Frequency and Pattern of Heteroplasmy in the Control Regionof Human Mitochondrial DNA

Cristina Santos Æ Blanca Sierra Æ Luis Alvarez Æ Amanda Ramos ÆElisabet Fernandez Æ Ramon Nogues Æ Maria Pilar Aluja

Received: 12 May 2007 / Accepted: 13 June 2008 / Published online: 11 July 2008

� Springer Science+Business Media, LLC 2008

Abstract In this work, we present the results of the

screening of human mitochondrial DNA (mtDNA) hetero-

plasmy in the control region of mtDNA from 210 unrelated

Spanish individuals. Both hypervariable regions of mtDNA

were amplified and sequenced in order to identify and

quantify point and length heteroplasmy. Of the 210 indi-

viduals analyzed, 30% were fully homoplasmic and the

remaining presented point and/or length heteroplasmy. The

prevalent form of heteroplasmy was length heteroplasmy in

the poly(C) tract of the hypervariable region II (HVRII),

followed by length heteroplasmy in the poly(C) tract of

hypervariable region I (HVRI) and, finally, point hetero-

plasmy, which was found in 3.81% of the individuals

analyzed. Moreover, no significant differences were found in

the proportions of the different kinds of heteroplasmy in the

population when blood and buccal cell samples were com-

pared. The pattern of heteroplasmy in HVRI and HVRII

presents important differences. Moreover, the mutational

profile in heteroplasmy seems to be different from the

mutational pattern detected in population. The results sug-

gest that a considerable number of mutations and,

particularly, transitions that appear in heteroplasmy are

probably eliminated by drift and/or by selection acting at

different mtDNA levels of organization. Taking as a whole

the results reported in this work, it is mandatory to perform a

broad-scale screening of heteroplasmy to better establish the

heteroplasmy profile which would be important for medical,

evolutionary, and forensic proposes.

Keywords mtDNA � Point heteroplasmy �Length heteroplasmy � Blood � Buccal cells

Introduction

The mitochondrial genome (mtDNA) of humans as well as of

other mammalians has been considered to be useful in pop-

ulation genetics, phylogeographic, and phylogenetic studies.

Besides the usually invoked mtDNA characteristics—high

copy number per cell, compact organization, and maternal

transmission—mtDNA has been widely used because it

provides easy access to an orthologous set of genes with little

or no recombination and rapid evolution. Moreover, from a

theoretical perspective, it has long been accepted that

mtDNA haplotype frequencies are controlled primarily by

migration and genetic drift and that most of the variation

within a species is selectively neutral (Ballard and Rand

2005). However, more recent reports have challenged these

traditional views and many works have been published

pointing out the possibility of recombination of mtDNA

(Kraytsberg et al. 2004; Zsurka et al. 2005), sustaining the

hypothesis that mtDNA frequency variation is due to natural

selection (Mishmar et al. 2003; Wallace et al. 2003; Ruiz-

Pesini et al. 2004) and that mtDNA heteroplasmy is not an

exceptional condition related to mitochondrial disease

(Monnat and Loeb 1985), and many cases of healthy het-

eroplasmic individuals have been described (Bendall et al.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-008-9138-9) contains supplementarymaterial, which is available to authorized users.

C. Santos (&) � B. Sierra � L. Alvarez � A. Ramos �E. Fernandez � R. Nogues � M. P. Aluja

Biological Anthropology Unit, Department BABVE,

Faculty of Sciences, Autonomous University of Barcelona,

08193 Bellaterra (Barcelona), Spain

e-mail: cristina.santos@uab.cat

C. Santos

Center for Research in Natural Resources (CIRN), University of

the Azores, 9500 Ponta Delgada, S. Miguel, Azores, Portugal

123

J Mol Evol (2008) 67:191–200

DOI 10.1007/s00239-008-9138-9

1996, 1997; Wilson et al. 1997; Grzybowski et al. 2003;

Brandstatter et al. 2004; Santos et al. 2005).

The problem of mtDNA heteroplasmy in healthy indi-

viduals has been investigated particularly in the context of

forensic genetics since it could suppose an obstacle for the

correct individual identification (Budowle et al. 2003;

Melton 2004) determined both for the presence of hetero-

plasmy per se and for the variation in the levels of

heteroplasmy between tissues (Paneto et al. 2007). How-

ever, in recent years it has merited more attention from

population and evolutionary genetics with the recognition

that heteroplasmy is an obligatory, if transient, phase in the

evolution of mtDNA (Rando et al. 1991; Howell et al.

2003; Santos et al. 2005, 2008).

Recently, we reported on the mtDNA mutation rate of the

D-loop (Santos et al. 2005), using an empirical approach and

we advocated that one of the key points in mtDNA evolution

is the transition of a new mutation from a heteroplasmic to a

homoplasmic state. Moreover, in this previous study we also

found some evidence for differences between the mutational

pattern observed at the population level and that observed in

heteroplasmy, however, our previous data did not enable us

to reach definitive conclusions and a broad population

analysis of heteroplasmy was necessary.

