Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

Results of a collaborative study of the EDNAP group regardingmitochondrial DNA heteroplasmy and segregation in hair shafts

G. Tullya,*, S.M. Barritti, K. Bendere, E. Brignonb, C. Capellij, N. Dimo-Simoninc,C. Eichmannf, C.M. Ernsti, C. Lamberta, M.V. Lareud, B. Ludesb, B. Mevagh,

W. Parsonf, H. Pfeifferg, A. Salasd, P.M. Schneidere, E. Staalstromh

aThe Forensic Science ServiceTM, Trident Court, Solihull Parkway, Birmingham Business Park, Solihull B37 7YN, UKbCODGENE Laboratory 11, Rue Humann 67085, Strasbourg, France

cInstitut Universitaire de Medecine Legale, Rue de Bugnon 21, CH-1005 Lausanne, SwitzerlanddFaculty of Medicine, Institute of Legal Medicine, c/San Fransisco s/n, 15705 Santiago de Compestela, Spain

eInstitute of Legal Medicine, University of Mainz, Am Pulverturm 3, D 55131 Mainz, GermanyfInstitut fuer Gerichtliche Medizin, DNA-Labor, Universitaet Innsbruck, Muellerstrasse 44, A-6020 Innsbruck, Austria

gInstitut fuer Rechtsmedizjn der Universitaet Muenster, Von-Esmarch Strasse 62, D-48149 Muenster, GermanyhRettsmedisinsk Institutt, Rikshospitalet, 0027 Oslo, Norway

iDepartment of the Army, Armed Forces Institute of Pathology, DNA Identification Laboratory,

1413 Research Boulevard, Rockville, MD 209850, USAjIstituto di Midicina Legale, Universita Cattoilca del sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy

Received 26 November 2002; received in revised form 5 March 2003; accepted 4 April 2003

Abstract

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution

of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and

buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using

differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group [Forensic Sci. Int. 97

(1998) 165]. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two

laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed.

The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated

differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying

ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093

and the poly(C) tract at 302–309, whilst a complete A–G transition was confirmed at position 16129 in one hair.

Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed

in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands

of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one

laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded.

Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other

laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be

associated with this technology.

The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

# 2003 Published by Elsevier Ireland Ltd.

Keywords: Mitochondrial DNA; Heteroplasmy; Mutation; Segregation; Hair

Forensic Science International 140 (2004) 1–11

* Corresponding author. Tel.: þ44-121-399-5041; fax: þ44-121-622-2051.

E-mail address: [email protected] (G. Tully).

0379-0738/$ – see front matter # 2003 Published by Elsevier Ireland Ltd.

doi:10.1016/S0379-0738(03)00181-6

1. Introduction

Mitochondrial DNA (mtDNA) analysis is widely used in

forensic examination of degraded remainsandsamples such as

hair shaftsdue to itshighcopy number [2]and hence its relative

abundance. The control region of human mtDNA, which is the

region most commonly analysed for forensic purposes, dis-

plays considerable sequence variation in human populations

(e.g. [3–9]). Comparison of mtDNA sequences from a ques-

tioned and a known sample can thus provide significant evi-

dence to exclude an individual as the source of an evidential

sample, or to support an association between the two. It is well

established that mtDNA mutates at a substantially higher rate

than that does nuclear DNA (e.g. [10–15]). Consequently, it is

not uncommon for differences to be observed in the mtDNA

sequence when comparing close maternal relatives (such as

mother and child) [15]. Substitution has also been observed in

somatic tissues, presumably due to segregation of existing

heteroplasmy within the individual. This means that differ-

ences may be observed between different hairs and/or tissues

within an individual (e.g. [16–19]). The clonal nature of hair

follicles and the high energy requirements of keratinizing hair

shaft cells are two features of hair histogenesis that could

contribute to the high observation of segregation of hetero-

plasmic variants in mtDNA from hair shafts [20].

Interpretation of mtDNA results in forensic casework

must take account of the possibility of sequence differences

within an individual [21–23]. This interpretation is aided by

accumulation of data concerning the occurrence and segre-

gation of mutation. The aim of this exercise was therefore

to add to the body of data concerning segregation of hetero-

plasmic mtDNA mutations in hairs. Additionally, the

exercise provided an opportunity to add to the earlier

reproducibility study undertaken by this group [1].

