YWHAE Variants And TAZ (G4.5) Variants In Patients With Left Ventricular Noncompaction

Objective1.Left ventricular noncompaction(LVNC)is a cardiomyopathy characterized by a pattern of prominent trabecular meshwork and deep intertrabecular recesses,and is thought to be due to arrest of normal endomyocardial morphogenesis.The genes contributing this myocardial development and regulating proliferation of embryonic cardiomyocytes remain poorly understood.14-3-3ε,encoded by YWHAE,is an isoform of 14-3-3 proteins.14-3-3ε is an adapter protein that plays important roles in neuronal development and is involved in Miller-Dieker syndrome.Mice lacking this gene develop LVNC.Therefore,we hypothesized that variants in YWHAE may contribute to the pathophysiology of LVNC in humans.2.TAZ(G4.5)was initially identified as the gene associated with Barth syndrome and LVNC.The purpose of this study was to investigate patients with LVNC for disease-causing mutations in TAZ.Materials and Methods1.Blood was obtained from 77 Japanese patients including 29 familial cases(12 females and 17 males)and 48 sporadic cases(15 females and 33 males),after informed consent,and then genomic DNA was extracted using Quick Gene DNA whole blood kits(FUJIFILM;JAPAN).Two hundred ethnicity-matched normal controls were recruited and DNA was extracted in an identical manner after informed consent.The study was approved by the Research Ethics Committee of Toyama University Hospital.After PCR amplification,the samples were purified,directly sequenced and analyzed according to the ABI Big Dye Terminator Cycle Sequencing protocol and an ABI 310 Automated Sequencer(Applied Biosystems;Foster City,California,United States).When a putative mutation was identified,the family members of the proband and at least 200 control chromosomes were screened for the sequence variations to recognize common polymorphisms.Blast search analysis was used to identify homology between the sequences obtained from patients and the published gene sequences.Mutations were confirmed by repeating the PCR from the genomic DNA template and sequencing the PCR products.After identified the mutation,the mutated fragment was subcloned and confirmed by DNA sequencing and was studied on the function.2.Blood was obtained from 124 Japanese patients with Barth syndrome or LVNC,comprising 50 familial cases(24 females and 26 males)and 74 sporadic cases(25 females and 49 males),after informed consent,and then genomic DNA was extracted using Quick Gene DNA whole blood kits.Two hundred ethnicity-matched normal controls were recruited and DNA was extracted in an identical manner after informed consent.The study was approved by the Research Ethics Committee of Toyama University Hospital.After PCR amplification,the samples were purified,directly sequenced and analyzed according to the ABI Big Dye Terminator Cycle Sequencing protocol and an ABI 310 Automated Sequencer.When a putative mutation was identified,the family members of the proband and at least 200 control chromosomes were screened for the sequence variations to identify common polymorphisms.Blast search analysis was used to identify homology between the sequences obtained from patients and the published gene sequences.Mutations were confirmed by repeating the PCR from the genomic DNA template and sequencing the PCR products.Results1.Seven novel variants were identified in this patient cohort.Four of these variants were located within the promoter,one was intronic,one was a synonymous substitution in exon 6 and the last was in the 3’ untranslated region.None of the variants were predicted to alter RNA splicing or exonic splice enhancer(ESE)sequences by multiple in silico analyses.Five of the variants were identified in the control samples at frequencies statistically indistinguishable from the patient cohort.Of the two variants that were not detected in the control cohort(-458G>T and c.64+9G>A)the former was noted to be located in a conserved CCAAT/enhancer binding protein(C/EBP)response element of the promoter while the latter was not predicted to alter m RNA splicing.The promoter variant was identified in two patients,including one familial and one sporadic patient.Both these patients were screened for mutations in other candidate genes of LVNC including TAZ,DTNA,LDB3,FKBP12,STN,CSX/Nkx2.5 and SCN5 A,but no mutations were found.Expression of a mutated promoter coupled to a luciferase reporter gene revealed that this variant reduced promoter activity by approximately 50%.-458 G is located within a regulatory CCAAT/enhancer binding protein(C/EBP)response element of the YWHAE promoter and C/EBPβ is a key regulator of 14-3-3ε expression.2.A hemizygous splice donor substitution(c.109+1 G>C)was identified in intron 1 of the TAZ gene in the proband(III-2)and his younger brother(III-3),their elder sister was heterozygous for the same mutation(III-1).This mutation was not identified in other members of the family,including the parents and maternal grandparents,all of whom wereasymptomatic.The remaining 123 patients and 200 normal control samples.This variant was considered deleterious based upon in silico analysis of the effect on RNA splicing.In addition,IVS+1 variants are categorized as deleterious based upon current recommendations for reporting sequence variations from the American College of Medical Genetics.In this cohort of 124 patients,2 additional variants were detected.These variants(IVS8-1G>C and 158 Ins C)were described previously.Conclusion1.These data suggest that this variant of YWHAE gene contributes to the abnormal myocardial morphogenesis that is characteristic of LVNC and YWHAE is a novel candidate gene for this disease.2.Due to the recurrent inheritance of this variant of G4.5 gene by each of the children we concluded that this was evidence of gonadal mosaicism in the obligate carrier mother,the first reported occurrence of this in Barth syndrome.