Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*^†

Abstract

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA "alleles" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Length heteroplasmy of the polyC-polyT-polyC stretch in the dog mtDNA control region

“…Studies of the human CR show that the abundance of length variants of heteroplasmic polynucleotide regions often varies within and among tissues in individuals [3,6,10,11]. In contrast to human mtDNA studies, an intra-individual comparison of DNA from five hair shafts and one buccal swab of 16 dogs displayed inter-and intra-tissue stability of the ma/mi pCpTpC combinations.…”

“…As studies of the human CR have shown intra-individual sequence variation of heteroplasmic polyC stretches among and within tissues (e.g., [3,6,10,11]), it has also been recommended not to use length variation of these polynucleotide stretches as the sole reason to exclude DNA samples having the same maternal origin [6,[20][21][22].…”

“…In human mtDNA studies, these differences range from apparent homoplasmy to various ratios of the heteroplasmic mtDNA sequences [2][3][4][5][6][7][8][9][10][11][12]. Several hypotheses have been proposed to explain these observations, such as (1) a genetic bottleneck, i.e., the proliferation of an isolated subset of mtDNA molecules, during oogenesis or early embryogenesis introducing variation between a mother and her offspring [13,14]; (2) somatic mutations leading to variation between and within tissues such as buccal cells, muscle tissue, and hairs [3,4,15]; and (3) a genetic bottleneck in each hair follicle causing variation between hairs and other tissues, among and within hairs [5,7,9,16].…”

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy

“…This is similar to the mechanism that results in stutter products associated with nuclear DNA testing of short tandem repeat (STR) markers [19]. Similarly, stutter of the dinucleotide AC repeats at nps 515-524 of the mtDNA genome is observed as LHP [4,20,21]. Replication slippage can occur in vivo, but it also may arise during in vitro replication events including PCR amplification.…”