Molecular Genetics And Clinical Research In Liddle Syndrome And PRKAG2 Cardiac Syndrome

Part 1:Genetic screening and genotype-phenotype correlation in Liddle syndromeBackground:Liddle syndrome is an autosomal dominant form of monogenic hypertension,caused by mutations in the gene encoding epithelial sodium channel(ENaC).It is characterized by early-onset hypertension,hypokalemia,suppression of plasma rennin activity and aldosterone secretion,metabolic alkalosis,and a clear-cut response to ENaC blockers but not glucocorticoid receptor antagonist treatment.Liddle syndrome has been considered as a rare-form disorder in the past,and most clinicians has a poor knowledge of it.As a consequence,Liddle's patients can be misdiagnosed with mistreatment,suffering early-onset severe target organ damage and even sudden death.Aims:This study was aimed to screen pathogenic mutations in SCNN1B and SCNN1G in three pedigrees and two sporadic cases with suspicious Liddle syndrome,to clarify the diagnosis and guide treatment.Furthermore,this study aimed at determining the screening target population for Liddle syndrome by systematically reviewing the reported cases.Subjects and methods:Three pedigrees and two sporadic cases with suspicious Liddle syndrome with a total of 46 participants were included in this study.Clinical investigation was conducted,including medical history,blood pressure,serum electrolyte,echocardiogram,plasma renin activity/concentrations,and plasma aldosterone concentrations.At first,Sanger sequencing was used to detect gene mutations in exon 13 of SCNN1B and SCNN1G in the five probands.Once a mutation was detected,family members were then performed with genetic screening.Patients with disease-causing mutations were treated with amiloride.And if one-month follow-up revealed improvement in blood pressure and serum potassium level,the diagnosis of Liddle syndrome was made.Additionally,the reported Liddle's cases confirmed by genetic testing from 1966 to March 2018 were collected by searching for Medline database,and the reported mutations were checked in HGMD.A database was made with the abstracted information about gene mutations and clinical presentations of the probands.Statistical analysis was performed using SPSSversion 19.0.Results:In the first pedigree,the proband(?-9)had a clear history of hypertension.His mother(?-6)was diagnosed with hypertension at 20,and died of hemorrhagic stroke at 43,while his grandpa(?-1)also died of hypertensive stroke.And his eldest sister presented with hypertension and hypokalemia.A known mutation causing Liddle syndrome in SCNN1B,c.1853C>A(p.P618H),was detected in five members in this family,including the proband and his eldest sister.In the second pedigree,the proband(?-16),his son(?-10)and his elder sister(?-15)presented with early-onset hypertension(<20 years),and the former two had hypokalemia.The proband's father(?-11),with a 19-year history of hypertension,died of hypertensive stroke at 39.Besides,?-4,?-7,?-4 and ?-8 had early-onset hypertension as well.A novel frameshift mutation,c.1806dupG(p.P603Afs*5),in SCNN1B was identified in a total of 8 members in this family.The proband(?-5)in the third pedigree was detected with a reported mutation causing Liddle syndrome,c.1696C>T(p.R566*).While this mutation was absent in her father who had normal blood pressure.This mutation could not be identified a de novo one,because other family members,including the proband's mother,were not available in this study.Two sporadic cases(S1,S2)had no history of hypertension.In SCNN1B,a novel frameshift mutation,c.1854dupC(p.N619Qfs*3),was identified in one,and a known disease-causing mutation,c.1853C>T(p.P618L),was identified in the other.No mutations were detected in their parents.All hypertensive patients were given oral administration of amiloride,and one-month follow-up revealed great improvement in blood pressure and serum potassium level.Then the diagnosis of Liddle syndrome was established.Through Medline databasesearch and HGMD database check,a total of 67 probands with Liddle syndrome were included in analysis.All probands had hypertension with early penetrance(? 30 years),while 93%of the probands had hypokalemia and 95%of the probands had suppressed plasma renin activity/concentrations.Conclusions:Five disease-causing mutations were identified in SCNN1B in suspicious Liddle's cases,two of which were novel mutations reported for the first time.It not only expanded the mutation spectrum of Liddle syndrome,but clarified the diagnosis and guide treatment as well.Genetic testing was the "golden standard" of the diagnosis of Liddle syndrome.Genetic screening for Liddle syndrome should be considered in hypertensive patients with early penetrance(? 