Voltage-gated Sodium Channel Gene Research Articles

Rapid evolution of a voltage-gated sodium channel gene in a lineage of electric fish leads to a persistent sodium current.

Most weakly electric fish navigate and communicate by sensing electric signals generated by their muscle-derived electric organs. Adults of one lineage (Apteronotidae), which discharge their electric organs in excess of 1 kHz, instead have an electric organ derived from the axons of specialized spinal neurons (electromotorneurons [EMNs]). EMNs fire spontaneously and are the fastest-firing neurons known. This biophysically extreme phenotype depends upon a persistent sodium current, the molecular underpinnings of which remain unknown. We show that a skeletal muscle–specific sodium channel gene duplicated in this lineage and, within approximately 2 million years, began expressing in the spinal cord, a novel site of expression for this isoform. Concurrently, amino acid replacements that cause a persistent sodium current accumulated in the regions of the channel underlying inactivation. Therefore, a novel adaptation allowing extreme neuronal firing arose from the duplication, change in expression, and rapid sequence evolution of a muscle-expressing sodium channel gene.

Effect of Relaxation of Deltamethrin Pressure on Metabolic Resistance in a Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) Strain Harboring Fixed P989P and G1016G kdr Alleles.

Mutation of the voltage-gated sodium channel genes or knockdown resistance (kdr) and metabolic resistance in Aedes aegypti (L.) (Diptera: Culicidae) are important resistance mechanisms against pyrethroids. The present study investigated the effect of relaxation of deltamethrin selection pressure on the level of mixed-function oxidases (MFO), when the allele frequency of S989P+V1016G mutations is fixed in a resistant Ae. aegypti strain (UPK-R) from Chiang Mai, Thailand. The mosquitoes were divided into two groups, exposure and nonexposure groups, and maintained for 12 generations in an insectary room. Adults of the exposure group (F3 to F12) were treated with 0.05% deltamethrin-impregnated papers. The median lethal concentrations (LC50) of deltamethrin of larvae were determined by World Health Organization (WHO) bioassay. MFO activity was determined in F0 and F12. The results revealed that there was a decreasing trend of adult mortality rates in the exposure group over time. The larval LC50 values of the exposure group were gradually increased, whereas those of the nonexposure group were gradually decreased. The level of MFO activity in the nonexposure group (F12) was lower than the parent and exposure groups (F12) by 1.5 and 4-fold in the larvae, respectively, and 1.5 and 2.5-fold in the adult females, respectively. However, the frequency of P989+G1016 alleles in both groups was 100% up to F12 when the experiment ended. This study indicates that there was a significant but small reduction in the activity levels of MFOs when pyrethroid selection pressure is relaxed in this kdr strain of Ae. aegypti.

Distribution and Frequency of Pyrethroid Resistance-Associated Mutations in Host Lineages of the Bed Bug (Hemiptera: Cimicidae) Across Europe.

For over two decades, the bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has been undergoing a dramatic global resurgence, likely in part to the evolution of mechanisms conferring resistance to insecticides. One such mechanism is knock-down resistance (kdr), resulting from nonsynonymous mutations within the voltage-gated sodium channel (VGSC) gene. To date, three mutations have been identified in C. lectularius, V419L, L925I, and I936F. Using Sanger sequencing, the frequency and distribution of these VGSC mutations across 131 populations collected from the bat-associated and human-associated lineages of C. lectularius found in Europe are documented. All populations from the bat-associated lineage lacked mutations at the three sites. In contrast, the majority of populations associated with humans (93.5%) possessed the mutation at the L925I site. The I936F mutation, previously only reported in Israel and Australia, was found in nine populations spread across several European countries, including the Czech Republic and Switzerland. The high frequency of kdr-associated resistance already reported in C. lectularius and the occurrence and broad geographic distribution of this additional VGSC mutation, questions the continued use of pyrethroids in the treatment of infestations.

