Boczek, Nicole J.

Dysferlinopathy is an uncommon, progressive muscular dystrophy that has a wide phenotypic variability and primarily supportive management (Nguyen et al., 2007; Narayanaswami et al., 2014). Amyloid myopathy is a distinct, rare disorder that can present similarly to inflammatory myopathies and requires a high clinical suspicion for early intervention to prolong survival. Amyloid myopathy is typically associated with other systemic manifestations of amyloidosis, but rare cases of isolated amyloid myopathy have been described (Mandl et al., 2000; Hull et al., 2001). Positive Congo red stains on tissue biopsy remain the gold standard for diagnosis (Spuler et al., 1998; Karacostas et al., 2005). A high clinical suspicion and meticulous diagnostic workup that includes novel techniques are necessary for identifying these rare disorders. We report a middle-aged man with progressive leg muscle weakness who was initially treated as having amyloid myopathy but was later diagnosed as having dysferlinopathy by Whole Exome Sequencing (WES) analysis. We also report a novel missense mutation (c.959G>C) to help correlate in any patient with presumed dysferlinopathy and to add to the already known genotype of this disorder.

Background— A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects. Methods and Results— Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in Ca V 1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current. Conclusions— Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype.

Rationale : Semaphorin 3A ( SEMA3A )-encoded semaphorin is a chemorepellent that disrupts neural patterning in the nervous and cardiac systems. In addition, SEMA3A has an amino acid motif that is analogous to hanatoxin, an inhibitor of voltage-gated K + channels. SEMA3A -knockout mice exhibit an abnormal ECG pattern and are prone to ventricular arrhythmias and sudden cardiac death. Objective : Our aim was to determine whether SEMA3A is a naturally occurring protein inhibitor of K v 4.3 (I to ) channels and its potential contribution to Brugada syndrome. Methods and Results : K v 4.3, Na v 1.5, Ca v 1.2, or K v 4.2 were coexpressed or perfused with SEMA3A in HEK293 cells, and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered K v 4.3 by significantly reducing peak current density without perturbing K v 4.3 cell surface protein expression. SEMA3A also reduced I to current density in cardiomyocytes derived from human-induced pluripotent stem cells. Disruption of a putative toxin binding domain on K v 4.3 was used to assess physical interactions between SEMA3A and K v 4.3. These findings in combination with coimmunoprecipitations of SEMA3A and K v 4.3 revealed a potential direct binding interaction between these proteins. Comprehensive mutational analysis of SEMA3A was performed on 198 unrelated SCN5A genotype–negative patients with Brugada syndrome, and 2 rare SEMA3A missense mutations were identified. The SEMA3A mutations disrupted SEMA3A’s ability to inhibit K v 4.3 channels, resulting in a significant gain of K v 4.3 current compared with wild-type SEMA3A. Conclusions : This study is the first to demonstrate SEMA3A as a naturally occurring protein that selectively inhibits K v 4.3 and SEMA3A as a possible Brugada syndrome susceptibility gene through a K v 4.3 gain-of-function mechanism.

The presence of 2 distinct populations of somatic or germline cells within a single individual harboring different genotypes is termed mosaicism. Recent reports suggest that parental mosaicism is involved in the heritability of type 1 Timothy syndrome (TS1), an extremely rare and life-threatening multisystem disorder characterized by severe QT interval prolongation, syndactyly, and several other complications. Although full TS1 is caused by a single missense mutation in the CACNA1C-encoded cardiac calcium channel, mosaic TS1 parents can display isolated syndactyly without additional phenotypic manifestations. A newborn boy presented with syndactyly at birth. The presence of syndactyly in his mother led to a diagnosis of benign familial syndactyly. However, at 9 months of age, during his first syndactyly-corrective surgery, intraoperative electrocardiograms revealed extreme QT prolongation and 2:1 atrioventricular block. A comprehensive cardiac evaluation was performed, and both mother and child were tested genetically, confirming a clinical suspicion of TS1. Only the patient tested positive for the TS1 mutation; however, more extensive molecular testing revealed a limited presence of the mutation in maternally-derived DNA. This case illustrates the potential of parental mosaicism to confound the diagnosis of potentially life-threatening genetic diseases, such as TS1. Here, a mother with a partial TS1 phenotype and genetically confirmed mosaicism transmitted the TS1-causative mutation to her son, resulting in fully expressive TS1. Thus, a shared partial phenotype should not be dismissed as a benign or insignificant finding, but should be evaluated further to rule out the possibility of parental mosaicism concealing a potentially fatal heritable disease.

Background— Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and Results— We combined whole-exome sequencing and bioinformatic/systems biology to identify the pathogenic substrate responsible for nonsyndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree, and we established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with “genotype-negative/phenotype-positive” LQTS. Whole-exome sequencing was used on 3 members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, cosegregated with the disease within the pedigree, was ranked by 3 disease-network algorithms as the most probable LQTS-susceptibility gene and involves a conserved residue localizing to the proline, gltamic acid, serine, and threonine (PEST) domain in the II-III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain of function with increased I Ca,L and increased surface membrane expression of the channel compared to wild type. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Ca v 1.2’s PEST domain. Conclusions— This study provides evidence that coupling whole-exome sequencing and bioinformatic/systems biology is an effective strategy for the identification of potential disease-causing genes/mutations. The identification of a functional CACNA1C mutation cosegregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.

AbstractThe stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases such as addiction and mood and anxiety disorders.

Abstract Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish ( Danio rerio ) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360°. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.