Aural CT characteristics of American Cocker Spaniels have features of both mesaticephalic and brachycephalic breeds

Otitis externa and otitis media are common in American Cocker Spaniels, however breed-specific aural CT descriptions are currently lacking. This prospective and retrospective, observational, analytical study aimed to describe quantitative CT characteristics of the horizontal ear canal and the tympanic bulla in American Cocker Spaniels versus similar-sized mesaticephalic dogs. We prospectively performed clinical examinations and aural CT scanning for 38 American Cocker Spaniels. Computed tomographic transverse area of the osseous horizontal ear canal (TA1), transverse area of the widest air-filled part of the cartilaginous horizontal ear canal (TA2), tympanic bulla volume, and tympanic bulla wall thickness were measured. The TA1 and TA2 measurements were compared with those of 23 retrospectively recruited, similar-sized mesaticephalic dogs that had undergone CT-scanning of the head for non-ear-related reasons. The TA1 and TA2 did not differ for healthy American Cocker Spaniels and mesaticephalic dogs. Severely affected American Cocker Spaniels had significantly smaller TA2 (P <.001). The intraclass correlation coefficient for intraobserver and interobserver repeatability was 0.972 and 0.983 for TA1 and 0.994 and 0.998 for TA2, respectively. Variation between individuals was subjectively noted in healthy and affected American Cocker Spaniels, but mean tympanic bulla volume was slightly smaller in relation to body weight, and the tympanic bulla wall was thicker than in previous reports for mesaticephalic dogs. The tympanic bulla wall appeared thicker rostro-ventrally than caudo-ventrally in 44% of the dogs. Our results imply that a relatively thick tympanic bulla wall may be a normal CT variation and should be interpreted cautiously in this breed.Peer reviewe

Ageing Contributes to Phenotype Transition in a Mouse Model of Periodic Paralysis

Background: Periodic paralysis (PP) is a rare genetic disorder in which ion channel mutation causes episodic paralysis in association with hyper- or hypokalaemia. An unexplained but consistent feature of PP is that a phenotype transition occurs around the age of 40, in which the severity of potassium-induced muscle weakness declines but onset of fixed, progressive weakness is reported. This phenotype transition coincides with the age at which muscle mass and optimal motor function start to decline in healthy individuals. We sought to determine if the phenotype transition in PP is linked to the normal ageing phenotype transition and to explore the mechanisms involved. Methods: A mouse model of hyperkalaemic PP was compared with wild-type littermates across a range of ages (13–104 weeks). Only male mice were used as penetrance is incomplete in females. We adapted the muscle velocity recovery cycle technique from humans to examine murine muscle excitability in vivo. We then examined changes in potassium-induced weakness or caffeine contracture force with age using ex vivo muscle tension testing. Muscles were further characterized by either Western blot, histology or energy charge measurement. For normally distributed data, a student's t-test (± Welch correction) or one- or two-way analysis of variance (ANOVA) was performed to determine significance. For data that were not normally distributed, Welch rank test, Mann Whitney U test or Kruskal–Wallis ANOVA was performed. When an ANOVA was significant (P < 0.05), post hoc Tukey testing was used. Results: Both WT (P = 0.009) and PP (P = 0.007) muscles exhibit increased resistance to potassium-induced weakness with age. Our data suggest that healthy-old muscle develops mechanisms to maintain force despite sarcolemmal depolarization and sodium channel inactivation. In contrast, reduced caffeine contracture force (P = 0.00005), skeletal muscle energy charge (P = 0.004) and structural core pathology (P = 0.005) were specific to Draggen muscle, indicating that they are caused, or at least accelerated by, chronic genetic ion channel dysfunction. Conclusions: The phenotype transition with age is replicated in a mouse model of PP. Intrinsic muscle ageing protects against potassium-induced weakness in HyperPP mice. However, it also appears to accelerate impairment of sarcoplasmic reticulum calcium release, mitochondrial impairment and the development of core-like regions, suggesting acquired RyR1 dysfunction as the potential aetiology. This work provides a first description of mechanisms involved in phenotype transition with age in PP. It also demonstrates how studying phenotype transition with age in monogenic disease can yield novel insights into both disease physiology and the ageing process itself

Excitability Properties of Mouse and Human Skeletal Muscle Fibres Compared by Muscle Velocity Recovery Cycles.

