High levels of mitochondrial DNA heteroplasmy in single hair roots ...

Electrophoresis 2003, 24, 1159–1165 1159 

Tomasz Grzybowski 1 

Boris A. Malyarchuk 2 

Jakub Czarny 1 

Danuta Miścicka-Śliwka 1 

Roman Kotzbach 3 

1 The Ludwik Rydygier 

Medical University, 

Forensic Medicine Institute, 

Bydgoszcz, Poland 

2 Institute of Biological 

Problems of the North, 

Genetics Laboratory, 

Magadan, Russia 

3 The Ludwik Rydygier 

Medical University, Department 

of Obstetrics Nursing, 

Bydgoszcz, Poland 

High levels of mitochondrial DNA heteroplasmy in 

single hair roots: Reanalysis and revision 

The present study demonstrates a reinvestigation of the mitochondrial DNA sequence 

heteroplasmy, which was previously found by the use of nested polymerase chain 

reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach 

was used for the amplification of mitochondrial DNA and a phylogenetic analysis 

was applied to both data sets for the verification of the authenticity of sequences. The 

comparative analysis of the sequencing results obtained from the same hair DNA 

extracts – but using two different techniques – shows that direct mitochondrial DNA 

amplification results in a considerably lower number of mixed positions. The majority 

of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA 

hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic 

mutations observed in four extracts after nested PCR significantly differs from the pattern 

of natural mutations. Some of these rare polymorphisms should be revised as 

inconsistent with phylogenetic expectations. The results of the present study contribute 

to the earlier reports by indicating that phylogenetic analysis is an effective tool 

in a posteriori quality check of mitochondrial DNA data. 

Nucleic acids 

Keywords: Heteroplasmy / Human identification / Mitochondrial DNA EL 5326 

1 Introduction 

The analysis of mitochondrial DNA (mtDNA) sequence polymorphism 

has been widely used in human population and 

molecular evolution studies [1–4]. In recent years, mtDNA 

typing has also become a useful tool in forensic applications 

[5]. Among several aspects of mtDNA genetics, the existence 

of heteroplasmy seems to be one of the most widely 

discussed. There is a considerable evidence that mtDNA 

sequence heteroplasmy exists at an appreciable frequency 

and segregates differentially in different tissues [6–10]. 

Furthermore, it was demonstrated that in some tissues, 

including hair, heteroplasmy tends to occur at highly variable 

levels [11–14]. In a majority of cases, new heteroplasmic 

mutations are observed preferentially at hypervariable 

sites of the human mtDNA control region [15]. However, a 

number of studies documented the existence of heteroplasmy 

at positions that had a lower-than-average rate of 

evolutionary substitution [9, 16–18]. It is also stressed that 

the detection of heteroplasmy varies with regard to the 

employed analytical techniques [14, 16, 19]. 

Correspondence: Tomasz Grzybowski, The Ludwik Rydygier 

Medical University, Forensic Medicine Institute, ul. M. Curie- 

Skl / odowskiej 9, PL-85-094 Bydgoszcz, Poland 

E-mail: tgrzyb@amb.bydgoszcz.pl 

Fax: +48-52-585-3553 

Abbreviations: HV1, first hypervariable segment; HV2, second 

hypervariable segment; mtDNA, mitochondrial DNA 

Heteroplasmy in the mtDNA control region is one of the 

chief issues in forensic DNA community. It appears to be 

particularly important for forensic testing, where mtDNA 

types are compared between unknown evidence and 

reference samples. If heteroplasmy indeed affects multiple 

bases within the mtDNA control region of an individual 

and segregates variably among different tissues, then an 

effective interpretation of cases where heteroplasmy is 

encountered becomes more complicated [5, 20, 21]. 

However, it is also possible that some detectable heteroplasmic 

events constitute a fingerprint of artificial “phantom” 

mutations, i.e., mutations generated in the mtDNA 

typing process itself. Latest observations of Bandelt et 

al. [22, 23] and Röhl et al. [4] suggest that phantom mutations 

are widespread and affect mtDNA data presented in 

both evolutionary and forensic studies. 