In this work, we screened heteroplasmy in the control

region of mtDNA from 210 unrelated Spanish individuals.

The study’s main goals were (a) to characterize the level

and pattern of heteroplasmy in the general population for

both HVRI and HVRII and (b) to compare the mutational

pattern of mtDNA in heteroplasmy with that observed at

the population level.

Materials and Methods

Samples and DNA Extraction

A total of 210 unrelated Spanish individuals were sampled.

For 146 individuals blood samples were collected, whereas

for the remaining 64 individuals buccal cells were

obtained. Total DNA was extracted using the JETQUICK

Blood DNA Spin Kit (Genomed). Voluntary donors were

asked for informed consent and to fill in an inquiry con-

cerning the birthplaces of all their known ancestors, to

attest that they were unrelated.

mtDNA Analysis

The mtDNA control region (from position 15907 to position

580, according to the Cambridge reference sequence [CRS])

was amplified using the primers L15907 (Santos et al. 2005),

50-ATACACCAGTCTTGTAAACC-30, and H580 (Heyer

et al. 2001), 50-TTGAGGAGGTAAGCTACATA-30. PCR

was performed as described by Santos et al. (2005). PCR

products were purified using the JETQUICK PCR Purifica-

tion Spin Kit (Genomed).

All samples were fully sequenced between positions

16024–16569 and positions 1–400. Seven internal primers

were used in sequencing reactions: three in the direct direc-

tion (L1_15978, 50-CACCATTAGCACCCAAAGCT-30,L1_16190, 50-CCCCATGCTTACAAGCAAGT-30; and L2_1

6485, 50-GAACTGTATCCGACATCTGG-30) and four

in the reverse direction (H1_16166, 50-GGTTTTGATGTG

GATTGGGT-30; H1_8, 50-GGTTAATAGGGTGATAG

ACC-3; H2_288, 50-GGGGTTTGGTGGAAATTTTT-30;and H2_481, 50-GATTAGTAGTATGGGAGTGG-30). All

the primers used in the sequence process, except L1_15978

(Pereira et al. 2000), were previously described by Santos

et al. (2005). Sequence reactions were carried out using the

sequencing kit BigDye Terminator v.3 (Applied Biosys-

tems) according to the manufacturer’s specifications and run

in an ABI Prism 3100 sequencer.

Detection and Authentication of Heteroplasmies

The detection and authentication of mtDNA heteroplasmy

were performed following a strategy similar to that used by

Santos et al. (2005, 2008), with six main steps.

1. DNA extraction, PCR amplification, and sequencing of

the control region of mtDNA are carried out; if

necessary, the amplification and sequencing process

was repeated to ensure sequences with a good intensity

and with almost imperceptible backgrounds.

2. Sequences obtained in the first step were analyzed using

the Sequencing Analysis software (Applied Biosys-

tems), considering a value of 2% in the option of Mixed

Base Identification. Moreover, all the sequences were

visually verified and compared with others from the

same run to detect the presence of heteroplasmy.

3. Individuals that appeared to present heteroplasmy in

step 1 were confirmed by a second PCR amplification

and sequencing.

4. Sequences obtained in the third step were analyzed as

described in step 2.

5. To authenticate results an independent DNA extraction,

PCR amplification, and sequencing were subsequently

performed for all individuals showing heteroplasmy.

6. The control region of those samples for which a more in-

depth study was of interest were cloned into the pCR 4-

TOPO vector, using the TOPO TA Cloning Kit for

Sequencing (Invitrogen). Forty clones were sequenced

using the previously explained methodology.

In previous works (Santos et al. 2005) we were able to

detect mix bases \5% using automated sequencing,

whereas cloning experiments enable the detection of levels

192 J Mol Evol (2008) 67:191–200

123

of heteroplasmy of 1%. Thus, in this work, we assume that

our methodology, based only on mtDNA automated

sequencing, enables the detection of levels of hetero-

plasmy [5%. Moreover, our authentication methodology

prevents the inclusion of false-positive results.

Data Analysis

Sequences without ambiguities were obtained between posi-

tions 16024–16569 and positions 1–400 and they were aligned

in relation to the CRS (Anderson et al. 1981), using BioEdit

(Hall 1999). Classification of all the samples into haplogroups

was performed according to the nomenclature summarized by

Richards et al. (2000) and Salas et al. (2004).

To establish the control region heteroplasmic profile,

previous works devoted to the analysis of heteroplasmy in

which both HVR regions were sequenced (Comas et al.

1995; Bendall et al. 1996; Wilson et al. 1997; Parsons

et al. 1997; Sigurðardottir et al. 2000; Tully et al. 2000;

Grzybowski et al. 2003; Howell et al. 2003; Brandstatter

et al. 2004; Santos et al. 2005 and present study) were

compiled. For comparison, the phylogenetically recon-

structed mutation spectra of HVRI and HVRII, defined by

Malyarchuk and Rogozin, (2004) were used.

