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Abstract

Brain-Lung-Thyroid syndrome (MIM 610978) is an autosomal dominant disorder resulting from disruption of the homeobox transcription factor gene NKX2-1 (MIM 600635). The disorder exhibits phenotypic variability, both between and within families, but is typically characterized by infant hypotonia that progresses to chorea and other movement abnormalities. Dysarthria (a motor speech disorder) and attentiveness disorder have recently been recognized as common neurological features of the syndrome. Most affected individuals also have thyroid dysfunction and/or lung problems such as neonatal respiratory distress and recurrent infections. Over 100 different lesions affecting the NKX2-1 gene have been reported in cases of Brain-Lung-Thyroid syndrome, including a number of missense variants. However there has been little functional investigation of the effects of these variants to elucidate the molecular mechanisms of disorder. Here we report the functional characterization of forty NKX2-1 variants, employing assay methodologies that could be extended for the high-throughput characterization of transcription factor gene variants identified by next-generation sequencing in disorders. By assessing the effects of NKX2-1 variants on protein expression, subcellular localization, protein-protein interactions and transcriptional regulatory activity, we uncovered diverse molecular-level effects for etiological variants, highlighting the importance of examining multiple aspects of protein function when characterizing putative disorder-related variants. In addition, we confirmed that NKX2-1 interacts with the forkhead transcription factor FOXP2 (MIM 605317) and show that NKX2-1 can also interact with the FOXP2 paralogs FOXP1 (MIM 605515) and FOXP4 (MIM 608924). The interaction between NKX2-1 and FOXP transcription factors may be of significance in brain and lung development. Heterozygous disruption of FOXP2 results in a severe speech/language disorder (MIM 602081). The presence of motor speech deficits as core features of both the NKX2-1 and FOXP2-related disorders points to roles for both genes in the development of speech-related motor circuitry. Strikingly, we find that disorder-related variants in the DNA-binding domains of both NKX2-1 and FOXP2 abrogate the interaction between these proteins.