Late-onset progressive retinal atrophy in the Gordon and Irish Setter breeds is associated with a frameshift mutation in C2orf71.

Progressive retinal atrophy (PRA) in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 14 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s) responsible have yet to be identified. Using genome-wide association with 16 Gordon Setter PRA cases and 22 controls, we identified a novel PRA locus, termed rod-cone degeneration 4 (rcd4), on CFA17 (Praw  = 2.22 × 10(-8) , Pgenome  = 2.00 × 10(-5) ), where a 3.2-Mb region was homozygous within cases. A frameshift mutation was identified in C2orf71, a gene located within this region. This variant was homozygous in 19 of 21 PRA cases and was at a frequency of approximately 0.37 in the Gordon Setter population. Approximately 10% of cases in our study (2 of 21) are not associated with this C2orf71 mutation, indicating that PRA in this breed is genetically heterogeneous and caused by at least two mutations. This variant is also present in a number of Irish Setter dogs with PRA and has an estimated allele frequency of 0.26 in the breed. The function of C2orf71 remains unknown, but it is important for retinal development and function and has previously been associated with autosomal recessive retinitis pigmentosa in humans.

Defective slow inactivation of sodium channels contributes to familial periodic paralysis.

OBJECTIVE: To evaluate the effects of missense mutations within the skeletal muscle sodium (Na) channel on slow inactivation (SI) in periodic paralysis and related myotonic disorders. BACKGROUND: Na channel mutations in hyperkalemic periodic paralysis and the nondystrophic myotonias interfere with the normally rapid inactivation of muscle Na currents following an action potential. This defect causes persistent inward Na currents that produce muscle depolarization, myotonia, or onset of weakness. Distinct from fast inactivation is the process called SI, which limits availability of Na channels on a time scale of seconds to minutes, thereby influencing muscle excitability. METHODS: Human Na channel cDNAs containing mutations associated with paralytic and nonparalytic phenotypes were transiently expressed in human embryonic kidney cells for whole-cell Na current recording. Extent of SI over a range of conditioning voltages (-120 to +20 mV) was defined as the fraction of Na current that failed to recover within 20 ms at - 100 mV. The time course of entry to SI at -30 mV was measured using a conditioning pulse duration of 20 ms to 60 seconds. Recovery from SI at -100 mV was assessed over 20 ms to 10 seconds. RESULTS: The two most common hyperkalemic periodic paralysis (HyperPP) mutations responsible for episodic attacks of weakness or paralysis, T704M and M1592V, showed clearly impaired SI, as we and others have observed previously for the rat homologs of these mutations. In addition, a new paralysis-associated mutant, I693T, with cold-induced weakness, exhibited a comparable defect in SI. However, SI remained intact for both the HyperPP/paramyotonia congenita (PMC) mutant, A1156T, and the nonparalytic potassium-aggravated myotonia (PAM) mutant, V1589M. CONCLUSIONS: SI is defective in a subset of mutant Na channels associated with episodic weakness (HyperPP or PMC) but remains intact for mutants studied so far that cause myotonia without weakness (PAM).

A proposed mutation, Val781Ile, associated with hyperkalemic periodic paralysis and cardiac dysrhythmia is a benign polymorphism.

Twenty different point mutations have been identified in the gene coding for the alpha subunit of the adult skeletal muscle sodium channel in families with hyperkalemic periodic paralysis, paramyotonia congenita, and the potassium-aggravated myotonias. One novel mutation (Val(781)Ile) was reported in an adopted boy with potassium-sensitive weakness and cardiac dysrhythmia. The confidence in establishing this rare amino acid substitution as a causative mutation was limited by the absence of family members for segregation analysis. Functional expression studies herein show that Val(781)Ile is most likely a benign polymorphism and not a disease-associated mutation.

Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis.