The exact tests were performed using the Struc program

by Genepop 3.3 (Raymond and Rousset 2001) and the

remaining statistical tests were performed using the pro-

grams SPSS v.15.0.1 (SPSS inc. 1989–2006) and

OpenStat2 v1.42 (Miller 2003).

Results and Discussion

Distribution of Heteroplasmies

Of the 210 analyzed samples, 30% of the individuals were

fully homoplasmic; the remaining subjects presented point

and/or length heteroplasmy distributed as shown in

Table 1. The prevalent form of heteroplasmy was length

heteroplasmy in the poly(C) tract of HVRII, which was

present in 64.76% (95% CI, 57.89–71.21%) of the total

analyzed individuals. Moreover, 17.14% (95% CI, 12.30–

22.93%) of the individuals presented length heteroplasmy

in the poly(C) tract of HVRI and 3.81% (95% CI, 1.66–

7.37%) carried a point heteroplasmy. The frequency of

point heteroplasmy obtained was slightly lower than that

reported by Brandstatter et al. (2004) for a Caucasian

population (7.4% [95% CI, 3.0–11.8%]), but the differ-

ences are not significant. There were 180 heteroplasmies

found in 147 individuals (Table 2), which means that in

this population there are many individuals who carry more

than one heteroplasmy.

Point heteroplasmy was found in eight individuals and

seven different positions were involved—16093, 16167,

16391, 132, 146, 152, and 246 (Table 3); position 152

appeared to be heteroplasmic in two individuals. Seven of

the point mutations detected in heteroplasmy were pyrim-

idine transitions and one was a purine transition; no

transversions were found.

In the total count of point heteroplasmy, the two poly(C)

tracts (16184–16193 and 303–315) were not considered

because their interpretation is complex. As an example of

this difficulty, we present the sequence of one individual

that carried a homoplasmic substitution in position 16189

Table 1 Classification of the

analyzed individuals depending

on the type(s) of heteroplasmy

they presented

Note: N, number of individuals;

PH, point heteroplasmy; LH,

length heteroplasmy; HVRI and

HVRII, hypervariable regions I

and II, respectively

Blood Buccal cells Total

N % N % N % 95% CI

Homoplasmy 41 28.08 22 34.38 63 30 23.87–36.69%

PH 2 1.37 0 — 2 0.95 0.11–3.40%

LH HVRI 5 3.42 3 4.69 8 3.81 1.66–7.37%

HVRII 75 51.37 32 50 107 50.95 43.98–57.90%

HVRI & HVRII 19 13.01 5 7.81 24 11.43 7.46–016.53%

PH & LH HVRI 1 0.68 0 — 1 0.48 0.01–2.62%

HVRII 1 0.68 1 1.56 2 0.95 0.11–3.34%

PH & LH, HVRI; LH, HVRII 2 1.37 1 1.56 3 1.43 0.30–4.12%

Total 146 64 210

Table 2 Distribution of the different types of heteroplasmies con-

sidering blood and buccal samples separately and globally

Blood Buccal cells Total

N % N % N %

PH 6 4.61 2 4 8 4.44

LH HVRI 27 20.77 9 18 36 20

HVRII 97 74.62 39 78 136 75.56

Total 130 50 180

Note: N, number of heteroplasmies; PH, point heteroplasmy; LH,

length heteroplasmy; HVRI and HVRII, hypervariable regions I and

II, respectively

J Mol Evol (2008) 67:191–200 193

123

and an apparent insertion of C before position 16192 and

the sequence of a wild-type individual (Fig. 1). To know

exactly the different mtDNA variants present in this indi-

vidual, the Dloop of this sample was cloned and 40 clones

were sequenced; the results are presented in Table 4. Four

different variants were found: (1) the prevalent variant,

which presents 16192T; (2) a variant that presents 16192C;

(3) another one with 16183C and 16192T; and (4) a variant

with an insertion of C in 16191. The identification of these

variants was impossible using only the electropheretogram

since their frequency is lower than the level of detection of

automated sequence and/or they are masked by the C

insertion. Thus, the quantification of length heteroplasmy,

presented afterward, may be underestimated.

Several publications have demonstrated the high rate of

length heteroplasmy in both hypervariable region I (HVRI)

and hypervariable region II (HVRII) (Hauswirth and

Clayton 1985; Bendall and Sykes 1995; Lee et al. 2004).

The different variants that we found in the poly(C) tracts of

HVRI and HVRII (based on the analysis of electrophere-

tograms) are listed in Table 5. As has been said before,

64.76% of the individuals carried a length heteroplasmy in

HVRII, the most frequent type being 309.1, 309.2 (61.02%

of the samples that carried length heteroplasmy in HVRII).

