Clinical application of whole exome sequencing reveals a novel compound heterozygous TK2-mutation in two brothers with rapidly progressive combined muscle-brain atrophy, axonal neuropathy, and status epilepticus
Introduction
A group of infantile or early-onset disorders, collectively termed mitochondrial DNA (mtDNA) depletion syndromes, is characterized by a massive reduction of mitochondrial DNA content. This is caused by a dysfunction of genes associated with mtDNA-synthesis and maintenance. Many of these patients suffer from a hepato-cerebral syndrome (Al-Hussaini et al., 2014). Encephalopathy concurs with myopathy in another subgroup of patients (Spinazzola et al., 2009), who carry mutations in SUCLA2 (ATP-dependent succinyl-CoA synthase), SUCLG1 (GTP-dependent succinyl-CoA synthase), RRM2B (p53-induced ribonucleotide reductase B subunit) and TK2 (thymidine kinase 2). The mitochondrial thymidine kinase 2 mediates the first step in the phosphorylation of deoxypyrimidine nucleosides required for mtDNA replication in the mitochondrial matrix. Disturbance of these phosphorylation steps results in overall mtDNA depletion (Zhou et al., 2008). The phenotypic spectrum of the TK2-related mtDNA-depletion syndrome (OMIM #609560) is broad and as many as 26 different missense and 9 InDel mutations (HGMD® public database, http://www.hgmd.cf.ac.uk) in 50 patients have been reported so far (Chanprasert et al., 2013). Patients typically present with a progressive muscle disease characterized by generalized hypotonia, proximal muscle weakness, loss of motor skills, feeding and respiratory difficulties (Saada et al., 2001). Respiratory failure due to weakness of the respiratory muscles leads to death in early childhood. TK2 mutations were initially thought to cause a purely myopathic form of mtDNA-depletion syndrome. However, subsequent reports have expanded the clinical spectrum including chronic external ophthalmoplegia, sensorineural hearing loss and early severe muscle weakness with encephalopathy and intractable epilepsy in single cases (Oskoui et al., 2006). Beyond that, milder forms with late- or adult-onset proximal muscle weakness and prolonged survival have been reported (Béhin et al., 2012, Chanprasert et al., 2013). The phenotypic range might be explained by the variability in the amount of residual activity of the mutant enzyme (Poulton et al., 2009).
Achieving an accurate diagnosis can be difficult and requires the coordinated interplay of clinical assessment, muscle histology, respiratory chain enzymology as well as molecular genetic analysis. Whole-exome sequencing (WES) has been used successfully in research to identify novel genes for many Mendelian disorders. Although still improving, WES has already arrived in the clinic and has become a powerful tool in the diagnosis of Mendelian disorders. Success of this approach is more frequent in consanguineous families since the search can be restricted to homozygous variants, but it is also possible for compound heterozygotes, especially if more than one affected patient can be investigated in a single family.
Here we report on two brothers with a fatal clinical course of early onset myopathy with profound loss of muscle mass, axonal neuropathy, respiratory failure as well as severe brain atrophy with status epilepticus. Although disease manifestation and course were dramatic, the diagnosis had been missed for several years because of the deviating phenotype. Finally, WES revealed the diagnosis of mitochondrial depletion syndrome due to a novel TK2 mutation.
Section snippets
Patient II-1
The boy was born as the first son of healthy non-consanguineous parents. Early motor and mental development was normal. At 5 months of age, the parents noticed loss of head control after an episode of acute illness with high fever two days after a vaccination. During the following weeks, generalized muscular hypotonia, weakness and a convergent squint became apparent. Deep tendon reflexes were attenuated. Serum CPK (3000–6500 U/l; normal range < 180) and blood lactate (35–45 mg/dl; normal range <
Identification of shared genotypes
In order to delineate regions of the genome that are shared by both affected individuals (‘identity-by-state’, IBS) and hence must harbor the disease mutation(s) (Nemesure et al., 1999), we first determined intervals with identical genotypes over the entire genome. The percentage of concordance was measured for all genotypes in the ‘variant calling format’ (*.vcf) file within a sliding window of 10 Mb, which was moved in 1 MB steps over the 22 autosomes (Fig. 2A). In a second step, we detected
Results
Identity-by-state analysis identified the chromosomal regions depicted on Fig. 2A and Supplementary Table 1, which comprised 274 Mb and 1781 protein-coding genes. The combination of WES and IBS-analysis with the results of MutationTaster revealed the following ‘disease-causing’ compound heterozygous variants (see also Supplementary Table 2): OR9G1: p.Y112C (rs4990194) | p.L286P (rs66943455), PYGL: p.I806L (rs34313873) | p.V600A, TK2: p.M117V | p.A139V (both not annotated in dbSNP).
OR9G1 encodes
Discussion
Here we investigated a family with two affected children with an extraordinary severe form of combined brain and muscular atrophy due to mtDNA depletion syndrome resulting from a novel compound heterozygous TK2 mutation.
Mitochondrial depletion syndromes are autosomal recessive disorders characterized by a reduction of mtDNA copy number in clinically affected tissues (Dimmock et al., 2010, Lesko et al., 2010) and by loss of enzymatic TK2 activity in isolated muscle or fibroblast mitochondria (
Acknowledgment
We thank the patients and their parents for participation at this study and Angelika Zwirner for excellent technical assistance. The project was funded by the Deutsche Forschungsgemeinschaft (DFG) via the SFB 665 TP C4. M.S. is a member of the NeuroCure Center of Excellence (DFG Exc 257) and of mitoNET (01GM1113D) financed by the German Bundesministerium für Bildung und Forschung.
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