Estimation of mutation rates in cultured mammalian cells

The factors that affect reliable estimations of mutation rates (μ) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (N_t) and the number of parallel cultures (C) on the variation in the rate estimates ($\text{μ}$) inferred from the P₀ method. The analysis can be made after the derivation of the variance of $\text{μ}$, which is a measure of variation of $\text{μ}$ for a given combination of N_t and C in a number of repeat experiments. The variance of $\text{μ}$ is inversely proportional to C and to the square of N_t. N_t determines the probability of occurrence of mutation in a cell culture. By influencing the size of P₀, N_t also determines whether a rate estimate is obtainable from the experiment. Since P_o is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of $\text{μ}$. The rate estimated from $\text{P}\text{?}{}_0$ is biased, but the bias is in general 2 orders of magnitude smaller than $\text{μ}$. By the selection of an appropriate combination of N_t and C for the experiment, this bias can be reduced even further.Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.     

Estimation of microsatellite mutation rates in Drosophila melanogaster

Microsatellite mutations were studied in a set of 175 mutation accumulation lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and maintained under strong inbreeding during 80 generations. We assayed 28 microsatellites and detected two mutations. One mutation consisted of a single addition of a dinucleotide repeat and the other was a deletion of five trinucleotide repeats. The average mutation rate was 5.1 x 10(-6), in full agreement with previous estimates from two different sets of mutation accumulation lines.     

Estimation of nucleotide diversity, disequilibrium coefficients, and mutation rates from high-coverage genome-sequencing projects

Recent advances in sequencing strategies have made it feasible to rapidly obtain high-coverage genomic profiles of single individuals, and soon it will be economically feasible to do so with hundreds to thousands of individuals per population. While offering unprecedented power for the acquisition of population-genetic parameters, these new methods also introduce a number of challenges, most notably the need to account for the binomial sampling of parental alleles at individual nucleotide sites and to eliminate bias from various sources of sequence errors. To minimize the effects of both problems, methods are developed for generating nearly unbiased and minimum-sampling-variance estimates of a number of key parameters, including the average nucleotide heterozygosity and its variance among sites, the pattern of decomposition of linkage disequilibrium with physical distance, and the rate and molecular spectrum of spontaneously arising mutations. These methods provide a general platform for the efficient utilization of data from population-genomic surveys, while also providing guidance for the optimal design of such studies.     

Estimation of the male and female mutation rates in Duchenne muscular dystrophy (DMD)

Summary We present the results of an international collaborative study aimed at estimating the ratio of male to female mutation rates in Duchenne muscular dystrophy based on the method of C. Müller and T. Grimm. With a sample size of 295, this ratio is found to be very close to 1, thus giving evidence for equal mutation rates in males and females in Duchenne muscular dystrophy.     

Environmental tuning of mutation rates

Through their life cycles, bacteria experience many different environments in which the relationship between available energy resources and the frequency and the nature of various stresses is highly variable. In order to survive in such changeable environments, bacteria must balance the need for nutritional competence with stress resistance. In Escherichia coli natural populations, this is most frequently achieved by changing the regulation of the RpoS sigma factor-dependent general stress response. One important secondary consequence of altered regulation of the RpoS regulon is the modification of mutation rates. For example, under nutrient limitation during stationary phase, the high intracellular concentration of RpoS diminishes nutritional competence, increases stress resistance, and, by downregulating the mismatch repair system and upregulating [corrected] the expression of the dinB gene (coding for PolIV translesion synthesis polymerase) increases mutation rates. The reduction of the intracellular concentration of RpoS has exactly opposite effects on nutritional competence, stress resistance and mutation rates. Therefore, the natural selection that favours variants having the highest fitness under different environmental conditions results in high variability of stress-associated mutation rates in those variants.     

Estimation of male to female ratio of mutation rates from carrier-detection tests in X-linked disorders.

A method is presented for the estimation of the ratio of male to female mutation rates from female carrier-detection test data from pedigrees containing an isolated male manifesting an X-linked necessive disorder. Pedigrees of any size and complexity (barring consanguinity) and containing any number of tested females can be utilized. The relative fitness of affected males and carrier females, and the segregation probability of the abnormal gamete in females, can be estimated simultaneously with the ratio of mutation rates in order to test specific hypotheses against given bodies of data. Here this method is applied to families containing isolated individuals affected with Lesch-Nyhan syndrome.     

Estimation of in-vivo miscoding rates

The replication of premutagenic DNA lesions generates mutant progeny in patterns that distinguish lesions that rarely produce a mutation per DNA replication from those that frequently do so. The quantitative aspects of this distinction were tested in studies of heat-mutagenized bacteriophage T4. Previous T4 studies had demonstrated that transition mutations produced at G.C base-pairs depended upon heat-induced DNA lesions distinct from those responsible for transversions at G.C pairs. In this study the transversion mutations are shown to arise in patterns predicted for mutations produced from lesions that miscode rarely (fewer than 10% per replication). In contrast, the transition mutations arise in patterns predicted for mutations produced from lesions that miscode at about 20 to 60% per replication. The fact that the two classes of DNA lesions are distinguishable as predicted by the quantitative model suggests that such studies may in general be useful in quantifying the behavior of mutation-generating DNA lesions. The method employed also estimates the frequency of premutagenic lesions in DNA.     

