Nicole J. Boczek

LSINCT5 is over expressed in breast and ovarian cancer and affects cellular proliferation

More than 98% of the human genome is comprised of non-protein coding sequences. Interestingly, a considerable fraction of these sequences is transcribed into non-protein coding RNA transcripts. These transcripts range in size from very small RNAs such as the miRNAs (20–25 base pairs) to transcripts that can range up to 100 kb or more. Some longer non-coding RNAs (lncRNAs) have been found to play important regulatory roles within cells. In this report, we demonstrate that LSINCT5 is a 2.6 Kb polyadenylated, long stress-induced non-coding transcript that is on the negative strand, localized in the nucleus and potentially transcribed by RNA polymerase III. LSINCT5 is overexpressed in breast and ovarian cancer cell lines and tumor tissues, relative to their normal counterpart. In addition, knocking down the expression of LSINCT5 in cancer-derived cell lines causes a decrease in cellular proliferation. Finally, we identified 95 genes with more than 2-fold changes when knocking down LSINCT5 expression by using the Affymetrix U133 Plus 2 array. We chose a subset of these genes to validate using qPCR and found that ten of these genes were indeed significantly affected by the LSINCT5 knockdown. Interestingly, two genes that were significantly downregulated were the lncRNA NEAT-1 and a protein coding gene PSPC1. We have therefore characterized a novel lncRNA that is overexpressed in breast and ovarian cancers, enhances cellular proliferation and may play a significant role in multiple processes.

Exome Sequencing and Systems Biology Converge to Identify Novel Mutations in the L-Type Calcium Channel, CACNA1C, Linked to Autosomal Dominant Long QT Syndrome

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 ICa,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 Cav1.2’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.

CALM3 mutation associated with long QT syndrome

Each year, more than 400,000 cases (>1,000 per day) of sudden cardiac death (SCD) are reported in the United States.1 The majority of these SCD cases and more than 10% of all natural deaths are due to cardiac arrhythmias.2 One condition that affects approximately 1 in 2,000 individuals and is known to cause these potentially lethal arrhythmias is long QT syndrome (LQTS).3 LQTS, a disorder of ventricular myocardial repolarization, is characterized by the prolongation of the heart rate-corrected QT interval (QTc) on an ECG, which can precipitate torsades de pointes (a subtype of polymorphic ventricular tachycardia) and as a result syncope, seizures, or SCD, in the setting of a structurally normal heart.2 LQTS is an inherited disorder, and approximately 80% of LQTS has been elucidated genetically. Accordingly, its genetic basis remains elusive in as many as 20% of patients.4 Here, we describe a case of severe LQTS in a young subject exhibiting profound QT prolongation (QTc > 650 ms) in which commercial genetic testing was negative. Recently, whole exome sequencing (WES) analysis has been used to successfully pinpoint the novel genetic cause of a variety of diseases when the genetic cause is still unknown.4, 5 Due to a negative family history, seemingly healthy parents, and normal parental ECGs, WES was performed on the trio (index case and his parents), and the results were filtered to include either novel sporadic or rare recessive variants. A sporadic p.D130G mutation in CALM3 was identified as the most likely cause of the patient’s LQTS.

Novel Timothy syndrome mutation leading to increase in CACNA1C window current

BACKGROUND Timothy syndrome (TS) is a rare multisystem genetic disorder characterized by a myriad of abnormalities, including QT prolongation, syndactyly, and neurologic symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel; however, some cases remain genetically elusive. OBJECTIVE The purpose of this study was to identify the genetic cause of TS in a patient who did not harbor a CACNA1C mutation in exon 8/A, and was negative for all other plausible genetic substrates. METHODS Diagnostic exome sequencing was used to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole-cell patch-clamp technique, and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. RESULTS Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiologic analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential as well as the development of spontaneous early afterdepolarizations. CONCLUSION Through expanded whole exome sequencing, we identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiologic experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted.

A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long QT Syndrome

Rationale: Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca2+/calmodulin (CaM)-dependent inactivation of L-type Ca2+ channels. Objective: To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS. Methods and Results: We generated and characterized the functional properties of induced pluripotent stem cell–derived cardiomyocytes from a patient with D130G-CALM2–mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived induced pluripotent stem cell–derived cardiomyocytes display (1) significantly prolonged action potentials, (2) disrupted Ca2+ cycling properties, and (3) diminished Ca2+/CaM-dependent inactivation of L-type Ca2+ channels. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only 1 of 6 redundant CaM-encoding alleles, we devised a strategy using CRISPR interference to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in induced pluripotent stem cell–derived cardiomyocytes with D130G-CALM2, as shown by the normalization of action potential duration and Ca2+/CaM-dependent inactivation after treatment. Moreover, CRISPR interference can be designed to achieve selective knockdown of any of the 3 CALM genes, making it a generalizable therapeutic strategy for any calmodulinopathy. Conclusions: Overall, this therapeutic strategy holds great promise for calmodulinopathy patients as it represents a generalizable intervention capable of specifically altering CaM expression and potentially attenuating LQTS-triggered cardiac events, thus initiating a path toward precision medicine.

Identification and Functional Characterization of a Novel CACNA1C-Mediated Cardiac Disorder Characterized by Prolonged QT Intervals with Hypertrophic Cardiomyopathy, Congenital Heart Defects, and Sudden Cardiac Death

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 CaV1.2 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.

Characterization of SEMA3A-Encoded Semaphorin as a Naturally Occurring Kv4.3 Protein Inhibitor and its Contribution to Brugada Syndrome

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 Kv4.3 (Ito) channels and its potential contribution to Brugada syndrome. Methods and Results: Kv4.3, Nav1.5, Cav1.2, or Kv4.2 were coexpressed or perfused with SEMA3A in HEK293 cells, and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered Kv4.3 by significantly reducing peak current density without perturbing Kv4.3 cell surface protein expression. SEMA3A also reduced Ito current density in cardiomyocytes derived from human-induced pluripotent stem cells. Disruption of a putative toxin binding domain on Kv4.3 was used to assess physical interactions between SEMA3A and Kv4.3. These findings in combination with coimmunoprecipitations of SEMA3A and Kv4.3 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 Kv4.3 channels, resulting in a significant gain of Kv4.3 current compared with wild-type SEMA3A. Conclusions: This study is the first to demonstrate SEMA3A as a naturally occurring protein that selectively inhibits Kv4.3 and SEMA3A as a possible Brugada syndrome susceptibility gene through a Kv4.3 gain-of-function mechanism.

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin Variants in Long QT Syndrome and Functional Characterization of a Novel Long QT Syndrome-Associated Calmodulin Missense Variant, E141G.

Background—Calmodulin (CaM) is encoded by 3 genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results—Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P<0.0001). When the clinical sequelae of these 5 CaM-positive cases were compared with the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 10 months, an average corrected QT interval of 676 ms, and a high prevalence of cardiac arrest. Functional characterization of 1 novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+-binding affinity and a functionally dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions—Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin (CaM) Variants in Long QT Syndrome (LQTS) and Functional Characterization of a Novel LQTS-Associated CaM Missense Variant, E141G

Background—Calmodulin (CaM) is encoded by 3 genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results—Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P<0.0001). When the clinical sequelae of these 5 CaM-positive cases were compared with the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 10 months, an average corrected QT interval of 676 ms, and a high prevalence of cardiac arrest. Functional characterization of 1 novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+-binding affinity and a functionally dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions—Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

A CACNA1C Variant Associated with Reduced Voltage-Dependent Inactivation, Increased CaV1.2 Channel Window Current, and Arrhythmogenesis

Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation.