RESEARCH ARTICLES
PCR-Induced Sequence Alterations Hamper the Typing of Prehistoric
Bone Samples for Diagnostic Achondroplasia Mutations
C. M. Pusch,* M. Broghammer,* G. J. Nicholson,à A. G. Nerlich,§ A. Zink,§
I. Kennerknecht,k L. Bachmann,{ and N. Blin*
*Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tübingen, Tübingen, Germany;
Institut für Ur- und Frühgeschichte, Abteilung Ältere Urgeschichte und Quartärökologie, University of Tübingen, Tübingen,
Germany; àInstitute of Organic Chemistry, University of Tübingen, Tübingen, Germany; §Institute of Pathology,
Division of Palaeopathology, Academic Teaching Hospital München-Bogenhausen, München, Germany;
kInstitute of Human Genetics, University of Münster, Münster, Germany; and {Department of Zoology,
Natural History Museums and Botanical Garden, University of Oslo, Oslo, Norway
Achondroplasia (ACH) is a skeletal disorder (MIM100800) with an autosomal dominant Mendelian inheritance and
complete penetrance. Here we report the screening of ancient bone samples for diagnostic ACH mutations. The
diagnostic G!A transition in the FGFR3 gene at cDNA position 1138 was detected in cloned polymerase chain reaction
(PCR) products obtained from the dry mummy of the Semerchet tomb, Egypt (first dynasty, ;4,890–5,050 BP [before
present]), and from an individual from Kirchheim, Germany (Merovingian period, ;1,300–1,500 BP), both of which had
short stature. However, these mutations were also reproducibly observed in four ancient control samples from
phenotypically healthy individuals (false-positives), rendering the reliable molecular typing of ancient bones for ACH
impossible. The treatment of a false-positive DNA extract with uracil N-glycosylase (UNG) to minimize type 2
transitions (G!A/C!T) did not reduce the frequency of the false-positive diagnostic ACH mutations. Recently, it was
suggested that ancient DNA extracts may induce mutations under PCR. Contemporary human template DNA from
a phenotypically healthy individual was therefore spiked with an ancient DNA extract from a cave bear. Again,
sequences with the diagnostic G!A transition in the FGFR3 gene were observed, and it is likely that the false-positive
G!A transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl2 indicate
that position 1138 of the FGFR3 gene is particularly sensitive for mutations. Our data are in line with previously
published results on the occurrence of nonrandom mutations in PCR products of contemporary human mitochondrial
HVRI template DNA spiked with ancient DNA extracts.
Introduction
The advent of polymerase chain reaction (PCR)
allowed the analysis of even traces of ancient nucleic acids
manifesting the era of palaeogenetic research. Given that
nucleic acids have survived in a particular sample, the
analysis of ancient DNA may provide valuable information
about diseases of the specimen under study. It was, for
example, possible to detect Mycobacterium tuberculosis in
Egyptian and pre-Columbian Peruvian mummies (Salo
et al. 1994; Nerlich et al. 1997). However, no research on
Mendelian diseases in old specimens has been reported so
far. This is mainly for three reasons: (1) Nuclear (i.e.,
single-copy) DNA has to be examined for this purpose. (2)
Inherited disorders often display substantial genetic and
phenotypic heterogeneity, and various different mutations
located in different exons may affect a particular gene. (3)
Only a limited number of inherited disorders are manifested
morphologically in the skeleton. With respect to the last two
arguments, achondroplasia is an exception from the rule.
Achondroplasia (ACH; MIM 100800) is the most
common form of short-limb skeletal disorder and occurs
with a frequency of 1:15,000. The disease has an autosomal dominant Mendelian inheritance with complete penetrance. The phenotype results from the disruption of the
Key words: ancient DNA extracts, ancient nuclear DNA, diagenesis, extract-induced mutations, manganese, PCR errors.
E-mail: bachmann@nhm.uio.no.