Likelihood-based estimation of microsatellite mutation rates

Microsatellites are widely used in genetic analyses, many of which require reliable estimates of microsatellite mutation rates, yet the factors determining mutation rates are uncertain. The most straightforward and conclusive method by which to study mutation is direct observation of allele transmissions in parent-child pairs, and studies of this type suggest a positive, possibly exponential, relationship between mutation rate and allele size, together with a bias toward length increase. Except for microsatellites on the Y chromosome, however, previous analyses have not made full use of available data and may have introduced bias: mutations have been identified only where child genotypes could not be generated by transmission from parents' genotypes, so that the probability that a mutation is detected depends on the distribution of allele lengths and varies with allele length. We introduce a likelihood-based approach that has two key advantages over existing methods. First, we can make formal comparisons between competing models of microsatellite evolution; second, we obtain asymptotically unbiased and efficient parameter estimates. Application to data composed of 118,866 parent-offspring transmissions of AC microsatellites supports the hypothesis that mutation rate increases exponentially with microsatellite length, with a suggestion that contractions become more likely than expansions as length increases. This would lead to a stationary distribution for allele length maintained by mutational balance. There is no evidence that contractions and expansions differ in their step size distributions.     

Statistical inference of sequence-dependent mutation rates

Several lines of research are now converging towards an integrated understanding of mutational mechanisms and their evolutionary implications. Experimentally, crystal structures reveal the effect of sequence context on polymerase fidelity; large-scale sequencing projects generate vast amounts of sequence polymorphism data; and locus-specific databases are being constructed. Computationally, software and analytical tools have been developed to analyze mutational data, to identify mutational hot spots, and to compare the signatures of mutagenic agents.     

Estimating mutation rates in low-replication experiments

Since mutation rate is a key biological parameter, its proper estimation has received great attention for decades. However, instead of the mutation rate, many authors opt for reporting the average mutant frequency, a less meaningful quantity. This is because the standard methods to estimate the mutation rate, derived from the Luria and Delbrück's fluctuation analysis, ideally require high-replication experiments to be applied; a requirement often unattainable due to constraints of time, budget or sample availability. But the main problem with mutant frequency, apart from being less informative, is its poor reproducibility; an especially marked defect when the chosen average is the arithmetic mean. Several authors tried to avoid this by employing other averages (such as the median or the geometric mean) or discarding outliers, though as far as we know nobody has evaluated which method performs best under low-replication settings. Here we use computer simulations to compare the performance of different methods used in low-replication experiments (≤4 cultures). Besides the customary averages of mutant frequency, we also tested two well-known fluctuation methods. Contrary to common practice, our results support that fluctuation methods should be applied in such circumstances, as they perform as well as or better than any average of mutant frequency. In particular, experimentalists will benefit from using MSS maximum likelihood in low-replication experiments because it: (i) provides more reproducible results, (ii) allows for direct estimation of mutation rate and (iii) allows for the application of conventional statistics.     

Fitness-dependent mutation rates in finite populations

Mutation rate may be condition dependent, whereby individuals in poor condition, perhaps from high mutation load, have higher mutation rates than individuals in good condition. Agrawal (J. Evol. Biol.15, 2002, 1004) explored the basic properties of fitness-dependent mutation rate (FDMR) in infinite populations and reported some heuristic results for finite populations. The key parameter governing how infinite populations evolve under FDMR is the curvature (k) of the relationship between fitness and mutation rate. We extend Agrawal's analysis to finite populations and consider dominance and epistasis. In finite populations, the probability of long-term existence depends on k. In sexual populations, positive curvature leads to low equilibrium mutation rate, whereas negative curvature results in high mutation rate. In asexual populations, negative curvature results in rapid extinction via 'mutational meltdown', whereas positive curvature sometimes allows persistence. We speculate that fitness-dependent mutation rate may provide the conditions for genetic architecture to diverge between sexual and asexual taxa.     

Sex-specific mutation rates in salmonid fish

If germline mutations arise because of replication errors, the mutation rate may differ between males and females given that they differ in their number of germ cell divisions. As males of many higher organisms produce more gametes than females, this has led to the idea of "male-driven evolution." The extent of such male bias to the mutation rate is currently debated. For human some recent data suggest a very low bias, at a factor 1.7 only, while other approaches have given values of alpha(m) (the male-to-female mutation rate ratio) of 5, which is more close to what might be expected from male and female germ cell biology. Comparative analyses of sex-specific mutation rates in other organisms may be necessary for understanding the generality of an effect of sex and the number of germline DNA replications on the mutation rate. In this study we estimate for the first time sex-specific mutation rates in fish. Comparing the intronic substitution rates of the autosomal GH- 2 gene and its duplicated Y-linked and male-specific copy GH- 2Y (447-468 bp of each gene), we estimate alpha(m) to be 5.35-6.60 in salmonid fish of the genus Oncorhynchus. To the observations previously made among mammals and birds, this adds evidence from another class of vertebrates showing that a majority of mutations are of paternal origin. This would suggest that replication errors play a major role for the generation of new mutations.