Identification And Functional Characterization Of An ISL1 Mutation Associated With Congenital Heart Defects

Background: Congenital heart defects（Congenital heart disease,CHD）represent the most frequent kind of birth defects in humans,with an estimated prevalence of 1% in all livebirths,and account for about one-third of all major congenital abnormalities.Congenital heart defects remain the main cause of infant death due to birth defects,with nearly 24% of newborns who died of birth deformities having congenital heart defects.Although enormous advance in medical and surgical treatments of congenital heart defects has allowed over 90% of neonates with congenital heart defects to survive into adulthood,it results in an ever-increasing population of adult patients with congenital heart defects,and now the adults with congenital heart diseases outnumber the children with congenital heart defects.In addition,the incidence rate and mortality rate of adult congenital heart defects are much higher than that of the general population.Therefore,it is of great significance to explore the etiology of congenital heart defects.In spite of important clinical significance,the molecular causes of congenital heart defects remain largely obscure.Recent researches have strongly demonstrated that embryonic heart development is a complex biological process,and both inherited and environmental pathogenic factors may interfere with the process,resulting in congenital heart defects.However,increasing researches highlight the major contribution of genetic determinants to congenital heart defects,especially to familial congenital heart defects,which are transmitted in an autosomal dominant,autosomal recessive or X-linked pattern in the family.Irrespective of chromosomal abnormalities,an increasing number of mutations in over 60 genes have been associated with the pathogenesis of congenital heart defects in humans.Among these CHD-causing genes,the majority encode cardiac core transcription factors。 As an important member of the homeobox transcription factor family,ISL1 is amply expressed in the embryonic heart and is key to normal cardiovascular morphogenesis.In mice,targeted deletion of the Isl1 gene led to embryonic demise due to severe developmental malformations of the heart,encompassing loss of the right ventricle,outflow tract,and most of the atria.In humans,multiple variants in ISL1 have been reported to confer an enhanced vulnerability to CHDs in diverse cohorts of patients.The key role of Isl1 gene in cardiac morphogenesis suggests that Isl1 may be an important susceptibility gene for congenital heart defects.The above-mentioned findings justified it to screen ISL1 as a prime candidate gene for congenital heart defects.Objectives:（1）To identify a new ISL1 mutation associated with congenital heart defects.（2）To investigate the mechanism by which the mutation leads to congenital heart defects at the molecular and cellular levels.Methods: Mutational screening of ISL1（1）Study subjects:Participants in this research include 114 unrelated patients with congenital heart defects（62 males and 52 females,with an average age of 3 years,ranging from 0 to 9 years of age）and 218 unrelated healthy subjects used as controls（118 males and 100 females,with a mean age of 3 years,varying from 1 to 9 years of age）.They were enrolled from the Chinese Han population.In addition,the available relatives of the index patient harboring a discovered ISL1 mutation were also included.All study participants experienced comprehensive clinical investigation,including review of medical history such as cardiac catheterization and/or surgery for congenital heart defects,detailed physical examination,two-dimensional echocardiogram with color Doppler flow and standard 12-lead electrocardiogram.Index patients with known chromosomal abnormalities or syndromic congenital heart defects,such as 22q11.2 microdeletion syndrome,Turner syndrome,Down syndrome,Noonan syndrome,Marfan syndrome and Holt-Oram syndrome,were excluded from the present research.This study follows the 1964 Helsinki declaration and its subsequent amendments.Informed consent was obtained from their legal guardians of both patients and control individuals prior to the investigation.（2）Purification of genomic DNA :About 2 μl of venous blood sample was collected from each study participants.Genomic DNAs from blood leukocytes were isolated using the QIAamp DNA Mini Kit following the manufacturer’s descriptions.（3）Primers for amplification of Isl1 gene :The primers for amplification of the coding exons and exon-intron boundaries as well as partial 3′-and 5′-untranslated regions of ISL1 by polymerase chain reaction were designed as described previously.（4）Amplification of Isl1 gene :All coding exons and flanking introns of ISL1 were amplified by polymerase chain reaction with Hot Star Taq DNA Polymerase on the Veriti Thermal Cycler.（5）PCR-DNA sequencing :The amplicons were purified and sequenced with the Big Dye（?） Terminator v3.1 Cycle Sequencing Kit under the ABI PRISM 3130 XL DNA Analyzer,according to the manufactuer′s instructions.（6）Identification of Isl1 gene mutation :For a detected genetic variant,the Single Nucleotide Polymorphism database,Exome Aggregation Consortium database,Human Gene Mutation Database,1000 Genomes Project database,Exome Variant Server database,and Genome Aggregation Database were retrieved to validate the novelty of an identified ISL1 variation.Functional analyses（1）Plasmid constructs: The wild-type ISL1-pc DNA3.1 and TBX20-pc DNA3.1 expression plasmids,and the reporter plasmid MEF2C-luciferase（MEF2C-luc）,which expresses firefly luciferase,were constructed as previously described.