Genetic Sampling Error of Distance ({delta}µ)2 and Variation in Mutation Rate Among Microsatellite Loci

Lev A. Zhivotovsky, David B. Goldstein and Marcus W. Feldman

Vavilov Institute of General Genetics, Moscow, Russia;
Department of Biology, University College London, London, England;
Department of Biological Sciences, Stanford University


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
An expression is obtained for the time-dependent variance of the microsatellite genetic distance ({delta}µ)2 when the mutation rate is allowed to vary randomly among loci. An estimator is presented for the coefficient of variation, Cw, in the mutation rate. Estimated values of Cw from genetic distances between African and non-African populations were less than 100%. Caveats to this conclusion are discussed.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
In order to estimate the time of divergence of two contemporary populations from a single ancestral lineage, a genetic distance that is a known function of this time is desirable. When the populations are assayed for microsatellite polymorphism, the genetic distance ({delta}µ)2, based on the average squared differences in the sizes of alleles sampled in pairs, one from each population, has an expectation that increases linearly with time at a rate equal to twice the mutation rate in the case of one-step mutations (Goldstein et al. 1995Citation ). For multistep mutations, the rate of increase is twice the effective mutation rate, which is the product of the mutation rate and the variance of changes in allele size due to mutation (Zhivotovsky and Feldman 1995Citation ).

The usual way to analyze a set of microsatellite loci from individuals sampled in two populations is to compute ({delta}µ)2 for each locus and average across loci. If the mutation rate (or the effective mutation rate) is the same at all loci, and is known, then simple division gives an estimate of the expected time since separation of the populations. Variation across loci in the mutation rate affects the variance of ({delta}µ)2 (but not its expectation).

The evolutionary process involves genetic sampling error due to random genetic drift and mutation, and thus the variance among the possible evolutionary replicates of the distance is an important issue. Zhivotovsky and Feldman (1995)Citation implied that among replicates, the distance follows a chi-square distribution. In fact, the variance of the distance does asymptotically satisfy the most important property of the chi-square distribution, namely, that its variance approaches twice the square of its expectation as time increases (Zhivotovsky, Feldman, and Grishechkin 1997Citation ), but the actual distribution is not exactly chi-square.

From their analysis of properties of ({delta}µ)2 in a study of more than 200 human microsatellite loci, Cooper et al. (1999)Citation found strong evidence for variation among loci in the mutation rate. Our purpose with this paper is to obtain an analytical expression for the variance of ({delta}µ)2 when the mutation rate is variable. An important application of this analytical expression could be estimation of the extent of variation in mutation rate among microsatellite loci. Our analysis also allows us to compute the time-dependent dynamics of the variance of ({delta}µ)2 and to assess how sensitive these dynamics are to the assumption of a fixed mutation rate that is constant across loci.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
Consider a randomly mating diploid population of constant size N with nonoverlapping generations and an autosomal microsatellite locus undergoing multiple-step mutation with mutation rate µ and, possibly, constant mutation bias, as measured by the difference between the mean size of mutations and the size of the parental allele. (There is no bias if the difference is zero). Let {eta}(2)m be the expectation of the square of mutational gains and losses (Di Rienzo et al. 1998Citation ), which in the case of no average mutation bias becomes the variances in mutation changes, {sigma}2m (Slatkin 1995Citation ). We call w = µ{eta}(2)m the effective mutation rate. Also, introduce k = µ{eta}(4)m, where {eta}(4)m is the fourth noncentral moment of mutational changes in repeat score; w = k = µ in the case of one-step symmetric mutation. Assume for a while that the mutation parameters do not vary between loci.

The within-population variation at a microsatellite locus can be characterized by the mean allele size (r), the variance of allele size (the second central moment) (V), and the unnormalized kurtosis (the fourth central moment) (K) (Zhivotovsky and Feldman 1995Citation ). The between-population variation can be measured by analogs of FST (Slatkin 1995Citation ; see also Michalakis and Excoffier 1996Citation ; Rousset 1996Citation ; Feldman, Kumm, and Pritchard 1999Citation ). For two populations, the ({delta}µ)2 distance is defined as the squared difference of the mean values of their repeat scores: ({delta}µ)2 = (r1 - r2)2 (Goldstein et al. 1995Citation ).

Suppose that two populations diverged from an ancestral population at initial time t = 0 at which the profile of allele frequencies was represented by 0 (i.e., 0 produced specific values of the variance V0, unnormalized kurtosis K0, etc.) and then evolved independently under random genetic drift and multistep mutation. Given 0, r{S | 0} is an expectation operator that averages the statistic S over all possible realizations (replicates) of the drift-mutation process. Averaging with r may then be followed by the operator 0, which averages over all possible genetic structures 0 of the unknown ancestral population, i.e., values of V0, K0, etc. Thus, r averages over loci having identical mutation parameters and identical starting conditions, and 0 averages over the different initial conditions. We assume that prior to divergence, the ancestral population had attained mutation-drift equilibrium, where the expectations of the within-locus variances, the between-locus variance of variances, and the unnormalized within-locus kurtosis are


respectively, with {kappa} = (2N - 1)k (Zhivotovsky and Feldman 1995Citation ); k is defined in equation (6) of the appendix. Recall that expressions (1) are valid if mutation parameters do not vary among loci (see eqs. 12 if they vary among loci).

After {tau} generations of divergence, the expected distance, 0r(({delta}µ)2), equals 2w{tau} (Zhivotovsky and Feldman 1995Citation ; see also Feldman, Kumm, and Pritchard 1999Citation ; Zhivotovsky 2001Citation ), which becomes 2µ{tau} with one-step symmetric mutation (Goldstein et al. 1995Citation ).

The square of the genetic sampling error of a statistic is its variance over replicates (Weir 1996Citation ). Therefore, the within-locus variance of ({delta}µ)2 is defined as


The between-locus variance,


is due to variation in initial conditions 0 and together with the within-locus variance makes up the total variance in the case of no variation in mutation rate among loci, VarT = VarW + VarB. VarT is in fact the quantity of interest in assessing the reliability of ({delta}µ)2 as a distance measure in the presence of variation across loci in the mutation rate. Analytical expressions for both variances are given in the appendix. It can be shown that the total variance is greater than 8w2{tau}2 near the beginning of the process, although this value is ultimately approached, as was shown by Zhivotovsky, Feldman, and Grishechkin (1997)Citation and has been observed in numerical simulations.

Suppose the effective mutation rate varies across loci with mean , variance {sigma}2w, and , the mean value of k over loci. It is proved in the appendix that


assuming mutation-drift equilibrium, which entails that the expected distance is 2w{tau} at generation {tau}. As time increases, this asymptotically approaches 82{tau}2 x [1 + 3{sigma}2w/22 + (1/{tau})]. In order to evaluate the accuracy of equation (2) , we carried out a simulation using coalescent techniques following the algorithm of Hudson (1990)Citation , modified to include the stepwise mutation process with and without variation among loci in the mutation rate. In figure 1 , we see that the simulated data produce values for VarT that are close to those expected from equation (2) .



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 1.—To evaluate the accuracy of equation (2) , we ran coalescent simulations following the algorithm of Hudson (1990)Citation including the stepwise mutation process with and without rate variation. Separation times are given in units of 2N. In the case of a constant mutation rate, {theta} is set to 3.5. For rate variation, the average {theta} is again 3.5, but the thetas are now drawn from a gamma distribution with a variance of 10. Each of 200 replications involves 30 loci and 30 sampled alleles at each locus. White triangles and circles represent analytical results for no variation in mutation and for variation in mutation among loci, respectively. Black triangles and circles are the corresponding simulated values

 
Rewrite equation (2) as

where is the expression in the first three lines of the right-hand side of equation (2) , and is the multiplier of {sigma}2w in the third line of equation (2) . Then, given the observed variance in genetic distances across loci, Varobs, the variance in mutation rates can be estimated as


As time increases, {sigma}2w and the coefficient of variation of w, Cw = {sigma}w/, asymptotically satisfy


where C({delta}µ)2 is the coefficient of variation of ({delta}µ)2. Expression (5) can also provide an upper estimate of C2w if the asymptote has not been approached (fig. 2 ).



