Abstract

Polymorphic Y-chromosomal short tandem repeats (Y-STRs) are being employed for phylogenetic and evolutionary studies as well as for forensic applications. Precise knowledge of mutation types and rates is essential and has hitherto been obtained from computer simulation or small-sized father/son pairs, or derived from the more intensively studied autosomal STRs, respectively. To establish more accurate values we analysed about 18 000 DNA sequences isolated from sperm cells of three donors, representing highly validated offspring. Two loci were examined, i.e. DYS19 and DYS390. The methodology applied was small pool PCR with automated laser-induced fluorescence detection. The mutation rates for single repeat gains were determined as 0.18% [95% confidence interval (CI) 0.11–0.31%] for DYS390 and 0.21% (95% CI 0.13–0.33%) for DYS19, and two-repeat changes occurred in the order of 0.01%. Assuming a similar rate for the loss of repeats, which could not be detected with our approach, we predict an overall mutation rate of ∼0.4% per gamete per generation for both Y tetranucleotide loci. Moreover, these results support the stepwise mutation mechanism based on replication slippage. We expect this approach to be useful for individual mutation risk determination, as well as for studies concerning male history.

Received 1 December 2000; Revised and Accepted 30 January 2001.

INTRODUCTION

Microsatellites or short tandem repeats (STRs), consisting of up to about 25 tandemly repeated 2–6 bp DNA stretches, are abundant on human chromosomes (1,2). Their variation in repeat numbers seems to have arisen from polymerase slippage (3,4). For phylogenetic and evolutionary studies as well as for forensic applications, a constantly growing set of polymorphic Y-chromosomal short tandem repeats (Y-STRs) is being employed, since Y-STR haplotype distribution differs widely among populations (57). Most Y-STRs do not recombine and are therefore ideal markers for forensic male identification (2,6) or for tracing paternal history in genetic studies (7,8). This is analogous to mtDNA studies concerning maternal lineages. However, mutation rates clearly influence forensic identification probabilities as well as characteristic population data, such as the age of the most recent common ancestor or division times of populations (9,10). Given that mutation rates of autosomal STRs depend on repeat numbers and parental ages and moreover vary between loci (3,11), forensic or anthropological conclusions drawn from Y-haplotypes require precise knowledge of mutation frequencies of all Y-STRs employed (12).

There are three approaches to determining mutation rates of microsatellite DNA: (i) The allelic pattern in confirmed parent–child trios or large pedigrees may be monitored by PCR and/or DNA sequencing. Deviations from Mendelian expectation can be attributed to mutations (4,13,14). (ii) Different populations with known histories may be analysed by PCR-based genotyping, leading under several assumptions to theoretically estimated mutation values (15,16). (iii) Sperm cell DNA either as single molecules or as in small pools may be subjected to PCR and Southern blotting in order to search for male-related mutational deviations (1722).

Only a few small-scale father/son genotyping studies have been performed applying the first approach to Y-STRs, thus resulting in rather imprecise mutation rates (13,14). With the second method, Caglià et al. (16) estimated an average mutation rate for Y-STR loci by analysing Italian and Sardinian populations. Using computer simulations, Cooper et al. (15) suggested that estimations of mutation rates of Y-STRs are rather imprecise. The third approach has been successfully employed for autosomal minisatellites (18,23) and gene-associated trinucleotide repeat polymorphisms showing large repeat expansions/deletions (2022). Despite experimental limitations due to the very small mutational changes in allele length when focussing on 4 bp single step mutations, the latter strategy seemed to be the most promising, for the following reasons. Sperm cells are available in nearly unlimited numbers, therefore allowing the determination of Y-STR mutation rates on a large scale. In addition, only DNA from known origins can be used, which in principle excludes the risk of unclear paternity. Finally, the mutation rates are taken as individual features, so that statistical comparisons between any given donor groups become possible.