Short Communication: Genotyping of kdr allele in insecticide resistant-Aedes aegypti populations from West Sumatra, Indonesia

Hasmiwati, Supargiyono. 2018. Short Communication: Genotyping of kdr allele in insecticide resistant-Aedes aegypti populations from West Sumatra, Indonesia. Biodiversitas 19: 552-558. Aedes aegypti, the primary vector of Dengue fever, is widely distributed in Indonesia including in West Sumatra. Chemical insecticide is an effective way of shedding the chain of Dengue fever transmission. Long-term applications of insecticides have resulted in the development of resistance in Ae. aegypti populations. Knockdown resistance (kdr) allele as a result of point mutations has been found in Voltage-Gated Sodium Channel (VGSC) gene. This study aimed to design allele-specific primers to detect the kdr allele mutations. Ae aegypti samples were collected from several dengue endemic areas in West Sumatra. The susceptibility of adult mosquitos to insecticides was determined by bioassays using impregnation paper test with 0.18% malathion and 0.75% permethrin. In this study, we successfully confirmed that the A. populations have point mutations in the VGSC gene corresponding to S989P and V1016G amino acid substitutions. To genotype S989P and V1016G alleles, we designed kdr allele-specific primers based on the sequence of VGSC gene (NCBI Accession number No. EU399179.1. PCR analysis using the kdr allele-specific primers successfully identified the genotype of Ae. aegypti populations resistant to malathion and permethrin in West Sumatra.

Evidence of multiple mechanisms of alphacypermethrin and deltamethrin resistance in ticks Rhipicephalus microplus in Benin, West Africa

Ticks are obligate haematophagous arthropods, causing heavy losses in affected livestock. The objective of this study is to investigate phenotypic and genotypic resistance in Rhipicephalus microplus populations from Benin. Engorged female adult ticks were collected from cattle in two districts of Benin. Bioassays, biochemical and molecular tests were carried out on these ticks to determine the phenotypic, enzymatic and genetic status of resistance. Results of bioassays showed high resistance factors (RF > 41). The molecular tests showing the presence of the domain II mutation and absence of the domain III mutation in the voltage-gated sodium channel gene. Biochemical tests showed increased activity of esterases, multifunction oxidases and glutathione transferases in resistant samples. Genotyping the samples showed high levels of heterozygous genotypes (73.36% and 63.30%) as compared to homozygous susceptible and resistant genotypes (23.3% and 10%) respective at Samiondji and Betecoucou. A correlation between phenotype resistance and presence of the domain II mutation at the voltage gated sodium channel gene was observed suggesting that this could be associated with resistance. Target site mutation and metabolic detoxification are mechanisms of resistance to pyrethroids in R. microplus tick populations from Benin.

Candidate-gene based GWAS identifies reproducible DNA markers for metabolic pyrethroid resistance from standing genetic variation in East African Anopheles gambiae

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype.

Polymorphisms in voltage-gated sodium channel gene and susceptibility of Aedes albopictus to insecticides in three districts of northern West Bengal, India.

BackgroundThe control and prevention of dengue largely depends on vector control measures, environmental management, and personal protection. Dengue control programmes are facing great challenges due to development of insecticide resistance among vector mosquitoes. Information on susceptibility status to different insecticides is important for national programmes to formulate vector control strategies.MethodsWe have studied the larval susceptibility of Aedes albopictus to temephos and adult susceptibility to 4% DDT, 0.05% deltamethrin, and 5% malathion as per WHO protocols in the northern districts of West Bengal. Polymorphisms in the VGSC gene were studied by direct sequencing of PCR products.ResultsThe Ae. albopictus larval population showed sensitive [Resistance Ratio (RR99)<3] to moderate levels of resistance (5<RR99>10) to temephos at different study sites. Adult bioassay results revealed that Ae. albopictus was highly resistant to DDT [Corrected Mortality (CM) < 90%] in all the study sites and susceptible to deltamethrin and malathion (CM > 98%), except in Dhupguri where a low level of resistance to deltamethrin (CM = 96.25%) was recorded. None of the six important kdr mutations (S953P, I975M/V, L978, V980G, F1474C, D1703Y) were found in the VGSC of studied mosquitoes, but we identified 11 synonymous and 1 non-synonymous mutation in the VGSC gene.ConclusionThe higher susceptibility level to deltamethrin and malathion, along with the absence of important kdr mutations indicates that these two insecticides are still effective against Ae. albopictus in the study areas. The susceptibility status of temephos should be monitored closely as low to moderate levels of resistance were observed in few sites. A similar study is recommended for monitoring and early detection of insecticide resistance in other parts of the country.