Mol. Biol. Evol. 21(11):2005–2011. 2004
doi:10.1093/molbev/msh208
Advance Access publication July 14, 2004
continuous process of enchondral ossification (Stanescu,
Stanescu, and Maroteaux 1990). The critical chromosome
region for ACH was localized to 4p16.3 by linkage analyses (Le Merrer et al. 1994; Velinov et al. 1994). In this
chromosomal region, the gene for the fibroblast growth
factor receptor 3 (FGFR3) described by Keegan et al.
(1991) is associated with ACH, hypochondroplasia (HCH),
and thanatophoric dysplasia (TD). It is noteworthy that
FGFR3 was originally considered a candidate gene for
the Huntington disease (Thompson et al. 1991). The vast
majority of ACH cases are caused by a G!A transition
(97%) at cDNA position 1138 causing a G380R amino acid
substitution; the remaining 3% are caused by a G!C
transversion at the same position resulting in the same
amino acid substitution of G380R (Shiang et al. 1994;
Rousseau et al. 1994; Wilkin et al. 1998).
Here we report on the identification and evaluation of
diagnostic mutations for achondroplasia in a mummified
specimen from Semerchet, Egypt, as well as in a Merovingian skeleton from Kirchheim, Germany, both of which
had short stature. A series of mock controls (i.e., modern
human blood and prehistoric bone samples from human
individuals not affected by ACH) and negative controls
were included to monitor sequence heterogeneity in PCR
products and to guarantee purity of the assays, respectively. Theoretically, the dominant mode of inheritance of ACH accompanied by its complete penetrance
will allow an unequivocal identification, because any
contamination of DNA extracts with DNA bearing the
Molecular Biology and Evolution vol. 21 no. 11 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
2006 Pusch et al.
Table 1
List of Prehistoric Samples Used in This Study
Samples
Geographic Origin
Dating of Sample
Short-statured individuals—suspected to suffer from achondroplasia
Semerchet
Egypt, Theben
4,890–5,050 BP
Qa’a
Egypt, Theben
4,890–5,050 BP
Den
Egypt, Theben
4,890–5,050 BP
Kirchheim
Germany
;1,300–1,500 BP
Mock controls—phenotypically healthy
Neresheim 1
Germany
Neresheim 2
Germany
Neresheim 3
Germany
Warburg 1
Germany
Warburg 2
Germany
Warburg 3
Germany
al Buhais A
United Arab Emirates,
Sharjah
al Buhais B
United Arab Emirates,
Sharjah
Nonhuman
Ursus spelaeus
Germany
;1,500
;1,500
;1,500
5,200
5,200
5,200
;6,000
BP
BP
BP
BP
BP
BP
BP
;6,000 BP
Pleistocene
diagnostic ACH point mutation must stem from shortstatured individuals with achondroplasia, which is very
unlikely.
Materials and Methods
Specimen and Sample Handling
In total, samples from 13 bones were processed (table
1). They include:
The skeletal remains of three short-statured individuals
from the necropolis of Abydos, Upper Egypt (predynastic 2 dynastic period) that belong to the tomb
complexes of the pharaohs Den, Semerchet, and Qa’a
(4,890–5,050 BP [before present]). These remains are
referred to as ‘‘dry mummies’’ because the individuals
were not embalmed and bandaged.
Bones of a soil-stored human individual with short stature
from Kirchheim/Ries, Germany, that was dated stratigraphically to the Merovingian period (;1,300–1,500
BP).
Eight samples from phenotypically healthy individuals
(mock controls) from Neresheim and Warburg, Germany, and from al Buhais, United Arab Emirates, that
are ;1,500, 5,200, and ;6,000 years old, respectively.
One nonhuman sample from Ursus spelaeus that was used
for spiking experiments of contemporary human DNA.
For all analyses, standard precautions against contamination of samples were taken. In particular, the
samples from the short-statured individuals and those of
phenotypically healthy individuals (mock controls) were
processed in different laboratories.
