Genetic Testing Of Congenital Long QT Syndrome And Study On The Correlation Between Genotype And Phenotype

BackgroundCongenital long QT syndrome (LQTS) has been understood as "cardiac channelopathy", caused by abnormal genes encoding ion channels in myocardium. LQTS is expressed as the prolongation of QT interval and abnormal morphology of T-wave. In clinical, it is characterized by recurrent syncope and ventricular arrhythmias, especially torsades de pointes (TdP). In certain circumstances, LQTS even cause sudden cardiac death. About1/2500patients has the risk of sudden death for a life. Children and adolescents have a high incidence of disease. Mortality rate often years is up to50%in untreated patients. In summary, LQTS has the characteristics of sudden onset, high mortality rate and high incidence in young people, with the incidence of1/2000to1/5000approximately in United States. It is estimated that there are about30thousands of LQTS patients in our country. LQTS has become a frontier and hot in genetic arrhythmia research.Since the sentinel discovery of the susceptibility locus on chromosome11p15.5by Keating in1991.There have been at least13genes identified for LQTS, including KCNQ1(LQT1), KCNH2(LQT2), SCN5A(LQT3), ANK2(LQT4), KCNE1(LQT5), KCNE2(LQT6), KCNJ2(LQT7), CACNA1C(LQT8), CAV3(LQT9), SCN4B(LQT10), AKAP9(LQT11), SCNA1(LQT12) and KCNJ5(LQT13). Depending on the different genes, LQTS is divided into13subtypes. Many types can lead to gene mutations, including missense, nonsense, insertion/deletion, frameshift and splice mutations. To date, More than900mutations of LQTS-causing mutations in13LQTS genes have been described all over the world. However, mutations being detected in most of families showed a specific distribution; only a small part of the mutant gene is widespread. Therefore, targeted genetic screening is more feasible. In2011, Heart Rhythm Society/European Heart Rhythm Society (HRS/EHRS) had published State of Genetic Testing for the Channelopathies and Cardiomyopathies. Genetic testing of the LQT1-3(KCNQ1, KCNH2and SCN5A) was particularly recommended. A clear clinical diagnosis or highly suspicious of LQTS patients should be carried by screening of these genes. Over the past decade, researcher in genetic testing for LQTS patients in our country has detected nearly50mutations.At present, the clinical diagnosis of the LQTS patients depends on resting ECG QTc values, syncope and family history of sudden cardiac death. LQTS is characterized by QT interval prolongation in resting electrocardiogram, however, about10-40%of patients and gene carriers have normal QTc. Therefore it is particularly important for targeted genetic screening. For these patients with obvious symptoms, the detection probability of mutation is up to75%. Tester and other researchers have made a series of studies about genotype-phenotype which revealed relatively specific ECG patterns, arrhythmogenic triggers, risk for sudden death, and responses to pharmacotherapy, especially in LQT1to LQT3. Now it is widely recognized that the typical T wave morphology of LQT1is expressed as broad-based pattern. LQT2is characterized by low-amplitude bifid T wave.The T wave of LQT3has the characteristics of late-onset peaked or biphasic T wave. Specific predisposing factors are also associated with genotypes. When patients are doing sports, swimming or doing something sympathetic, LQT1are prone to syncope and sudden death. LQT2and LQT3have fewer symptoms during exercise, with the cardiovascular events often occurred during rest or sleep. It is noteworthy that the auditory stimulus is a specific triggering factor to LQT2, especially awakened from sleep. The correlation of genotype and phenotype is critical to the diagnosis of LQTS. Genetic testing can help to genotyping for risk stratification and specific treatment. In addition, different genotypes respond differently to the epinephrine provocation test. Genotype is closely related to specific drugs treatment. β blockers, as the preferred medication, can significantly reduce the occurrence of syncope and cardiac sudden death in LQT1. In terms of LQT2, β blockers may cause syncope recurrence. LQT3with bradycardia are not sensitive to adrenaline, in addition to the occurrence of cardiac events during the rest or sleep, should be preferred to pacemaker or ICD therapy. Therefore, it is more feasible to screening targeted gene according to the different manifestations of particular genotype of LQTS. LQTS is a kind of hereditary disease, including two genetic types:autosomal dominant Romano-Ward syndrome (RWS) and autosomal recessive Jervell and Lange-Nilsen syndrome (JLNS). The most common genes of RWS are KCNQ1and KCNH2, while JLNS are KCNQ1and KCNE1. According to the guidelines, probands should be carried genetic screening first, then their family members.Clinical datas of the3probands and their family were collected and analyzed in this study, trying to clear the association between particular genotype and phenotype of LQTS. At the same time, genetic screening was carried in probands. When mutations were detected, genetic screening was carried in their family members at the same sites for new mutation.Objective1. Clinical datas of3LQTS probands and their family members(a total of22persons), including disease