Evaluating sequence-derived mtDNA length heteroplasmy by amplicon size analysis.
Bottom Line: The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise.However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types.In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases.
Affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, 6020 Innsbruck, Austria.Show MeSH
Mentions: Length heteroplasmy (LH) describes the co-existence of at least two populations of mitochondrial (mt)DNA molecules in a DNA extract that differ in the number of nucleotides and therefore in their length. This generally leads to difficulties when Sanger sequencing data are to be interpreted, as the individual nucleotide sequences overlay after LH regions and appear as blurred pattern. While LH is usually observed in homopolymeric tracts when the number of identical adjacent nucleotides is greater than eight (for C-tracts), it has also been found in the AC-repeat and less often in shorter length variants of the control region (CR) [1–5]. In rare cases LH has even been observed in non-repetitive sequences . The human mtDNA CR comprises five regions where LH occurs regularly and it is most frequently observed in the polycytosine tracts of the two hypervariable regions, HVS-I and HVS-II (Fig. 1) [3,5,7–10]. More than half the samples of a systematic study on 5015 individuals exhibited length heteroplasmy somewhere in the control region , whereas typical frequencies for LH to appear in polycytosine tracts are 12–20%, 45%, and 3–5% for HVS-I, II and III, respectively [11–15]. Note that the frequency of LH in HVS-I can also reach much higher levels exceeding 50% in populations where the transition T16189C that leads to an uninterrupted polycytosine tract represents a common and haplogroup (hg)-specific signature mutation (e.g. hg B in East Asia and Native America ).
Affiliation: Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, 6020 Innsbruck, Austria.