De novo TLK1 and MDM1 mutations in a patient with a neurodevelopmental disorder and immunodeficiency

Abstract

The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants underlie the neurodevelopmental disorder (NDD) 'Intellectual Disability, Autosomal Dominant 57' (MRD57), characterized by intellectual disability and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400∗). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions. Our study provides the first functional characterization of NDD-associated TLK1 variants and suggests that, such as TLK2, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Keywords: genetics; immunology; neuroscience.

Graphical abstract

Figure 1

Neurodevelopmental defects in a patient with TLK1 and MDM1 variant alleles (A–D) T2/FLAIR axial (A, B), sagittal (C), and coronal (D) sections from age 14 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 Table S2. (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 Table S1. (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 and D). Data are presented as mean ± SEM.

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 Table S2. 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). Data are presented as mean ± SEM.

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 h 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 the 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). Scale bar = 50 μM. (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). Data are presented as mean ± SEM.

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.^, (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. Full results provided in Table S4 and additional data in Figure S4.