5.2. 5656A>G is not a pathogenic mutation but a common variant in haplogroup U (II)

The nucleotide at position 5656 in mtDNA is a single non-coding nucleotide between the tRNA genes for alanine and asparagine (Anderson et al. 1981). The position is conserved between species, and in most cases it is occupied by an A. The transition 5656A>G has been reported at a low frequency in patients with diabetes mellitus (Thomas et al. 1996) and in a patient with familial progressive tubulointerstitial nephritis (Zsurka et al. 1997). The transition has been thought to be homoplasmic (Thomas et al. 1996). Interestingly, a heteroplasmic 5656A>G has been found in association with another mutation at nt 10010 in a patient with severe mitochondrial encephalomyopathy (Bidooki et al. 1997). The heteroplasmy at nt 5656 was verified by demonstrating a novel NheI restriction site in the presence of the mutant variant 5656G when DNA was amplified in the presence of a mismatched oligonucleotide primer (Bidooki et al. 1997).

We found that three out of six patients with migraine-associated occipital stroke harboured 5656A>G, but it was also found in 14 out of 83 healthy population samples from northern Finland, suggesting a population frequency of 17%. 5656A>G was exclusively associated with mtDNA haplogroup U and was found in 58% of all the haplogroup U samples. It appeared to be heteroplasmic when studied by restriction fragment analysis following NheI digestion. Surprisingly, we failed to detect the wild type genome when cloning DNA with apparent heteroplasmy. The nuclear-embedded mtDNA pseudogenes did not contribute to the erroneous identification of 5656A>G heteroplasmy. Finally, the apparent heteroplasmy was found to be due to inhibition of NheI by NaCl.