Several heritable forms of myotonia and hyperkalemic periodic paralysis (HyperPP) are caused by missense mutations in the alpha subunit of the skeletal muscle Na channel (SkM1). These mutations impair fast inactivation or shift activation toward hyperpolarized potentials, inducing persistent Na currents that may cause muscle depolarization, myotonia, and onset of weakness. It has been proposed that the aberrant Na current and resulting weakness will be sustained only if Na channel slow inactivation is also impaired. We therefore measured slow inactivation for wild-type and five mutant Na channels constructed in the rat skeletal muscle isoform (rSkM1) and expressed in HEK cells. Two common HyperPP mutations (T698M in domain II-S5 and M1585V in IV-S6) had defective slow inactivation. This defect reduced use-dependent inhibition of Na currents elicited during 50-Hz stimulation. A rare HyperPP mutation (M1353V in IV-S1) and mutations within the domain III-IV linker that cause myotonia (G1299E) or myotonia plus weakness (T1306M) did not impair slow inactivation. We also observed that slow inactivation of wild-type rSkM1 was incomplete; therefore it is possible that stable membrane depolarization and subsequent muscle weakness may be caused solely by defects in fast inactivation or activation. Model simulations showed that abnormal slow inactivation, although not required for expression of a paralytic phenotype, may accentuate muscle membrane depolarization, paralysis, and sensitivity to hyperkalemia.

Three novel mutations and two variants in the gene for Cu/Zn superoxide dismutase in familial amyotrophic lateral sclerosis.

Autosomal dominant inheritance is exhibited by about 10% of cases of amyotrophic lateral sclerosis (ALS), a paralytic disorder characterized by the death of motor neurons in the brain and spinal cord. A subgroup of these familial cases are linked to mutations in the gene which codes for Cu/Zn superoxide dismutase (SOD1). We report three additional mutations occurring in the SOD1 gene in ALS patients and two single base pair variant changes. The single base pair change in an ALS family causes a glycine 93 to valine substitution, which is the fifth distinct amino acid change reported for the glycine 93 residue. One missense mutation in exon 5 would substitute neutral valine for the negatively-charged aspartate 124 (aspartate 124 to valine). An individual with an apparently sporadic case of ALS carries a three base pair deletion in exon 5 of the SOD1 gene. These three mutations bring to 38 the total number of distinct SOD1 mutations associated with familial ALS.

Inactivation defects caused by myotonia-associated mutations in the sodium channel III-IV linker.

Missense mutations in the skeletal muscle Na+ channel alpha subunit occur in several heritable forms of myotonia and periodic paralysis. Distinct phenotypes arise from mutations at two sites within the III-IV cytoplasmic loop: myotonia without weakness due to substitutions at glycine 1306, and myotonia plus weakness caused by a mutation at threonine 1313. Heterologous expression in HEK cells showed that substitutions at either site disrupted inactivation, as reflected by slower inactivation rates, shifts in steady-state inactivation, and larger persistent Na+ currents. For T1313M, however, the changes were an order of magnitude larger than any of three substitutions at G1306, and recovery from inactivation was hastened as well. Model simulations demonstrate that these functional difference have distinct phenotypic consequences. In particular, a large persistent Na+ current predisposes to paralysis due to depolarization-induced block of action potential generation.

Adhesion of Aeromonas sp. to cell lines used as models for intestinal adhesion.

Adhesion to HEp-2 cells has been shown to correlate with enteropathogenicity for Aeromonas species. Such adhesion is thought to reflect the ability of strains to adhere to human intestinal enterocytes, although HEp-2 cells are not of intestinal origin. In this study strains of Aeromonas veronii biotype sobria isolated from various sources were investigated in parallel assays for their ability to adhere to HEp-2 cells and to an intestinal cell line (Caco-2). Quantitative assays showed identical adhesion values were obtained with both cell lines. Adhesion was best when bacteria were grown at 22 degrees C compared with 37 degrees C and 7 degrees C. Some environmental isolates showed greater adhesion when grown at 7 degrees C than when grown at 37 degrees C. Filamentous structures on these strains are also optimally expressed under the above conditions (reported elsewhere). Mechanical shearing or trypsin treatment to remove surface structures from several adhesive strains grown at 22 degrees C decreased adhesion to cell lines by 50-80% providing further indirect evidence that filamentous adhesins may play a role in cell adhesion for this Aeromonas species.

Sodium channel inactivation is impaired in equine hyperkalemic periodic paralysis.