2. Materials and methods

2.1. Samples

Reference blood and buccal scrape samples from a donor

were extracted using Chelex100 resin (BioRad, Hemel

Hempstead, UK) following published protocols [24].

Multiple hairs with roots were obtained from the donor

and numbered sequentially. Each was cut into at least

5 cm � 2 cm sections, with the root section being given

the suffix ‘‘.1’’, the next section ‘‘.2’’ and so on.

Two sections from each of 10 hairs were sent to each

participating laboratory, such that each hair was analysed by

at least two laboratories; the root sections were retained for

subsequent analysis where required. Each laboratory used

their own protocols for the analysis.

2.2. Methods

The methods employed for washing the hairs, extracting,

amplifying and sequencing the DNA and for electrophoresis

and analysis of the sequenced products are summarised in

Table 1.

As shown in the table, a wide variety of methodologies

were employed. The wash procedures applied to the hairs

before digestion ranged from mild washing using only

ethanol and/or water to more vigorous washing using deter-

gents and/or proteinase K (pK) digestion from the external

surface of the hair.

For extraction, chemical digestion was employed, fol-

lowed by organic or resin-based purification and concentra-

tion by ethanol precipitation or spin columns. Where the root

section was analysed, a preferential extraction method was

used to first remove sheath material by a mild pK digestion

and washing, before the hair material itself was chemically

digested (pK, DTT, SDS) and the DNA organically purified

and ethanol precipitated.

PCR strategies included direct amplification of either

single or overlapping fragments, semi-nested PCR or nested

PCR. The majority of laboratories used an electrophoretic

assessment stage prior to sequencing, with some laboratories

also employing a spin column purification step.

Both dye primer and dye terminator sequencing chemis-

tries were employed, with a variety of dye chemistries.

Electrophoresis was performed using both slab-gels and

capillaries; all laboratories used real-time fluorescent

detection.

3. Results and discussion

The results are summarised in Table 2.

In instances where sequence was not obtained by two

different laboratories, or where the hair seemed to show a

graduation of heteroplasmy along its length, the root section

of the hair was extracted and analysed. For this analysis, the

sheath material was digested using proteinase K and dis-

carded prior to digestion of the hair itself using additional pK

plus dithiothreitol (DTT). The rationale of removing the soft

tissue adhering to the root in this manner was to remove the

possibility of mesoderm and endoderm cells from such

tissue, which may contain a different mtDNA population

[20], contributing to the mtDNA analysed.

The existence and differential segregation of sequence

heteroplasmy at positions 16093 and 16234 and of length

heteroplasmy at the polycytosine region between 302 and

310 was confirmed by the present study. Within some hairs,

graduation of heteroplasmy along the length of the hair was

apparent; for example, in hair 17, the section closest to the

root showed a T at position 16093, the next section showed a

T with a small C underneath, the next showed C at greater

peak height than T and the section nearest the tip showed C

with a small T beneath (Fig. 1). In other hairs, the change

along the length of the hair did not show a gradual variation;

an example is seen at position 16234 in hair 43, where

apparently homoplasmic T is observed in the two sections

nearest the root and the two sections at the tip, whilst the

2 G. Tully et al. / Forensic Science International 140 (2004) 1–11

Table 1

Methods employed by participating laboratories

Laboratory Hair washing method Hair extraction method PCR primers PCR strategy Post-PCR assessment

and/or purification

Sequencing

strategy

Electrophoresis

instrument

Analysis software

1 2� 10% SDS washes, 2� extraction

buffer washes, all with vortexing

Chemical digestion

(pK, SDS and DTT) then

phenol chloroform extraction

and EtOH precipitation

First round: L15933/H00575;

second round: L15997/H16401;

L00029/H00408

Nested PCR

(2 � 25 cycles)

and direct PCR

(35 cycles)

Agarose gel electrophoresis

to quantify

BigDye primer

cycle sequencing

377 Sequence analysis v. 3.0,

sequence navigator

2 1% SDS washes, water washes,

ethanol washes, all with vortexing

Chemical digestion (DTT, pK)

then phenol chloroform

extraction and purification

with microcon

L15997; H16401; L29; H408 Direct PCR Agarose gel electrophoresis

followed by microcon

(Millipore) purification

dRhodamine dye

primer cycle

sequencing

373A Sequence analysis v. 3.0,

sequence navigator v. 1.0.1

3 1� ethanol wash, 2� water washes,

all with vortexing

Chelex, pK and DTT with

Centricon 100 purification

and concentration

L15926/H00580;