Impact of pest management practices on the frequency of insecticide resistance alleles in Bemisia tabaci (Hemiptera: Aleyrodidae) populations in three countries of West Africa

In West Africa, the use of organophospates and pyrethroid insecticides to control cotton pests has led to the evolution of resistance in field populations of Bemisia tabaci Gennadius. Three pest management programs have been commonly recommended: the Conventional Program (CP) where 6 treatments are applied, the use of Bt cotton plants for which only 2 applications of neonicotinoids are required and that has been adopted in many countries, and a biological program (BP) without any chemical treatment. The present study aimed to determine the influence of these practices on the frequency of mutations that confer resistance to pyrethroids (mutation L925I in the para-type voltage-gated sodium channel gene) and organophosphates (mutation F331W in the acetylcholinesterase enzyme ace1: allele Ace1R) in B. tabaci populations using Bt cotton and CP areas in Pô and Saria (Burkina Faso), CP and BP areas in Kandi (Benin) and only CP areas in Tové and Infa (Togo). All individuals sampled belonged to the MED (biotypes MED-Q1) and Africa Silver Leafing (ASL) species. MED-Q1 was found in sympatry with ASL in Burkina Faso both on CP and Bt cotton areas at variable frequencies. In Togo and Benin, only ASL was found, except in Tové where MED-Q1 was also detected, but at low frequency. Frequencies of mutations that confer resistance varied between localities and species but we did not find any strong evidence of a relationship between the pest management program and these frequencies except for the allele Ace1R in Burkina Faso for which the frequencies decrease when chemical applications are reduced. This study provides valuable information for the development of efficient integrated pest management programs.

Genotyping acaricide resistance profiles of Rhipicephalus microplus tick populations from communal land areas of Zimbabwe

Acaricide resistance is one of the greatest threats towards the successful control of vector ticks worldwide. Communal farmers of Zimbabwe use amitraz as the most common acaricide with occasional usage of pyrethroids and organophosphates. As a strategy towards developing an effective acaricide resistance management system in Zimbabwe, screening was done by genotyping Rhipicephalus microplus tick populations using molecular markers associated with resistance to these chemicals. The frequency of the mutant allele for the octopamine/tyramine receptor marker associated with amitraz resistance was high (0.55) and a large proportion 78.5% (288/367) of heterozygote genotypes were observed indicating balancing selection. Of the communal dipping tanks where R. microplus occurred 37.8% (39/103) showed complete resistance genotypes for amitraz. The carboxylesterase marker that has been associated with resistance to organophosphate and pyrethroids indicated no selection pressure in these chemical groups with a low frequency (0.052) of the mutant allele and 89.6% (329/367) sampled ticks showing homozygous susceptibility genotypes. Heterozygous genotypes were present at 27.2% (28/103) of the dipping tanks. The L641 mutation in the voltage–gated sodium channel gene associated with pyrethroid resistance was not detected in Zimbabwean samples. This would suggest a different mechanism of resistance to pyrethroids in these tick populations. Sequence analysis of the octopamine/tyramine receptor gene revealed the presence of other mutations in this region, it will be important to investigate their association with amitraz resistance. These results present the first molecular genotyping of resistance profiles of R. microplus tick populations from Zimbabwe.

