Study On The Pathogenesis Of ISL2 Mutation Underpinning Congenital Heart Disease

Backgrounds: Congenital heart disease（CHD）is the most frequent type of birth defect globly in humans.Despite significant clinical importance,the molecular etiologies underlying CHD remain largely elusive.Previous researches have demonstrated that cardiac morphogenesis during embryogenesis is a complex biological process,which is finely regulated by a regulatory network,including signaling molecules,transcription factors,and epigenetic modifiers.However,accumulating studies highlight the strong genetic basis for CHD,an increasing number of pathogenic mutations in more than 100 genes,including those encoding cardiac transcription factors,cellular signaling molecules and myocardial structural proteins,have been identified to cause CHD in humans.Among these well-established disease-causing genes for CHD,the majority code for cardiac core transcription factors,including TBX5,TBX20,TBX1,TBX3,NKX2.5,GATA4,GATA6,GATA5,ISL1,MEF2 C,HAND1 and HAND2.Nevertheless,due to the substantial genetic heterogeneity of CHD,the genetic determinants underpinning CHD in the vast majority of cases remain obscure.Now that the gene ISL2 and the established CHD-causative gene ISL1 belong to the same family of genes（both code for the LIM homeodomain-containing transcription factors）,and the expression profiles and functional characteristics of the genes ISL1 and ISL2 overlap at least in part,playing important mediatory roles in the cardiac development,Hence,as an important candidate gene for CHD,ISL2,when mutated,may result in CHD by interfering in normal development of the heart.Objectives:1.To identify an ISL2 mutation underpinning CHD.2.To unveiling the mechanism by which the ISL2 mutation led to CHD at the molecular and cellular levels.Methods:Part I: Discovery of an ISL2 Mutation Underpinning Congenital Heart Disease1.Study SubjectsIn the current research,a total of 230 unrelated patients with CHD（122 male cases and 108 female cases,with an average age of 4years,ranging from 1year to 12 years of age）and 276 unrelated healthy individuals employed as controls（150 males and 126 females,with a mean age of 4years,varying from 1 year to 12 years of age）.All the included CHD patients and healthy subjects were enlisted from the Chinese Han population of Shanghai.Additionally,the available family members of the proband who carrys an identified ISL2 variation were also enrolled.Approximately 1 m L of venous blood specimen was collected from every study participant.Genomic DNAs were extracted from blood leukocytes using a DNA purification kit.2.Amplification and Sequencing Analysis of the ISL2 GeneThe primers to specifically amplify the ISL2 gene were designed and synthesized.Amplification of all coding exons and partial flanking introns as well as 5’-and 3’-untranslated regions of ISL2 were performed by polymerase chain reaction on a thermal cycler,with the designed primer pairs and Hot Star Taq DNA Polymerase as well as other reagents.The amplified products（DNA fragments）were purified,then polymerase chain reaction for sequencing was carried out on a thermal cycler with a cycle sequencing kit.The amplified DNA fragments were purified and sequenced under a DNA Analyzer by electrophoresis.3.Discovery of an ISL2 Mutation Underpinning Congenital Heart DiseaseBy comparative analysis of the sequenced ISL2 sequences,an ISL2 mutation was likely to be discovered.For an identified ISL2 mutation,the Single Nucleotide Polymorphism database,1000 Genomes Project database,Genome Aggregation Database,Pub Med database and Wanfang database were retrieved to verify the novelty of a found ISL2 mutation.The disease-causing potential of a novel ISL2 sequence variation was predicted by Mutation Taster（an online programat http://www.mutationtaster.org）,and MUSCLE was applied for analysis of the conservation of altered amino acids to verify the ISL2 mutation underpinning congenital heart disease.Part II: Functional Investigation of an ISL2 Mutation Underpinning Congenital Heart Disease1.Plasmid ConstructionThe wild-type ISL2 was cloned.The ISL2-pc DNA3.1 plasmid expressing wild-type ISL2,the GATA4-p SSRa plasmid expressing wild-type GATA4 as a transcriptionally operative partner of ISL2,the TBX20-luciferase（TBX20-luc）plasmid where the promoter of TBX20 as a target gene of ISL2 drove the expression of firefly luciferase were constructed.2.Site-Directed MutagenesisThe identified CHD-causative mutation c.598G>T was introduced into the wild-type ISL2-pc DNA3.1 plasmid by site-targeted mutagenesis（polymerase chain reaction）with a site-directed mutagenesis kit,using a complementary pair of primers（forward primer:5′-CGGACTGTGCTGAACTAGAAGCAGCTGCACA-3′;backward primer: 5′-TGTGCAGCTGCTTCTAGTTCAGCACAGTCCG-3′）,and the E200 Xmutant ISL2-pc DNA3.1 plasmid was obtained by digestion of wild-type ISL2-pc DNA3.1 template with DNA enzyme Dpn I,and validated by DNA sequencing.3.Cell Culture and TransfectionHuman cervix carcinoma（He La）cells were maintained in Dulbecco′s modified Eagle′s media,supplemented with 10% fetal bovine serum as well as 100 U/m L penicillin and 100 μg/m L streptomycin,in an incubator with an atmosphere of 5% CO2 at a constant temperature of 