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 2.—Dynamics of the coefficient of variation (%) of the effective mutation rate Cw (equation 5 ). The parameters are w = 0.001, {sigma}w = 0.0005 (hence, Cw = 0.5), and 2N = 5,000. Mutation is single-step and symmetric

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
We can use expression (5) to estimate Cw from data. Table 1 shows the estimates for different sets of di- and tetranucleotide loci based on genetic distances between African and non-African human populations. Two of three sets show substantial values of Cw. However, probably not more than 10,000 generations have passed since the divergence of Africans and non-Africans, and thus the values of Cw in table 1 are overestimated (see fig. 1 ). Therefore, on average, variation in mutation rate does not seem to be very extensive, although it is not excluded that some microsatellite loci can show much higher or lower mutation rates than an average locus. For example, Forster et al. (2000)Citation found that the average mutation rate at the Y-chromosome loci could be taken as 0.26 x 10-3 if locus DYS392 was omitted because of its unusual behavior; otherwise, it was about 10 times as high. However, we should emphasize that our findings concern the effective mutation rate, i.e., the product of mutation rate and the variance in the number of repeats due to mutation, while Forster et al. (2000)Citation considered only the mutation rate.


View this table:
[in this window]
[in a new window]
 
Table 1 Estimates of the Coefficient of Variation Among Loci of the Effective Mutation rate, Cw, Based on Genetic Distances Between African and Non-African Populations for Different Sets of Data

 
Two caveats should be noted in connection with the above remarks on the size of Cw. First, our estimates were made under the assumption of constant population size, which is surely erroneous for humans in the last 4,000 generations. Second, since the variance of Cw is likely to be large over this time range and with the number of loci considered here, our confidence that Cw is indeed small cannot be great.

Earlier, Zhivotovsky and Feldman (1995)Citation pointed out that hundreds of loci are required to estimate the genetic distance ({delta}µ)2 with reasonable accuracy, and with variable mutation rates, the number of loci must be even greater. Indeed, as follows from equation (5) , the coefficient of variation of genetic distance ({delta}µ)2 averaged over L loci, which can be used as a measure of the relative accuracy (R) of estimation of the genetic distance, is approximated by [(2 + 3C2w)/L]1/2, or L = (2 + 3C2w)/R2. For instance, if the relative accuracy is 10%, i.e., R = 0.1, then 200 loci with identical mutation rates would be needed, whereas 500 loci are required to estimate genetic distance with the same precision if the relative variation in mutation rates is 100%, i.e., if Cw = 1. As an example, using combined data on 131 di-, tri-, and tetranucleotide microsatellite loci, Zhivotovsky (2001,Citation table 1 ) estimated approximately 14% for the accuracy of genetic distances between African and non-African populations. It should be noted, however, that in the analyses of Jin et al. (2000)Citation , ({delta}µ)2 was not able to reliably distinguish continental groups in trees made using the 28 loci of Bowcock et al. (1994)Citation , although its performance was comparable with other distance measures with 64 microsatellite loci. Again, this reinforces our view that several hundred loci would be needed to produce satisfactory estimates of ({delta}µ)2 and Cw.

It should be strongly emphasized that expression (2) , as well as expressions (4) and (5) , derived from it, are only valid for reproductively isolated populations of constant size at mutation-drift equilibrium. Otherwise, if we consider a process of subdivision of a parental population into two populations that subsequently evolve under mutation and genetic drift, the genetic distance ({delta}µ)2 becomes a nonlinear function of time; in particular, it underestimates the divergence time if the two populations are growing in size and/or are connected by gene flow (Zhivotovsky 2001Citation ). Therefore, our estimates in table 1 have to be regarded with caution.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
We are indebted to two anonymous reviewers for helpful comments and constructive suggestions. This research was supported in part by the National Institutes of Health (grants GM 28016, GM 28428, and 1 R03 TW005540), the Russian Foundation of Basic Research (grants 01-04-48441 and 01-07-90197), and the Russian State Program "Human Genome" (grant 26/01).