A multiplex\xa0PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti

BackgroundMutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale.MethodsA multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR).ResultsThe results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory.ConclusionsOur multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Pyrethroid target site resistance in Greek populations of the honey bee parasite Varroa destructor (Acari: Varroidae)

The mite Varroa destructor is the most serious pest of honey bees worldwide and its control is mainly based on the use of synthetic acaricides, which, in many cases, has led to the development of resistance. In Greece, evidence of resistance to the pyrethroid tau-fluvalinate goes back to the early 1990s. In the present study we examined the presence of resistance mutations at the voltage gated sodium channel (VGSC) gene, encoding the target of pyrethroids. The frequency of resistance mutations was examined by genotyping 113 V. destructor individuals from seven localities in Greece The nucleotide sequencing of the IIS4-IIS5 region of the VGSC gene revealed two amino acid replacements at position 925: a leucine to valine substitution (L925 V) and a leucine to isoleucine substitution (L925I). This is the first report of L925I mutation in European populations of V. destructor that has so far been identified only in USA. No mutations at two other “hot spot” resistance positions, 918 and 929 of the VGSC gene encompassing position 925 were identified. Resistant alleles were predominant (53%) and most of them (83%) corresponded to the L925I substitution. Genotyping results showed the presence of homozygous wild type 925L (44.2%) individuals as well as homozygous resistant 925I (39.8%) and 925 V (7.1%), while heterozygous individuals (925I/L, 925I/V, 925 V/L) were present at low frequency (5.3, 2.7 and 0.9 respectively). Our results clearly show the presence of pyrethroid resistant mutations in high frequency in Greek Varroa populations.

Voltage-gated sodium channel gene repertoire of lampreys: gene duplications, tissue-specific expression and discovery of a long-lost gene.

Studies of the voltage-gated sodium (Nav) channels of extant gnathostomes have made it possible to deduce that ancestral gnathostomes possessed four voltage-gated sodium channel genes derived from a single ancestral chordate gene following two rounds of genome duplication early in vertebrates. We investigated the Nav gene family in two species of lampreys (the Japanese lamprey Lethenteron japonicum and sea lamprey Petromyzon marinus) (jawless vertebrates-agnatha) and compared them with those of basal vertebrates to better understand the origin of Nav genes in vertebrates. We noted six Nav genes in both lamprey species, but orthology with gnathostome (jawed vertebrate) channels was inconclusive. Surprisingly, the Nav2 gene, ubiquitously found in invertebrates and believed to have been lost in vertebrates, is present in lampreys, elephant shark (Callorhinchus milii) and coelacanth (Latimeria chalumnae). Despite repeated duplication of the Nav1 family in vertebrates, Nav2 is only in single copy in those vertebrates in which it is retained, and was independently lost in ray-finned fishes and tetrapods. Of the other five Nav channel genes, most were expressed in brain, one in brain and heart, and one exclusively in skeletal muscle. Invertebrates do not express Nav channel genes in muscle. Thus, early in the vertebrate lineage Nav channels began to diversify and different genes began to express in heart and muscle.

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Investigating knockdown resistance (kdr) mechanism against pyrethroids/DDT in the malaria vector Anopheles funestus across Africa

BackgroundUnderstanding the molecular basis of insecticide resistance is key to improve the surveillance and monitoring of malaria vector populations under control. In the major malaria vector Anopheles funestus, little is currently known about the role of the knockdown resistance (kdr) mechanism. Here, we investigated the presence and contribution of knockdown resistance (kdr) to pyrethroids/DDT resistance observed in Anopheles funestus across Africa.ResultsPyrosequencing genotyping and sequencing of the voltage gated sodium channel (VGSC) gene did not detect the common L1014F mutation in field collected An. funestus across Africa. Amplification and cloning of the full-length of the sodium channel gene in pyrethroid resistant mosquitoes revealed evidences of alternative splicing events with three transcripts of 2092, 2061 and 2117 amino acids (93% average similarity to An. gambiae). Several amino acid changes were detected close to the domain II of the protein such as L928R, F938 W, I939S, L802S and T1008 M. However, all these mutations are found at low frequency and their role in pyrethroid resistance could not be established. The presence of the exclusive alternative splicing at exon 19 was not associated with resistance phenotype. Analysis of patterns of genetic diversity of the VGSC gene revealed a high polymorphism level of this gene across Africa with no evidence of directional selection suggesting a limited role for knockdown resistance in pyrethroid resistance in An. funestus. Patterns of genetic differentiation correlate with previous observations of the existence of barriers to gene flow Africa-wide with southern population significantly differentiated from other regions.ConclusionDespite an apparent limited role of knockdown resistance in An. funestus, it is necessary to continue to monitor the contribution of the mutations detected here as increasing selection from insecticide-based interventions may change the dynamic in field populations as previously observed in other vectors.

