De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder

Abstract

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

Keywords: Tousled-like; facial averaging; haploinsufficiency; intellectual disability; kinase.

Figure 1

Intragenic Variants and Balanced Translocation Identified in TLK2 (A) Location of TLK2 (GenBank: NM_006852.3) on chromosome 17q23.2 (see Supplemental Subjects and Methods for discussion about different TLK2 spliceforms). Vertical marks in TLK2 represent the 22 exons. Green arrow indicates region enlarged in panel below. (B) Schematic view (not to scale) of exons 11–22 and locations of 12 identified splice site mutations (green crosses). The splice site mutation inherited from an affected parent is shown in bold and green. The variant subjected to cDNA analysis is shown in the dark green rectangle. (C) Overview of TLK2 protein with the protein kinase domain (dark green) and three coiled-coil motifs (light green). Loss-of-function variants (24 total, including 8 nonsense, 4 frameshift, and 12 splice site mutations) are shown above the protein with green crosses indicating positions of splice site mutations. Other variants (11 missense variants and 2 nonsense variants causing a premature stop codon in the last exon) are shown below the protein. The frameshift mutation inherited from an affected parent is shown in bold and green. The variants subjected to cDNA analysis are shown in the dark green rectangles. (D) Balanced translocation between chromosomes 4 and 17, with the breakpoint disrupting TLK2 between exons 2 and 3, identified in one individual: 46,XX,t(4;17)(27;q23.2).seq[GRCh37]t(4;17)g.[chr4:pter_cen_122332907:: chr17:60,581,319_qter]g.[chr17_pter_cen_60,581,315::chr4:122,332,920_qter]. (E) Pedigrees of individuals with inherited variants and photographs of probands and their affected mothers. Both mothers have facial dysmorphism similar to their children. WT, wild-type at variant position.

Figure 2

Clinical Spectrum Associated with TLK2 Variants Overview of clinical features observed in individuals with TLK2 variants.

Figure 3

Facial Dysmorphism of Individuals with TLK2 Variants (A) Photographs of 21 unrelated individuals with a loss-of-function variant in TLK2, showing overlapping facial dysmorphism. Most frequently reported by clinicians were blepharophimosis, telecanthus, prominent nasal bridge, broad nasal tip, thin vermilion upper lip, and upward slanted palpebral fissures. Pointed and tall chin, epicanthal folds, narrow mouth, high palate, microtia, posteriorly rotated ears, long face, ptosis, and asymmetric face were observed in fewer than half of the individuals. (B) Photographs of seven unrelated individuals with a missense or C-terminal truncating variant in TLK2. Variant c.2170C>T (p.Arg724^∗) is assigned to this subgroup, since a premature stop codon is introduced in the last exon. Facial dysmorphisms overlapped with dysmorphism observed in individuals with loss-of-function variants. (C) Computational averaging of 33 facial photographs of 22 subjects with LOF variants in TLK2 (left) compared with 22 gender- and age-matched control subjects (right). (D) Computational averaging of 11 facial photographs of 8 subjects with missense or C-terminal truncating variants in TLK2 (left) compared with 8 gender- and age-matched control subjects (right).

Figure 4

Analysis of TLK2 Transcripts in Cell Lines (A) Analysis of transcripts encoding nonsense mutations c.989C>A (p.Ser330^∗) and c.2092C>T (p.Arg698^∗) in cell lines of affected individuals. Left panel shows reverse transcriptase-PCR (RT-PCR) products of cDNA prepared from fibroblast and lymphoblastoid cell lines of subject with p.Ser330^∗ variant, either in the presence (+C) or absence (−C) of cycloheximide and incubated with ApoI (digests wild-type allele). Central panel shows RT-PCR of cDNA prepared from lymphoblastoid cell line of subject with p.Arg698^∗ variant, in the presence (+C) or absence (−C) of cycloheximide and incubated with Hpy99I (digests wild-type allele). Right panel shows proportion (±standard deviation) of variant alleles quantified by deep sequencing of triplicate samples. Statistical testing of differences: ^∗p = 0.046; ^∗∗p = 0.011; NS, not significant. (B) Analysis of transcripts with canonical splice-site mutation c.1720+1G>T. A wild-type fragment at 300 bp in c.1720+1G>T lymphoblastoid cells is observed as well as a second fragment at 130 bp, which is absent in control cDNA. An increase of mutant transcript in cells was present when treated with cycloheximide (+C), indicating that the aberrant transcript was subject to NMD. Sequencing of the 300 bp (white box) and 130 bp (green box) fragments demonstrated skipping of exon 18 in the lower cDNA product. Abbreviations: Fibs, fibroblasts; EBV, lymphoblastoid cells; C/CHX, cycloheximide; WT, control cDNA.