（2）Site-targeted mutagenesis : The mutation identified in the patients was introduced into the wild-type ISL1-pc DNA3.1 plasmid by site-directed mutagenesis with the Quik Change（?） XL Site-Directed Mutagenesis Kit,using a complementary pair of primers（forward primer: 5′-TTATATCAGGTTGTAGGGGATCAAATGCGCC-3′;reverse primer: 5′-GGCGCATTTGATCCCCTACAACCTGATATAA-3′）according to the manufacturer′s protocol,and was confirmed by sequencing.（3）Cell culture and transient transfection: Chinese hamster ovary（CHO）cells and 10T1/2 cells were cultivated in Dulbecco′s modified Eagle′s media added with 10% fetal bovine serum as well as 100 U/ml penicillin and 100 μg/ml streptomycin in an incubator with an atmosphere of 5% CO2 at 37°C.Cells were grown in 12-well plates at a density of 1 × 105 cells per well.After 48 h,cellular transfection was carried out with Effetene（?） transfection reagent following the manufacturer′s mnual.Cellular transfections with various plasmids were performed as previously described.The p GL4.75 vector,which expresses a renilla luciferase,was co-transfected into the cells as an internal control to normalize transfection efficiency.Briefly,in transient transfection experiments,CHO cells were transfected with 1.0 μg of wild-type ISL1-pc DNA3.1,1.0 μg of Tyr75*-mutant ISL1-pc DNA3.1,0.5 μg of empty pc DNA3.1 plus 0.5 μg of wild-type ISL1-pc DNA3.1,or 0.5 μg of wild-type ISL1-pc DNA3.1 in combination with 0.5 μg of Tyr75*-mutant ISL1-pc DNA3.1,in the presence of 1.0 μg of MEF2C-luc and 0.04 μg of p GL4.75.For analysis of the synergistic effect between ISL1 and TBX20 on the MEF2 C promoter,10T1/2 cells were co-transfected with 0.25 μg of wild-type ISL1-pc DNA3.1,or 0.25 μg of Tyr75*-mutant ISL1-pc DNA3.1,or 0.25 μg of wild-type TBX20-pc DNA3.1,or 0.25 μg of wild-type ISL1-pc DNA3.1 plus 0.25 μg of wild-type TBX20-pc DNA3.1,or 0.25 μg of Tyr75*-mutant ISL1-pc DNA3.1 plus 0.25 μg of wild-type TBX20-pc DNA3.1,in the presence of 1.0 μg of MEF2C-luc and 0.04 μg of p GL4.75.Cells were cultured at 37°C and collected 48 h after transfection.（4）Luciferase analysis: Luciferase activity of the cell lysates was measured in a Glo Max（?） 96 Microplate Luminometer,with the Dual-Luciferase Reporter Assay System according to the manufacturer′s protocol.The results were expressed as the ratios of the activities of firefly luciferase to renilla luciferase.For each expression plasmid,three independent transfection experiments were performed in triplicate,and the MEF2 C promoter activity was given as mean ± standard deviation.（5）Statistical analysis: The statistical analyses were made with the SPSS for Windows statistical software package（version 18）.Continuous variables were compared between two groups with Student′s unpaired t-test,while categorical variables were compared between two groups with Pearson′s χ2 test or Fisher’s exact test,as indicated.A two-tailed P-value of <0.05 was considered significantly different.Results:（1）In this research,a cohort of 114 unrelated patients was clinically evaluated in contrast to a total of 218 unrelated control individuals.Patients were matched with control individuals for gender,age and ethnicity.The patients had congenital heart defects documented by echocardiogram,of whom roughly 32% had positive family history of congenital cardiovascular anomalies.The control subjects had a negative family history of congenital heart defect,and their echocardiograms displayed normal cardiac images with no structural cardiac defects.（2）Sequence analysis of the whole coding regions and splicing donors/ acceptors of the ISL1 gene performed in 114 unrelated patients affected with congenital heart defects revealed a mutation,NM₀02202.2: c.225C>G;p.（Tyr75*）,in an index case who was one year old.Specifically,a substitution of guanine for cytosine at the third nucleotide of codon 75（c.225C>G）,predicting the change of the codon encoding tyrosine at amino acid position 75 into a premature termination codon,p.（Tyr75*）,was identified in a girl affected with double outlet right ventricle and ventricular septal defect,who had a positive family history of congenital heart defects.（3）Sequencing analysis of the ISL1 gene in the proband’s relatives available showed that the mutation was present in all the affected relatives,but absent in unaffected relatives.Genetic analysis of the proband’s pedigree unveiled that the truncating mutation co-segregated with the disease phenotype,which was transmitted in an autosomal dominant pattern,with complete penetrance.The nonsense mutation was neither observed in the 218 control persons nor found in the Single Nucleotide Polymorphism database,Exome Aggregation Consortium database,Human Gene Mutation Database,1000 Genomes Project database,Exome Variant Server database,or the Genome Aggregation Database.（4）Biological measurements showed that 1.0 μg of wild-type ISL1-pc DNA3.1 plasmid and 1.0 μg of Tyr75*-mutant ISL1-pc DNA3.1 plasmid transactivated the MEF2 C promoter by ～13 folds and ～1 fold,respectively.In addition,when 0.5 μg of wild-type ISL1-pc DNA3.1was utilized in combination with 0.5 μg of empty pc DNA3.1 or 0.5 μg of Tyr75*-mutant ISL1-pc DNA3.1,the resultant transactivation of the MEF2 C promoter was the same of ～7-fold.When the wild-type TBX20-pc DNA3.1 plasmid was co-transfected,the wild-type and Tyr75*-mutant ISL1 transactivated the MEF2 C promoter by ～23 folds and ～9 fold,respectively.These functional findings indicate that the truncated ISL1 protein has no transcriptional activity,and mutation nullifies the synergistic transactivational activation between ISL1 and TBX20.Conclusions:（1）This study for the first time reveals that ISL1 loss-of-function mutation predisposes to double outlet right ventricle in humans.（2）This study helps further understand the molecular mechanism underlying congenital heart defects,implying potential implications for genetic counseling and individualized management of the patients affected with congenital heart defects.