Mouse models of skeletal muscle channelopathies are not phenocopies of human disease. In some cases (e.g. Myotonia Congenita) the phenotype is much more severe, whilst in others (e.g. Hypokalaemic periodic paralysis) rodent physiology is protective. This suggests a species' difference in muscle excitability properties. In humans these can be measured indirectly by the post-impulse changes in conduction velocity, using Muscle Velocity Recovery Cycles (MVRCs). We performed MVRCs in mice and compared their muscle excitability properties with humans. Mouse Tibialis Anterior MVRCs (n = 70) have only one phase of supernormality (increased conduction velocity), which is smaller in magnitude (p = 9 × 10-21), and shorter in duration (p = 3 × 10-24) than human (n = 26). This abbreviated supernormality is followed by a period of late subnormality (reduced velocity) in mice, which overlaps in time with the late supernormality seen in human MVRCs. The period of late subnormality suggests increased t-tubule Na+/K+-pump activity. The subnormal phase in mice was converted to supernormality by blocking ClC-1 chloride channels, suggesting relatively higher chloride conductance in skeletal muscle. Our findings help explain discrepancies in phenotype between mice and humans with skeletal muscle channelopathies and potentially other neuromuscular disorders. MVRCs are a valuable new tool to compare in vivo muscle membrane properties between species and will allow further dissection of the molecular mechanisms regulating muscle excitability

Exome Sequencing Reveals a Phenotype Modifying Variant inZNF528in Primary Osteoporosis With aCOL1A2Deletion

We studied a family with severe primary osteoporosis carrying a heterozygous p.Arg8Phefs*14 deletion in COL1A2, leading to haploinsufficiency. Three affected individuals carried the mutation and presented nearly identical spinal fractures but lacked other typical features of either osteogenesis imperfecta or Ehlers-Danlos syndrome. Although mutations leading to haploinsufficiency in COL1A2 are rare, mutations in COL1A1 that lead to less protein typically result in a milder phenotype. We hypothesized that other genetic factors may contribute to the severe phenotype in this family. We performed whole-exome sequencing in five family members and identified in all three affected individuals a rare nonsense variant (c.1282C > T/p.Arg428*, rs150257846) in ZNF528. We studied the effect of the variant using qPCR and Western blot and its subcellular localization with immunofluorescence. Our results indicate production of a truncated ZNF528 protein that locates in the cell nucleus as per the wild-type protein. ChIP and RNA sequencing analyses on ZNF528 and ZNF528-c.1282C > T indicated that ZNF528 binding sites are linked to pathways and genes regulating bone morphology. Compared with the wild type, ZNF528-c.1282C > T showed a global shift in genomic binding profile and pathway enrichment, possibly contributing to the pathophysiology of primary osteoporosis. We identified five putative target genes for ZNF528 and showed that the expression of these genes is altered in patient cells. In conclusion, the variant leads to expression of truncated ZNF528 and a global change of its genomic occupancy, which in turn may lead to altered expression of target genes. ZNF528 is a novel candidate gene for bone disorders and may function as a transcriptional regulator in pathways affecting bone morphology and contribute to the phenotype of primary osteoporosis in this family together with the COL1A2 deletion. (c) 2020 The Authors.Journal of Bone and Mineral Researchpublished by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).Peer reviewe