A recent report on the very high levels of mtDNA heteroplasmy 

in the human mitochondrial DNA hypervariable 

region I (HV1) from single hairs [24] has been one of the 

most frequently debated. That report presents a list of 

24 heteroplasmic positions found in hairs of 13 individuals, 

employing a nested PCR strategy (hereinafter 

called “nested PCR”, 30132 cycles). This protocol was 

originally proposed and widely used by members of the 

forensic DNA community [25–28]. It was suggested that 

the very high levels of the observed heteroplasmy might 

had been caused by contamination and/or possibly by 

the amplification of nuclear pseudogenes [20, 21, 29]. It 

was also stressed that the interpretation of instances of 

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/07-0804–1159 $17.501.50/0

1160 T. Grzybowski et al. Electrophoresis 2003, 24, 1159–1165 

the observed heteroplasmy depended on the models of 

mtDNA mutation and of human population genetics [29, 

30]. In reply to recent studies concerning the high level 

of heteroplasmy, the authors have reinvestigated the earlier 

data [24]. Heteroplasmic samples have been retested 

and phylogenetic analysis has been applied to both data 

sets in order to verify the authenticity of sequences. 

2 Materials and methods 

2.1 Amplification and sequencing conditions 

Thirteen samples previously reported as heteroplasmic 

were reanalyzed. For the amplification of the hair DNA 

extracts, prepared in the previous study [24, 41], a direct 

PCR approach was used, amplifying the HV1 in two overlapping 

fragments (hereinafter called “direct PCR”). This 

technique is widely employed by forensic mtDNA laboratories 

[14, 28, 31–33]. The following two sets of primers 

were used for reanalysis: L15990 (5’ TTA ACT CCA CCA 

TTA GCA CC 3’); H16239 (5’ TGG CTT TGG AGT TGC 

AGT TG 3’); L16209 (5’ CCC CAT GCT TAC AAG CAA GT 

3’); H16391 (5’ GAG GAT GGT GGT CAA GGG AC 3’). In 

order to generate templates for sequencing both strands 

of the PCR products, the primers were “tailed” with a 

221M13 universal primer sequence, as described previously 

[24]. Amplification was performed in 50 mL reactions 

using a GeneAmp System 9600 thermal cycler (Perkin 

Elmer, Norwalk, CT, USA). Each amplification reaction 

comprised 16Promega buffer, 1.5 mM MgCl 2 , 200 mM of 

each of the dNTPs (Promega, Madison, WI, USA), 0.4 mM 

of each primer, 2.5 U of Taq Polymerase (Promega) and 

1 ng of the hair DNA extract. Thermal cycling conditions 

for primer pair L15990/H16239 were 2 min at 947C, 

followed by 38 cycles at 947C for 20 s, 627C for 20 s, 

and 727C for 30 s. The amplification with the primer pair 

L16209/H16391 was carried out under the cycling conditions 

given above, except for the annealing temperature 

and time, which in this case was 567C for 10 s. Negative 

PCR controls and reagent blank controls were always 

used. The amplification products were purified, sequenced 

and analyzed as described previously [24]. 

2.2 Phylogenetic classification of the HV1 

sequences 

The sequencing results obtained by a direct PCR were 

compared with those obtained by nested PCR (presented 

earlier) [24]. The phylogenetic analysis was applied to 

both data sets in order to verify the authenticity of mtDNA 

sequences. HV1 sequences were classified into haplogroups, 

according to the nomenclature suggested by 

Richards et al. [1, 3] and Macaulay et al. [34] for West Eurasian 

mtDNAs. All available data published on HV1-RFLP 

[3] or HV1 variability alone (summarized by Röhl et al. [4]) 

in West Eurasian populations were used for a comparative 

analysis. Data on the HV1/HV2 sequence variability 

in Poles were taken from an earlier study [35]. Variability 

patterns observed for hair samples in the original experiments 

and in the reanalysis were compared with data on 

molecular stability for each site in the HV1 region of West 

Eurasian populations [36]. A list of the HV1 mutational hotspots 

was used in accordance with Malyarchuk et al. [36]. 

