George W. Forsyth

Differential Gene Expression of TRPM1, the Potential Cause of Congenital Stationary Night Blindness and Coat Spotting Patterns (LP) in the Appaloosa Horse (Equus caballus)

The appaloosa coat spotting pattern in horses is caused by a single incomplete dominant gene (LP). Homozygosity for LP (LP/LP) is directly associated with congenital stationary night blindness (CSNB) in Appaloosa horses. LP maps to a 6-cM region on ECA1. We investigated the relative expression of two functional candidate genes located in this LP candidate region (TRPM1 and OCA2), as well as three other linked loci (TJP1, MTMR10, and OTUD7A) by quantitative real-time RT–PCR. No large differences were found for expression levels of TJP1, MTMR10, OTUD7A, and OCA2. However, TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) expression in the retina of homozygous appaloosa horses was 0.05% the level found in non-appaloosa horses (R = 0.0005). This constitutes a >1800-fold change (FC) decrease in TRPM1 gene expression in the retina (FC = −1870.637, P = 0.001) of CSNB-affected (LP/LP) horses. TRPM1 was also downregulated in LP/LP pigmented skin (R = 0.005, FC = −193.963, P = 0.001) and in LP/LP unpigmented skin (R = 0.003, FC = −288.686, P = 0.001) and was downregulated to a lesser extent in LP/lp unpigmented skin (R = 0.027, FC = −36.583, P = 0.001). TRP proteins are thought to have a role in controlling intracellular Ca2+ concentration. Decreased expression of TRPM1 in the eye and the skin may alter bipolar cell signaling as well as melanocyte function, thus causing both CSNB and LP in horses.

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ2=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.

Congenital stationary night blindness is associated with the leopard complex in the miniature horse

OBJECTIVE   To determine if congenital stationary night blindness (CSNB) exists in the Miniature Horse in association with leopard complex spotting patterns (LP), and to investigate if CSNB in the Miniature Horse is associated with three single nucleotide polymorphisms (SNPs) in the region of TRPM1 that are highly associated with CSNB and LP in Appaloosas. ANIMALS STUDIED   Three groups of Miniature Horses were studied based on coat patterns suggestive of LP/LP (n=3), LP/lp (n=4), and lp/lp genotype (n=4). PROCEDURES   Horses were categorized based on phenotype as well as pedigree analysis as LP/LP, LP/lp, and lp/lp. Neurophthalmic examination, slit-lamp biomicroscopy, indirect ophthalmoscopy, and scotopic flash electroretinography were performed on all horses. Hair samples were processed for DNA analysis. Three SNPs identified and associated with LP and CSNB in the Appaloosa were investigated for association with LP and CSNB in these Miniature Horses. RESULTS   All horses in the LP/LP group were affected by CSNB, while none in the LP/lp or lp/lp groups were affected. All three SNPs were completely associated with LP genotype (χ(2) = 22, P << 0.0005) and CSNB status (χ(2) =11, P<0.0005). CONCLUSIONS   The Miniature Horse breed is affected by CSNB and it appears to be associated with LP as in the Appaloosa breed. The SNPs tested could be used as a DNA test for CSNB until the causative mutation is determined.

Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.

Inhibition of ileal brush-border chloride conductance by specific antibody.

SummaryAntibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.

Redundant contribution of a Transient Receptor Potential cation channel Member 1 exon 11 single nucleotide polymorphism to equine congenital stationary night blindness.

BackgroundCongenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts.ResultsRetinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression.ConclusionsThese finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

Relative quantification of white blood cell mitochondrial DNA and assessment of mitochondria by use of transmission electron microscopy in English Springer Spaniels with and without retinal dysplasia.

OBJECTIVE To compare relative amounts of WBC mitochondrial DNA (mtDNA; assessed via real-time PCR assay) and morphology of lymphocyte mitochondria (assessed via transmission electron microscopy [TEM]) in blood samples collected from English Springer Spaniels with and without retinal dysplasia. ANIMALS 7 and 5 client-owned English Springer Spaniels (1 to 11 years old) with and without retinal dysplasia, respectively. PROCEDURES Blood samples were obtained from affected and unaffected dogs via venipuncture. Genomic DNA was extracted from WBCs of the 7 affected and 5 unaffected dogs, and relative quantification of the cytochrome c oxidase subunit 1 gene (COX1) was determined via analysis of real-time PCR results. White blood cells from 3 affected and 4 unaffected dogs were embedded in epoxide resin for TEM; cross sections were examined for lymphocytes, which were measured. The mitochondria within lymphocytes were quantified, and the mitochondrial surface area per lymphocyte cross section was calculated. A masked technique was used to compare mitochondrial morphology between the 2 groups. RESULTS Compared with the smallest measured quantity of mtDNA among unaffected dogs, mtDNA amounts varied among unaffected (1.08- to 4.76-fold differences) and affected dogs (1- to 2.68-fold differences). Analysis of lymphocyte measurements and mitochondrial surface area, morphology, and quantity revealed no significant differences between affected and unaffected dogs. CONCLUSIONS AND CLINICAL RELEVANCE No significant differences were detected in relative amounts of WBC mtDNA or the size, number, or morphology of lymphocyte mitochondria in English Springer Spaniels affected with retinal dysplasia, compared with results for unaffected control dogs.