M13(-21)L15997/M13revH16401;

M13revL00029/M13(-21)H00408

Nested PCR

(2 � 30 cycles)


to quantify and spin

column purification

BigDye terminator

cycle sequencing

(nested sequencing)


sequence navigator v. 1.02b3

4 1� 80% ethanol wash, 1� water,

all with vortexing

Chelex, pK and DTT L15997/H16236; L16159/H16401;

L047/H285; L172/H408

Overlapping

fragments

PAGE assessment and spin

column purification

dRhodamine

terminator cycle

sequencing

377 Sequence analysis,

sequence navigator

5 1� ethanol wash, 2� water washes,

all with vortexing

Chemical digestion (pK, SDS

and DTT) then phenol

chloroform extraction and

amicon30 concentration

L15997/H16401; L291/H408 Direct PCR

(35 cycles)


to quantify and spin

column purification

BigDye terminator

cycle sequencing

310 Sequence analysis v. 2.1.2,

sequence navigator v. 1.0.1

6 Mild pK digestion Chemical digestion (pK,

SDS and DTT) then phenol

chloroform extraction and EtOH

precipitation

L15971/H16410; L15/H484 Direct PCR

(35 cycles)

PAGE gel (sliver stained)

for assessment

BigDye terminator

cycle sequencing

310/3700 Sequence analysis v. 3.0

and v. 3.6

7 30 min 70% ethanol followed by

30 min HPLC-water

Chemical digestion proK, DTT,

PCR buffer, HPLC-water

phenol chloroform extraction,

microcon

L15997/H16401; L0029/H00389 Direct PCR

(30 cycles)

Qiagen BigDye terminator

cycle sequencing


sequence navigator

8 1� water wash, 5% Tergazym 60 8Cfor 20 min, 1� water 37 8C for

10 min, 1� ethanol left to air dry

Chemical digestion (DTT, pK,

Laureth 10), phenol chloroform

extraction and EtOH precipitation

L15997/H16405; H16169,

H16209; L00029; H00408;

H00266

Semi-nested Agarose gel electrophoresis

and spin column purification

TaqFS dye primer

cycle sequencing


sequence navigator

9 4� wash with 5% detergent solution,

wash with ethanol and water

Micro tissue grinder and chemical

digestion (proK, DTT) organic

extraction, Centricon 30 concentration

L15989/H16251; L16190/H16410;

L15/H285; L155/H389

Overlapping

fragments

(38 cycles)


and spin column purification

BigDye terminator

cycle sequencing


sequence navigator

10 1� ethanol wash Chemical digestion (pK,

SDS and DTT) and silica purification

L15990/H16401; L34/H370 Direct PCR

(38 cycles)

Agarose gel electrophoresis TaqFS dye primer

cycle sequencing

ALF express ALF win 1.0

G.

Tu

llyet

al./F

oren

sicS

cience

Intern

atio

na

l1

40

(20

04

)1

–1

13

Table 2

Results, expressed as differences from the Anderson reference sequence (‘‘AND’’) [33]


Table 2 (Continued )

G. Tully et al. / Forensic Science International 140 (2004) 1–11 5



intermediate section has C as the major component with only

a small T peak underneath (Fig. 2). The same laboratory,

using the same methodology, sequenced two of the mid-

sections that demonstrated the dramatic shift in proportions.

This switch is therefore unlikely to be attributable to differ-

ences in sequencing chemistry. Linch et al. in their review of

hair histogenesis for the mtDNA forensic scientist [20],

highlight that a significant proportion of the mitochondria

in hair shafts are likely to originate from a relatively small

number of melanocytes, the remainder originating from

keratinocytes. Melanocytes, whilst not undergoing mitosis,

continue to produce mitochondria until later in life, when

grey hairs are produced. They therefore represent a bottle-

neck, through which heteroplasmic ratios could shift mark-

edly, both within and between individual hair shafts [20].

Intuitively, gradual changes in heteroplasmic ratios along the

length of a hair shaft would be expected rather than appar-

ently homoplasmic switches between one base and another

in adjacent sections of hair, arbitrarily cut into 2 cm lengths.