2.3 Amplification of nuclear pseudogenes 

In order to further verify experimentally the suggestion 

that coamplification of nuclear pseudogenes may be a 

possible explanation of the high levels of heteroplasmy 

observed when using nested PCR, that amplification 

technique was applied to semen samples enriched in 

nuclear DNA. Semen samples were obtained from seventeen 

healthy donors and a differential lysis of sperm cells 

was performed according to Zischler et al. [37]. This resulted 

in obtaining a pelleted fraction of sperm nuclei 

and the remaining fraction enriched in mtDNA. DNA was 

isolated from both fractions by way of a standard organic 

extraction protocol. Both DNA extracts were amplified for 

HV1 and HV2, using the nested PCR strategy. Next, the 

amplification products were purified, sequenced and analyzed 

as described earlier [24]. The obtained sequences 

were inspected for possible unusual polymorphisms and 

subjected to a phylogenetic classification as described 

above. 

3 Results and discussion 

3.1 Comparative analysis of heteroplasmy 

levels obtained by two operational 

approaches 

A comparative analysis of mtDNA sequencing results, 

which were obtained from the same hair extracts by using 

the nested and direct PCR, shows a considerably lower 

number of mixed positions found after direct mtDNA 

amplification (Table 1). Out of the 13 samples reported previously 

as heteroplasmic, 5 were confirmed as such by a 

direct PCR (samples No. 2, 3, 4, 7, 11; see Table 1). Out 

of these, four sequence types have been found mostly 

in different West Eurasian populations, including Poles. 

Sample No. 11 reveals the sequence type which appears 

to be extremely rare among human populations, although 

homologous sequence types (e.g., profile 16063C- 

16069T-16126C) have been traced in Poles, Germans, Russians 

and Karelians [35, 38, 39]. The confirmation of the 

sequences by two independent techniques and the phylo-

Electrophoresis 2003, 24, 1159–1165 Reanalysis of mitochondrial DNA heteroplasmy 1161 

Table 1. Comparative analysis of mtDNA sequencing results obtained from the same hair extracts by the use of nested 

or direct PCR 

Sample 

Methods 

(PCR) 

HV1 sequence Haplogroup Frequency a) 

Heteroplasmy revealed in both direct and 

nested PCR 

Poland West Eurasia 

(n = 436) b) (n = 4072) c) 

2 Both 16183C, 16189C, 16223T, 16278C/T X 1 (0.2) 25 (0.6) 

3 Both 16129A, 16172C, 16223C/T, 16311C/T I 2 (0.5) 12 (0.3) 

4 Both 16129A/G, 16316A/G H 0 5 (0.1) 

7 Both 16129A, 16172C, 16223C/T, 16311C/T I 2 (0.5) 12 (0.3) 

11 Both 16063C, 16069T, 16126C/T, 16303A J* 0 N.A. 

Heteroplasmy revealed only after nested PCR 

9 Direct 16311C H or HV* 7 (1.6) 75 (1.8) 

Nested 16311C/T 

cont. Cambridge Reference Sequence (CRS) H or HV* 69 (15.8) 539 (13.2) 

5 Direct 16192T, 16256T, 16270T U5a 1 (0.2) 31 (0.8) 

Nested 16192C/T, 16256T, 16270T 

cont. 16256T, 16270T U5a 2 (0.5) 24 (0.6) 

8 Direct 16168T, 16343G U3 1 (0.2) 5 (0.1) 

Nested 16051A/G, 16168T, 16343G 

cont. 16051G, 16168T, 16343G U3 0 N.A. 

