 channels by capsaicin and the activation of TRPM8 channels by menthol. We sought to identify additional plant compounds that are active on other types of TRP channels. We screened a library of extracts from 50 Chinese herbal plants using a calcium-imaging assay to find compounds active on TRPV3 and TRPV4 channels. An extract from the plant Andrographis paniculata potently activated TRPV4 channels. The extract was fractionated further, and the active compound was identified as bisandrographolide A (BAA). We used purified compound to characterize the activity of BAA on certain TRPV channel subtypes. Although BAA activated TRPV4 channels with an EC(50) of 790-950 nm, it did not activate or block activation of TRPV1, TRPV2, or TRPV3 channels. BAA activated a large TRPV4-like current in immortalized mouse keratinocytes (308 cells) that have been shown to express TRPV4 protein endogenously. This compound also activated TRPV4 currents in cell-free outside-out patches from HEK293T cells overexpressing TRPV4 cDNA, suggesting that BAA can activate the channel in a membrane-delimited manner. Another related compound, andrographolide, found in abundance in the plant Andrographis was unable to activate or block activation of TRPV4 channels. These experiments show that BAA activates TRPV4 channels, and we discuss the possibility that activation of TRPV4 by BAA could play a role in some of the effects of Andrographis extract described in traditional medicine.

Genetic variants in epigenetic genes and breast cancer risk.

Epigenetic events, resulting changes in gene expression capacity, are important in tumour progression, and variation in genes involved in epigenetic mechanisms might therefore be important in cancer susceptibility. To evaluate this hypothesis, we examined common variants in 12 genes coding for DNA methyltransferases (DNMT), histone acetyltransferases, histone deacetyltransferases, histone methyltrasferases and methyl-CpG binding domain proteins, for association with breast cancer in a large case-control study (N cases = 4474 and N controls = 4580). We identified 63 single nucleotide polymorphisms (SNPs) that efficiently tag all the known common variants in these genes, and are also expected to tag any unknown SNP in each gene. We found some evidence for association for six SNPs: DNMT3b-c31721t [P (2 df) = 0.007], PRDM2-c99243 t [P (2 df) = 0.03] and t105413c [P-recessive = 0.05], EHMT1-g-9441a [P (2df) = 0.05] and g41451t (P-trend = 0.04), and EHMT2-S237S [P (2df) = 0.04]. The most significant result was for DNMT3b-c31721t (P-trend = 0.124 after adjusting for multiple testing). However, there were three other results with P < 0.05. The permutation-based probability of this occurring by chance was 0.335. These significant SNPs were genotyped in 75 human cancer cell lines from different tumour types to assess if there was an association between them and six epigenetic measures. No statistically significant association was found. However, a trend was observed: homozygotes for the rare alleles of the EHMT1, EHMT2 and PRDM2 had a mean value for both trimethylation of K9 and K27 of histone H3 remarkably different to the homozygotes for the common alleles. Thus, these preliminary observations suggest the possible existence of a functional consequence of harbouring these genetic variants in histone methyltransferases, and warrant the design of larger epidemiological and biochemical studies to establish the true meaning of these findings.

The effect of substance P on the spontaneous discharge from injured inferior alveolar nerve fibres in the ferret.

Previous studies in our laboratory have shown that, at a site of inferior alveolar nerve (IAN) injury, there is a close association between the development of spontaneous neural activity and the accumulation of neuropeptides in the damaged axons. As this ectopic activity may contribute to the development of sensory disturbances after injury, we have examined further this relationship by determining the potential role of one neuropeptide, substance P (SP), in the initiation or modulation of the spontaneous discharge. Thirty-six adult ferrets were anaesthetised, the IAN sectioned, and the animals allowed to recover for 3-4 days. In terminal experiments under general anaesthesia, electrophysiological recordings were made from axons in fine filaments dissected from the nerve, central to the injury site. The effect of SP (2 x 10(-12), 10(-6) and 10(-4) M) and SP-antagonist (10(-4) M) applied either close-arterially or topically to the injury site was determined. Of the 101 units studied, 59% were spontaneously active. Close-arterial administration of SP increased the level of spontaneous discharge in a dose-dependent manner, with higher concentrations affecting more units (2 x 10(-12) M, 14%; 10(-6) M, 58%; 10(-4) M, 85%). SP also initiated spontaneous discharge in some previously silent units. Activity in 46% of units also increased in response to the SP-antagonist. None of the units responded to topical application of either SP or SP-antagonist. This study shows that SP can both initiate and modulate the spontaneous discharge from damaged axons, and this mechanism may be a potential therapeutic target in the management of sensory disturbances after nerve injury.

Allelic association of the human homologue of the mouse modifier Ptprj with breast cancer.

Human homologues of mouse cancer modifier genes may play a role in cancer risk and prognosis. A proportion of the familial risk of common cancers may be attributable to variants in such genes, each contributing to a small effect. The protein tyrosine phosphatase receptor type J (PTPRJ) has been recently identified as being the protein encoded by the Scc1 mouse gene (susceptibility to colon cancer-1). In addition, the PTPRJ gene has been shown to be somatically altered in several human cancer types such as colon, lung and breast cancers and to have the characteristics of a tumour-suppressor gene. The purpose of this study was to determine whether common variants in the PTPRJ gene represent low penetrance breast cancer susceptibility alleles. To test this hypothesis, we assessed single nucleotide polymorphisms (SNPs) tagging the common SNPs and haplotypes of the gene in 4512 cases and 4554 controls from the East Anglian population. We observed a difference in the haplotype frequency distributions between cases and controls (P = 0.0023, OR = 0.81 [0.72-0.92]). Thus, carrying a specific PTPRJ haplotype confers a protective effect on the risk of breast cancer. This result establishes the principle that mouse cancer modifier genes are candidates for low penetrance human breast cancer susceptibility genes.