RESULTS

In this study, we precisely quantified DNA concentrations from sperm cell lysates of three males aged 30–35 years and digested the DNA with restriction enzymes. Then PCR amplification was performed using fluorescence labelled primers flanking the DYS19 and DYS390 loci. Fluorescence detection revealed an efficiency of >50% for single template molecule PCR. Next, mixtures of DNA from semen donors, with the admixed DNA differing by one 4 bp repeat, were amplified by PCR. Electropherograms derived from such mixes showed a major and a minor peak at the expected lengths according to the ratios of mixtures. Finally, one single deviant molecule could be resolved in up to 40 original template molecules if the admixed molecule was longer than the major component.Theefficiency observed was the same as after corresponding single template molecule PCR (data not shown).

For mutation rate determination, a multiplicity of assays, each with 25 template molecules, was analysed. Besides the main peak there infrequently occurred a minor peak at distances of +4 or +8 bp (Fig. 1). This was considered a one-repeat or a two-repeat gain in one mutant sperm cell. Since stutter peaks always occurred in the region smaller than the main peak, repeat losses were not considered at this stage of the analysis.

In a total of 9223 and 8802 sperm cells analysed for DYS19 and DYS390, respectively, we observed 20 and 17 mutations, respectively. All mutations observed were one-repeat gains, except for one in each system, which were found to be two-repeat gains.

An overall mutation rate of 0.43% [95% confidence interval (CI) 0.26–0.66%] at DYS19 and 0.39% (95% CI 0.22–0.61%) at DYS390 per gamete per generation was calculated under the assumption of repeat losses and gains being nearly at equilibrium (3) by simple duplication of the observed rates (Tables 1 and 2). Recently, Xu et al. (24) also observed no significant difference between the numbers of expansion and contraction mutations at autosomal tetrameric STRs.

DISCUSSION

Comparison of the three donors revealed small differences at individual mutation rates varying between 0.27 and 0.61%. The fact that even the longest alleles in this series were associated with even longer mutant alleles with frequencies similar to the short ones strongly opposes the possible explanation that deviant peaks were due to contamination. This is especially so because the elongated alleles were not investigated with the same methodology in our laboratory. Moreover, as a safeguard, we had designed new primers for this study which had previously not been used in the laboratory.

Both loci examined have a compound repeat structure with relatively long stretches of uninterrupted repeats, i.e. the highly variable segment (Fig. 2). In these proportions, GATA repeats (DYS19) vary between 10 and 14 and CTAT repeats (DYS390) range from 8 to 15, whereas in our study the number of uninterrupted repeats in the three donors was 10 or 11 (DYS19) and 12 or 13 (DYS390). For conclusions concerning the influence of the number of uninterrupted repeats as one of the three proposed major factors besides age and sex (3), further determinations of mutation rates at different loci would be necessary (Fig. 2).

Our mean mutation rate of 0.4% (95% CI 0.22–0.62%) is in good accordance with that of similarly structured autosomal loci (3). They are also well in the range of the figure of 0.21% as determined by Weber and Wong (4) for autosomal tetranucleotide STRs, but they exceed that of Caglià et al. (16), who theoretically predicted frequencies for six Y-STRs ranging between 0.03 and 0.11% after comparing historical population data. However, our data would be compatible with the Y-STR mutation rates from father/son pairs or deep-rooting pedigrees samples. Published mean values are 0.3% (95% CI 0.04–0.67%; two mutations out of 626 allelic transfers) and 0.2 % (95% CI 0.03–0.68%; two mutations out of 996 allelic transfers) for DYS19 (6,25), 0.2% (four mutations out of 1917 allelic transfers) for nine Y tetranucleotide loci including DYS19 and DYS390 (13) (95% CI 0.05–0.55%), and 0.86% (95% CI 0.25–2.3%; four mutations out of 426 allelic transfers) (25). The narrower CI of our data may easily be explained by the larger sample size that was analysed by the small pool PCR approach and this may also indicate an improved accuracy of the observed value for the particular mutation rate.

Moreover, our results support the single step mutation model based on replication slippage as previously suggested (26) since we have directly observed gains of exactly one repeat or—by at least one order of magnitude less frequently—two repeats.

Interestingly, donor 3 shows at both loci the lowest rates (0.26% at DYS19 and 0.30% at DYS390). Since this individual is a strict non-smoker and a teetotaller—in contrast to the other donors—we cannot exclude that individual lifestyle influences the individual mutation frequencies.