Status of insecticide resistance in Anopheles mosquitoes in Ubon Ratchathani province, Northeastern Thailand

BackgroundMalaria is common in hilly, forested areas along national borders in Southeast Asia. Insecticide resistance in malaria vectors has been detected in a few countries in the Greater Mekong sub-region (GMS), representing a threat to malaria control and prevention. This study aims to determine the insecticide resistance status of Anopheles mosquitoes in Ubon Ratchathani province, northeastern Thailand, where increasing number of malaria cases were reported recently.MethodsMosquitoes were collected in 2013–2015 using human landing and cattle bait collections in six sites during both the rainy and dry seasons. Mosquitoes were first morphologically identified to species and their susceptibility status to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) investigated, according to WHO guidelines. Bioassays with the synergists PBO and DEF were carried out to address the role of detoxifying enzymes in insecticide resistance. DNA sequencing of a fragment of the voltage-gated sodium channel gene was carried out to detect knock-down resistance (kdr) substitutions at position 1014 in resistant species.ResultsDue to low vector abundance, complete bioassays (n ≥ 100 mosquitoes) were only achieved for Anopheles hyrcanus s.l., which was resistant to all insecticides tested (mortality ranged from 45 to 87%). Suspected resistance to DDT was found in Anopheles barbirostris s.l. (mortality 69%), but it was susceptible to deltamethrin (mortality 97–100%) and permethrin (mortality 100%). Although insufficient number of primary vectors were collected, results showed that Anopheles dirus s.l. and Anopheles maculatus s.l. were susceptible to deltamethrin (mortality 100%). Anopheles nivipes and Anopheles philippinensis were susceptible to all three insecticides. PBO significantly increased mortality to deltamethrin and permethrin in pyrethroid-resistant An. hyrcanus s.l. None of the sequenced specimens presented the L1014F or L1014S mutation.DiscussionThis study shows that insecticide resistance is present in potential malaria vectors in northeastern Thailand. The absence of kdr mutations in all Anopheles species tested suggests that metabolic resistance is the main mechanism of pyrethroid resistance. This study provides new findings about insecticide susceptibility status of potential malaria vectors in northeastern Thailand that are deemed important to guide malaria vector control.

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

Rare variants of small effect size in neuronal excitability genes influence clinical outcome in Japanese cases of SCN1A truncation-positive Dravet syndrome

Dravet syndrome (DS) is a rare, devastating form of childhood epilepsy that is often associated with mutations in the voltage-gated sodium channel gene, SCN1A. There is considerable variability in expressivity within families, as well as among individuals carrying the same primary mutation, suggesting that clinical outcome is modulated by variants at other genes. To identify modifier gene variants that contribute to clinical outcome, we sequenced the exomes of 22 individuals at both ends of a phenotype distribution (i.e., mild and severe cognitive condition). We controlled for variation associated with different mutation types by limiting inclusion to individuals with a de novo truncation mutation resulting in SCN1A haploinsufficiency. We performed tests aimed at identifying 1) single common variants that are enriched in either phenotypic group, 2) sets of common or rare variants aggregated in and around genes associated with clinical outcome, and 3) rare variants in 237 candidate genes associated with neuronal excitability. While our power to identify enrichment of a common variant in either phenotypic group is limited as a result of the rarity of mild phenotypes in individuals with SCN1A truncation variants, our top candidates did not map to functional regions of genes, or in genes that are known to be associated with neurological pathways. In contrast, we found a statistically-significant excess of rare variants predicted to be damaging and of small effect size in genes associated with neuronal excitability in severely affected individuals. A KCNQ2 variant previously associated with benign neonatal seizures is present in 3 of 12 individuals in the severe category. To compare our results with the healthy population, we performed a similar analysis on whole exome sequencing data from 70 Japanese individuals in the 1000 genomes project. Interestingly, the frequency of rare damaging variants in the same set of neuronal excitability genes in healthy individuals is nearly as high as in severely affected individuals. Rather than a single common gene/variant modifying clinical outcome in SCN1A-related epilepsies, our results point to the cumulative effect of rare variants with little to no measurable phenotypic effect (i.e., typical genetic background) unless present in combination with a disease-causing truncation mutation in SCN1A.