37°C.After 48 h of cellular culture,cellular transfections of various plasmids were performed with Lipofectamine TM 2000 transfection reagent（the p GL4.75 plasmid expressing a renilla luciferase was co-transfected as an internal contrast to normalize transfection efficiency）.Specifically,for analysis of transcriptional activation by ISL2,He La cells were transfected with 1.0 μg of the wildtype ISL2-pc DNA3.1 plasmid,1.0 μg of the E200X-mutant ISL2-pc DNA3.1 plasmid,0.5 μg of the empty pc DNA3.1plasmid plus 0.5 μg of the wild-type ISL2-pc DNA3.1 plasmid,or 0.5 μg of the wild-type ISL2-pc DNA3.1 plasmid in combination with 0.5 μg of the E200X-mutant ISL2-pc DNA3.1 plasmid,together with 1.0 μg of the TBX20-luc plasmid and 0.04 μg of the p GL4.75 plasmid.For analysis of the synergistic transactivation between ISL2 and GATA4,He La cells were transfected with 0.25 μg of the wild-type ISL2-pc DNA3.1 plasmid,or 0.25 μg of the E200X-mutant ISL2-pc DNA3.1 plasmid,or 0.25 μg of the wild-type GATA4-p SSRa plasmid,or 0.25 μg of the wild-type ISL2-pc DNA3.1 plasmid plus 0.25 μg of the wild-type GATA4-p SSRa plasmid,or 0.25 μg of the E200X-mutant ISL2-pc DNA3.1 plasmid plus 0.25 μg of the wild-type GATA4-p SSRa plasmid,in the presence of 1.0 μg of the TBX20-luc and 0.04 μg of the p GL4.75 plasmid.He La cells were cultured at a constant temperature of 37°C,and collected and lysed 48 h after transfection.4.Reporter Gene AnalysisLuciferase activity（fluorescent intensity）of the cell lysates was measured in a quantitative luminometer,utilizingadual-luciferase reporter assay system.Results were expressed as the ratios of the fluorescent intensities of firefly luciferase to renilla luciferase.For each kind of plasmid,three independent transfection experiments were conducted in triplicate,and the promoter activity was expressed as mean ± standard deviation of the results of three independent transfection experiments.5.Statistical AnalysisStatistical analysis was made by using the SPSS for Windows statistical software package（version 17.0）.Continuous variables between two groups were compared with Student’s unpaired t test,while categorical variables between two groups were compared with Pearson′s χ2 test or Fisher’s exact test.A two-tailed P <0.05 indicated statistical difference.Results:1.Identification of a Novel ISL2 MutationIn the present investigation,sequencing analysis of all coding exons of the ISL2 gene was completed in 230 unrelated cases inflicted with CHD and 276 unrelated subjects utilized as controls,and an ISL2 mutation,NM₁45805.3: c.598G>T;p.（Glu200*）,was identified in a proband.Specifically,a transversion of guanine into thymine at the first nucleotide of codon 200（c598G>T）,resulting in the transition of the codon encoding glutamic acid at amino acid position 200 into a premature termination codon,p.（Glu200*）,namely E200 X,was detected in a 2-year-old girl suffering from patent ductus arteriosus as well as ventricular septal defect,who possessed a confirmed family history for CHD.Sequencing assay of the ISL2 gene in the index patient’s available family members unveiled that the mutation was in complete co-segregation with CHD.The nonsense mutation was not detected in 276 control people and not retrieved in the Single Nucleotide Polymorphism database,the 1000 Genomes Project database,the Genome Aggregation Database,the Pub Med database,or the Wanfang database.2.Disease-Causing Potential of the Identified ISL2 Sequence VariationThe ISL2 sequence variation of c.598G>T was predicted by Mutation Taster to be pathogenic,with a p value of 1.A cross-species alignment of multiple ISL2 protein sequences by MUSCLE displayed that the affected amino acid of E200 X was completely conserved evolutionarily,suggesting that the amino acid is functionally important.3.Diminished Transcriptional Activity of the Mutant ISL2Functional explorations demonstrated that the same amount（1.0 μg）of the wild-type ISL2-pc DNA3.1 plasmid and the E200X-mutant ISL2-pc DNA3.1 plasmid transcriptionally activated the TBX20 promoter by ¹0-fold and ¹-fold,respectively;while when the same amount（0.5 μg）of the wild-type ISL2-pc DNA3.1 plasmid was used together with the E200X-mutant ISL2-pc DNA3.1 plasmid,the induced transcriptional activationon the TBX20 promoter was ⁶-fold.When the wild-type GATA4-p SSRa plasmid was co-transfected,the wild-type ISL2-pc DNA3.1 plasmid and the E200X-mutant ISL2-pc DNA3.1 plasmid transactivated the TBX20 promoter by ²0-fold and ⁸-fold,respectively.These results of functional assays indicate that the truncated ISL2 protein loses transcriptional activation function,and the nonsense mutation disrupts the transcriptional activation of ISL2 in synergy with GATA4.Conclusions:1.The current research firstly reveals that ISL2 loss-of-function mutation leads to CHD in humans.2.Glu124*-mutant ISL2 lost transcriptional activity on the promoters of TBX20.3.The Glu124* mutation abolished the synergistic transcriptional activation of ISL2 in synergy with GATA4.4.The current research not only reveals the new molecular etiology and its pathogenic mechanism underpinning CHD,but also implies potential implications for genetic counseling and precisive prophylaxis and therapeutics of the patients affected with CHD.