Functional characterization of TRPM4 variants identified in sudden unexpected natural death
| Autor: | Ekaterina Subbotina, Nori Williams, Barbara A. Sampson, Yingying Tang, William A. Coetzee |
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| Rok vydání: | 2018 |
| Předmět: |
Adult
Forensic Genetics Male 0301 basic medicine Adolescent Mutation Missense New York TRPM Cation Channels 030204 cardiovascular system & hematology Transfection Pathology and Forensic Medicine Death Sudden Young Adult 03 medical and health sciences 0302 clinical medicine Humans Protein Isoforms Child Sequence Analysis RNA Myocardium Infant Alternative Splicing HEK293 Cells 030104 developmental biology Child Preschool Mutagenesis Site-Directed Channelopathies Female Law |
| Zdroj: | Forensic Science International. 293:37-46 |
| ISSN: | 0379-0738 |
| Popis: | The TRPM4 gene encodes the subunit of the CaWe have identified thirteen uncommon missense VUS in TRPM4 by testing 95 targeted genes implicated in channelopathy and cardiomyopathy in 330 cases of SUD. In several cases there were co-existing VUS in one or more other genes that were tested. We selected four TRPM4 VUS (C20S, A380V, L595V and I1082S) for functional characterization, since these cases lacked detectable variants in other genes of our testing panel. Two of the cases were infants, one was a child and one an adult. RNA-seq data analysis showed that the longer TRPM4b splice variant is predominantly expressed in adult and fetal human heart. We therefore used site-directed mutagenesis to introduce these variants in a TRPM4b cDNA. HEK293 cells were transfected with the cDNAs and patch clamping was performed to assess the functional consequences of the TRPM4 mutants. The TRPM4 current was recorded in excised patches and was significantly reduced by each of the mutants. The total protein level of TRPM4-C20S was markedly decreased, whereas the A380V and L595V mutants exhibited decreased surface expression. The TRPM4-A380V current rapidly desensitized following patch excision.Each of the VUS tested caused a defect in TRPM4 channel function via distinctly different mechanisms, hence, it lays the foundation for further co-segregation family studies and animal studies of the TRPM4 variants. |
| Databáze: | OpenAIRE |
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