Knockdown resistance (kdr) of the voltage-gated sodium channel gene of Aedes aegypti population in Denpasar, Bali, Indonesia

BackgroundAedes aegypti is the main vector of several arthropod-borne viral infections in the tropics profoundly affecting humans, such as dengue fever (DF), West Nile (WN), chikungunya and more recently Zika. Eradication of Aedes still largely depends on insecticides, which is the most cost-effective strategy, and often inefficient due to resistance development in exposed Aedes populations. We here conducted a study of Ae. aegypti resistance towards several insecticides regularly used in the city of Denpasar, Bali, Indonesia.MethodsAedes aegypti egg samples were collected with ovitraps and thereafter hatched in the insectary of the Gadjah Mada University. The F0 generation was used for all bioassay-related experiments and knockdown resistance (kdr) assays.ResultsResults clearly showed resistance development of Ae. aegypti against tested insecticides. Mortalities of Ae. aegypti were less than 90% with highest resistance observed against 0.75% permethrin. Mosquitoes from the southern parts of Denpasar presented high level of resistance pattern in comparison to those from the western and northern parts of Denpasar. Kdr analysis of voltage-gated sodium channel (Vgsc) gene showed significant association to S989P and V1016G mutations linked to resistance phenotypes against 0.75% permethrin. Conversely, Ae. aegypti F1534C gene mutation did not result in any significant correlation to resistance development.ConclusionsPeriodically surveillance of insecticide resistances in Ae. aegypti mosquitoes will help local public health authorities to set better goals and allow proper evaluation of on-going mosquito control strategies. Initial detection of insecticide resistance will contribute to conduct proper actions in delaying mosquito resistance development such as insecticide rotation or combination of compounds in order to prolong chemical efficacy in combating Ae. aegypti vectors in Indonesia.

Classification of Involuntary Movements in Dogs: Myoclonus and Myotonia

Myoclonus is a sudden brief, involuntary muscle jerk. Of all the movement disorders, myoclonus is the most difficult to encapsulate into any simple framework. On the one hand, a classification system is required that is clinically useful to aid in guiding diagnosis and treatment. On the other hand, there is need for a system that organizes current knowledge regarding biological mechanisms to guide scientific research. These 2 needs are distinct, making it challenging to develop a robust classification system suitable for all purposes. We attempt to classify myoclonus as “epileptic” and “nonepileptic” based on its association with epileptic seizures. Myotonia in people may be divided into 2 clinically and molecularly defined forms: (1) nondystrophic myotonias and (2) myotonic dystrophies. The former are a group of skeletal muscle channelopathies characterized by delayed skeletal muscle relaxation. Many distinct clinical phenotypes are recognized in people, the majority relating to mutations in skeletal muscle voltage‐gated chloride (CLCN1) and sodium channel (SCN4A) genes. In dogs, myotonia is associated with mutations in CLCN1. The myotonic dystrophies are considered a multisystem clinical syndrome in people encompassing 2 clinically and molecularly defined forms designated myotonic dystrophy types 1 and 2. No mutation has been linked to veterinary muscular dystrophies. We detail veterinary examples of myotonia and attempt classification according to guidelines used in humans. This more precise categorization of myoclonus and myotonia aims to promote the search for molecular markers contributing to the phenotypic spectrum of disease. Our work aimed to assist recognition for these 2 enigmatic conditions.

Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome.

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced byelevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2aQ54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a+/- Dravet phenotype. Scn1a+/- mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a+/- epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.