1 Direct 16311C H or HV* 7 (1.6) 75 (1.8) 

Nested 16024C/T, 16105C/T, 16243C/T, 16311C 

cont. 16024C, 16105C, 16243C, 16311C ? 0 0 

6 Direct 16311C H or HV* 7 (1.6) 75 (1.8) 

Nested 16126C/T (T .. C), 16311C 

cont. 16126C, 16311C ? 0 0 

10 Direct 16311C, 16354T H 0 2 (0.05) 

Nested 16088C/T, 16090C/T, 16140C/T, 16311C, 16354T 

cont. 16088C, 16090C, 16140C, 16311C, 16354T ? 0 0 

12 Direct 16111T, 16167C/T (T .. C), 16288C, 16362C H 0 0 

Nested 16111C/T, 16167C/T, 16288C/T, 16294C/T, 

16296C/T, 16362C/T 

cont. 16294T, 16296T ? 0 0 

13 Direct 16126C, 16163G, 16186C, 16189C, 16294C T1 6 (1.4) 65 (1.6) 

Nested 16125A/G, 16126C, 16163A/G, 16186C/T, 

16189C/T, 16294C/T, 16296C/T 

cont. 16125A, 16126C, 16296T ? 0 0 

Sample nomenclature is maintained according to the previous study [24]. HV1 sequences are given together with their 

haplogroup assignment and frequencies determined in human populations. Wherever discrepancies are observed between 

the results obtained by the use of both protocols, the supposedly contaminating sequence type (denoted as 

“cont.” ) is given with its frequency in the databases. This type is supposed to be the sequence of a hypothetical second 

homoplasmic DNA that would account for the heteroplasmy if it were present as a contaminant. 

a) The number of observations and frequency (in %) of the HV1 type revealed by direct PCR. “?” denotes that the phylogenetic 

affiliation of the HV1 sequence cannot be determined without additional data. “N.A.” denotes a sequence type 

which is not available in the West Eurasian database owing to the different sequencing range (16090–16365). 

b) Data are from Malyarchuk et al. [35] 

c) Data are from Richards et al. [3] 

genetic plausibility of the mtDNA profiles obtained, lead 

to the conclusion that the mixed positions revealed for 

the hair samples described are authentic and appear to 

be truly heteroplasmic in mitochondria. It should be 

emphasized that the maximum number of heteroplasmic 

sites within single individual for HV1 was two positions, 

which is in complete agreement with the findings of other 

authors who used similar analytical methods [14].


The second group of HV1 sequences includes those 

sequence types which are characterized by heteroplasmy 

revealed only after nested PCR analysis (Table 1). In the 

case of four of such samples (No. 9, 6, 5 and 8), phylogenetically 

related HV1 sequences were identified after the 

direct and nested PCR. The limited additional variability, 

appearing here after nested PCR, occurs against the 

background of defined haplogroups. Hence, it does not 

change the haplogroup assignment of sequences. Therefore, 

the results obtained after nested PCR are plausible 

and can be attributed to a unique sensitivity of the technique. 

It is generally accepted that even the hair of an individual 

with normally undetectable levels of heteroplasmy 

may show different mtDNA sequences [12, 40]. 

Due to the high number of cycles employed in nested 

PCR, it is likely that the existing minority variant has been 

amplified to a degree which made its detection possible 

by direct sequencing. It is noteworthy that similar observations 

(i.e., detection of additional heteroplasmic 

sites in sequences belonging to H or HV haplogroups) 

have recently been made by other forensic mtDNA laboratories 

[14]. 

The four remaining samples (No. 1, 10, 12 and 13) were 

characterized by patterns of heteroplasmic positions 

observable only after nested PCR. While corresponding 

sequences derived from direct PCR show no heteroplasmy 

and can be easily affiliated into mtDNA haplogroups 

(Table 1), the specific mutation patterns revealed 

after nested PCR make it impossible to localize the 

respective sequences as a part of phylogeny. Thus, this 

incompatibility would suggest that the mixed positions 

revealed in those samples after nested PCR could be artificial. 