This result is similar to that published by Lee et al. (2004)

for a Korean population, using size-based separation of

fluorescently labeled polymerase chain reaction products

by capillary electrophoresis; they found length hetero-

plasmy in this tract in 69% of the analyzed cases. In HVRI

the heteroplasmy rate was lower; 17.14% of the individuals

presented length heteroplasmy, and the most frequent type

was 16193.0, 16193.1 (50.00% of the samples that carried

length heteroplasmy in HVRI). Lee et al. found hetero-

plasmy in the poly(C) tract of HVRI in 36% of individuals,

a rate considerably higher than that described in this study.

However, this discrepancy is probably due to the method of

analysis, reinforcing the idea that automated sequence may

underestimate length heteroplasmy.

Comparison of Point Heteroplasmy by Tissues

In Tables 1 and 2 the distribution of heteroplasmies, in two

independent groups of individuals for which blood samples

or buccal cells were obtained, is presented. When the two

Table 3 Heteroplasmic point positions found in both hypervariable region I and hypervariale region II in 210 individuals analyzed, excluding

poly(C) tracts (16184–16193 and 303–315) For each position the mtDNA haplotype and the haplogroup is also reported

Individual Position Tissue CRS Haplotype Hg

1 16093, T/c B T 16093 (T/C), 16183, 16189, Het poly(C) (16193.0, 16193.1, 16193.2), 16223, 16278, 16290,

16519, 73G, 143, 195, 225, 226, 263, Het poly(C) 303–309 (309.1, 309.2), 315.1

X

2 16167, C/t B C 16162, 16167 (C/T), 16209, 16519, 73, 263, 315.1 H

3 16391, g/A B G 16172, 16183, 16189, Het poly-C (16193.0, 16193.1, 16193.2),16219, 16278,16391 (A/G),

73G, 195, 263, 315.1, 499

U6

4 132, C/T B C 16183, 16189, Het poly(C) (16193.0, 16193.1, 16193.2), 16223, 16278, 16408, 16519,73G,

132 (C/T), 153, 195, 225, 226, 263, Het poly(C) 303–309 (309.1, 309.2, 309.3), 315.1

X

5 146, T/c B T 16519, 146 (T/C), 263, Het poly(C) 303–309 (309.0, 309.1), 315.1 H

6 246, T/c B T 16519, 246 (T/C), 263, 315.1 H

7 152, C/T BC C 16291, 16519, 152 (C/T), 263, Het poly(C) 303–309 (309.1, 309.2, 309.3), 315.1, 319 H

8 152, C/t BC C 16172, 16189, 16192, 16270, 16311, 16519, 73G, 150, 152 (C/T), 194, 263, Het poly(C)

303–309 (309.1, 309.2), 315.1

U5

Note: B, blood; BC, buccal cells; Hg, haplogroup. For each position the mtDNA haplotype and the haplogroup are also reported. In the

heteroplasmic positions, the predominant variant is represented by a capital letter

Fig. 1 (a) Sequence showing length heteroplasmy in the poly(C)

tract of hypervariable region I. (b) Sequence of a wild-type individual

without signs of length heteroplasmy in the poly(C) tract of

hypervariable region I

194 J Mol Evol (2008) 67:191–200

123

groups of individuals were compared (group of blood

samples vs group of buccal cell samples), no significant

differences were found in the proportions of heteroplasmy

between blood and buccal cell samples: neither in the

distribution at the individual level (Table 1) (exact test,

p = 0.8416) nor in the count of heteroplasmies at the

population level (Table 2) (p = 0.7373). Even though our

results are derived from independent samples of blood and

buccal cells, they are similar to those obtained by Bendall

et al. (1997). The authors examined the levels of a het-

eroplasmic point mutation in HVRI of nine members of a

maternal lineage. They found significant levels of point

heteroplasmy in three individuals, the proportions of each

variant being similar in both blood and buccal cells,

whereas single hair roots showed highly variable levels of

heteroplasmy, even among roots from the same individual.

In our work, two point heteroplasmies were found in

buccal cell samples, representing 3.13% (95% CI, 0–7.4%)

of these samples, while the other six were from blood

samples, which means 4.11% (95% CI, 0.9–7.3%) of blood

samples (Table 1).

Pattern of Point Heteroplasmy Across the D-Loop

Point heteroplasmic positions described in previous works

(Comas et al. 1995; Bendall et al. 1996; Wilson et al.

1997; Parsons et al. 1997; Sigurðardottir et al. 2000; Tully

et al. 2000; Grzybowski et al. 2003; Howell et al. 2003;

Brandstatter et al. 2004; Santos et al. 2005; present study)

were compiled and are presented in Table 6.

In both hypervariable regions of human mtDNA, there

are 63 positions described as presenting point hetero-

plasmy. Globally there are 92 heteroplasmies in the HVRI

and 24 in the HVRII. As shown in Table 6, heteroplasmic

positions are distributed across both hypervariable regions.