Taken together, with 0.4% per gamete per generation, we have determined a relatively high mutation frequency that may render the utility of Y-STR loci in general, or at least of DYS19 and DYS390 for evolutionary research, even more difficult. On the other hand, the mutation rates and mechanisms determined here provide an update of former results for population genetic studies and for forensic applications. Moreover, the single molecule/small pool PCR method presented to determine individual mutation rates derived from single step mutations is useful for evaluating even unspecific health risks in particular concerned groups, at least in males.

MATERIALS AND METHODS

DNA preparation

Semen samples were obtained from three healthy Caucasoid volunteers who were known to carry alleles at the loci investigated representing almost 60% of the alleles that are present in the German population, i.e. alleles 23 and 24 at DYS390, 25.7 and 34.4 %, respectively; alleles 13 and 14 at DYS19, 7.6 and 48.2%, respectively (http://www.uni-duesseldorf.de/WWW/MedFak/Serology/database.html). Subsequent manipulations were performed in a laminar flow hood to minimize the risk of contaminations. DNA was prepared each from 5 ml of sperm with QIAamp Tissue kit (Qiagen) followed by two subsequent phenol-chloroform extractions (27). DNA concentration was measured spectrophotometrically within values spanning A260 nm = 0.3–0.5 for accurate determination. 5 µg DNA aliquots from each donor were digested with 50 U EcoRV in 0.1 ml volumes. The enzyme was then heat-deactivated by incubation at 70°C for 30 min. Five 1:10 dilution steps prior to PCR led to a final DNA concentration of 0.5 pg DNA/µl.

DNA amplification

All primers were purchased from Eurogentec. DYS19 allele amplification was performed with primers DYS19-P1-FAM and DYS19-RA, and DYS390 alleles were amplified with primers DYS390-FA-Hex and DYS390-P2 (sequences are underlined in Figure 2). The use of new primers (DYS19-P1-FAM and DYS390-FA-Hex) that were designed for regions outside the established primer sites enhanced PCR sensitivity and excluded contamination with ‘old’ PCR products.

Reactions were performed in a total volume of 25 µl with 5–125 pg DNA corresponding to 1–25 Y-containing sperm cell nuclei, assuming 2.5 pg DNA per haploid genome and an equilibrium of X and Y chromosome-carrying sperm cells. The following reagents were added: primers (1 µM each, DYS390-P2; 2 µM), MgCl2 (1.5 mM), BSA (3 µM), reaction buffer [10 dNTPs (each 0.2 mM); Applied Biosystems] and AmpliTaq Gold DNA polymerase (2.5 U; Applied Biosystems). Ten DNA-free controls were similarly prepared for each assay using the water from former DNA dilution steps. All PCR reactions were overlaid with paraffin oil. PCR conditions were 94°C for 10 min, then 40 cycles of 94°C for 90 s, 54°C (DYS19) and 50°C (DYS390) for 40 s and 72°C for 80 s, followed by a final extension step of 72°C for 10 min. This final step was optimized to yield two peaks differing by 1 bp (incomplete 3′ adenylation) to easily distinguish amplicon peaks from artefacts. Amplification was performed in a Trioblock thermalcycler (Biometra). Polyacrylamide gel electrophoresis with subsequent silver-staining served as a first control of PCR products. Fragment length analysis was performed on an ABI PRISM 373A automated DNA sequencer (Applied Biosystems). For further control all samples containing mutations were reanalysed by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Sequencing

PCR products from 5 pg template DNA originating from each donor and amplified for both loci were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol.

Analysis

For fragment length analysis the internal standard GeneScan-500 ROX was used with GS analysis version 1.2.2-1 (on the ABI373A) and version 2.1 (on the ABI310), and sequencing analysis 3.0 was applied for DNA sequencing.