30 years),after exclusion of common secondary causes of hypertension.Part 2:Identification of a novel gene mutation associated with PRKAG2 cardiac syndrome by high-throughput sequencing and analysis of PRKAG2 cardiac syndrome phenotypeBackground:Hypertrophic cardiomyopathy(HCM)is a myocardial disorder characterized by cardiac hypertrophy that is not solely explained by abnormal loading conditions.Besides mutations in cardiac sarcomere protein genes,other genetic and non-genetic conditions,such as congenital metabolic disorders,neuromuscular diseases,mitochondrial diseases,and systemic amyloidosis,can cause HCM as well.PRKAG2 cardiac syndrome is a rare disorder in an autosomal dominant fashion,with the characteristic of cardiac hypertrophy,Wolff-Parkinson-White syndrome(or ventricular pre-excitation),and progressive conduction system abnormalities.Nearly half of patients with PRKAG2 cardiac syndrome received pacemaker implantation,and sudden cardiac death(SCD)and heart failure were not uncommon.The mutation in PRKAG2 gene which encodes AMP-activated protein kinase(AMPK),is the main contributor to this disorder.To date,21 mutations have been reported to be related to PRKAG2 cardiac syndrome.Although cardiac magnetic resonance imaging(CMR)has been widely used in the diagnosis and prognostic evaluation of HCM,only a few cases with PRKAG2 cardiac syndrome were reported with CMR findings.Aims:This study was aimed to screen pathogenic mutations in a three-generation family with cardiac hypertrophy and ventricular pre-excitation,determining the genetic defect.Moreover,CMR features were analyzed in several patients in this pedigree,evaluating the clinical value of CMR in PRKAG2 cardiac syndrome.Subjects and methods:Eleven members from a three-generation pedigree with cardiac hypertrophy and ventricular pre-excitation were included in this study.Clinical investigation was conducted,including medical history,physical examination,electrocardiogram(ECG),echocardiogram,chest X-ray,and 24h-holter ECG and CMR(performed in several members).Target region sequencing was performed in the proband,screening disease-causing mutations in 30 pathogenic genes related to HCM.After excluding the filtered variant,whole exome sequencing was initiated in three patients and one normal control in the pedigree.Sanger sequencing was used to validate cosegregation of the candidate variant with the disease in all affected family members.Three hundred unrelated healthy control samples were also subjected to variant examination.In vivo cDNA splicing assay was performed to examine the effect of the candidate variant on normal mRNA splicing.The cardiac structure and function of 5 affected members were evaluated with CMR images.Results:The pedigree investigation showed that SCD occurred in a 24-year old affected member with HCM,and another deceased member had a history of HCM,atrial fibrillation,stroke and pacemaker implantation.L.eft ventricular thickness was found in 5 members,while ventricular pre-excitation was observed in 4 members and conduction system abnormalities were recorded in 2 members.A young female had a hypertension history.A variant filtered from target region sequencing was excluded due to lack of segregation.A novel candidate variant,c.1006G>T(p.V336L),was detected in PRKAG2 by whole exome sequencing.It was validated to segregate in affected members of the family by Sanger sequencing,and to be absent in 300 unrelated healthy control samples.Abnormal mRNA splicing was not detected by cDNA sequencing.According to ACMG guideline,it was a likely disease-causing mutation of the investigated family.And in terms of clinical features and the genetic defect,the preliminary diagnosis of PRKAG2 cardiac syndrome was made.Through CMR assessment of 5 affected members,it was found that left ventricular wall was widely thickened(? 10 segments)in a concentric pattern.In 2 affected members with enlarged left ventricle and the one with SCD,late gadolinium enhancement(LGE)was observed widely distributed in the mid-distal hypertrophic regions,exhibiting global subendocardial pattern,as well as transmural pattern in the apical portion.And CMR images from the affected member with SCD also showed LGE between endocardium and epicardium.Conclusions:A novel likely disease-causing variant related to PRKAG2 cardiac syndrome was identified.Functional study is needed to validate its pathogenicity in the future.Molecularscreening for PRKAG2 mutations should be considered in patients with cardiac hypertrophy and ventricular pre-excitation to confirm or exclude the diagnosis of PRKAG2 cardiac syndrome.LGE might correlate to the risk of adverse events in patients with PRKAG2 cardiac syndrome.CMR may offer potential clinical value in the diagnosis and prognostic evaluation of PRKAG2 cardiac syndrome.