ISL1 loss-of-function mutation contributes to congenital heart defects

Ma, Lan; Wang, Juan; Li, Li; Qiao, Qi; Di, Ruo-Min; Li, Xiumei; Xu, Ying‐Jia; Zhang, Min; Li, Ruogu; Qiu, Xing‐Biao; Li, Xun; Yang, Yi‐Qing

doi:10.1007/s00380-018-1289-z

Cited by 21 publications

(20 citation statements)

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“…The mutant ISL1 protein lost transactivation of the MEF2C promoter: Previous experiments in vivo and in vitro have demonstrated that ISL1 transcriptionally activates the promoter of MEF2C. [116][117][118] As displayed in Figure 2, 1.0 μg of wild-type ISL1-pcDNA3.1 plasmid (ISL 1) and 1.0 μg of Tyr75*-mutant ISL1-pcDNA3.1 plasmid (Tyr75*) transactivated the MEF2C promoter by 13folds and 1-fold, respectively. In addition, when 0.5 μg of wild-type ISL1-pcDNA3.1 (ISL1) was utilized in combination with 0.5 μg of empty pcDNA3.1 or 0.5 μg of Tyr75*-mutant ISL1-pcDNA3.1 (Tyr75*), the resultant transactivation of the MEF2C promoter was the same of 7-folds.…”

Section: Clinical Features Of the Study Subjectsmentioning

confidence: 84%

“…The primers for amplification of the coding exons and exonintron boundaries as well as partial 3'-and 5'-untranslated regions of ISL1 by polymerase chain reaction (PCR) were designed as described previously. 116) All coding exons and flanking introns of ISL1 were amplified by PCR with Hot-Star Taq DNA Polymerase (Qiagen) on a Veriti Thermal Cycler (Life Technologies, Carlsbad, CA, USA). The purified amplicons were sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) under an ABI PRISM 3130 XL DNA Analyzer (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

“…The wild-type ISL1-pcDNA3.1 and TBX20-pcDNA3.1 expression plasmids and the reporter plasmid MEF2C-luciferase (MEF2C-luc), which expresses Firefly luciferase, were constructed as previously described. 116) The mutation identified in CHD patients was introduced into the wild-type ISL1 MUTATION IN DORV ISL1-pcDNA3.1 plasmid by site-directed mutagenesis with the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), using a complementary pair of primers (forward primer: 5'-TTAT ATCAGGTTGTAGGGGATCAAATGCGCC-3 ' ; reverse primer: 5'-GGCGCATTTGATCCCCTACAACCTGATATA A-3') according to the manufacturer's protocol, and was confirmed by sequencing. To construct a control expression plasmid, the most common polymorphism (rs 2303751) of c.504A>G (p.Pro168Pro), with an allele gene frequency of 0.3739 in the general population reported in the ExAC database (http://exac.broadinstitute.org/variant/5-50685505-A-G), was introduced into the wild-type ISL1-pcDNA3.1.…”

Section: Plasmid Constructs and Site-targeted Mutagenesismentioning

confidence: 99%

“…Cellular transfections with various plasmids were performed as previously described. 116) The pGL4.75 vector (Promega, Madison, WI, USA), which expresses a Renilla luciferase, was co-transfected into the cells as an internal control to normalize transfection efficiency. As two negative controls, the empty pcDNA3.1 plasmid with no ISL1 expression and the pcDNA3.1 plasmid expressing Pro168Pro-ISL1 were used.…”

Section: Plasmid Constructs and Site-targeted Mutagenesismentioning

confidence: 99%

“…110) In humans, multiple variants in ISL1 have been reported to confer an enhanced vulnerability to CHDs in diverse cohorts of patients. [114][115][116] These results make it justifiable to scan ISL1 for mutations underlying CHDs in another cohort of patients.…”

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A New <i>ISL1</i> Loss-of-Function Mutation Predisposes to Congenital Double Outlet Right Ventricle

et al. 2019

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Occurring in about 1% of all live births, congenital heart defects (CHDs) represent the most frequent type of developmental abnormality and account for remarkably increased infant morbidity and mortality. Aggregating studies demonstrate that genetic components have a key role in the occurrence of CHDs. Nevertheless, due to pronounced genetic heterogeneity, the genetic causes of CHDs remain unclear in most patients. In this research, 114 unrelated patients affected with CHDs and 218 unrelated individuals without CHDs served as controls were recruited. The coding regions and splicing donors/acceptors of the ISL1 gene, which codes for a transcription factor required for proper cardiovascular development, were screened for mutations by sequencing in all study participants. The functional characteristics of an identified ISL1 mutation were delineated with a dual-luciferase reporter assay system. As a result, a new heterozygous ISL1 mutation, NM_002202.2: c.225C>G; p.(Tyr75*), was discovered in an index patient with double outlet right ventricle and ventricular septal defect. Analysis of the proband's family unveiled that the mutation co-segregated with the CHD phenotype. The nonsense mutation was absent in the 436 control chromosomes. Biological analysis showed that the mutant ISL1 protein had no transcriptional activity. Furthermore, the mutation nullified the synergistic activation between ISL1 and TBX20, another CHD-associated transcription factor. This research for the first time links an ISL1 loss-of-function mutation to double outlet right ventricle in humans, which adds insight to the molecular pathogenesis underpinning CHDs, suggesting potential implications for timely personalized management of CHD patients. (Int Heart J 2019; 60: 1113-1122

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Section: Clinical Features Of the Study Subjectsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Section: Plasmid Constructs and Site-targeted Mutagenesismentioning

confidence: 99%

Section: Plasmid Constructs and Site-targeted Mutagenesismentioning

confidence: 99%

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confidence: 95%

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