Amino Acid Racemization
The amino acid content of the samples was determined quantitatively by enantiomer labeling (Frank,
Nicholson, and Bayer 1978). The degree of racemization
of alanine, aspartic acid, leucine, phenylalanine, and serine
was determined according to Gerhardt and Nicholson
(1994). For this purpose, ;1.0 mg of dry pulverized
compacta was hydrolyzed in 6 N DCl in D2O. Ethyl ester/
TFA derivatives of the released amino acids were
subsequently analyzed by GC/MS (Agilent 6890/5973)
with SIM-detection on a 20 m 3 0.25 mm fused silica
capillary (30% Lipodex E / 70% PS255, film thickness of
0.13 l). The degree of racemization of serine and
phenylalanine indicated that bone glue treatment can be
excluded as a source for exogeneous contaminating DNA
(Nicholson et al. 2002).
DNA Extraction
Extraction of DNA from bone samples was done as
described in Pusch and Scholz (1997). Modern human
control DNA was isolated from white blood cells (Miller,
Dykes, and Polesky 1988).
PCR Cloning and Sequencing
A 164 bp stretch of the FGFR3 gene (position 1121–
1284 of GenBank entry M58051) was amplified using the
primers ACHF and ACHR of Shiang et al. (1994). Alternatively, the primer pair ACHvF 59-GTGTATGCAGGCATCCTCAG-39 (position 1153–1172 in M58051) and
ACHR was used to amplify a shorter 132 bp stretch of the
FGFR3 gene. The high fidelity Pfu polymerase (Stratagene,
La Jolla, Calif.) with proofreading property was used for
amplifications of ancient DNA templates and the spiking
experiments. Taq polymerase (Roche, Basil, Switzerland)
was used for control amplifications of modern template
DNA and for the screening of buffers for contamination.
Spiking experiments were done by adding 5 ll DNA
extract from an Ursus spelaeus (Pleistocene) sample to
50 ng modern human template DNA.
Cloning of PCR products into plasmid vectors
followed the temperature cycle-ligation protocol of Pusch,
Schmitt, and Blin (1997). DNA sequencing was performed
on an automated DNA sequencer using BigDye chemistry
(Applied Biosystems, Foster City, Calif.). All sequences
are provided as Supplementary Material online.
Uracil N-glycosylase (UNG) Treatment
The DNA extractions from the phenotypically healthy
Neresheim 1 individual were treated with UNG from
Escherichia coli as recently described (Hofreiter et al.
2001). To test for a UNG-mediated reduction of deaminated sites at cDNA position 1138 of the FGFR3 gene
the obtained clones of Neresheim 1 were screened with
SfcI. SfcI allows the differentiation between wild type
alleles and mutated sequences that are altered by the
predominant G!A transition.
Software
The Lasergene/Seqscape packages were employed for
sequence analyses and ClustalX (Thompson et al. 1997)
was used for multiple sequence alignments.
Results
Amino Acid Profiling
Amino acid racemization is frequently used to assess
the likelihood of DNA survival in bone samples (Poinar
Achondroplasia Typing of Prehistoric Bone Samples 2007
Table 2
Amino Acid Preservation of Three Prehistoric Samples
and a Modern Bone
Modern
Bone
Amino acids
(mg/g)
Protein
preservation (%)a
D/L aspartic acid
D/L alanine
D/L leucine
Neresheim
1
Kirchheim
277
217
66
100
0.02
,0.01
,0.01
78.3
0.04
,0.01
,0.01
24.1
0.04
0.01
0.02
Semerchet
113
40.1
0.23
0.01
0.01
1996; Krings et al. 1997) and in all samples the D/L
alanine and D/L leucine ratios were smaller than the D/L
asp ratio (Poinar et al. 1996).