3.2 Analysis of the HV1 mutational spectrum 

revealed in heteroplasmic samples 

Further insight into mutational spectra detected in heteroplasmic 

samples may be helpful in evaluating some of 

the results obtained by the use of nested PCR. A recent 

report by Malyarchuk et al. [36] provides a list of the 

HV1 mutational hotspots, which is in concordance with 

nucleotide positions that were described as rapidly evolving 

in the previous studies. If one assumes that heteroplasmy 

in somatic tissues has the same root cause as 

population-level polymorphism, then heteroplasmic point 

mutations should occur preferentially at mtDNA hypervariable 

sites (mutational hotspots). 

Table 2 shows heteroplasmic sites revealed both in the 

previous hair study and in this reanalysis, accompanied 

by their estimated variability. These positions can be subdivided 

into three groups. The first group includes posi- 

Table 2. Heteroplasmic sites revealed in the previous hair 

study [24] and the reanalysis presented, accompanied 

by their estimated variability 

Position 

Classification of 

heteroplasmy a) Population variability b) 

I II III Poland 

(n = 436) c) West 

Eurasia 

(n = 4072) d) 

16024 1 0 – 

16051 8 9.4 13.3 e) 

16088 10 0 0 e) 

16090 10 0 0 e) 

16105 1 0 0 e) 

16111 12 3.1 26.5 

16125 13 0 2.9 

16126 f) 11 6 3.1 41.2 

16129 f) 4 21.9 64.7 

16140 10 9.4 5.9 

16163 13 3.1 8.8 

16167 12 12 3.1 17.6 

16186 13 3.1 23.5 

16189 f) 13 40.6 94.1 

16192 f) 5 18.8 35.3 

16223 3, 7 6.3 29.4 

16243 1 3.1 14.7 

16278 f) 2 18.8 50.0 

16288 12 6.3 17.6 

16294 f) 13 12 12.5 32.4 

16296 12, 13 3.1 11.8 

16311 f) 3, 7 9 34.4 64.7 

16316 4 0 5.9 

16362 f) 12 28.1 64.7 

Heteroplasmy is classified into three groups, depending 

on the analytical technique by which it was revealed. 

a) Group I, heteroplasmy revealed after nested PCR and 

confirmed by direct PCR; Group II, nucleotide positions 

of sequence types revealed by direct PCR which 

were found to be heteroplasmic after nested PCR; 

Group III, new heteroplasmic variants revealed only 

after nested PCR. Sample numbers according to 

Table 1 indicate the presence of heteroplasmic position 

in the respective group. 

b) Frequency (in %) of parallel mutations which arose 

independently in different haplogroups of the previously 

reconstructed mtDNA phylogenetic tree. 

c) Data from Malyarchuk et al. [35] 

d Data from Richards et al. [3] 

e) Data from Malyarchuk and Derenko [50] 

f) Mutational hotspots 

tions observed after both direct and nested PCR. The 

second group includes nucleotide positions of sequence 

types revealed by direct PCR, which were found to be 

heteroplasmic after nested PCR. The third group includes 

newly arisen mutations revealed exclusively after nested


PCR. As shown in Table 2, the revealed heteroplasmic 

positions are characterized by a different molecular stability. 

Sites 16126, 16129, 16189, 16192, 16278, 16294, 

16311, 16362, indicated by Malyarchuk et al. as mutational 

hotspots [36], were found to be heteroplasmic 

mostly in the first and the second groups of positions. 

Additionally, sites 16051, 16111, 16140, 16163, 16186, 

16223, 16288, 16296, 16316 were detected as heteroplasmic 

and occurring independently in all three groups 

of nucleotide positions. According to Bandelt et al. [23], 

transitions for these sites were estimated to occur at a 

rate as high as the average HV1 transitional rate. Yet, the 

third group of mutations, newly arisen after nested PCR, 

includes a whole list of rare positions 16024, 16088, 

16090, 16105, 16125, which essentially differ from the 

mutational spectra reconstructed phylogenetically on the 

basis of population data (Table 2). As presented above, 

these observations on molecular stability of particular 

heteroplasmic sites are consistent with the evaluation of 

entire HV1 sequences. The pattern of heteroplasmic 

mutations, observed in samples No. 1, 10, 12 and 13 by 

the use of nested PCR, significantly differs from that of 

natural mutations. It is noteworthy that a number of previous 

studies [9, 16–18] documented also the existence of 

heteroplasmy at rare positions (e.g., 16104, 16205, 

16222, 16257, 16262, 16301). Therefore, in the light of 

the current data, the relative lack of population variability 

at particular mtDNA positions should not be raised as an 

argument to question any single heteroplasmic site. 

