Reliable pre-implantation sex determination and genetic screening enables informed embryo transfer decisions in equine breeding while avoiding later interventions. We developed a streamlined workflow that couples rapid whole genome amplification (WGA) with a multiplex real-time PCR targeting ETSTY5 as a Y-specific marker and UBC as an autosomal control. On purified equine DNA, sex was correctly assigned down to 10 pg gDNA and to a single fibroblast cell. Direct testing of embryo biopsies without WGA yielded inconsistent results, whereas introducing a short WGA step produced tight allelic-discrimination clusters and 100% diagnostic calls, including in cloned embryos of known sex. The same WGA product supported targeted genotyping for inherited disease screening of hyperkalemic periodic paralysis (HYPP) and hereditary equine regional dermal asthenia (HERDA) alleles. This WGA plus real-time PCR pipeline supports robust and practical embryo sexing and targeted pre-implantation genetic diagnostics within in vitro produced (IVP) equine embryo and embryo transfer workflows.

Skeletal muscle channelopathies are rare genetic disorders caused by mutations in voltage-gated ion channel genes that regulate sarcomere excitability, including the CLCN1 gene encoding ClC-1, the KCNJ2 gene encoding Kir2.1, the SCN4A gene encoding Nav1.4, and the CACNA1S gene encoding Cav1.1. More than one hundred heterozygous missense mutations have been identified in SCN4A, representing a broad spectrum of clinical phenotypes, including sodium channel myotonia (SCM), paramyotonia congenita (PMC), hyperkalemic periodic paralysis (HyperPP) and hypokalemic periodic paralysis (HypoPP). In addition, recent case reports have shown that compound heterozygous mutations or homozygous mutations in SCN4A are associated with congenital myopathy or congenital myasthenic syndrome. Regarding the pathological mechanisms of SCM/PMC and HyperPP, a large number of electrophysiological analyses have shown an association between the functional alteration of the mutant Nav1.4 and the clinical phenotype. On the other hand, HypoPP has long been a mysterious disorder. In 2007, the recent discovery of aberrant leak currents, called "gating pore currents", brought a breakthrough in the field of HypoPP research and contributed to the elucidation of the structure-function relationship of the voltage sensing domain of voltage-gated ion channels. However, there has been little progress in the discovery of the therapeutics. Recently, we have generated HEK293T-based HypoPP model cell lines aiming to establish the in vitro platform for the high-throughput drug screening. Our HypoPP model cells would provide new insight into the development of novel therapeutics for channelopathies.

Primary periodic paralysis (PPP) comprises inherited ion channelopathies, marked by episodic or sustained muscle weakness. Dichlorphenamide has been investigated as a treatment option, however existing studies report inconsistent and variable findings regarding its efficacy. To clarify these inconsistencies, we conducted a meta-analysis and systematic review evaluating its effectiveness in managing PPP. A comprehensive literature exploration was undertaken across multiple databases encompassing studies disseminated up to July 2025, assessing efficacy of dichlorphenamide in PPP. Primary outcomes encompassed mean changes in weekly attack frequency and severity-weighted scores. Meta-analysis was performed using RevMan, reporting mean differences (MD), statistical significance was ascribed to p-values < 0.05. Evidence certainty was evaluated using the GRADE framework. Clinically significant reductions in the frequency of weekly periodic paralytic attacks were observed in both the hyperkalemic and hypokalemic subgroups (MD -1.72, P-value: 0.01), as well as across the age subgroups, adolescents and adults (MD -0.95, P-value: <0.00001). Analysis of severity-weighted attack scores also revealed significant improvements across both etiological hyperkalemic and hypokalemic categories (MD -1.37, P-value: 0.002), and age-based subgroups (MD -1.28, P-value: <0.00001). Along with therapeutic benefits, some adverse effects were also associated with dichlorphenamide, such as paresthesias (p < 0.00001), cognitive disturbances (p: 0.008), dysgeusia (p: 0.01), and rash (p: 0.006). The GRADE analysis confirmed high-certainty evidence for primary outcomes, supporting dichlorphenamide's consistent efficacy across etiologies and ages, though its side effects necessitate careful monitoring and counseling.

Hyperkalemic periodic paralysis (hyperKPP) is characterized by attacks of transient weakness. A subset of hyperKPP patients suffers from transient involuntary contraction of muscle (myotonia). The goal of this study was to determine mechanisms causing myotonia in hyperKPP. Intracellular electrophysiology, single-fiber Ca2+ imaging, and whole muscle contractility studies were performed in a mouse model of hyperKPP. Myotonia in hyperkPP was caused by both involuntary myogenic action potentials (AP myotonia) lasting less than 5 min and action potential-independent myotonia (non-AP myotonia) lasting over 1 h. Non-AP myotonia was caused by prolonged subthreshold depolarization and elevated intracellular Ca2+ in the absence of action potentials. Treatment with dantrolene effectively mitigated non-AP myotonia, suggesting that the source of Ca2+ was the sarcoplasmic reticulum. Although non-AP myotonia occurred in the absence of action potentials, Na+ channel blockers were effective as therapy. We propose myotonia in hyperKPP occurs via two mechanisms: (1) suprathreshold depolarization triggering action potentials that are detectable with EMG and (2) sustained subthreshold depolarization resulting in Na+ overload and Ca2+ leak from the sarcoplasmic reticulum. Notably, clinical diagnostics such as EMG cannot detect the second mechanism as it occurs in the absence of action potentials. Currently, only a minority of patients with hyperKPP are treated with Na+ channel blockers and none are treated with dantrolene. Our data suggest hyperKPP patients, as well as patients with a number of other neuromuscular disorders, may benefit from trials of these therapies, even if they do not have myotonia detectable clinically or by EMG.

Andersen-Tawil syndrome (ATS) is an ion channelopathy with variable penetrance for the triad of periodic paralysis, arrhythmia, and dysmorphia. Dominant-negative mutations of KCNJ2 encoding the Kir2.1 potassium channel subunit are found in 60% of ATS families. As with most channelopathies, episodic attacks in ATS are frequently triggered by environmental stresses: exercise for periodic paralysis or stress with adrenergic stimulation for arrhythmia. Fluctuations in K+, either low or high, are potent triggers for attacks of weakness in other variants of periodic paralysis (hypokalemic periodic paralysis or hyperkalemic periodic paralysis). For ATS, the [K+] dependence is less clear; with reports describing weakness in high-K+ or low-K+. Patient trials with controlled K+ challenges are not possible, due to arrhythmias. We have developed two mouse models (genetic and pharmacologic) with reduced Kir currents, to address the question of K+-sensitive loss of force. These animal models and computational simulations both show K+-dependent weakness occurs only when Kir current is <30% of wildtype. As the Kir deficit becomes more severe, the phenotype shifts from high-K+-induced weakness to a combination where either high-K+ or low-K+ triggers weakness. A K+ channel agonist, retigabine, protects muscle from K+-sensitive weakness in our mouse models of the skeletal muscle involvement in ATS.