Genetic alterations of ion channels in cardiac and sensorineural pathologies in Argentina

DIONISIO, LEONARDO

Buenos Aires

Encuentro; Humboldt Colloquium "Shaping the future of German-Argentinian scientific cooperation; 2018

Alexander von Humboldt Foundation

Channelopathies are genetic diseases triggered by alterations in functionality of ion channels. Molecular biology techniques have allowed analyse the functioning of most of the channels involved in channelopathies and identify the causes of several inheritable diseases. In this project we will focus our study in two of them: cardiac and auditory disorders. Cardiovascular diseases are the main cause of death all over the world. Different causes lead to a heart attack. One of them, is the sudden death which consist in a sudden stop of the heart beating in a healthy individual caused by a ventricular fibrillation. People under 40 years old, rarely show ventricular fibrillation and, when that happens, is caused by inheritable disorders. 40% of them have an arrhythmogenic cause, as is the case in Long QT (LQT), Short QT (SQT) and Brugada Syndromes (BS). LQT syndrome is an inheritable arrhythmogenic disease in which the cardiac repolarization phase is extended (evidenced as a prolonged QT interval on electrocardiogram (ECG)) leading to ventricular fibrillation, syncope and sudden death. This syndrome can emerge at any time in life and is responsible for thousands of death in Europe and North America. It can manifest in young individuals, even among children. LQT syndrome is mostly caused by genetic anomalies present in KCNQ1, HERG or Nav1.5 ion channels, responsible for LQT type-1, -2 and -3, respectively. Mutations in KCNQ1, KCNH2 and SCN5A genes are responsible for about 80% of cases of LQT syndrome. These mutations conduct to a prolonged QT interval by increasing depolarization current INa (gain of function, LQT3) or decreasing repolarization current Iks o Ikr (loss of function, LQT1 and LQT2). Due to its high heterogeneity, individuals with LQT syndrome may not show any symptoms even the prolonged QT interval on the ECG. Genotype determine the clinical manifestations. Sudden death risk is higher in patients who have mutations in SCN5A than in KCNQ1 and KCNH2 genes. Clinical manifestations may be triggered by specific factors particular to each LQT type and they have different pharmacological responses. For example, in LQT1, syncope events are produced during sympathetic stimulation, therefore β-blockers drugs must be used. Moreover, these patients are forbidden from doing sport activities particularly swimming. Thus, genetic testing is of great importance in LQT syndrome evaluation. Identification of mutations allow to establish a correct diagnosis, which leads to a personalized drug treatment and a proper evaluation of prognosis and life-style changes in order to avoid arrhythmogenic gating events and drugs. Moreover, genetic tests allow to do genetic counselling to first order relatives of the index-case. On the other hand, deafness is a reduction of the hearing function. It can be caused by alterations in the middle (conductive) or in the inner ear (sensorineural). The inherited ones can be classified as autosomal recessive (75-85%), autosomal dominant (15-25%) or X-linked (1-2%). autosomal dominant nonsyndromic hearing loss (DFNA2) is a sensorineural disease characterized by late starting hearing loss that progress slowly. Mutations in the potassium channel KCNQ4 are the principal causes responsible for DFNA2 disorder. 10 different mutations have been reported that code for mutant malfunctioning channels with a dominant negative effect (dn). Heterozygous patients, carrying the mutant allele, have a very low K+ current in cochlear hair cells (HC). The hearing process requires the fine movement of the potassium ion between the different components of the cochlea. Wave sound causes the opening of ion channels in Inner and Outer Hair Cells (IHC and OHC, respectively) leading the entrance of K+ and depolarizing the cell. To recover the membrane potential, KCNQ4 channels open and extrude K+ toward extracellular space. The loss of function of mutated channels cause a chronic depolarization of HC that conduct to impairment in hearing functioning. The molecular events triggered by chronic depolarization conduct to cell degeneration and OHC death, causing slow progressive hearing loss. In transgenic animal models that carry the same mutation detected in humans, slow and progressive hair cell loss at old ages are observed. Different drugs that activate KCNQ channels, have also been effective in vitro, on opening KCNQ4 channels carrying the DFNA2 related mutations. Thus, they could be effective for the treatment of this disorder. Recently, retigabine, a KCNQ2-5 activator, has been approved for the treatment of epilepsy in humans it has been proposed for treating tinnitus. Therefore, retigabine and other related drugs could be a pharmacological alternative for DFNA2 deafness. LQT syndrome and DFNA2 deafness, both related to KCNQ channel family, are two disorders that are not investigated in our country. Besides, a complete clinical research and genetic diagnosis tests are not also done in Argentina. So, the prevalence of these diseases are unknown in our region. Moreover, this data help physicians and patients to establish rational pharmacological treatments and to obtain diagnostic certainty. Taking all together, the main objective of our project is to identify reported and novel mutations in genes associated to LQT syndrome and DFNA2 deafness; and generate a database that allow categorize the risk for the patient that carry the detected mutation. In addition, we will analyse drugs acting as modulators of channel activity to evaluate its use as a novel drug therapy. To achieve this objective we plan the following activities: 1.1Developing of methods for sequencing the exons of KCNQ1, KCNH2 and SCN5A genes responsible for LQT1, 2 and 3 syndromes, respectively and the exons of KCNQ4 related to DFNA2 deafness. We will isolate genomic DNA from blood cells obtained from healthy individuals. Blood samples were provided by Hospital Municipal de Agudos de Bahía Blanca. Using specific primers flanking each exon we will perform PCR experiments and, by agarose gel electrophoresis, the expected bands will be separated, purified and sent to sequencing. 