    Appendix
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 
A Case of Constant Mutation Bias
We permit a constant bias in mutation; that is, the expected average repeat score in progeny may be larger (or smaller) than the size of a parental allele by a constant value that is independent of the parental allele. As noted by Di Rienzo et al. (1998)Citation and Zhivotovsky (2001)Citation , if {eta}(2)m and {eta}(4)m are the second and fourth noncentral moments of mutational changes in repeat score, equations (3)–(8) of Zhivotovsky and Feldman (1995)Citation , as well as the expectation of ({delta}µ)2 (namely, 2w{tau}), remain valid with the parameters


neglecting terms of order µ2 and smaller. In the case of no mutation bias, {eta}(2)m is {sigma}2m, the variance in mutation changes. In particular, the relationships (eq. 1 ) that were obtained by Zhivotovsky and Feldman (1995)Citation under the assumption of no mutation bias remain valid in the case of constant bias if the moments are taken with respect to zero instead of with respect to the mean (Zhivotovsky 2001Citation ). Expressions for and Var(V) were extended to the case of constant mutation bias by Kimmel and Chakraborty (1996)Citation and Di Rienzo et al. (1998)Citation .

The Within-Locus Variance of ({delta}µ)2
Using the expression for the within-locus variance Var{{Delta}(t)} (Zhivotovsky, Feldman, and Grishechkin 1997Citation , p. 932, right column), which remains valid with the moments taken with respect to zero, and taking the limit as the regression coefficient ß -> +0, we obtain


(Note that in Zhivotovsky, Feldman, and Grishechkin [1997Citation , p. 932, right column], the expressions Var{{Delta}({tau})} and ({{Delta}({tau})})2 in the above notation are 0{(Varr{{Delta}({tau})}} and 0{r{{Delta}({tau})})2}, respectively, and the symbol k should read km.) Zhivotovsky, Feldman, and Grishechkin (1997)Citation showed that this variance is approximated as

when {tau}/2N either is small or increases infinitely. (Earlier, Zhivotovsky and Feldman [1995Citation , corollaries 1 and 2] had suggested that the variance was twice the squared distance expected at equilibrium, namely, 2(2w{tau})2 = 8w2{tau}2. However, the latter is 2[0{r{({delta}µ)2}}]2, which is not the right-hand side of eq. 8 ).

The Between-Locus Variance of ({delta}µ)2
From equations (4) and (14) of Zhivotovsky and Feldman (1995)Citation , the changes in the expected values of the distance and the variance are r(({delta}µ)2({tau} + 1) | 0) - r(({delta}µ)2({tau}) | 0) {approx} (1/N)r(V({tau}) | 0), and r(V({tau} + 1) | 0) - r(V({tau}) | 0) {approx} w - (1/N)r(V({tau}) | 0)), neglecting terms of order less than 1/N and recalling that w is defined by equation (6) . Replacing the differences in the left-hand sides of these approximations with corresponding differentials and solving the resulting linear differential equations, we have


As follows from the definition of the between-locus variance, VarB is equal to the expectation 0 of the square of 2(V0 - )(1 - e-{tau}/2N). Then, using equation (1) , we obtain


Variation in Mutation Rate
The well-known partitioning of conditional variance (e.g., Rice 1995Citation ) can be extended to the case of three random values: for an arbitrary function f(x, y, z), its variance, zyx(f - zyxf)2, is


Now, consider x, y, and z, respectively, as r, 0, and the expectation operator averaging over varying values of the mutation parameters, m, and take the distance ({delta}µ)2 as function f. The first two terms in the right-hand side of equation (11) represent the expectation m of VarW in equation (7) and VarB in equation (10) , respectively. The third term is Varm(0(({delta}µ)2)), the variance of the expected distance in equation (9) with respect to mutation parameters. Taking the expectations and summing in equation (11) , we obtain equation (2) .

Additionally, note that at mutation-drift equilibrium, the within-locus variance, the unnormalized within-locus kurtosis, and the between-locus variance of variances in the case of varying mutation rate become (using the same notation as in eq. 1 )


Di Rienzo et al. (1998)Citation obtained the same expression for Var(V).


