Identification And Functional Analylsis Of A De Novo NKX2.5 Mutation And Translational Readthrough Analysis Of NKX2.5 Nonsense Mutations By Aminoglycosides

NKX2.5, located on human chromosome 5q34, contains two exons and codes for a protein of 324 amino acids. The NKX2.5 protein contains three conserved domains:the TN domain, the Homeodomain (HD) and the NK2-specific domain (NK2-SD). NKX2.5 is an important cardiac transcriptional factor, and plays key roles both in cardiogenesis and adult heart maintainance. In the mouse, NKX2.5 begins to express strongly in the nucleus of the cardiogenes endoderm and mesoderm at embryogenesis day E7.5. The cardiogenesis of NKX2.5-/- mice arrested on cardiac looping, and the embryoes died on E10.5. In humans, NKX2.5 mutation causes congenital heart disease (CHD), such as aterial septal defects, the conduction system abnormality. The reported NKX2.5 mutations included missense, nonsense, insertion and deletion mutations. So far, the geneotype-phenotype correlation of NKX2.5 is still not clear, and the molecular mechanism of NKX2.5 in heart development is not well understood. Identification of more NKX2.5 mutations and functional analysis of the mutations will be helpful to elucide the role of NKX2.5 in cardiogenesis and may provide a potential therapy for patients with NKX2.5 mutations. Therefore, I have proposed to identify new NKX2.5 mutations, and to perform functional analysis of NKX2.5 mutations as the major goals for my thesis. My thesis consists of three parts.Partâ… :I collected a congenital heart disease family. The phenotypes of the family include atrial septal defects, atrioventricular block, left ventricular noncompaction, synocope and sudden death. I aimed to find the pathogenic gene in the family and direct sequencing analysis of NKX2.5 was used for the family members. I found a heterozygous 2 bp insertion at codon-512 (named L171RfsX5), which was co-segregated with the disease and, did not exist in healthy people, suggesting that it is a mutation. This mutation leads to frameshift strating with amino acid 171, and codes for a prematurely terminated protein of 175 amino acids. The online Swiss Model Service predicted the three dimension structure of the HD of the truncated protein made from L171RfsX5, and found that the mutant protein lost half of the second helix and the entire third helix. Then, I constructed mammalian expression plasmids for both the wild type and mutant-NKX2.5 (pWT-NKX2.5 and pNKX2.5-L171RfsX5), transfected them into HeLa cells, and functional analysis showed that the wild type NKX2.5 protein localized in the nucleus of HeLa as expected and increased the transactivation of the ANFp-Luc by about 5-fold, but the mutant NKX2.5 protein dispersed into both the cytoplasm and nucleus, and abolished the transactivation activity of NKX2.5. All these results suggest that L171RfsX5 is a functional mutation. This is the first time that a human NKX2.5 mutation is correlated with ventricular noncompaction, which expands the spectrum of NKX2.5 mutations causing CHD.Part II:I collected 125 sporadic Han CHD patients and 105 healthy people. The sporadic patients were affected with atrial septal defects, ventricular septal defects, tetralogy of fallot and other congenital heart defefcts. I performed mutation analysis of NKX2.5 in the sporadic CHD patients. In the sporadic CHD samples, I identified a SNP, rs2277923, which had a higher frequency in the patient population than in healthy people (controls). The difference was statistically significant (p=0.04), which implicated SNP rs2277923 as being associate with CHD. Due to the limited small sample size, this result may need further research to be confirmed. In addition, the functional analysis showed that the mutant protein with SNP rs2277923 (63a>g) localized in the nucleus and decreased the transactivation activity of NKX2.5 by about 20%.Part III:I performed structure-function analysis and translational readthrough assays by aminoglycosides for the eight NKX2.5 in-frame premature mutations (PTCs) reported to date. First, I constructed all PTCs in pcDNA3.1 (designed pE109X, pQ149X, pQ170X, pQ187X, pQ198X, pY256X, pY259X and pC264X) and three mutations (E109X, Q149X and C264X) in pEGFP-N1. Mutations Y259X, Y256X, and Q198X reduced transcriptional activity of NKX2.5 by about 2-fold, whereas mutations Q187X, Q170X, Q149X, and E109X eliminated the transcriptional activity. Immunostaining showed that mutant NKX2.5 produced by C264X, Y259X, Y256X, and Q198X were localized in the nucleus, but those generated by Q187X, Q170X, Q149X, and E109X had disrupted nuclear localization. Qualitative assays for translational readthrough with NKX2.5-EGFP fusion reporters with E109X, E149X, and C264X showed successful suppression of these PTCs by gentamicin and G418. Quantitative luciferase assays with an ANFp-Luc reporter for all eight nonsense mutations showed that gentamicin and G418 increased transcriptional activation activity of NKX2.5 by up to 13.8% and 32.2%, respectively. In conclusion, this study establishes the C-terminal domain of NKX2.5 downstream of Q198 is required for transcriptional activation, and an R/K-rich sequence motif from Q187 to Q197 (QNRRYKCKRQR, a portion of the homeodomain) is required for nuclear localization of NKX2.5. Furthermore, NKX2.5 nonsense mutations can be suppressed by aminoglycosides, which may provide a potential therapeutic option for patients with NKX2.5 nonsense mutations.