1. Equine hyperkalemic periodic paralysis (E-HPP) is a dominantly inherited disorder of muscle that causes recurrent episodes of stiffness (myotonia) and weakness in association with elevated serum K+. Affected horses carry a mutant allele of the skeletal muscle isoform of the Na channel alpha-subunit. To understand how this mutation may cause the disease phenotype, the functional defect in Na channel behavior was defined physiologically by recording unitary currents from cell-attached patches on normal and affected equine myotubes. 2. The presence of the mutation was confirmed in our cell line by restriction digest of polymerase chain reaction (PCR)-amplified genomic DNA. Myotubes from the affected horse were heterozygous for the point mutation that codes for a Phe to Leu substitution in S3 of domain IV. This assay provides a rapid technique to screen for the mutation in horses at risk. 3. The primary physiological defect in mutant Na channels was an impairment of inactivation. This defect was manifest as bursts of persistent activity during which the channel closed and reopened throughout a maintained depolarization. Disrupted inactivation slowed the decay of the ensemble-averaged current and produced an eightfold increase in the steady-state open probability measured at the end of a 40-ms pulse. This point mutation identifies a new region of the alpha subunit that is important for rapid inactivation of the channel. 4. The persistent Na current was produced by a distinct mode of gating. Failure of a mutant channel to inactivate was infrequent and occurred in groups of consecutive trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic examination of factors influencing the expression of filamentous surface structures on clinical and environmental isolates of Aeromonas veronii Biotype sobria.

Strains of Aeromonas veronii biotype sobria isolated from clinical and environmental sources were examined for their expression of surface structures under a variety of culture conditions. When grown on solid media at 37 degrees C, more than 95% of bacteria from the majority of strains isolated from human diarrheal feces and chicken carcasses were non-piliated or expressed only a few pili of long, flexible morphology per cell. Strains isolated from water or other foods were much more likely to express pili. Heavily piliated strains (all sources) possessed pili of several morphological types, including long, flexible pili of varying widths and rigid pili of varying lengths. Expression of Pili was favored by growth at temperatures ca. 20 degrees C and below and growth in liquid medium. Most fecal strains expressed some pili under these conditions. In addition, other surface structures (fibrillar aggregates, fibrillar networks bundle-forming pili) were seen on some strains from most sources. These were also seen most frequently when bacteria were grown in liquid media at temperatures ca. 20 degrees C and below. Pili expression was not dramatically influenced by growth under anaerobic conditions, or in iron-depleted media, or by combinations of the above conditions. The role of the above surface structures in Aeromonas pathogenicity remains to be elucidated.

Distribution of Aeromonas hydrophila hybridization groups and their virulence properties in Australasian clinical and environmental strains.

A total of 182 Aeromonas hydrophila strains isolated from environmental (food and water) and clinical (stool and other sources) samples taken in mainland Australia, Tasmania and New Zealand were assigned to one of three DNA/DNA hybridization groups (HGs) on the basis of biochemical characteristics, and tested with regard to their ability to produce virulence factors. Strains from HG2 were rarely isolated; strains from HG1 were most commonly isolated from clinical sources; and strains from HG3 formed the majority of environmental strains. There was no correlation of HG to geographic source. Strains from HG2 infrequently produced virulence factors. Strains from HG1 were more likely to produce virulence factors if they came from a clinical source. Overall, strains from mainland Australia produced virulence factors more frequently than those from Tasmania or New Zealand. Strains from HG1 may be of more clinical significance than strains from the other two HGs.

The growth and expression of virulence factors at refrigeration temperature by Aeromonas strains isolated from foods.

A potentially significant subset (10%, 6/61) of Aeromonas strains isolated from food (milk, lamb, chicken, seafood), all A. veronii biotype sobria, were able to produce two or more exotoxins (haemolysin, enterotoxin, and cytotoxin) at 37 degrees C, and grow well at 43 degrees C. Although mesophilic organisms, they grew at 5 degrees C. In addition, they could adhere to HEp-2 cells when grown at 37 degrees C, or at 5 degrees C, and expressed flexible pili (possible colonization factors) in greater numbers at the lower temperature. These strains, as well as other exotoxin-producing strains (A. veronii biotype sobria and A. hydrophila) (33%, 20/61) lacking adhesive ability, were able to produce cytotoxins in broth cultures over a seven to 10-day period at 5 degrees C. One strain in particular, an A. hydrophila isolated from goats' milk, grew rapidly at low temperature. This psychrotrophic strain produced all three exotoxins within 3 days in broth cultures at 5 degrees C. The properties of the above strains suggest they could be of public health significance in food products that have an extended shelf-life at refrigeration temperature.

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression.

Specific DNA fragments complementary to the 3' untranslated regions of the beta-, alpha-cardiac, and alpha-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the alpha-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the alpha-cardiac actin gene probe. Since virtually all of the bipolar cells express alpha-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, alpha-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the alpha-cardiac actin gene. Accumulation of alpha-skeletal but not alpha-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of alpha-cardiac and then alpha-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the beta-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different alpha-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle beta-actin gene.

Sequential expression of chicken actin genes during myogenesis.

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.