    Footnotes
 
Keith Crandall, Reviewing Editor

Keywords: microsatellite loci mutation rate genetic distance Back

Address for correspondence and reprints: Marcus W. Feldman, Department of Biological Sciences, Stanford University, Stanford, California 94305. marc{at}charles.stanford.edu . Back


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Acknowledgements
 Appendix
 References
 

    Bowcock A. M., A. Ruiz-Linares, J. Tomfohrde, E. Minch, J. R. Kidd, L. L. Cavalli-Sforza, 1994 High resolution of human evolutionary trees with polymorphic microsatellites Nature 368:455-457[ISI][Medline]

    Cooper G., W. Amos, R. Bellamy, M. R. Siddiqui, A. Frodsham, A. V. S. Hill, D. C. Rubinsztein, 1999 An empirical exploration of the ({delta}µ)2 genetic distance for 213 human microsatellite markers Am. J. Hum. Genet 65:1125-1133[ISI][Medline]

    Di Rienzo A., P. Donnelly, C. Toomajian, B. Sisk, A. Hill, M. L. Petzl-Erler, G. K. Haines, D. H. Barch, 1998 Heterogeneity of microsatellite mutations within and between loci, and implications for human demographic histories Genetics 148:1269-1281[Abstract/Free Full Text]

    Feldman M. W., J. Kumm, J. K. Pritchard, 1999 Mutation and migration in models of microsatellite evolution Pp. 98–115 in D. G. Goldstein and C. Schlotterer, eds. Microsatellites: evolution and applications. Oxford University Press, Oxford.

    Forster P., A. Rohl, P. L. Lunnermann, C. Brinkmann, T. Zerjal, C. Tyler-Smith, B. Brinkmann, 2000 A short tandem repeat-based phylogeny for the human Y chromosome Am. J. Hum. Genet 67:182-196[ISI][Medline]

    Goldstein D. B., A. R. Linares, L. L. Cavalli-Sforza, M. W. Feldman, 1995 Genetic absolute dating based on microsatellites and the origin of modern humans Proc. Natl. Acad. Sci. USA 92:6723-6727[Abstract]

    Hudson R. R., 1990 Gene genealogies and the coalescent process Oxf. Surv. Evol. Biol 7:1-45

    Jin L., M. L. Baskett, L. L. Cavalli-Sforza, L. A. Zhivotovsky, M. W. Feldman, N. A. Rosenberg, 2000 Microsatellite evolution in modern humans: a comparison of two data sets from the same populations Ann. Hum. Genet 64:117-134[ISI][Medline]

    Jorde L. B., A. R. Rogers, M. Bamshad, W. S. Watkins, P. Krakowiak, S. Sung, J. Kere, H. Harpending, 1997 Microsatellite diversity and the demographic history of modern humans Proc. Natl. Acad. Sci. USA 94:3100-3103[Abstract/Free Full Text]

    Kimmel M., R. Chakraborty, 1996 Measures of variation at DNA repeat loci under a general stepwise mutation model Theor. Popul. Biol 50:345-367[ISI][Medline]

    Michalakis Y., L. A. Excoffier, 1996 Generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci Genetics 142:1061-1064[Abstract/Free Full Text]

    Rice J. A., 1995 Mathematical statistics and data analysis. 2nd edition Duxbury Press, Belmont, Calif

    Rousset F., 1996 Equilibrium values of measures of population subdivision for stepwise mutation processes Genetics 142:1357-1362[Abstract/Free Full Text]

    Slatkin M., 1995 A measure of population subdivision based on microsatellite allele frequencies Genetics 139:457-462[Free Full Text]

    Weir B. S., 1996 Genetic data analysis II Methods for discrete population genetic data. Sinauer, Sunderland, Mass

    Zhivotovsky L. A., 2001 Estimating divergence time with the use of microsatellite genetic distances: impacts of population growth and gene flow Mol. Biol. Evol 18:700-709[Abstract/Free Full Text]

    Zhivotovsky L. A., M. W. Feldman, 1995 Microsatellite variability and genetic distances Proc. Natl. Acad. Sci. USA 92:11549-11552[Abstract]

    Zhivotovsky L. A., M. W. Feldman, S. A. Grishechkin, 1997 Biased mutations and microsatellite variation Mol. Biol. Evol 14:926-933[Abstract]

Accepted for publication June 6, 2001.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Request Permissions
Google Scholar
Articles by Zhivotovsky, L. A.
Articles by Feldman, M. W.
PubMed
PubMed Citation
Articles by Zhivotovsky, L. A.
Articles by Feldman, M. W.