Racemization values of aspartic acid indicate a good
DNA preservation in the bones from Warburg and
Neresheim, Germany, while it is unlikely to expect DNA
suitable for PCR experiments in the samples from al
Buhais, United Arab Emirates (data not shown).
Amplification of the FGFR3 Sequence
NOTE.—The Kirchheim and Semerchet samples are short-statured individuals.
Neresheim 1 is phenotypically healthy but shows the diagnostic ACH mutation, i.e.,
a false-positive ACH typing.
a
For determining the relative degree of protein preservation in the prehistoric
samples the absolute value of the modern control was set to 100%.
et al. 1996). The absolute amount of amino acids and the
degree of racemization of aspartic acid, alanine, and
leucine in the Kirchheim and Semerchet samples as well as
in the Neresheim 1 sample are listed in table 2. In these
particular samples the diagnostic ACH G!A transition in
the FGFR3 was detected, although the Neresheim 1
individual is phenotypically healthy (see below). Survival
of DNA that can be successfully subjected to PCR is likely
for both the short-stature Kirchheim individual (D/L asp:
0.04) and the healthy Neresheim 1 individual (D/L asp:
0.04), whereas it is unlikely for the Semerchet material
(D/L asp: 0.23), according to the criteria of Poinar et al.
(1996). Contamination of the samples with amino acids
from other sources is unlikely, because the relative concentrations of the individual amino acids correspond to
those of collagen from contemporary samples (Poinar et al.
A 164 bp segment of the FGFR3 gene (1121–1284 of
GenBank entry M58051) was amplified by using the
primer pair ACHF–ACHR. Amplicons of the expected
size were obtained for six mock controls and the
Semerchet sample but the other five samples failed even
in repeated rounds of PCR (table 3).
The PCR products were cloned and several clones per
sample were subsequently sequenced (see Supplementary
Material online). The diagnostic G!A transition at cDNA
position 1138 (position 1177 of GenBank entry M58051)
was found in the Semerchet sample. Surprisingly, the same
substitution also occurred in a number of clones derived
from the four phenotypically healthy mock controls
Neresheim 1 and 2, Warburg 2, and al Buhais A. In these
four cases the results must be considered as false-positives,
because ACH has an autosomal dominant inheritance with
complete penetrance and the resulting phenotype cannot be
overlooked. The diagnostic G!A transition at cDNA
position 1138 occurred in the PCR products from the falsepositives almost in the same frequency as in those obtained
from the short-statured individuals (table 3).
A reduction of the size of the target sequence to
132 bp (1153–1284 of GenBank entry M58051) by using
the primer pair ACHvF–ACHR yielded amplicons also in
Table 3
Observed Sequence Variability of Cloned FGFR3 Amplicons
Sample
164 bp Fragment (No. of
Clones/No. Different
Sequences)
132 bp Fragment (No. of
Clones/No. Different
Sequences)
1 (7/4)
—
—
—
1 (7/5)
1 (7/3)
1 (9/4)
1 (8/3)
1 (7/2)
—
1 (6/4)
—
n.d.
n.d.
1
n.d.
n.d.
n.d.
1 (50/35)
—
—
1 (37/32)
1 (5/5)
1 (8/7)
1 (5/5)
1 (8/5)
1 (8/6)
—
1 (6/5)
—
1 (26/25)
1 (12/n.d.)
1
1 (10/9)
1 (22/n.d.)
1 (10/9)
Semerchet
Qa’a
Den
Kirchheim
Neresheim 1
Neresheim 2
Neresheim 3
Warburg 1
Warburg 2
Warburg 3
al Buhais A
al Buhais B
Neresheim 3 UNG-treated
Neresheim 1 UNG-treatedc
Blood DNA
Blood DNA spiked
Blood DNA spikedc
Blood DNA, 0.25 mM
MnCl2
NOTE.—n.d., not determined.
a
132 bp and 164 bp amplicons pooled.
b
50% expected.
c
As determined by ScfI digestion.
