PCR-Induced Sequence Alterations Hamper the Typing of Prehistoric Bone Samples for Diagnostic Achondroplasia Mutations
http://www.100md.com
分子生物学进展 2004年第11期
* Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tübingen, Tübingen, Germany; Institut für Ur- und Frühgeschichte, Abteilung ?ltere Urgeschichte und Quart?r?kologie, University of Tübingen, Tübingen, Germany; Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany; Institute of Pathology, Division of Palaeopathology, Academic Teaching Hospital München-Bogenhausen, München, Germany; || Institute of Human Genetics, University of Münster, Münster, Germany; and ? Department of Zoology, Natural History Museums and Botanical Garden, University of Oslo, Oslo, Norway
E-mail: bachmann@nhm.uio.no.
Abstract
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 GA 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 (GA/CT) 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 GA transition in the FGFR3 gene were observed, and it is likely that the false-positive GA 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.
Key Words: ancient DNA extracts ? ancient nuclear DNA ? diagenesis ? extract-induced mutations ? manganese ? PCR errors
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 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 GA transition (97%) at cDNA position 1138 causing a G380R amino acid substitution; the remaining 3% are caused by a GC 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 diagnostic ACH point mutation must stem from short-statured 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 – 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.
Table 1 List of Prehistoric Samples Used in This Study
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 x 0.25 mm fused silica capillary (30% Lipodex E / 70% PS255, film thickness of 0.13 μ). 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 5'-GTGTATGCAGGCATCCTCAG-3' (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 μl 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 GA 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 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 GA 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. 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).
Table 2 Amino Acid Preservation of Three Prehistoric Samples and a Modern Bone
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
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).
Table 3 Observed Sequence Variability of Cloned FGFR3 Amplicons
The PCR products were cloned and several clones per sample were subsequently sequenced (see Supplementary Material online). The diagnostic GA 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 GA transition at cDNA position 1138 occurred in the PCR products from the false-positives 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 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 x 10–2 to 1.6 x 10–2 per position (ratio 0.62), for the Neresheim 2 sample from 4.5 x 10–3 to 2.3 x 10–2 per position (ratio 0.19), and for the al Buhais A sample from 6.7 x 10–3 to 1.8 x 10–2 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 GA 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 (H?ss 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 μl Semerchet extract amplified almost as good as 10 initial competitor molecules. Thus, the number of template molecules is 1–2 per μl Semerchet extract. This means that the PCR experiments on the Semerchet extracts certainly started from less than 20 template molecules.
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 μl 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 μl of a blank extraction; M: DNA size standard; W: PCR water control.
UNG Treatment of Ancient DNA Extracts
The most frequent base alterations observed in our sample collection were type 2 (GA/CT) 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 GA transition at FGFR3 cDNA position 1138 through UNG treatment was thus not possible.
Spiking Experiments
As suggested by Pusch and Bachmann (2004), the observed GA 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 GA 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 GA 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 sequence alteration as in the spiking experiments. The diagnostic GA 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 GT transversion (see Supplementary Material online).
Discussion
We report the first screening of ancient bone samples for diagnostic ACH mutations. The diagnostic GA 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 GA 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 (P??bo 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 GA transitions occurred frequently. The application of UNG was recently proposed to reduce or exclude artificial type 2 (GA/CT) 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 GA 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 occurrence of sequence alterations via the deoxyuracil pathway that are introduced after the UNG treatment, thus during PCR.
Low Number of Template Molecules, PCR Jumping, and Contamination of Samples
Quantitation experiments gave an estimate of 1–2 template molecules per μl 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 (P??bo 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 false-positive GA transition. However, this could be ruled out because all amplicons from modern blood controls of healthy individuals (no diagnostic GA 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.
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 GA transition at cDNA position 1138 as well as some other 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 GA transition was observed at this particular position (in addition, 20% of the obtained sequences had a GT 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 GA 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 extract-induced 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 GA transition at position 1138 of the FGFR3 gene (or a GC 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 GA 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.
Acknowledgements
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 (F?. 01KS9602), the Interdisciplinary Center of Clinical Research (IZKF-Q3), Tübingen, and the German Science Foundation (Co226/6–1).
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E-mail: bachmann@nhm.uio.no.
Abstract
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 GA 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 (GA/CT) 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 GA transition in the FGFR3 gene were observed, and it is likely that the false-positive GA 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.
Key Words: ancient DNA extracts ? ancient nuclear DNA ? diagenesis ? extract-induced mutations ? manganese ? PCR errors
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 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 GA transition (97%) at cDNA position 1138 causing a G380R amino acid substitution; the remaining 3% are caused by a GC 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 diagnostic ACH point mutation must stem from short-statured 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 – 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.
Table 1 List of Prehistoric Samples Used in This Study
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 x 0.25 mm fused silica capillary (30% Lipodex E / 70% PS255, film thickness of 0.13 μ). 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 5'-GTGTATGCAGGCATCCTCAG-3' (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 μl 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 GA 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 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 GA 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. 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).
Table 2 Amino Acid Preservation of Three Prehistoric Samples and a Modern Bone
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
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).
Table 3 Observed Sequence Variability of Cloned FGFR3 Amplicons
The PCR products were cloned and several clones per sample were subsequently sequenced (see Supplementary Material online). The diagnostic GA 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 GA transition at cDNA position 1138 occurred in the PCR products from the false-positives 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 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 x 10–2 to 1.6 x 10–2 per position (ratio 0.62), for the Neresheim 2 sample from 4.5 x 10–3 to 2.3 x 10–2 per position (ratio 0.19), and for the al Buhais A sample from 6.7 x 10–3 to 1.8 x 10–2 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 GA 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 (H?ss 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 μl Semerchet extract amplified almost as good as 10 initial competitor molecules. Thus, the number of template molecules is 1–2 per μl Semerchet extract. This means that the PCR experiments on the Semerchet extracts certainly started from less than 20 template molecules.
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 μl 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 μl of a blank extraction; M: DNA size standard; W: PCR water control.
UNG Treatment of Ancient DNA Extracts
The most frequent base alterations observed in our sample collection were type 2 (GA/CT) 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 GA transition at FGFR3 cDNA position 1138 through UNG treatment was thus not possible.
Spiking Experiments
As suggested by Pusch and Bachmann (2004), the observed GA 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 GA 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 GA 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 sequence alteration as in the spiking experiments. The diagnostic GA 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 GT transversion (see Supplementary Material online).
Discussion
We report the first screening of ancient bone samples for diagnostic ACH mutations. The diagnostic GA 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 GA 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 (P??bo 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 GA transitions occurred frequently. The application of UNG was recently proposed to reduce or exclude artificial type 2 (GA/CT) 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 GA 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 occurrence of sequence alterations via the deoxyuracil pathway that are introduced after the UNG treatment, thus during PCR.
Low Number of Template Molecules, PCR Jumping, and Contamination of Samples
Quantitation experiments gave an estimate of 1–2 template molecules per μl 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 (P??bo 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 false-positive GA transition. However, this could be ruled out because all amplicons from modern blood controls of healthy individuals (no diagnostic GA 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.
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 GA transition at cDNA position 1138 as well as some other 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 GA transition was observed at this particular position (in addition, 20% of the obtained sequences had a GT 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 GA 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 extract-induced 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 GA transition at position 1138 of the FGFR3 gene (or a GC 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 GA 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.
Acknowledgements
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 (F?. 01KS9602), the Interdisciplinary Center of Clinical Research (IZKF-Q3), Tübingen, and the German Science Foundation (Co226/6–1).
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