However, both the proportion of heteroplasmic positions

(HVRI, 20.1%; HVRII, 6.27%; proportion test, Z =

- 4.916, p \ 0.001) and the mean number of heteroplas-

mies per position (HVRI, 0.32; HVRII, 0.085; Mann-

Whitney test, Z = - 3.6, p \ 0.001) are significantly

higher in the HVRI region, even when the mutational

hotspots are excluded from the analysis (HVRI, 0.09;

HVRII, 0.04; Mann-Whitney test, Z = - 2.287,

p \ 0.022).

Concerning the kind of mutations that cause hetero-

plasmy, there are 85 pyrimidine transitions in both

hypervariable regions (72 in HVRI and 13 in HVRII), and

29 purine transitions (19 in HVR1 and 10 in HVRII), while

there are only 2 transversions (1 in each HVR). The pro-

portions of the three types of mutation observed in the

heteroplasmy mutational pattern are similar to that reported

in the phylogenetic mutational spectrum (Malyarchuk and

Rogozin 2004) of HVRII (v2 = 1.192, df = 2, p = 0.551)Ta

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J Mol Evol (2008) 67:191–200 195

123

and different for HVRI (v2 = 10.69, df = 2, p = 0.005).

The differences reported for HVRI essentially result from

the 10 times higher proportion of transversions observed at

the phylogenetic level, compared with the proportion

observed in the heteroplasmy spectrum. In conformity with

the previously reported data, the transition/transversion rate

is 57 for the two hypervariable regions, 91 for the HVRI

and 23 for HVRII, and these results are considerably

higher, especially for HVRI, than those published by other

authors using phylogenetic approaches (Meyer et al. 1999;

Malyarchuk and Rogozin 2004). The resultant pyrimidine/

purine rate is 3.79 for HVRI, 1.3 for HVRII, and 2.93 when

both hypervariable regions are considered. The pyrimidine/

purine rates are similar to those reported by other authors

(Meyer et al. 1999; Malyarchuk and Rogozin 2004). These

results provide evidence for the existence of an excess of

transitions (or a deficit in transversions), particularly in

HVRI, in the mutational spectrum in heteroplasmy.

Considering the probability of mutation in the hetero-

plasmy spectrum (Table 6), once again a different pattern

emerges for HVRI and HVRII; considering all of the posi-

tions, a significantly higher probability of mutation was

obtained for HVRI (Mann-Whitney test: Z = - 3.24,

p = 0.001). However, when mutational hotspot positions

were analyzed separately from the remaining positions it was

observed that HVRII presents a significantly higher proba-

bility of heteroplasmy than HVRI (Z = - 2.216,

p = 0.027); on the other hand, HVRI presents a significantly

higher probability of heteroplasmy than HVRII when more

stable positions are considered (Z = - 2.168, p = 0.03).

To investigate whether there is a relation between the

heteroplasmic and the phylogenetic mutational patterns, we

correlated the probability of mutation obtained for each

spectrum. For both hypervariable regions a significant

positive correlation was obtained (HVRI, Rspearman = 0.420,

p \ 0.001; HVRII, Rspearman = 0.329, p \ 0.001); although

the relation between the two probabilities was significant, the

variation in the phylogenetic mutational pattern only

explains a small percentage of the variation observed in the

heteroplasmy spectrum (18% and 11% for HVRI and HVRII,

respectively). Thus, we also compare whether there are dif-

ferences between the two probability distributions. We note

that the mean range of differences between the two proba-

bilities is higher in those positions that have a higher

probability of mutation in the heteroplasmy spectrum than in

the mutational spectrum and significant differences were

found for the two distributions in both HVRI (Wilcoxon

test: Z = - 4.877, p \ 0.001) and HVRII (Z = - 5.135,

p \ 0.001). Moreover, combining the information of

Meyer et al. (1999) and Malyarchuk and Rogozin (2004) for

the site-specific mutation rate and the probability of mutation

of each heteroplasmic point position (Table 6), we found that

there are 3 positions with a site specific-rate of zero that are

present in heteroplasmy, and 16 of the positions detected in

heteroplasmy present very low (\1) values for the site-spe-

cific mutation rate.

Table 5 mtDNA variants

found by sequencing 210

individuals for the poly(C)

tracts in hypervariable region I

(HVRI) and hypervariable

region II (HVRII) (results

according to electropheretogram

analysis)