1.2Determination of single nucleotide polymorphism (SNP) for LQT syndrome in healthy individuals of our community. In order to establish a genetic study in the population of our community, we will evaluate the most expressed allelic variations of the indicated genes. 124 SNPs and more than 400 mutations are reported to generate LQT syndrome, but is still unclear if all of them can produce the disease. In our country, population and genetic studies of pathological variations of the genes associated with LQT syndrome, have not been reported yet, even though about 10% of new born that suffer sudden death could have some of these allelic variations. We will study samples from healthy individuals provided by Hospital Municipal de Agudos de Bahía Blanca according to Ethical Committee. Results obtained from sequencing of each exon will be analysed using the BLAST platform of NIH, by comparing with reported sequences. Detected modifications in the sequence will be recorded and searched in international databases for LQT syndrome. If the mutation has been reported, it will be categorized as pathological, no pathological or with uncertain effect. Thus, we will start to register the population data for the detected variations in order to establish a regional LQT data-base. 1.3Evaluation of reported and novel genetic variations of genes related to LQT in patients with arrhythmia. We will provide genetic studies to patients that were diagnosed only by their ECG alterations or have suffered an episode of arrhythmia. Even in relatives of individuals who have suffered sudden death under 40 years old. As mentioned before, genetic diagnosis allow to stratify the risk to suffer severe arrhythmia. Different affected genes have different risks. In addition, according to the mutated gene, the pharmacological treatment varies. That is why, molecular diagnosis helps to establish a personalized and more effective drug treatment. Moreover, it may prevent the use of some drugs prescribed for others pathologies that can affect the patients who carry a certain mutation. An additional important point is the genetic counselling that could be provided to the first grade relatives of the patient in order to take action to avoid dangerous situations. The Service of Cardiac Electrophysiology of the Hospital Privado del Sur de Bahía Blanca have identified patients with clinical diagnosis of QT syndrome that we are analysing in this moment. Samples will be processed as was indicated before. The detected mutation will be referred to the local and the international data bases to determine if it was reported or not, the associated risk to suffer sudden death and the recommended pharmacological treatment. 1.4Study of novel mutations not reported in bibliography for LQT syndrome. In case that a detected mutation was not reported in LQT databases, we will evaluate it effect of it on the transport to membrane or the functionality of the channel. To do that, we will introduce the mutation by directed mutagenesis into the cDNA of the altered gene. By heterologous expression, HEK293 cells will be transfected and using immunocytochemistry we will evaluate membrane expression. To evaluate functionality of the mutated channel we will perform patch clamp experiments. 1.5Evaluation of KCNQ channels drugs modulators. Activators of KCNQ channels can revert in vitro the effect of the mutations that generate loss of function. That is why this drugs could be a new line of pharmacological treatment for some kinds of hearing loss. We will evaluate the effect in vivo of retigabine in transgenic mice model of DFNA2 deafness. We have 2 transgenic mice lines: Kcnq4-/-, which is knock out for kcnq4 channel, and Kcnq4dn/dn, which carries a point mutation with dominant negative effect observed in human patients. We will administer the drug to the mice at different ages and then the cochlea will be extracted and hair cell death will be evaluated. On the other hand, we will use a model for evaluating modulator drugs of cardiac KNCQ channels to revert the loss of function caused by mutations associated with LQT syndromes. KCNQ1 and HERG channels have their homologous in the fruit fly D. melanogaster. For example, fly sei channel is similar to human HERG channel. Sei is also expressed in neurons and we will introduce a mutation (seits2/ts2) that produce loss of function of the channel similar to the mutation founded in LQT syndrome. This mutation turn neurons overexcited and an increase in temperature produce uncoordinated movements which leads to paralysis in less than a minute. In wild type flies this paralysis takes 4-5 minutes, so we will evaluate if exposed flies to channel activators can revert temperature sensitivity to longer times as wild type flies. This objective will be done with the help of Dr. A. Garelli who works with D. melanogaster in his lab. This project was approved by the National Council of Scientific and Technical Research (CONICET) of Argentina for my application as Researcher of the Scientific Research Career. I started to work in it since February of the current year at the Laboratory of Ion Channels under the supervision of Dr. Guillermo Spitzmaul at the INIBIBB in Bahía Blanca, Argentina. Currently, we have implemented the procurement of KCNQ1 and KCNH2 exons by multiplex PCR. 16 exons of KCNQ1 and 3 exons of KCNH2 were fine-tuned. Last month we received the first samples from patients with clinical suspect of LQT syndrome. In first place, we decided to amplify exons 3, 4, 6, 7 and 8 of KCNQ1 and exons 6, 7 and 8 of KCNH2 because they carry about 75% of the reported mutations. Then we will continue with the remaining exons of both genes. At the moment, we are analysing the obtained sequences from those exons. Recently we received the mutant flies and we started to evaluate the functioning of the mutation in temperature sensitivity experiments.