Early onset or syndromic epilepsy

Gene: DTYMK

Green List (high evidence)

The rating of this gene has been updated to Green and the mode of inheritance set to 'BIALLELIC, autosomal or pseudoautosomal' following NHS Genomic Medicine Service approval.

Created: 1 Feb 2023, 9:39 a.m. | Last Modified: 1 Feb 2023, 9:39 a.m.

Panel Version: 3.29

Green List (high evidence)

Comment on list classification: There is enough evidence for this gene to be made green at the next major review.

Created: 14 Apr 2022, 12:12 p.m. | Last Modified: 14 Apr 2022, 12:12 p.m.

Panel Version: 2.513

Four variants have been reported in three unrelated cases (total of four cases), together with supportive functional studies (PMID:31271740; 34918187; 35346037). Seizures were seen in two of the cases (PMID:31271740; 34918187).

Created: 14 Apr 2022, 12:04 p.m. | Last Modified: 14 Apr 2022, 12:04 p.m.

Panel Version: 2.512

Green List (high evidence)

4 individuals (from 3 families) harboring biallelic DTYMK pathogenic variants have been reported.

Consider inclusion in the current panel with green rating given consistent and relevant phenotype and evidence provided to date [effect of variants (LoF), pathogenesis, similar phenotypes in zebrafish model, etc].

Relevant studies are summarized below.
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Lam et al (2019 - PMID: 31271740) described two siblings aged 25m and 7y, harboring biallelic DTYMK variants.

The phenotype consisted of hypotonia, congenital microcephaly, DD, severe ID. Other shared features included raised serum lactate, pyruvate and alanine. The phenotype was more pronounced in the younger one (epilepticus during febrile illness, epilepsy on multiple anti-convulsants, evidence of regression, etc). Brain MRI revealed marked cerebral atrophy among the findings while a lactate peak was present in spectroscopy. The elder brother developed an episode of sudden onset coma with respiratory failure at the age of 7y.

Quartet WES identified compound heterozygosity for a fs and a missense DTYMK variant (NM_012145.3:c.287_320del / p.Asp96Valfs*8 - c.295G>A / p.Ala99Thr). There were no additional findings. Previous genetic panel analysis for epilepsy was unremarkable for the 1st sib.

There are two pathways for synthesis of dNTPs, the de novo pathway operating in the cytosol only and the salvage operating in both cytosol and mitochondria. DTYMK encodes (deoxy)thymidylate kinase which catalyzes conversion (phosphorylation) of dTMP to dTDP - a step right after convergence of both pathways - in the dTTP synthesis pathway.

Mutations in TK2, an enzyme phosphorylating thymidine in mitochondria to dTMP have been associated with mitochondrial DNA depletion syndrome (MDDS).

Given this and as the 2 sibs had raised serum lactate and pyruvate, the authors performed in silico analyses to calculate mtDNA/nDNA ratio dividing the respective read depths for mitochondrial and nuclear DNA obtained from WGS data of the two sibs (blood).

This ratio was shown to be reduced in the more severely affected sib (65.5% of control) although this was not the case for the mildly affected brother (114.6%). As a control a non-MDDS mitochondrial cytopathy sample (corresponding to m.8993T>G) was used. The respective ratio which was calculated for a known POLG-related MDDS case was 15.6%.
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Vanoevelen et al (2022 - PMID: 34918187) describe two unrelated children with hypotonia, absence of developmental progress, microcephaly, seizures (recurrent febrile seizures/myoclonic jerks). Severe cerebral atrophy (with unaffected cerebellum) was observed upon brain imaging. Other findings included puffy body/extremities. Both had complications following respiratory illness leading to demise. CNS pathology in the 1st individual revealed massive neuronal dropout, with sparing of dentate nucleus and brainstem.

CMA in both cases was normal. This was also the case for extensive metabolic investigations (which provided no evidence of eventual mitochondrial dysfunction).

WES revealed compound heterozygosity for 2 missense variants in the first individual (NM_012145.3:c.382G>A - p.Asp128Asn and c.242C>T - p.Pro81Leu). The second individual, born to consanguineous parents, was homozygous for c.242C>T / p.Pro81Leu.

In silico predictions varied although each variant were (mostly) suggestive of a deleterious effect.

Variants were both ultrarare without homozygotes in ExAC,.

The authors generated a dtymk ko zebrafish model (hmz for a frameshift variant). Zebrafish exhibited markedly smaller eyes and pericardiac edema (3dpf-), twitching movements somewhat reminiscent of epilepsy (at 3dpf), prominent edema of brain and intestine. Head size was significantly smaller at a timepoint prior to brain edema (also after correction for length). Histology provided evidence of empty spaces in brain, suggestive of neurodegeneration, with high amounts of apoptotic cells.

dTMPK activity was measured in zebrafish (at 5dpf) as well as in fibroblasts from one individual and in both cases, it was barely detectable and significantly lower compared to wt/htz zebrafish or to the activity in fibroblasts from the parents of the individual tested.

In fibroblasts from the same individual with comparison to his parents, the authors demonstrated that DNA replication was impaired (using pulse-EdU staining to quantify cells in S-phase).

Assessment of cell proliferation in the brain of dtymk ko zebrafish using phospo-Ser10-Histone H3 (pH3) staining was suggestive of severe proliferation defects in forebrain.

Impaired biosynthesis of nucleotides for DNA synthesis/repair would be predicted to result in nucleotide pool imbalance, leading to incorporation of ribonucleotides in genomic DNA with - in turn - impairment of DNA replication and genomic instability (sensitivity to strand breakage).

In line with this, genomic DNA of ko zebrafish following alkaline hydrolysis and alkaline gel electrophoresis was shown to migrate at lower position and to be more fragmented indicating increased sensitivity (due to incorporation of ribonucleotides).

Visualization of DNA breakage by γH2AX staining, following UV-irradiation of zebrafish embryos revealed persistence of elevated γH2AX levels and DNA damage response signaling, interpreted as increase in unrepaired DNA breaks.

mtDNA copy numbers in fibroblasts from the affected individual was somewhat but not significantly lower compared to his parents. Importantly, the copy numbers were similar to controls (N=5) which overall does not support mtDNA depletion as a consequence of DTYMK deficiency.

Integrity of mtDNA did not appear to be compromised , with the mitochondrial genome migrating at the expected length of 16,5 kb with no indications of mtDNA deletions for both affected individual and his parents.

Activity of the mitochondrial respiratory complexes I-V in fibroblasts from the affected individual was comparable to that of his parents.

Overall, there was no evidence for mtDNA depletion (although not studied in muscle biopsy) while functional studies failed to demonstrate mitochondrial dysfunction.

The authors discuss other disorders of impaired dTTP metabolism due to mutations in TYMP, RRM2B or CAD.
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In a recent study using zebrafish model, Hu Frisk et al (2022 - PMID: 35346037) further demonstrate that Dtymk is essential for neurodevelopment providing evidence for expression of a compensatory thymidylate kinase-like enzyme at later stages of development (explaining survival of ko dtymk zebrafish despite the central role of this enzyme in dTTP generation). [Not further reviewed]
Sources: Literature

Created: 10 Apr 2022, 11:33 p.m.

Mode of inheritance
BIALLELIC, autosomal or pseudoautosomal

Phenotypes
Global developmental delay; Intellectual disability; Microcephaly; Seizures; Global brain atrophy; Cardiorespiratory arrest

Publications

• 31271740
• 34918187
• 35346037