The ambiguous role of NKX2-5 mutations in thyroid dysgenesis

Abstract

NKX2-5 is a homeodomain-containing transcription factor implied in both heart and thyroid development. Numerous mutations in NKX2-5 have been reported in individuals with congenital heart disease (CHD), but recently a select few have been associated with thyroid dysgenesis, among which the p.A119S variation. We sequenced NKX2-5 in 303 sporadic CHD patients and 38 families with at least two individuals with CHD. The p.A119S variation was identified in two unrelated patients: one was found in the proband of a family with four affected individuals with CHD and the other in a sporadic CHD patient. Clinical evaluation of heart and thyroid showed that the mutation did not segregate with CHD in the familial case, nor did any of the seven mutation carriers have thyroid abnormalities. We tested the functional consequences of the p.A119S variation in a cellular context by performing transactivation assays with promoters relevant for both heart and thyroid development in rat heart derived H10 cells and HELA cells. There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in activation of the investigated promoters in both cell lines. Additionally, we reviewed the current literature on the topic, showing that there is no clear evidence for a major pathogenic role of NKX2-5 mutations in thyroid dysgenesis. In conclusion, our study demonstrates that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the wealth of published evidence, we suggest that NKX2-5 mutations do not play a major pathogenic role in thyroid dysgenesis, and that genetic testing of NKX2-5 in TD is not warranted.

Figure 1. Family pedigrees, aminoacid alignments, nuclear localization of NKX2-5 protein and electro mobility shift assay.

A. Pedigrees of the two families with the p.A119S NKX2-5 mutation. Individuals with congenital heart defects are indicated with a filled black symbol, while individuals with normal echocardiography are indicated with a white symbol. Grey symbols represent individuals that have not been evaluated clinically. A slash denotes a deceased individual; the proband is indicated by an arrow. None of the evaluated family members showed thyroid abnormalities. Heterozygous carriers of p.A119S are represented by +/− and non-carriers by −/−. B. Multiple alignments of aminoacids of the region surrounding p.A119 for various species. C. Nuclear localization of either wildtype or p.A119S NKX2-5 protein in COS7 cells. Nuclei are stained in green, red represents either the wildtype or mutant protein, orange indicates nuclei that are positive for wildtype or mutant protein. D. Electro mobility shift assay in cos7 cells, using the published nkx2.5 binding site . Wildtype nkx2.5 protein and p.A119S Nkx2.5 protein bind equally well, – is untransfected control.

Figure 2. Relative activation of the Nppa, Dio2, Tg and TPO promoters in combination with wildtype NKX2-5, p.A119S NKX2-5, p.N188K NKX2-5 or TBX5.

A. In H10 cells. B. In HELA cells. Significant differences between vector and condition tested are marked with *, P<0.05. # denotes a significant difference between conditions tested with and without TBX5, P<0.05. Error bars represent standard deviation (SD). Each condition has been tested at least in three independent triplicate experiments.