% Clones with the 1138
G!A Transitiona
% Transitions of All
Sequence Alterationsa
33.3b
64.0
32.4b
58.3
40
0
0
20
50.5
86.7
80.9
77.8
42.8
54.5
41.7
66.7
0
33.3
0
50
22.7
40
41.1
n.d.
0
66.7
n.d.
56.0
2008 Pusch et al.
competitor molecules. Thus, the number of template molecules is 1–2 per ll Semerchet extract. This means that the
PCR experiments on the Semerchet extracts certainly
started from less than 20 template molecules.
UNG Treatment of Ancient DNA Extracts
FIG. 1.—Quantitation of genomic DNA in the Semerchet extract.
Targeting the 164 bp segment of the FGFR3 gene (primer pair ACHF–
ACHR), a series of a diluted FGFR3 competitor sequence with 55 bp
deleted was added to 5 ll DNA extract. ACHF and ACHR are expected
to equally amplify competitor and Semerchet DNA templates. The
number of 109 bp competitor molecules is indicated above the respective
lanes. C: control that contained neither competitor nor Semerchet extract
but 5 ll of a blank extraction; M: DNA size standard; W: PCR water
control.
the case of the Kirchheim sample, which was refractory
to PCR amplification in the previous experiments. The
diagnostic transition was detected in the Semerchet and
Kirchheim samples, but again false-positives were found
in the four phenotypically healthy mock controls Neresheim 1 and 2, Warburg 2, and al Buhais A (see Supplementary Material online).
When looking at the sequences one sees that
replacing primer ACHF with ACHvF to amplify the 132
bp fragment increased the mutation rate for some template
DNAs, e.g., for the Neresheim 1 sample from 1.0 3 1022
to 1.6 3 1022 per position (ratio 0.62), for the Neresheim
2 sample from 4.5 3 1023 to 2.3 3 1022 per position (ratio
0.19), and for the al Buhais A sample from 6.7 3 1023 to
1.8 3 1022 per position (ratio 0.37). However, the specific
mutation rate at cDNA position 1138 is almost the same,
i.e., 57% versus 60% of clones for Neresheim 1, 42.8%
versus 37.5% of clones for Neresheim 2, and 33.3% versus
50% for al Buhais A. Therefore, we conclude that the
length of the amplified fragment and possible primer
effects on the mutation rate per position are not relevant
for the frequency of false-positive G!A transitions at
cDNA position 1138.
Quantitation of Ancient DNA Molecules
Sequence alterations that occur in only a few
sequences of a cloned PCR product may result from
misincorporation of nucleotides at various stages of a
PCR. Degradation and damage of ancient DNA such as
lesions (Hansen et al. 2001) or miscoding bases (Höss et al.
1996) are likely to increase the frequency of such misincorporations. It is also known that PCR amplifications
that start from less than 1,000 template molecules tend to
yield inconsistent results (Handt et al. 1996).
As an example, we determined the number of initial
template molecules in the Semerchet sample. The 164 bp
segment of the FGFR3 gene (primer pair ACHF–ACHR)
was amplified quantitatively from the Semerchet extract in
the presence of defined numbers of competitor molecules
that had 55 bp deleted (fig. 1). It turned out that 5 ll
Semerchet extract amplified almost as good as 10 initial
The most frequent base alterations observed in our
sample collection were type 2 (G!A/C!T) transitions
(37.7%). Such transitional mutations can occur artificially
when using ancient DNA templates in PCR (Hansen et al.
2001), and it was suggested that a treatment of the extracts
with UNG from Escherichia coli can reduce such artifacts
(Hofreiter et al. 2001). We tested this enzyme on the
Neresheim 1 (phenotypically healthy but false-positive)
sample. Cloned PCR products from the UNG treated
Neresheim 1 extract were tested by SfcI analysis that allows
differentiation between wild type alleles and mutated ACH
sequences. Four of 12 analyzed clones still contained the
ACH mutation as determined by the correct length of the
resulting DNA fragments after electrophoresis (data not
shown). Elimination of the G!A transition at FGFR3
cDNA position 1138 through UNG treatment was thus not
possible.
