Identification Of Pathogenic Genes And Clinical Research In Monogenic Inherited Hypertension

Part 1:The identification of the novel pathogenic mutations in epithelial sodium channel genes and clinical research in Liddle syndromeBackground:Liddle syndrome(LS)is an autosomal dominant form of monogenic hypertension.LS is caused by pathogenic mutations in epithelial sodium channel(ENaC)genes.It is mainly characterized by salt-sensitive hypertension,hypokalemic metabolic alkalosis,decreased plasma concentration of aldosterone and renin.Early-onset hypertension is common in pediatrics with LS.On account of variable and nonspecific phenotypes of LS,it is susceptible to clinically misdiagnosis and missed diagnosis.Genetic testing provides the"gold standard" for the diagnosis of LS.Treatment is based on the use of ENaC inhibitors.Aims:In this study,we performed a mutational screening of pathogenic genes in two families with LS,and provided guidance on the precision diagnosis and specific treatment of LS patients.In addition,a systematic review was conducted to conclude the clinical genetic features of ENaC-gene-positive LS cases in childhood.Subjects and methods:Clinical data and peripheral blood samples were collected from the subjects in these two families.Genomic DNA was extracted from peripheral blood.Next-generation sequencing and in silico analysis were performed in the probands to discover candidate variants.Sanger sequencing was used to identify the predicted likely pathogenic variant.In order to reveal co-segregation between the genotype and the phenotype,candidate variants were also verified in their respective family members.To exclude common genetic polymorphisms,candidate variants were identified in 100 unrelated hypertension patients and 100 normotensive subjects.Apart from the designed panel,we also solely detected SCNN1A gene for two probands.LS patients were treated with amiloride and followed up one month after treatment.In order to conclude reported phenotypic and genetic features of ENaC-gene-positive LS patients in childhood,papers on LS published in English on Medline and the Human Gene Mutation Database were retrieved before March 2021.Results:Genetic analysis of the proband revealed a novel frameshift mutation(c.1838de1C,p.P613Qfs*63)in SCNN1B gene.This heterozygous mutation generated deletion of the important PY motif and elongation of the ?-ENaC subunit.Other 12 affected family members were also detected the identical mutation.There was no SCNN1A or SCNN1G gene mutation in this family.Amiloride was effective in alleviating LS for patients.A novel SCNN1G gene missense variant(c.1874C>T,p.P625L)was identified by genetic screening in the proband with LS in childhood.In silico analysis demonstrated that this heterozygous variant was highly conserved and deleterious.Further genetic screening revealed three other relatives with LS in this family.All LS patients were treated with amiloride.Follow-up results showed significant improvements in blood pressure and serum potassium levels after one month of tailored therapy.A total of 39 publications reported 54 pediatric LS cases associated with 25 pathogenic variants in SCNN1B and SCNN1G genes.Despite the phenotypic heterogeneity,the most common feature was early-onset hypertension.After treatment with ENaC inhibitors,these cases had significantly improvements in hypertension and hypokalemia.Conclusions:In this study,two novel pathogenic mutations of ENaC genes(SCNN1B:c.1838delC and SCNN1G:c.1874C>T)were identified in two families with LS,which enriched the genetic mutation spectrum of LS and provided the new molecular targets for genetic diagnosis and clinical intervention.This study highlights the importance of ENaC genes screening for precision diagnosis of LS.ENaC inhibitors can effectively improve the clinical symptoms of patients with LS.Therefore,for young patients with Liddle syndrome(especially children),early precision diagnosis and treatment is necessary to avoid the occurrence of adverse cardiovascular events.Part 2:Apparent mineralocorticoid excess syndrome caused by the compound heterozygous mutations in HSD11B2 gene and related clinical researchBackground:Apparent mineralocorticoid excess syndrome(AME)is an ultrarare autosomal recessive monogenic hypertension.It results from deficiency of 11?