The efficiency of amplification was determined for every PCR assay from 30 samples (10 for every donor) containing each 5 pg DNA corresponding to one Y chromosome-carrying molecule (28). The efficiencies in mixtures of 5 pg of foreign DNA in a 125 pg DNA template sample were found to be the same and decreased only slightly over a period of 6 months. These donor-related 5 pg efficiencies (E) were used to estimate the number of detectable Y-containing sperm cells (Ndet) in each of the 125 pg test samples (i.e. Ndet = E × Ntheor) with the theoretical number of templates (Ntheor) = 25. The observed number of mutations (Mobs) is related to the number of theoretical mutations (Mtheor) by Mobs = E × Mtheor. The mutation rate (µ) was calculated as the ratio of observed mutations/number of detectable template molecules, i.e. µ = Mobs/Ndet. These values were also employed for the calculation of CIs (http://member.aol.com/johnp71/confint.html).

ACKNOWLEDGEMENTS

This work was supported by grant Br-1-1-I/97-16 from the fund ‘Innovative Medizinische Forschung’.

+

Present address: Institut für Rechtsmedizin, Universität München, Frauenlobstraβe 7a, D-80337 München, Germany

§

Present address: The McDonald Institute for Archaeological Research, University of Cambridge, Downing Street, Cambridge CB2 3ER, UK

To whom correspondence should be addressed. Tel: +49 251 83 55160; Fax: +49 251 83 55158; Email: brinkma@uni-muenster.de

Figure 1. Electropherograms depicting mutated alleles (arrows) at the loci DYS19 (B) and DYS390 (D). Samples with no mutations at the respective loci are shown in (A) and (C). For each, 125 pg sperm cell DNA was subjected to small pool PCR and capillary gel electrophoresis with automated fluorescence detection on an ABI PRISM 310 Genetic Analyzer.

Figure 1. Electropherograms depicting mutated alleles (arrows) at the loci DYS19 (B) and DYS390 (D). Samples with no mutations at the respective loci are shown in (A) and (C). For each, 125 pg sperm cell DNA was subjected to small pool PCR and capillary gel electrophoresis with automated fluorescence detection on an ABI PRISM 310 Genetic Analyzer.

Figure 2. Schematic structure of the loci DYS19 and DYS390. Primer sequences and variable repeats are underlined.

Figure 2. Schematic structure of the loci DYS19 and DYS390. Primer sequences and variable repeats are underlined.

Table 1.

Mutational events at the DYS19 locus

Donor Allele no./fragment size (bp) No. of +1/+2 repeat mutations No. of random tests Mutation rate for repeat gains (%) Mutation rate per generation (%) 95% CI 
13/211 9/0 2930 0.31 0.61 0.28–1.06 
14/215 6/1 3290 0.21 0.43 0.18–0.88 
14/215 4/0 3003 0.13 0.27 0.08–0.68 
Total  19/1 9223 0.22 0.43 0.26–0.66 
Donor Allele no./fragment size (bp) No. of +1/+2 repeat mutations No. of random tests Mutation rate for repeat gains (%) Mutation rate per generation (%) 95% CI 
13/211 9/0 2930 0.31 0.61 0.28–1.06 
14/215 6/1 3290 0.21 0.43 0.18–0.88 
14/215 4/0 3003 0.13 0.27 0.08–0.68 
Total  19/1 9223 0.22 0.43 0.26–0.66 
Table 2.

Mutation events at the DYS390 locus

Donor Allele no./fragment size (bp) No. of +1/+2 repeat mutations No. of random tests Mutation rate for repeat gains (%) Mutation rate per generation (%) 95% CI 
23/238 5/0 3338 0.15 0.30 0.10–1.70 
23/238 7/1 2809 0.28 0.57 0.24–1.12 
24/242 4/0 2655 0.15 0.30 0.08–0.78 
Total  16/1 8802 0.19 0.39 0.22–0.61 
Donor Allele no./fragment size (bp) No. of +1/+2 repeat mutations No. of random tests Mutation rate for repeat gains (%) Mutation rate per generation (%) 95% CI 
23/238 5/0 3338 0.15 0.30 0.10–1.70 
23/238 7/1 2809 0.28 0.57 0.24–1.12 
24/242 4/0 2655 0.15 0.30 0.08–0.78 
Total  16/1 8802 0.19 0.39 0.22–0.61 

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