Spiking Experiments
As suggested by Pusch and Bachmann (2004), the
observed G!A transition at FGFR3 cDNA position 1138
in the false-positive samples as well as some other
sequence alterations may be introduced during PCR as
a consequence of the presence of ancient DNA extracts in
the reaction mixture. We tested this hypothesis by adding
DNA extracts from Pleistocene Ursus spelaeus bones to
50 ng modern human DNA template from a phenotypically
healthy individual and amplified the 132 bp FGFR3
fragment. The diagnostic G!A transition at FGFR3
cDNA position 1138 was detected in five out of 10 clones
(50%) as well as other sequence alterations. In a replication
experiment 22 clones were screened by SfcI cleavage for
the diagnostic G!A transition and five clones (22.7%)
were positive as determined by the correct length of the
resulting DNA fragments after electrophoresis (table 3).
The unspiked modern human template DNA yielded the
wild type sequence without alterations. Furthermore, the
control amplification using the Ursus spelaeus DNA
extract yielded no 132 bp FGFR3 amplicon, i.e., contamination with human DNA was not detectable.
Amplification of the 132 bp FGFR3 Fragment Under
the Presence of MnCl2
Pusch and Bachmann (2004) suggested that agents
coextracted with DNA such as multivalent metal ions that
accumulate postmortem during diagenesis may induce
mutations under PCR. In particular, manganese in combination with magnesium is known to induce mutations
(e.g., Svetlov and Cooper 1998; Kunichika, Hashimoto,
and Imoto 2002). We amplified 50 ng of the same modern
DNA template that was used for the spiking experiment
under the presence of 0.25 mM MnCl2 and sequenced
10 clones. We obtained basically the same pattern of
Achondroplasia Typing of Prehistoric Bone Samples 2009
sequence alteration as in the spiking experiments. The
diagnostic G!A transition at FGFR3 cDNA position
1138 was detected in four out of 10 clones (40%) as well
as other sequence alterations. In two clones position 1138
was altered by a G!T transversion (see Supplementary
Material online).
occurrence of sequence alterations via the deoxyuracil
pathway that are introduced after the UNG treatment, thus
during PCR.
Discussion
Quantitation experiments gave an estimate of 1–2
template molecules per ll for the Semerchet sample. In
contrast, 35 different sequences (out of 50 clones
analyzed) were generated from the Semerchet extract
(132 bp PCR product). This indicates that miscoding
lesions in the ancient template DNA cannot account for the
observed sequence heterogeneity. Most of the heterogeneity of PCR products must, therefore, be artificially
induced de novo. One may assume that this effect is due to
low template numbers, a general problem when subjecting
ancient DNA extracts to PCR. However, PCR experiments
on diluted modern DNA templates always yielded the
expected authentic sequences. We therefore conclude that
the observed sequence heterogeneity is independent of
the number of template molecules. PCR jumping (Pääbo
1989) as a possible source for the observed sequence
variation can be ruled out through the spiking experiments
(i.e., cave bear extracts added to contemporary human
DNA template). The obtained sequences are without any
doubt human and there is no indication that damaged cave
bear DNA was involved in the amplification process. The
Ursus spelaeus sample itself did not yield any amplicon
and was, therefore, not contaminated with human DNA.
Further support comes from the amplification of the 132
bp stretch of the FGFR3 gene from the same contemporary
human template DNA that was used for the spiking experiment under the presence of MnCl2 (see below). Contamination through previously cloned fragments may also
be a possible explanation for the occurrence of the falsepositive G!A transition. However, this could be ruled out
because all amplicons from modern blood controls of
healthy individuals (no diagnostic G!A transition in the
FGFR3 gene at cDNA position 1138 observed) and some
of the mock controls never yielded the diagnostic ACH
mutation. Moreover, the water and blank extraction
controls never yielded PCR products.