Blood Buccal cells Total

HVRI Homoplasmic 119 81.50% 55 85.94% 174 82.86%

16193.(-1), 16193.0 1 0.68% 0 – 1 0.48%

16193.(-1), 16193.0, 16193.1 1 0.68% 0 – 1 0.48%

16193.0, 16193.1 13 8.90% 5 7.81% 18 8.57%

16193.0, 16193.1, 16193.2 10 6.85% 3 4.69% 13 6.19%

16193.0, 16193.1, 16193.2, 16193.3 1 0.68% 0 – 1 0.48%

16193.1, 16193.2 0 – 1 1.56% 1 0.48%

16193.1, 16193.2, 16193.3 1 0.68% 0 – 1 0.48%

Total 146 64 210

HVRII Homoplasmic 315.1 49 33.56% 23 35.94% 72 34.29%

315.2 0 – 2 3.13% 2 0.95%

309.0, 309.1 1 0.68% 1 1.56% 2 0.95%

309.0, 309.1, 309.2 2 1.37% 2 3.12% 4 1.90%

309.0, 309.1, 309.2, 309.3 1 0.68% 0 – 1 0.48%

309.1, 309.2 63 43.15% 20 32.81% 83 39.52%

309.1, 309.2, 309.3 20 13.70% 15 23.44% 35 16.67%

309.1, 309.2, 309.3, 309.4 5 3.42% 0 – 5 2.38%

309.2, 309.3 0 – 1 1.56% 1 0.48%

309.2, 309.3, 309.4 5 3.42% 0 – 5 2.38%

Total 146 64 210

196 J Mol Evol (2008) 67:191–200

123

Table 6 Point heteroplasmic positions described in the literature and in the present study

Position CRSa/

consensusb/

chimpanzeec

No.

