Identification of a de novo mutation in TLK1 associated with a neurodevelopmental disorder and immunodeficiency

Abstract

Background: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with 'Intellectual Disability, Autosomal Dominant 57' (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown.

Methods: A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Single cell gel electrophoresis, western blot, flow cytometry and RNA-seq were performed in patient-derived lymphoblast cell lines. In silico, biochemical and proteomic analysis were used to determine the functional impact of the p.Q479E variant and previously reported NDD-associated TLK1 variant, p.M566T.

Results: Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling. Cells expressing the p.Q479E variant exhibited reduced cytokine responses and higher levels of spontaneous DNA damage but not increased sensitivity to radiation or DNA repair defects. The p.Q479E and p.M566T variants impaired kinase activity but did not strongly alter localization or proximal protein interactions.

Conclusion: Our study provides the first functional characterization of TLK1 variants associated with NDDs and suggests potential involvement in central nervous system and immune system development. Our results indicate that, like TLK2 variants, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Keywords: Neurodevelopmental disorder; STAT3; TLK1; TLK2; immunodeficiency; microcephaly.

Figure 1:

T2/FLAIR axial (A, B), sagittal (C), and coronal (D) sections from age 10–15 years shows symmetric periventricular and subcortical leukoencephalopathy with mild cystic white matter encephalomalacia, ex vacuo ventriculomegaly, and thin corpus callosum.

Figure 2:. TLK1 and MDM1 variant alleles and impact on protein levels.

A. Normalized read counts for the indicated genes from RNA-seq of transformed LCLs of the Sibling or Proband. N=2, mean and standard deviation are shown. Full details in online supplementary table S3. B. Allele specific expression of the indicated genes in the Proband inferred from RNA-seq data. N=2, mean and standard deviation are shown. Full details in online supplementary table S4. C. Representative blot of protein levels of TLK1. Quantification of western blots (N=5) is shown in right panel normalized to Vinculin or Actin. D. Representative blot of protein levels of MDM1 and quantification of western blots (N=4) is shown in right panel normalized to Vinculin or Actin. E. Representative blot of ASF1A and ASF1A-pS166 (ASF1A-p) levels and quantification of western blots (N=2) is shown in right panel. F. Representative blot NEK1-pS141 (P-NEK1) levels and quantification of western blots (N=3) is shown in right panel. N represents biological replicates, statistical significance was determined using an unpaired t test with Welch’s correction (*P<0.05) in panels C,D.

Figure 3:. Gene expression differences identify defects in STAT3 signaling in patient-derived cell lines.

A. Volcano plot of RNA-seq data depicting DEGs in the Proband compared to Sibling LCLs. Duplicate samples from each LCL line were analyzed. Full data in online supplemental Table S3. Genes in each enriched category identified in IPA analysis (B) are shown in the indicated color. Hepatic fibrosis genes overlap with Osteoarthritis and are therefore not shown. B. Ingenuity Pathway Analysis (IPA) of RNA-seq data is shown. C. Heatmap of individual genes from the indicated enriched pathway are shown. D. Analysis of STAT3 phosphorylation on Y705 in response to IL6 in LCLs. Quantification of 4 independent experiments is shown below the western blot. STAT3-p (Y705)/STAT3 was normalized to vinculin and samples were normalized to Sibling -IL6. Statistical significance was determined using an unpaired t test with Welch’s correction (*P<0.05).

Figure 4:. Analysis of cell cycle and DNA damage in patient cell lines.

A. Relative cell growth of the Proband and Sibling LCLs. N=6. B. Example of representative flow cytometry data to analyze cell cycle in LCLs. Cells were pulsed with 10 μM EdU for 1 hour and stained with DAPI for DNA content. C. Quantification of cell cycle phases from N=2 independent experiments with 2 biological replicates each. Replicates from same experiment indicated with a triangle or circle. D. Western blot analysis of p53 and p21 levels in LCLs. E. Quantification of p53 (N=4) and p21 (N=3) levels from biological replicates. F. Representative images of alkaline comet assays untreated or treated with the indicated dose of ionizing radiation (IR). G. Analysis of tail moment with the indicated treatments and recovery times. Representative of N=2 independent experiments. H. Analysis of tail moment in neutral comet assays. Representative of N=2 independent experiments. At least 100 comets were analyzed per condition and experiment. Statistical significance was determined using an unpaired t test with Welch’s correction (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

Figure 5:. Conservation analysis, kinase activity and proximal interactions of NDD associated TLK1 variants.

A. Consurf was used to analyze the level of conservation of the amino acids of human TLK1, with both Q479 and M566 scoring as highly conserved. The predicted structure of the TLK1 kinase domain (Alphafold) is shown with the location of the two residues highlighted in pink(42,43). B. Representative in vitro kinase assay of Streptavidin purified TLK1-WT or NDD variants on purified ASF1A. TLK2-KD is used as a negative control. C. Quantification of N=4 independent kinase assay experiments. D. Western blotting of transfected BioID constructs and biotin labeling imaged with Streptavidin. Ponceau is provided as a transfer control. E. Network depiction of the proximal interactors identified with TLK1 with physical interactions indicated by solid lines. Proteins identified on nascent DNA at replication forks by iPOND-MS and proteins in the Simon’s Foundation Autism Research Initiative (SFARI) or DECIPHER databases are indicated(44). Full results provided in online supplementary table S6 and additional data in online supplementary figure S4.