Unexpectedly, the combination of rare heteroplasmic 

sites was revealed in the mutation patterns of four samples 

tested by way of nested PCR. Together with the nonreproducibility 

of the results, this would suggest that 

some of those mutations might be artifacts resulting from 

specific conditions of nested PCR. 

3.3. Consideration of sample contamination 

There are several potential explanations of the apparent 

differences between the results obtained when using 

nested or direct PCR. First, contamination must be taken 

into consideration as a possible explanation of the nested 

PCR results. Although appropriate safety and control 

measures were taken in the previous hair study [41], 

nested PCR is known to be highly sensitive to contamination 

problems due to the large number of amplification 

cycles. However, Table 1 shows that all HV1 sequences, 

revealed after direct PCR, can be affiliated to certain mitochondrial 

haplogroups and almost all of these sequences 

are spread among human populations. If one suggests 

that it is due to mtDNA contamination that heteroplasmy 

after nested PCR appeared, then the contaminating 

sequence types should be observed in human populations. 

However, for a great majority of samples, the supposedly 

contaminating sequences do not match any population 

HV1 sequence types (Table 1). The only exception 

is the type corresponding to the most frequent European 

Cambridge Reference Sequence [42]. This may explain 

the appearance of heteroplasmy at position 16311 in 

sample No. 9 (Table 1). Therefore, given the combinations 

of rare positions occurring in the supposedly contaminated 

sequences, sample contamination does not seem 

to be a sufficient explanation of the obtained nested PCR 

results. 

3.4 Possibility of inadvertent pseudogene 

amplification 

Coamplification of nuclear pseudogenes can be another 

possible explanation of discrepancies observed between 

the results of nested and direct PCR. A recent analysis by 

Woischnik and Moraes [43] shows the presence of 612 

independent integrations of mtDNA fragments into the 

human genome. As it is reported, the amplification of 

nuclear DNA corresponding to the mtDNA HV1 region 

occurs under specific conditions [16, 44]. 

Since the efficiency of the possible pseudogene amplification 

may vary between individual primer pairs, an 

attempt to verify experimentally the potential of pseudogene 

amplification was made using primer pairs from the 

previous hair study [24]. To accomplish this, nuclear DNAenriched 

sperm nuclei fractions and mtDNA-enriched 

fractions were prepared by a preferential lysis of semen 

obtained from 17 subjects. Real-time DNA quantitation 

revealed that the quantity of mtDNA in the nuclear DNAenriched 

fraction was more than three times lower than 

that observed in the mtDNA-enriched fraction (data not 

shown). However, irrespective of the specific conditions 

favoring pseudogene amplification, mtDNA amplified efficiently 

from all nuclear DNA-enriched fractions. There 

were no differences found between sequences obtained 

from nuclear- and mtDNA-enriched preparations. It is 

important that no unique, unusual polymorphisms were 

revealed and the mtDNA sequences can be easily 

assigned to specific mtDNA haplogropups. This again 

demonstrates an extremely high sensitivity but also specificity 

of the nested PCR technique. To conclude, these 

results do not support the hypothesis that nuclear pseudogene 

amplification contributed to rare heteroplasmic 

positions observed after nested PCR. 