heteroplasmies

Probability of

heteroplasmydHET Site-

specific

ratee

No. hits

in the

phylogenyf

Site-

specific

rateg

Probability

of

mutationh

Distribution

in population

databasei

16024 T/T/T 1 n.a. T/C \1 n.a. n.a. n.a. n.a.

16051 A/A/A 1 n.a. A/G 4 n.a. n.a. n.a. n.a.

16086 T/T/T 1 n.a. T/C \1 n.a. n.a. n.a. 98.9%T; 1.1%C

16088 T/T/T 1 n.a. T/C 0 n.a. n.a. n.a. 100%T

16090 T/T/T 1 0.011364 T/C 0 0 0 0.0000 100%T

16092 T/T/T 1 0.011364 T/C \1 20 2.5 0.0091 99.5%T; 0.5%C

16093 T/T/T 18 0.204545 T/C 3 69 8.6 0.0313 93.3%T; 6.7%C

16104 C/C/C 2 0.022727 C/T \1 6 0.8 0.0027 99.7%C; 0.2%T; 0.1%A

16105 T/T/T 1 0.011364 T/C 0 1 0.1 0.0005 99.9%T; 0.1%C

16111 C/C/C 2 0.022727 C/T; G/A 2.5 35 4.4 0.0159 99.1%C; 0.1%A; 0.1%G;

0.7%T

16124 T/T/C 1 0.011364 T/C \1 9 1.1 0.0041 96.6%T; 3.4%C

16125 G/G/G 1 0.011364 G/A \1 1 0.1 0.0005 100%G

16126 T/T/T 2 0.022727 T/C 4 20 2.5 0.0091 82.1%T; 17.9%C

16129 G/G/A 7 0.079545 A/G *5 54 6.8 0.0245 89.1%G; 10.6%A; 0.3%C

16150 C/C/C 1 0.011364 C/T \1 10 1.3 0.0045 99.4%C; 0.5%T; 0.1%A

16162 A/A/A 1 0.011364 A/G *1 9 1.1 0.0041 98.3%A; 1.7%G

16167 C/C/C 1 0.011364 C/T \1 12 1.5 0.0054 99.1%C; 0.9%T

16172 T/C/C 1 0.011364 T/C *4 50 6.3 0.0227 92.5%T; 7.5%C

16185 C/C/C 1 0.011364 C/T \1 8 1.0 0.0036 99.7%C; 0.3%T

16189 T/C/A 2 0.022727 T/C *5 62 7.8 0.0281 77.1%T; 22.7%C; 0.1%A

16192 C/C/C 4 0.045455 C/T *4.5 36 4.5 0.0163 95%C; 4.9%T; 0.1%A

16216 A/A/A 1 0.011364 A/G \1 3 0.4 0.0014 99.9%A; 0.1%G

16220 A/A/A 1 0.011364 A/G \1 5 0.6 0.0023 99.9%A; 0.1%C

16222 C/C/T 1 0.011364 C/T \1 10 1.3 0.0045 97.9%C; 1.9%T; 0.1%A

16223 C/T/C 1 0.011364 C/T *5 12 1.5 0.0054 75%C; 25%T

16232 C/C/C 1 0.011364 C/G \1 4 0.5 0.0018 99.9%C; 0.1%T

16234 C/C/A 1 0.011364 C/T *1 35 4.4 0.0159 97.7%C; 2.3%T

16239 C/C/A 2 0.022727 C/T \1 24 3 0.0109 98%C; 1.5%T; 0.4%G;

0.1%A

16243 T/T/C 1 0.011364 T/C \1 13 1.6 0.0059 98%T; 2%C

16256 C/C/A 3 0.034091 C/T *2 34 4.3 0.0154 95.4%C; 4.6%T

16257 C/C/C 1 0.011364 C/T \1 15 1.9 0.0068 98.5%C; 1.5%T

16262 C/C/C 1 0.011364 C/T \1 6 0.8 0.0027 100%C

16264 C/C/C 1 0.011364 C/T \1 17 2.1 0.0077 97.4%C; 2.6%T

16270 C/C/C 1 0.011364 C/T *3.5 15 1.9 0.0068 90.4%C; 9.6%T

16278 C/C/C 1 0.011364 C/T *5 34 4.25 0.0154 85.2%C; 14.8%T

16288 T/T/T 1 0.011364 T/C \1 10 1.25 0.0045 99.8%T; 0.2%C

16293 A/A/C 3 0.034091 A/G *4 23 2.9 0.0104 96%A; 3.3%G; 0.7%T

16294 C/C/C 2 0.022727 C/T *5 27 3.4 0.0122 85.9%C; 14%T; 0.1%A

16295 C/C/C 1 0.011364 C/T \1 17 2.1 0.0077 99.5%C; 0.5%T

16296 C/C/C 2 0.022727 C/T *1.5 8 1.0 0.0036 95.3%C; 4.7%T

16301 C/C/C 2 0.022727 C/T \1 18 2.25 0.0082 99.5%C; 0.5%T

16309 A/A/A 2 0.022727 A/G 4 12 1.5 0.0054 98.9%A; 1.1%G

16311 T/T/T 4 0.045455 T/C *5 70 8.8 0.0317 76.2%T; 23.8%C

16316 A/A/A 1 0.011364 A/G \1 8 1 0.0036 99.4%A; 0.6%G

16355 C/C/C 2 0.022727 C/T *2 30 3.8 0.0136 98%C; 2%T

J Mol Evol (2008) 67:191–200 197

123

Results of phylogenetic studies have suggested a

complex pattern of mtDNA control region evolution. It

was found that the base composition in the HVRI and

HVRII regions is not uniform; transitions occur at

higher frequencies compared to transversions, the

number of pyrimidine transitions in the L-stand exceeds

the number of purine transitions, the substitution rates

vary among nucleotide positions, and HVI and HVII

show different mutation rates (2.08 and 1.48 for HVRI

and HVRII, respectively) (Meyer et al. 1999; Horai

et al. 1995).

Although it would be desirable to increase the number of

samples analyzed for the presence of heteroplasmy, the

results obtained in heteroplasmy are in agreement with the

previously reported differences between HVRI and HVRII.

It seams that at both the phylogenetic and the heteroplas-

mic level there are very pronounced differences between

the two hypervariable regions. It is well known that there

are several segments in the HVII region which are involved

in the functional aspects related to the replication and

translation in the mtDNA molecule (Crews et al. 1979;

Fisher et al. 1987; Chang and Clayton 1985), and the

Table 6 continued

Position CRSa/

consensusb/

chimpanzeec

No.

heteroplasmies

Probability of

heteroplasmydHET Site-

specific

ratee

No. hits

in the

phylogenyf

Site-

specific

rateg

Probability

of

mutationh

Distribution

in population

databasei

16362 T/T/C 4 0.045455 T/C *5 54 6.8 0.0245 93.9%T; 6.1%C

64 C/C/C 1 n.a. C/A *3 n.a. n.a. n.a. 97%C; 3%T

94 G/G/A 1 0.043478 G/A 0 1 0.5 0.0019 99.8%G; 0.2%A

132 C/C/C 1 0.043478 C/T \1 0 0 0.0000 99.9%C; 0.1%G

146 T/T/C 1 0.043478 T/C *6 36 18 0.0682 86.1%T; 13.9%C

150 C/T/T 1 0.043478 T/C *6 32 16 0.0606 87.5%C; 12.5%T

152 T/T/T 6 0.26087 T/C *6 56 28 0.1061 72.4%T; 26.6%C

153 A/A/A 1 0.043478 A/G 4 6 3 0.0114 97.1%A; 2.9%G; 0.1%C

154 T/T/T 1 0.043478 T/C 0 0 0 0.0000 100%T

185 G/G/C 1 0.043478 G/A *5 10 5 0.0189 93.5%G; 4.3%A; 2%T;

0.2%C

189 A/A/G 1 0.043478 A/G *6 17 8.5 0.0322 93.4%A; 5.1%G; 1.5%C

194 C/C/C 1 0.043478 C/T *1 11 5.5 0.0208 99.2%C; 0.8%T

195 T/C/C 1 0.043478 T/C *6 46 23 0.0871 78.1%T; 21.8%C; 0.1%A

207 G/G/G 2 0.086957 G/A *3 17 8.5 0.0322 95.7%G; 4.2%A; 0.1%C

215 A/A/A 1 0.043478 A/G \1 7 3.5 0.0133 99.4%A; 0.6%G

234 A/A/A 2 0.086957 A/G \1 5 2.5 0.0095 99.1%A; 0.1%G

238 A/A/A 1 0.043478 A/G 0 1 0.5 0.0019 100%A

246 T/T/A 1 0.043478 T/C 0 1 0.5 0.0019 99.7%T; 0.3%C

Note: n.a., not available. For each position, the state in the human mtDNA reference sequences and in the chimpanzee sequence, the number of