However, present sequencing technologies do not detect all

of the variability present, as has been demonstrated by using

more sensitive techniques such as DGGE [25]. Thus, if the

detection threshold of sequencing technologies is in the

order of 20%, apparently homoplasmic changes between


The sequence referred to as ‘‘REF’’ was determined from blood and saliva, both of which gave the same result. Lower case indicates that

sequence was determined from only one strand. In such instances, as per the ISFG recommendations [22], if heteroplasmy was suspected, the

position was marked as not determined. In order to clarify the heteroplasmic proportions observed, the following nomenclature was adopted.

C�: C peak with small T peak underneath; C > T: C peak stronger than T peak; C � T: C and T peaks of approximately equal intensity; C ¼ T:

C and T peaks of equal intensity; T > C: T peak stronger than C peak, etc. NI was used to denote the absence of an inserted base; (�) was used

to denote an undetermined base; DEL was used to denote a deletion.


adjacent sections of hair can be explained by an approxi-

mately 60% shift in the proportions of the heteroplasmic

variants (Fig. 3). Comparing fragments within each hair, a

shift in the proportions of heteroplasmic variants was

observed within 37 hairs, and an apparently homoplasmic

change observed within 11. Such changes in heteroplasmic

proportions within individual hairs has been documented

previously [19]. Wherever possible, analysis of two separate

segments of an evidential hair is considered by the EDNAP

group to be optimal [23]. Whilst this recommendation was

primarily aimed at ensuring that erroneous results due to

contamination are never reported, the results of the present

study show that sequencing more than one fragment of a hair

can give additional information to the forensic scientist to

aid interpretation. In only 13 of the hairs from this study

would a single fragment from each hair have given as much

information about the occurrence of heteroplasmy within the

donor individual as did the multiple hair segments.

An apparently complete base change was observed at

position 16129 in hair 39; no heteroplasmy was detected in

any other hair at this position. One potential explanation for

this result is that heteroplasmy at position 16129 exists

within the donor individual at a low level, and has

segregated, via a bottleneck with a very low number of

segregating units, into the melanocytes contributing mtDNA

to this one hair. An alternative explanation is that a de novo

mutation has arisen during the course of the formation of this

hair; study of maternal family members of the donor indi-

vidual would assist in determining which of these possibi-

lities is the more likely. In concordance with family studies,

phylogenetic analyses have shown that position 16129 is a

rapidly mutating base position [26,27]; similarly, position

16093 is a quickly mutating position, and 16148 has a higher

than average mutation rate. Moreover, positions 16093 and

16129 have been identified as by far and away the most

prominent sites for heteroplasmy in HV1 [25], to an extent

even in excess of their high relative mutation rates that have

been inferred phylogenetically. The demonstration that 1 of

55 hairs shows an apparently complete change at this posi-

tion underlines the need for careful consideration in the

interpretation of mtDNA evidence, especially in cases where

only one or a few bases differ between the reference and

unknown samples. Sequencing of multiple reference hairs

has been suggested in such instances (e.g. [21]); whilst often

providing useful information, the result in hair 39 shows that

even if additional hairs are typed, instances are likely to

Fig. 1. Gradual variation in the distribution of heteroplasmy at position 16093 along the length of hair 17. Section 17.2 is the section 2 cm

proximal to the root; sections 0.3–0.5 are progressively further away from the root. The reference blood and saliva show C and T in

approximately equal proportions, whilst this hair shows an increasing proportion of C along its length. In section 17.3, electrophoretic

‘‘shouldering’’ of each peak is present, but the heteroplasmy is distinguishable from this shouldering as it is raised in height, and is not

adjacent to a blue peak. This shouldering was not present in the sequence from the reverse strand, but the heteroplasmy was clearly

distinguishable.


Fig. 2. Variation in the distribution of heteroplasmy at position 16234 along the length of hair 43. Section 43.1 is the root section, with

sections 43.2–43.5 being progressively further away from the root. The reference blood and saliva are apparently homoplasmic for the C

variant, section 43.3 shows C > T heteroplasmy, whilst all other sections are apparently homoplasmic T. Sections 43.3 and 43.4 were

sequenced by the same laboratory, using the same chemistry.

Fig. 3. Theoretical heteroplasmic proportions at position 16234 in the fragments of hair 43. Assuming a detection threshold of 20%, any point

above the upper dashed line would be observed as a C base, whilst any point below the lower dashed line would be observed as a T base; points

between the lines would be observed to exhibit C/T heteroplasmy.


occur in which the heteroplasmy or base change is

not sufficiently common within the individual to be detected.