Clinical and genetic spectrum of a Chinese cohort with SCN4A gene mutations

Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. However, each phenotype has been reported in few cases of Chinese origin. We present detailed phenotype and genotype data from a cohort of 40 cases with SCN4A gene mutations seen in neuromuscular diagnostic service in Huashan hospital, Fudan University. Cases were referred from 6 independent provinces from 2010 to 2018. A questionnaire covering demographics, precipitating factors, episodes of paralysis and myotonia was designed to collect the clinical information. Electrodiagnostic studies and muscle MRI were retrospectively analyzed. The clinical spectrum of patients included: 6 Hyperkalemic periodic paralysis (15%), 18 Hypokalemic periodic paralysis (45%), 7 sodium channel myotonia (17.5%), 4 paramyotonia congenita (10%) and 5 heterozygous asymptomatic mutation carriers (12.5%). Review of clinical information highlights a significant delay to diagnosis (median 15 years), reports of pain and myalgia in the majority of patients, male predominance, circadian rhythm and common precipitating factors. Electrodiagnostic studies revealed subclinical myotonic discharges and a positive long exercise test in asymptomatic carriers. Muscle MRI identified edema and fatty infiltration in gastrocnemius and soleus. A total of 13 reported and 2 novel SCN4A mutations were identified with most variants distributed in the transmembrane helix S4 to S6, with a hotspot mutation p.Arg675Gln accounting for 32.5% (13/40) of the cohort. Our study revealed a higher proportion of periodic paralysis in SCN4A-mutated patients compared with cohorts from England and the Netherlands. It also highlights the importance of electrodiagnostic studies in diagnosis and segregation studies

Exome sequencing of Finnish isolates enhances rare-variant association power.

Exome-sequencing studies have generally been underpowered to identify deleterious alleles with a large effect on complex traits as such alleles are mostly rare. Because the population of northern and eastern Finland has expanded considerably and in isolation following a series of bottlenecks, individuals of these populations have numerous deleterious alleles at a relatively high frequency. Here, using exome sequencing of nearly 20,000 individuals from these regions, we investigate the role of rare coding variants in clinically relevant quantitative cardiometabolic traits. Exome-wide association studies for 64 quantitative traits identified 26 newly associated deleterious alleles. Of these 26 alleles, 19 are either unique to or more than 20 times more frequent in Finnish individuals than in other Europeans and show geographical clustering comparable to Mendelian disease mutations that are characteristic of the Finnish population. We estimate that sequencing studies of populations without this unique history would require hundreds of thousands to millions of participants to achieve comparable association power

Dysfunction of NaV1.4, a skeletal muscle voltage-gated sodium channel, in sudden infant death syndrome: a case-control study.

BACKGROUND: Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant death in high-income countries. Central respiratory system dysfunction seems to contribute to these deaths. Excitation that drives contraction of skeletal respiratory muscles is controlled by the sodium channel NaV1.4, which is encoded by the gene SCN4A. Variants in NaV1.4 that directly alter skeletal muscle excitability can cause myotonia, periodic paralysis, congenital myopathy, and myasthenic syndrome. SCN4A variants have also been found in infants with life-threatening apnoea and laryngospasm. We therefore hypothesised that rare, functionally disruptive SCN4A variants might be over-represented in infants who died from SIDS. METHODS: We did a case-control study, including two consecutive cohorts that included 278 SIDS cases of European ancestry and 729 ethnically matched controls without a history of cardiovascular, respiratory, or neurological disease. We compared the frequency of rare variants in SCN4A between groups (minor allele frequency <0·00005 in the Exome Aggregation Consortium). We assessed biophysical characterisation of the variant channels using a heterologous expression system. FINDINGS: Four (1·4%) of the 278 infants in the SIDS cohort had a rare functionally disruptive SCN4A variant compared with none (0%) of 729 ethnically matched controls (p=0·0057). INTERPRETATION: Rare SCN4A variants that directly alter NaV1.4 function occur in infants who had died from SIDS. These variants are predicted to significantly alter muscle membrane excitability and compromise respiratory and laryngeal function. These findings indicate that dysfunction of muscle sodium channels is a potentially modifiable risk factor in a subset of infant sudden deaths. FUNDING: UK Medical Research Council, the Wellcome Trust, National Institute for Health Research, the British Heart Foundation, Biotronik, Cardiac Risk in the Young, Higher Education Funding Council for England, Dravet Syndrome UK, the Epilepsy Society, the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, and the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis

