No-Cost Genetic Testing Program for Thymidine Kinase 2 Deficiency

Mitochondrial DNA (mtDNA) Depletion Syndromes (MDSs) are a group of clinically and genetically heterogeneous diseases characterized by a quantitative abnormality of the mitochondrial genome in specific tissues (Suomalainen and Isohanni. 2010. PubMed ID: 20444604; El-Hattab and Scaglia. 2013. PubMed ID: 23385875). The myopathic form of MDS has a wide clinical spectrum, with a typical age of onset that ranges from infancy to early childhood (Wang et al. 2012. PubMed ID: 23230576).

The most severe presentation of TK2-related MDS is characterized by infantile myopathy with motor regression, resulting in early death from respiratory failure (Oskoui et al. 2006. PubMed ID: 16908738). Clinical features may include hypotonia, proximal muscle weakness, decreased physical stamina, poor feeding, and respiratory difficulties. Other severe phenotypes have been reported, including a spinal muscular atrophy-like presentation, rapidly progressive proximal muscle weakness with elevated transaminases in liver tissue, and progressive myopathy with dystrophic changes (Oskoui et al. 2006. PubMed ID: 16908738; Lesko et al. 2010. PubMed ID: 20083405; Zhang et al. 2010. PubMed ID: 19815440; Collins et al. 2009. PubMed ID: 19736010).

TK2d is different from other mitochondrial diseases in that it predominantly, and sometimes exclusively, manifests as myopathy (muscle disease) (Wang et al. 2012. PubMed ID: 23230576). This severe muscle weakness affects different parts of the body and often manifests as respiratory weakness, ptosis, and/or progressive external ophthalmoplegia. Eventually, patients may lose the ability to walk, eat, and breathe independently.

In contrast, milder phenotypes of this disease have also been described, such as late-onset proximal muscle weakness and adult-onset progressive myopathy (Cámara et al. 2015. PubMed ID: 25948719).

The myopathic form of MDS is an autosomal recessive disorder caused by defects in the TK2 gene (Saada et al. 2001. PubMed ID: 11687801). TK2 has 10 exons that encode thymidine kinase, a mitochondrial deoxyribonucleoside kinase required for mtDNA synthesis. Pathogenic missense, nonsense, and splicing site variants, in addition to small deletion and insertion events, have been found across the whole coding region of TK2. Arg130Trp is a pathogenic Finnish founder variant associated with one of the most severe phenotypes (Götz et al. 2008. PubMed ID: 18819985, referred to as R172W). One large intragenic deletion (encompassing exons 1 and 2 of TK2) and one complex rearrangement have also been documented (Zhang et al. 2010. PubMed ID: 19815440; Stewart et al. 2011. PubMed ID: 21138766). Compound heterozygous TK2 pathogenic variants have also been reported to result in a rare form of adult-onset autosomal recessive progressive external ophthalmoplegia (arPEO), which is more frequently caused by defects in the POLG gene (Tyynismaa et al. 2012. PubMed ID: 21937588). Additionally, pathogenic variants in RNASEH1 or RRM2B have also been reported as a rare cause of arPEO (Reyes et al. 2015. PubMed ID: 26094573; Pitceathly et al. 2012. PubMed ID: 23107649).

These tests are performed via Sequencing and CNV Detection via NextGen Sequencing using exome sequencing.

The single gene test (TK2 only, Test 12029) provides full coverage of all coding exons of the TK2 gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing.

TK2 deficiency may be difficult to diagnose due to symptoms that may overlap with other conditions, including the limb girdle muscular dystrophies and mitochondrial depletion syndromes. Therefore, a larger next generation panel test is also offered. This test (55 genes, Test 12031) typically provides ≥98% coverage of all coding exons of the genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions where pathogenic variants have been identified. We define coverage as ≥20X NGS reads or Sanger sequencing.

Candidates for TK2 testing (Test 12029):

• Patients with suspicious features of TK2 deficiency
• Patient has not had previous genetic testing for TK2 (either as single gene or as part of any previous panel testing)

Candidates for larger panel testing (Test 12031) must have both criteria above for TK2 testing. In addition candidates must have:

• Patient has not yet had previous testing for Limb Girdle Muscular Dystrophy or Mitochondrial Depletion Syndromes
• Patient has clinical features suggestive of either Limb Girdle Muscular Dystrophy or Mitochondrial Depletion Syndrome

Specimen Collection

WHOLE BLOOD

Collect 3 ml - 5 ml of whole blood in EDTA (purple top tube) or ACD (yellow top tube).

Saliva

Oragene™ or GeneFiX™ Saliva Collection kit used according to manufacturer instructions. DNA from saliva specimens is invariably contaminated with microbial and food DNA, which can impact specimen quality and may result in delayed testing and/or the need for a second specimen.

OCD-100 Buccal Swab

OCD-100 Buccal Swab used according to manufacturer instructions.

Buccal

Specimens may be shipped at room temperature.

Specimen collection kits: Buccal specimen collection kits, which contain the TRF and the shipping label, may be requested through the kit order form or via the online order form.

Shipping and Handling Instructions

Label all specimen containers with the patient's name, date of birth, and/or ID number. At least two identifiers should be listed on specimen containers. Specimen deliveries are accepted Monday-Saturday for all specimen types. Holiday schedules will be posted on our website at least one week prior to major holidays.

WHOLE BLOOD

At room temperature or refrigerated, a blood specimen is stable for up to 8 days. Include a refrigerated gel pack in the shipping container. Fresh blood specimens are preferred. If frozen, a blood specimen is stable for up to 1 month before shipping. Frozen blood specimens should be shipped frozen (preferably on dry ice) overnight.