history, predisposing factors, resting ECG QTc values and T wave morphology, were collected and analyzed. We try to clear the correlation between genotype and phenotype in LQT1and LQT2patients.2. In our study, genetic screening of KCNQ1and KCNE1were carried to a JLNS proband, while genetic screening of KCNQ1and KCNH2were carried to two RWS probands.After the mutation was discovered, family members were detected the same gene, expecting to find LQTS new mutation.Subjects 1. According to the diagnostic criteria of LQTS proposed by Schwartz, diagnosis of>≥4points for LQTS met the inclusion criteria.3probands were confirmed in Nanfang Hospital from December2007to December2011, including1male and2female. All of them were Han from Guangdong Province. Combined with the recommendation of genetic screening for LQTS from HRS/EHRS in2011, the family members including the proband’s children, brothers, sisters and parents, a total of22persons were collected.100of healthy cases were selected as a control study group. The above studies were informed consent.2. All subjects have excluded from vasovagal syncope, orthostatic hypotension, arrhythmogenic right ventricular cardiomyopathy, catecholamine ventricular tachycardia, and etiologies of acquired long QT syndrome, including1. drug-induced QT interval prolongation:antiarrhythmic drugs, antibiotics, antipsychotics, chemotherapy drugs and so on;2. electrolyte disorders and systemic diseases: hypokalemia, hypocalcemia, diabetes, connective tissue disease, renal failure, pheochromocytoma and so on;3. organic heart disease:coronary heart disease, cardiac hypertrophy, heart failure, cardiomyopathy, mitral valve prolapse and so on.Methods1. Clinical data of probands and their family members were collected.12-lead ECG was recorded. According to the classification criteria, specific T wave morphology was recorded and analyzed to genotype, proposed by Zhang.24h Holter or exercise test were performed if necessary. The clinical indicators were consisted of the syncopal attacks, the time of first onset, predisposing factors (motion, agitation or quiet, sleep, etc.), family history of sudden cardiac death, previous treatment. All of the subjects were extracted peripheral blood5ml, anticoagulant by EDTA and saved at-80℃.2. Genomic DNA from all subjects was extracted from peripheral blood leukocytes. All the exons of KCNQ1, KCNE1and KCNH2were amplified by polymerase chain reaction (PCR) and the PCR products were then sequenced directly. The PCR reaction system are as follows:the Platinum Taq reaction system25ul including lul template DNA and24ul of supermix as follows:2.5ul10x PCR buffer, 2u±2.5mM of dNTP mixture,0.75ul50mM MgCl2,0.2ul Platinum Taq DNA polymerase and5uM upstream and downstream primers lul respectivly, the rest of the volume was made up by adding sterilized distilled water. The amplification conditions of the reaction system as follows:predenaturation at94℃for5min, then denaturation at94℃for30s, annealing at58℃for45s, extension at72℃for90s, there were35cycles from denaturation to extension, extension at72℃again for5min after the cycles, and keep the products at4℃forever. The products were identified using a1.5%agarose electrophoresis. The products were sequenced in Invitrogen Biotechnology Co. by3730XL type DNA sequencing instrument. The abnormal gene base which could make amino acid sequence change was detected by comparing in NCBI database, while the same exon was detected in100nomal control people.Results1. The3LQTS probands who diagnosed by Schwartz were consisted of1male and2female, aged from23to46years. The age of onset ranged from15to46years, QTc values from486to582ms. Two cases were suspected patients, scoring from2to4. The remaining family members were≤1point. Family member B2, scoring1, was diagnosed as pathogenic gene carrier by genetic testing. Among all members, five cases suffered from syncope, including four cases caused by emotional stimulation or exercises and one case at asleep. One case had the family history of sudden death. No one had sudden death during the follow-up. According T wave morphology to predict all members’genotypes, of which two cases were LQT1, two cases were LQT2and one case could not be predicted. Proband A and his child had congenital deafness, belonging to JLNS while proband B and C belonging to RWS.(3blocker agent was effective to A1and C1, but less effective to B1.2. DNA sequencing showed a gene mutation of KCNQ1in proband B1. A nucleotide substitution changing from Cytosine (C) to Thymine (T) was found in the exon6, resulting in the830Mino acid codon of Iks a-subunit protein became TTG for Leucine instead of TCG for Serine. The same exon of their family members and100control cases were screened. The missense mutation of S277L was located in transmembrane segment of KCNQ1channel and has not been announced in the NCBI SNP database of KCNQ1. It had been reported that the same variat was found in Chinese LQTS patient.Conclusion1. There has been correlation between the specific genotype (LQT1and LQT2type) and phenotype of Chinese LQTS patients, and basically the same as reports.2. A mutation site in exon6of KCNQ1gene exists in a Chinese LQTS family, resulting in a substitution of Leucine by Serine at codon830(S277L). RWS may be caused by this heterozygous mutation.