Thus, although any difference between unknown and

reference sample must diminish the strength of the evidence

[23], a single such difference cannot provide conclusive

evidence that the two samples are from different maternal

origins.

An intriguing suggestion of Linch et al. is the potential

link between degree of pigmentation and degree of hetero-

plasmy [20]. This link is postulated to be due to the

melanocyte contribution of mitochondria to hair shaft being

coincident with contribution of melanosomes, although the

authors note that there have been no reports in the literature

relating degree of pigmentation to observed mtDNA het-

eroplasmy. The donor of the hairs examined in the present

study is of Asian descent (belonging to the typically Asian

haplogroup F1a, characterised in the hypervariable regions

by positions 16129, 16172, 16304, 249del [28]) and as such

has extremely darkly pigmented hair. The EDNAP group

is currently undertaking a similar exercise, using an indi-

vidual in whose reference sample no heteroplasmy

was evident. This second donor individual has mid brown

hair; this and similar studies may cast more light on the

hypothesised link between pigmentation and heteroplasmy

in hair shafts. A range of hair colours was studied by

Huhne et al., but no heteroplasmy was recorded in their

study [29].

Heteroplasmy has been observed at multiple positions

within a single individual in several previous studies

[18,19,25,30]. The findings of Grzybowski [30] have been

challenged previously [31,32], largely on the basis of poten-

tial contamination or amplification of nuclear pseudogenes.

With respect to contamination, the EDNAP group has pre-

viously stated that ‘‘in the event of duplication failing, or

where insufficient sample is present, the report should

clearly state the limitations of an unconfirmed result. It is

possible to have apparently clear negative controls and yet to

obtain an erroneous result due to contamination’’ [23].

Following this guideline, the potential heteroplasmy at

position 16095 in hair fragment 34.2 (this position is not

a common variant in human population studies), at position

16148 in hair fragment 44.3, at position 73 in fragment 60.3,

and the potential base changes at positions 249 in fragments

58.3 and 60.3, and position 315.1 in fragments 57.3, 58.4,

59.3 and 60.3 would be treated with caution. The results

from hair fragment 60.3 are worthy of particular considera-

tion: three differences to the results from the other two

fragments of this hair were observed. These results may

suggest that contamination has been encountered in the

analysis of this hair fragment. Overall, the results from

laboratory 10 show several anomalies in comparison to

the results from laboratories 1–9 (positions 73, 249,

315.1) and the possibility of contamination cannot be ruled

out. This contamination may have been introduced during

the analysis, or may have been present on the surface of the

hair and have been inefficiently removed; of all the methods

used for washing the hairs, that of laboratory 10 was the least

stringent. More detailed examination of the electrophero-

grams from laboratory 10 has highlighted that the resolution

was poor in some regions, particularly around position 249

and the poly(C) stretch in HVII. This laboratory was the only

laboratory in this study using sequencing technology requir-

ing the four reactions (A, C, G and T) to be electrophoresed

in separate lanes; such technology increases the potential for

error in sequencing, particularly in polynucleotide regions,

and should not therefore be used to assess the length of

polynucleotide stretches in forensic analyses.

Nevertheless, the results from the remaining laboratories

show a high level of consistency. In particular, it is note-

worthy that despite the relatively high levels of heteroplasmy

observed in this study, the maximum number of homoplas-

mic differences between any single hair and the reference

blood and saliva was found to be one (excluding results from

laboratory 10). While it is clear that in rare cases two or more

homoplasmic differences would be possible, this study

demonstrates that the magnitude of mutational segregation

within hair samples is tractable, and follows predictable

patterns of increasing rarity with increasing heteroplasmic

and homoplasmic differences. If interpreted with care, fol-

lowing published guidelines [21–23], such results can pro-

vide valuable and reliable information to assist in forensic

examinations.

Acknowledgements

The authors wish to thank all of the members of the

EDNAP group for useful comments and discussion. Parti-

cular thanks for helpful comments on the manuscript are due

to Angel Carracedo and Tom Parsons. The EDNAP group

worked in the period 1997–2000 within the framework of the

Standardisation of DNA Profiling in Europe (STADNAP)

consortium, a network project of the European Commis-

sion—DG XII programme ‘‘Standards, Measurement and

Testing’’ (Contract 97-7506).

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Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

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