individuals with heteroplasmy, the site-specific rate, the number of hits on the mtDNA phylogeny, the probability of mutation, and the frequency

in a population database are reported. Mutational hotspots were defined as by Malyarchuk and Rogozin (2004) and are in boldface. An extended

version of this table is available as online Supplementary Materiala From Anderson et al. (1981)b From Ingman et al. (2000); GenBank accession number NC_001807c From Horai et al. (1995); GenBank accession number D38113d Regions considered: 16090–16365 and 70–339. Calculated as: number of heteroplasmies/total number of heteroplasmies. Total heteroplas-

mies: hypervariable region I (HVRI), 88; hypervariable region II (HVRII), 23e From Meyer et al. (1999)f From Malyarchuk and Rogozin (2004)g Regions considered: 16090–16365 and 70–339. Based on data of Malyarchuk and Rogozin (2004). Calculated as: number of hits/mean number

of hits across all positions. Mean number of hits: HVRI, 8; HVRII, 2h Regions considered: 16090–16365 and 70–339. Based on data of Malyarchuk and Rogozin (2004). Calculated as: number of hits/total number

of hits. Total hits: HVRI, 2207; HVRII, 528i Frequency of each nucleotide in a population database of 1215 European sequences and 288 African sequences, as described by Santos et al.

(2005)

198 J Mol Evol (2008) 67:191–200

123

functional aspects of those sites could constrain the amount

of neutral variation.

Our data provide evidence that there is a significant

relation between the phylogenetic and the heteroplasmic

mutation patterns, however, this relation explains only a

small amount of the variation observed. On the other hand,

there are significant differences between the probability of

mutation when the two spectra are compared, and the tran-

sition/transversion rate value observed in the heteroplasmy

mutation pattern is much higher than the values obtained by

phylogenetic estimations (Vigilant et al. 1991; Tamura and

Nei 1993). These results may suggest that a considerable

number of mutations and, particularly, transitions that appear

in heteroplasmy are probably eliminated by drift and/or by

selection acting at different mtDNA levels of organization

(the molecule, organelle, cell, and tissue). For example, the

transversion in position 215 described by Santos et al. (2005)

presents, according to the authors, a pattern of segregation

across generations that is not compatible with neutrality; we

further analyzed the implications of this mutation and real-

ized that it is located in the conserved sequence block 1

located in the D-loop near the 30 termini of the most promi-

nent DHP-RNA species (Chang and Clayton 1985).

Moreover, the prediction of the secondary structure of

HVRII indicates that the G(215) variant can induce an

important conformational change in HVRII and could

interfere with mtDNA replication efficiency. So, to under-

stand the causes that explain the differences between

phylogenetic and heteroplasmic mutational spectrums, it

would be necessary to understand the process by which a

mutation passes from heteroplasmy to homoplasmy. Previ-

ous studies (Howell et al. 1992; Bendall et al. 1996; Lutz

et al. 2000) have focused their interest on this question,

paying particular attention to the effect of the number of

segregating units during the bottleneck. However, Santos

et al. (2005) argue that in some families the variation in

heteroplasmy across generations cannot be explained by the

number of segregating units during the bottleneck. The

authors propose that other factors, such as the proportions of

mtDNA variants in the heteroplasmic mothers and the effect

of selection at different mtDNA levels of organization, must

also be considered.

Conclusion

In recent years several works have been published that

question the characteristics (homoplasmy, nonrecombina-

tion, and paternal inheritance) of mtDNA (Schwartz and

Vissing 2002; Kraytsberg et al. 2004; Zsurka et al. 2005).

To improve knowledge of homoplasmy/heteroplasmy we

analyzed a high number of blood and buccal samples,

confirming the theory that heteroplasmy in the control

region of the mtDNA is much more common in a general

healthy population than traditionally was supposed. The

size of the sample is large enough to affirm that blood and

buccal cells present similar heteroplasmic profiles.

The pattern of heteroplasmy in HVRI and HVRII presents

important differences. Moreover, the mutational profile in

heteroplasmy seems to be different from the mutational

pattern detected in the population.

Considering the results reported in this work as a whole,

it is mandatory to perform a broad-scale screening of het-

eroplasmy to better establish the heteroplasmy profile,

which is important for medical, evolutionary, and forensic

proposes.

Acknowledgments This work was supported by MCYT (BOS

2002-00724) and MEC (CGL2006-07374). C. Santos was a post-

doctoral fellow of the Fundacao para a Ciencia e a Tecnologia

(SFRH/BPD/20944/2004).

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