We report the first screening of ancient bone samples
for diagnostic ACH mutations. The diagnostic G!A
transition in the FGFR3 gene at cDNA position 1138 was
detected in cloned PCR products obtained from the dry
mummy of the Semerchet tomb, Egypt (first dynasty,
;4,890–5,050 BP), and from an individual from Kirchheim, Germany (Merovingian period, ;1,300–1,500 BP),
that both had short stature. However, these mutations were
also reproducibly observed in four ancient control samples
from phenotypically healthy individuals (false-positives).
Thus, a reliable typing of ancient samples for diagnostic
ACH mutations is impossible.
There might be various reasons to explain the
occurrence of the false-positive G!A transition at cDNA
position 1138 in the FGFR3 gene. Here, three lines of
arguments will be discussed.
Post Mortem Damage of Template DNA
After the death of an organism, natural degeneration
of bio-/macromolecules begins and the speed of the decay
depends on the particular conditions of the environment.
Moisture, temperature, and soil chemistry are important
parameters in this context. Under certain conditions of
preservation with low to moderate levels of hydrolysis and
oxidation, various sized fragments of the DNA (in
particular those with a low molecular weight) may survive
in a bone. High salt concentrations at neutral pH and/or
fast dehydration/mummification are expected to favor
DNA survival (Pääbo and Wilson 1991; Lindahl 1993;
Burger et al. 1999; Ovchinnikov et al. 2000). In addition to
extensive enzymatic and physical DNA fragmentation, the
most important route of decay for hydrated DNA is
depurination (Lindahl 2000), leading, in part, to nicked
double strands (i.e., partially single-stranded DNA; Pusch,
Giddings, and Scholz 1998; Di Bernardo et al. 2002). It
was suggested that sequence alterations may originate
from such gaps or lesions in the DNA template (Hansen
et al. 2001).
In our study, false-positive diagnostic FGFR3 1138
G!A transitions occurred frequently. The application of
UNG was recently proposed to reduce or exclude artificial
type 2 (G!A/C!T) transitions (Hofreiter et al. 2001).
UNG is expected to cleave specifically uracil bases in the
ancient DNA to generate apyrimidinic sites (Pu and Struhl
1992). Such affected/altered DNA strands are no longer
suitable template molecules for subsequent PCR experiments because they will fall apart during the first denaturation step. A treatment of the Neresheim 1 extract did
not eliminate the occurrence of the false-positive diagnostic FGFR3 1138 G!A alterations. It should be noted
that the first denaturation step of a PCR will also inactivate
the UNG. UNG treatment can, therefore, not rule out the
Low Number of Template Molecules, PCR Jumping,
and Contamination of Samples
PCR-Induced Sequence Alterations
Pusch and Bachmann (2004) showed that ancient
DNA extracts can induce mutations in a nonrandom
fashion. They have amplified a 148 bp stretch of the
mitochondrial HVRI from contemporary human template
DNA in PCR reactions spiked with ancient DNA extracts.
In total, 34 positions of a 103 bp alignment were affected
and most mutations occurred repeatedly in independent
PCR amplifications. The spiking experiments presented
here (i.e., cave bear extracts added to contemporary human DNA template) support the hypothesis that the phenomenon of extract-induced mutations is responsible for
the unreliable typing of achondroplasia. The obtained
sequences—although derived from a healthy individual—
displayed frequently (31.3%) the diagnostic G!A transition at cDNA position 1138 as well as some other