Mutations in the skeletal muscle channel (SCN4A), encoding the Nav1.4 voltage-gated sodium channel, are causative of a variety of muscle channelopathies, including non-dystrophic myotonias and periodic paralysis. The effects of many of these mutations on channel function have been characterized both in vitro and in vivo. However, little is known about the consequences of SCN4A mutations downstream from their impact on the electrophysiology of the Nav1.4 channel. Here we report the discovery of a novel SCN4A mutation (c.1762A>G; p.I588V) in a patient with myotonia and periodic paralysis, located within the S1 segment of the second domain of the Nav1.4 channel. Using N-ethyl-N-nitrosourea mutagenesis, we generated and characterized a mouse model (named draggen), carrying the equivalent point mutation (c.1744A>G; p.I582V) to that found in the patient with periodic paralysis and myotonia. Draggen mice have myotonia and suffer from intermittent hind-limb immobility attacks. In-depth characterization of draggen mice uncovered novel systemic metabolic abnormalities in Scn4a mouse models and provided novel insights into disease mechanisms. We discovered metabolic alterations leading to lean mice, as well as abnormal AMP-activated protein kinase activation, which were associated with the immobility attacks and may provide a novel potential therapeutic target

Circulating inflammatory cytokines and risk of five cancers: a Mendelian randomization analysis

BACKGROUND: Epidemiological and experimental evidence has linked chronic inflammation to cancer aetiology. It is unclear whether associations for specific inflammatory biomarkers are causal or due to bias. In order to examine whether altered genetically predicted concentration of circulating cytokines are associated with cancer development, we performed a two-sample Mendelian randomisation (MR) analysis. METHODS: Up to 31,112 individuals of European descent were included in genome-wide association study (GWAS) meta-analyses of 47 circulating cytokines. Single nucleotide polymorphisms (SNPs) robustly associated with the cytokines, located in or close to their coding gene (cis), were used as instrumental variables. Inverse-variance weighted MR was used as the primary analysis, and the MR assumptions were evaluated in sensitivity and colocalization analyses and a false discovery rate (FDR) correction for multiple comparisons was applied. Corresponding germline GWAS summary data for five cancer outcomes (breast, endometrial, lung, ovarian, and prostate), and their subtypes were selected from the largest cancer-specific GWASs available (cases ranging from 12,906 for endometrial to 133,384 for breast cancer). RESULTS: There was evidence of inverse associations of macrophage migration inhibitory factor with breast cancer (OR per SD = 0.88, 95% CI 0.83 to 0.94), interleukin-1 receptor antagonist with endometrial cancer (0.86, 0.80 to 0.93), interleukin-18 with lung cancer (0.87, 0.81 to 0.93), and beta-chemokine-RANTES with ovarian cancer (0.70, 0.57 to 0.85) and positive associations of monokine induced by gamma interferon with endometrial cancer (3.73, 1.86 to 7.47) and cutaneous T-cell attracting chemokine with lung cancer (1.51, 1.22 to 1.87). These associations were similar in sensitivity analyses and supported in colocalization analyses. CONCLUSIONS: Our study adds to current knowledge on the role of specific inflammatory biomarker pathways in cancer aetiology. Further validation is needed to assess the potential of these cytokines as pharmacological or lifestyle targets for cancer prevention

Cross-trait analyses with migraine reveal widespread pleiotropy and suggest a vascular component to migraine headache

Background: Nearly a fifth of the world's population suffer from migraine headache, yet risk factors for this disease are poorly characterized. Methods: To further elucidate these factors, we conducted a genetic correlation analysis using cross-trait linkage disequilibrium (LD) score regression between migraine headache and 47 traits from the UK Biobank. We then tested for possible causality between these phenotypes and migraine, using Mendelian randomization. In addition, we attempted replication of our findings in an independent genome-wide association study (GWAS) when available. Results: We report multiple phenotypes with genetic correlation (P < 1.06 × 10-3) with migraine, including heart disease, type 2 diabetes, lipid levels, blood pressure, autoimmune and psychiatric phenotypes. In particular, we find evidence that blood pressure directly contributes to migraine and explains a previously suggested causal relationship between calcium and migraine. Conclusions: This is the largest genetic correlation analysis of migraine headache to date, both in terms of migraine GWAS sample size and the number of phenotypes tested. We find that migraine has a shared genetic basis with a large number of traits, indicating pervasive pleiotropy at migraine-associated loci.Peer reviewe