3.5 A possible PCR amplification error 

The accumulation of errors by Taq DNA polymerase during 

the template synthesis constitutes another possible 

mechanism which may explain some of the rare hetero-


plasmic positions revealed after nested PCR. This mechanism 

was previously considered by other authors reporting 

mtDNA heteroplasmy data [10, 16]. There are three 

reasons for which enzyme errors might contribute to 

some of the nested PCR results. The first reason is the 

lack of reproducibility of results for samples No. 1, 10, 12 

and 13 in which the group III mutations occurred (Table 2). 

The second reason is that specific conditions of nested 

PCR favor the occurrence of PCR replication errors. One 

cannot exclude these mechanisms where errors introduced 

by low-fidelity Taq polymerase in the first round 

of PCR and normally undetectable by direct sequencing, 

are eventually made visible due to the subsequent 

amplification for additional 32 cycles. One must also take 

into consideration the length of the first D-loop amplicon 

(1333 bp), which may not represent the most abundant 

size class of mtDNA available for amplification from the 

hair extracts. As it was demonstrated earlier, small amounts 

of template DNA, longer amplicons, additional amplification 

cycles and high enzyme concentrations may contribute 

to the increase of the error rate of a conventional 

PCR [45–47]. The third reason is that group III mutations 

do not respect phylogenetic hierarchy of the data (see 

Table 2). Moreover, Table 3 shows that the spectrum of 

group III mutations is biased toward transitions T–C, one 

of the most frequent errors made by the wild-type Taq 

polymerase I [48]. Finally, the majority of T–C transitions 

occurred on the 5’-GTand 5’-CTcontext, which may influence 

the rate of substitutions made by wild-type and 

mutants of Taq DNA polymerase [47–49] (Table 3). 

It must be stressed, however, that misincorporation during 

PCR is not proposed as a general explanation of 

all rare heteroplasmic events. This mechanism may contribute 

to a limited number of observations. On the other 

hand, heteroplasmy at rare positions does occur, as 

demonstrated by this study and other reports. In the face 

Table 3. Spectra of three groups of mutations revealed in 

the HV1 sequences from hair and the effect of 

5’-sequence context in T–C transitions 

Transitions Group I Group II Group III 

T–C 2 4 7 

C–T 3 5 2 

G–A 1 0 1 

A–G 1 1 1 

% of T–C out of 28.6 40.0 63.6 

all transitions 

5’-Sequence context in T–C transitions 

CT 0 2 2 

GT 2 2 3 

AT 0 0 2 

of these uncertainties, the exclusion of nested PCR from 

the techniques employed in forensic casework would 

be a more conservative approach. The interpretation of 

sequence data obtained by direct PCR (i.e., lower number 

of cycles) appears to be more straightforward and leads 

to unambiguous conclusions even in the presence of heteroplasmy. 

4 Concluding remarks 

While the general conclusions of the previous study on 

highly variable levels of heteroplasmy in hair are still warranted, 

this reanalysis strongly emphasizes a different 

ability of current protocols to reveal mixed sequences. 

The uniquely sensitive nested PCR technique leads to 

the detection of an additional variability, which may be difficult 

to interpret or in some instances, may appear to be 

an artifact. At the same time, this reinvestigation into the 

widely discussed data shows that phylogenetic analysis 

may be of a considerable value in a posteriori evaluation 

of authenticity and plausibility of the newly obtained 

mtDNA sequences. Phylogenetic rules, which are similar 

to those recently demonstrated as the effective tools in a 

quality control of mtDNA population data, can also be 

helpful in interpreting the difficult forensic cases in which 

heteroplasmy is encountered. 

Supplementary information. The electropherograms illustrating 

the differences between the detection of heteroplasmic 

positions with nested and direct PCR strategies 

are available online, http://www.zms-bydgoszcz. 

w.pl. This site also includes control region sequences 

found in nuclear- and mtDNA-enriched fractions of the 

semen samples. 

We would like to thank in particular Dr. Miroslava V. 

Derenko who has offered her valuable critical comments 

and constructive suggestions during the numerous discussions 

we have had together. 

Received October 26, 2002 

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High levels of mitochondrial DNA heteroplasmy in single hair roots ...

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