The mean mtDNA content in blood samples was reduced to 78% of the control value in samples from individuals with POLG mutations, 52% in those with DGUOK or MPV17 mutations, 79% in those with TK2 mutations, 70% in those with TYMP mutations, and 39% in those with RRM2B mutations (see Table 2A in the online Data Supplement).
The mtDNA content was not reduced in blood samples from 97 symptomatic individuals with a single mutation or unclassified variant in POLG, DGUOK, TK2, or TYMP (mean mtDNA content 105%, 98%, 118%, and 141% of the age-matched pooled control value, respectively) (Fig.
We sequenced DNA from 1 liver, 9 blood, and 10 muscle samples with an mtDNA content <30% of the matched pooled control by the Sanger method for mutations in the POLG, DGUOK, MPV17, and TK2 genes.
A sequencing analysis of the POLG, TYMP, TK2, RRM2B, DGUOK, and SUCLA2 genes from a blood sample revealed no deleterious mutations.
Most of these samples had been screened for mutations in the POLG, DGUOK, MPV17, and TK2 genes and were negative for deleterious mutations.
Notable is the mtDNA depletion (19% of control) we observed in both the liver sample and the muscle sample from a patient with myopathic MDDS caused by TK2 mutations.
New mutations in TK2 gene associated with mitochondrial DNA depletion.
To check that no TK2 enzyme was present in the crude extract, we performed rod electrophoresis, followed by REA detection with the prolifigen[R] TK REA assay (DiaSorin AB), which detects both TK1 and TK2 (32).
The cross-determination of human recombinant TK2 activity, with AZT as a substrate, compared with hrTK1 was <0.
More importantly, the substrate specificity of TK1 differs from that of TK2; AZT is much less efficiently phosphorylated by TK2 than TK1 (4,5).
Our estimates of cross-determination of TK2 activity (<0.
4] Nonstandard abbreviations: TK1 and TK2, thymidine kinase 1 and 2, respectively; AZT, 3'-azido-2',3'-dideoxythymidine; REA, radioenzyme assay; AZTMP, 3'-azido-2',3'-deoxythymidine 5'-monophosphate; AZXMP, 3'-azido2',3'-deoxy-5'-[monophosphate-.