-hydroxysteroid dehydrogenase type 2(11?HSD2)due to mutations in HSD11B2 gene.The clinical features of AME are hypertension,hypokalemia,low plasma renin activity and aldosterone concentration,plus responsiveness to spironolactone.Aims:The purpose of this study was to identify pathogenic HSD11B2 mutations in a Chinese family with AME and explore individualized treatment for the proband with AME.A systematic review was conducted for evaluating the AME clinical features associated with HSD11B2 mutations.Subjects and methods:A total of nine subjects were enrolled in this study.Next-generation sequencing was performed in the proband for detecting the candidate pathogenic variants.Using Sanger sequencing,the candidate variants were identified in other family members,unrelated 100 hypertensives,and 100 healthy controls.A predicted structure of 11PHSD2 was constructed by in silico modeling.The AME patient was treated with spironolactone and low dose dexamethasone,followed up regularly and adjusted the treatment protocols in time.A systematic review was conducted for H5D11B2-related AME cases.We extracted genotypic and phenotypic data for analysis.Results:The novel compound heterozygous mutations(c.343₃48del and c.1099₁101del)in HSD11B2 gene were found by next-generation sequencing and genetic analysis in the proband with early-onset hypertension and hypokalemia.The monoallelic form of the same mutations were verified in five other relatives,but not in 100 hypertensives or 100 healthy subjects.In silico structural modeling predicted that compound mutations may simultaneously perturb the substrate and coenzyme binding pocket.The combination of spironolactone and low dose dexamethasone treatment returned blood pressure and serum potassium to normal levels in the proband with AME.The systematic review included 54 HSD11B2 mutations in 102 AME patients.It revealed that early-onset hypertension,hypokalemia and homozygous mutations were common features.The low birth weight correlated with homozygous HSD11B2 mutations(r=0.285,P=0.02).Conclusions:In this study,compound heterozygous HSD11B2 mutations(c.343₃48del and c.1099₁101 del)were identified for young AME proband with hypertension and hypokalemia by genetic testing,which enriched the genetic mutation spectrum of AME.Spironolactone and low dose dexamethasone were effective for the proband with AME.Genetic testing is beneficial to the early diagnosis and treatment of AME patients and has important clinical significance for the prevention of adverse cardiovascular events.Part 3:Identification of a PDE3A gene mutation and genotype-phenotype association analyses in Bilginturan syndromeBackground:Bilginturan syndrome is a rare autosomal dominant monogenic hypertension.It is characterized by severe salt-independent hypertension and brachydactyly.Hence,Bilginturan syndrome is also called hypertension and brachydactyly syndrome(HTNB).Untreated HTNB patients mostly die from stroke before the age of 50.Aims:The aims of the present study were to identify the mutation and analyze the correlation between genotypes and clinical phenotypes in HTNB patients.Subjects and methods:Peripheral blood samples and genomic DNA were collected and extracted from all participants in this family.The PDE3A mutation was identify by next-generation sequencing and Sanger sequencing.We also performed a comparative overview in the PDE3A-related probands with HTNB for analyzing the association between phenotype and genotype.Results:A missense PDE3A mutation(c.1346G>A)was identified in the HTNB proband by genetic testing and analysis.Different bioinformatics tools predicted that this mutation was highly conserved and damaging.The proband may inherited the identified mutation from her father through cosegregation analyses in this family.In addition,the proband with HTNB was associated with vertebral artery dysplasia.And antihypertensive treatment was effective for the proband.Comparative overview of PDE3A-related HTNB probands revealed that all pathogenic mutations were located in the "hot spot" of PDE3A gene,and c.1346G>A was the most common mutation.Phenotypic heterogeneity was present among patients with HTNB,even if they carry the same pathogenic mutation.Conclusions:In this study,a case of HTNB patient with vertebral artery dysplasia was reported for the first time in China,and the pathogenic mutation(c.1346G>A,p.G449D)of PDE3A gene was identified,which enriched the genetic mutation spectrum and clinical phenotypic spectrum of HTNB.Patients with hypertension and brachydactyly should be tested for PDE3A gene to detect "hot spot" mutations.Phenotype of HTNB is heterogeneous,so this study emphasizes the necessity of early diagnosis and early intervention for HTNB to prevent stroke and other cardiovascular and cerebrovascular complications.