2010 Pusch et al.
mutations. This indicates that the observed sequence alterations were introduced during PCR amplification. In other
words, it was possible to generate human ACH sequences
from modern human nucleic acids due to the presence of
ancient cave bear extracts in the PCR. We therefore have
to conclude that ancient DNA extracts contribute currently
unidentified components to the PCR mixture which either
alter template molecules directly or reduce the fidelity of
the Pfu polymerase. Pusch and Bachmann (2004) have
suggested that multivalent metal ions such as manganese
that accumulate postmortem during diagenesis may induce
mutations under PCR. PCR amplifications of contemporary human template DNA under the presence of MnCl2
indicate that cDNA position 1138 of the FGFR3 gene is
indeed particularly sensitive to mutagenesis. In 40% of the
obtained sequences the diagnostic G!A transition was
observed at this particular position (in addition, 20% of the
obtained sequences had a G!T transversion). It is still not
understood why particular nucleotides are more prone to
erroneous misincorporation of nucleotides than others. The
frequent occurrence of the diagnostic ACH mutation in
false-positive ancient samples may be the result of
processes affecting CG dinucleotides. For contemporary
nucleic acids it is known that such CG dinucleotides are
preferential targets for spontaneous point mutations. They
account for one-third of single-site mutations observed
in inherited human diseases and are among the major
types of miscoding lesions in the genome of living human cells (Cooper and Youssoufian 1988; Rideout et al.
1990).
To summarize, the experiments presented here show
that the G!A transitions at position 1138 of the FGFR3
gene, which is diagnostic for achondroplasia, occurred
artificially in amplicons from ancient DNA extracts of
phenotypically healthy individuals and render a reliable
molecular typing of ancient samples for the disease
impossible. We conclude that this is due to extract-induced
mutations under PCR as described by Pusch and Bachmann
(2004). They present the analyses of 547 sequences from
cloned amplicons of a 148 bp stretch of the mitochondrial
HVRI from contemporary human template DNA generated
in spiked PCR reactions. The authors observed that extractinduced mutations occurred in a nonrandom fashion in
independent PCR amplifications and in total 34 positions of
a 103 bp alignment were affected. It is noteworthy that
15.7% of all sequences analyzed by Pusch and Bachmann
(2004) differed from the closest human match in GenBank
by nine substitutions and three gaps but shared seven out of
11 mutations that are in combination characteristic for the
Neandertal sequence AF011222 (Krings et al. 1997). Their
data might challenge the authenticity of several published
sequences. However, showing that a combination of nucleotide substitutions can be generated artificially does not
necessarily imply that similar published sequences are not
authentic. In the example of Neandertal sequences, it might
be difficult to tell apart authentic nucleotide substitutions
from extract-induced mutations, because no contemporary
Neandertal control DNA exists. Theoretically, ACH offers
a more straightforward test system. ACH is a skeletal
disorder with an autosomal dominant Mendelian inheritance and complete penetrance and the resulting phenotype
cannot be overlooked. This means that the diagnostic
G!A transition at position 1138 of the FGFR3 gene (or
a G!C transversion at the same position) is expected in
short-statured individuals but must not occur in phenotypically healthy individuals. The observed false-positives are,
therefore, unambiguously artifacts. However, since this
particular mutation can occur as a false-positive there is no
reason to conclude that the occurrence of the diagnostic
G!A transitions at position 1138 of FGFR3 in PCR products of DNA extracts from short-statured individuals is
authentic. If so, we were biased to believe authenticity,
because the sequence data meet the phenotype. Thus, the
unreliable molecular typing of ancient bones for ACH
provides an example that even in a test system with phenotypic control proof of authenticity might be impossible.
Acknowledgments
We are grateful to G. Dreyer and A. Czarnetzki for
providing access to the skeletal remains studied here. This
work was supported in part by grants from the Research
Council of Norway (‘‘National Centre for Biosystematics,’’ 146515/420), the German Federal Ministry of
Education, Science, Research and Technology (Fö.
01KS9602), the Interdisciplinary Center of Clinical Research (IZKF-Q3), Tübingen, and the German Science
Foundation (Co226/6–1).
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Arndt von Haeseler, Associate Editor
Accepted June 1, 2004