A New and More Accurate Estimate of the Rate of Concurrent Tandem‐Base Substitution Mutations in the Human Germline: ∼0.4% of the Single‐Nucleotide Substitution Mutation Rate

Determining the rate of spontaneous germline mutation in the human genome has always been a central theme in genetics [Campbell and Eichler, 2013]. The rate of single-nucleotide substitution (SNS) mutation has so far been the most extensively studied with the earlier gene-specific estimates being validated by more recent genome-wide approaches. In particular, the sequencing of the entire genomes of 78 Icelandic parent-offspring trios yielded an average de novo SNS mutation rate of 1.20 × 10 per nucleotide per generation [Kong et al., 2012]. Although nucleotide substitution mutations are often assumed to occur independently, there is accumulating evidence that SNS mutation can sometimes occur simultaneously at adjacent or closely spaced sites (for reviews, see [Drake, 2007; Chen et al., 2012]). The accurate estimation of the rate of this particular type of mutation is of paramount importance if we are to obtain a better understanding of the driving force underlying both human genome evolution [Averof et al., 2000] and genetic disease [Chen et al., 2013]. We recently estimated the rate of simultaneously generated double-nucleotide mutation affecting adjacent sites (alternatively known as concurrently generated double tandem-base substitution [TBS] mutations) in the human germline to be 0.2–1.2 × 10 per base per generation [Chen et al., 2013]. This was calculated in accordance with the formula illustrated in Figure 1. With hindsight, the sixfold variation in the estimated rate was probably due to the inappropriate inclusion of nonrelevant somatic data from some tumors. Here, by integrating data from a diverse set of genetic systems, we have attempted to provide a more accurate estimate of the rate of concurrent double TBS mutations in the human germline. Using sequence comparison of coding and noncoding DNA from diverse organisms (including humans), Averof et al. (2000) reported a high frequency of double TBS mutations, ∼2% of the SNS rate in terms of Rd/s (see Fig. 1 for term definition). This study was however subject to two basic limitations. On the one hand, in the context of coding sequences, only switches between TCN and AGY codons were analyzed. On the other, in the context of noncoding sequences, only one 7-kb sequence from a pseudogene was used for the analysis; this may account for the Rd/s value being considerably higher than those reported in subsequent studies. Using a new method that allows for the effect of chromosome regional variation in evolutionary rates and applying it to >1 Mb of human–chimpanzee–baboon genomic DNA alignments and >1 million human single-nucleotide polymorphisms (SNPs), Smith et al. (2003) reported an Rd/s of 0.3%. Using data from 896 nuclear genes that were resequenced in between 90 and 95 human individuals, Hodgkinson and Eyre-Walker (2010) showed that ∼0.89% of all distinct nucleotide substitutions were generated from double TBS mutations. Given that a double TBS mutation comprises two SNS mutations, this ratio corresponds to an Rd/s of 0.45% (i.e., ∼0.89%/2). Comparing noncoding sequences from several species of primate (including humans) demonstrated that simultaneous double-nucleotide mutations spanning distances of up to 10 nucleotides accounted for at least 2.3% of all observed nucleotide substitutions [Terekhanova et al., 2013]; since double TBS mutations account for 36% of these double-nucleotide mutations, this corresponds to an Rd/s of 0.41% (2.3%/2 × 36%). The analysis of SNP data from a Han Chinese individual revealed that 1.93% of all nucleotide substitutions were attributable to simultaneous multiple mutations that occurred within a 20-bp interval; 50% of these simultaneous multiple mutations were located within four bases of one another [Schrider et al., 2011]. To obtain some idea as to the proportion of double TBS mutations/multiple mutations that were located within four bases of one another, we surveyed such multiple mutations logged in the Human Gene Mutation Database (HGMD, www.hgmd.org; [Stenson et al., 2014]); the resulting proportion was 79% (479/604; as of June 12, 2013). We also surveyed the de novo mutation data from the whole-genome sequencing of individuals with autism, where the corresponding figure was 75% [Michaelson et al., 2012]. Applying the average value of 77% to the aforementioned data [Schrider et al., 2011] yielded an Rd/s of 0.37% (1.93%/2 × 50% × 77%). It should be noted that this ratio was biased in the direction of overestimation because all multiple mutations were treated equally as double mutations (e.g., a quadruplet TBS mutation comprises four nucleotide substitutions). However, this is unlikely to deviate much from the actual value for two reasons. On the one hand, the frequency of TBSs appears to be inversely correlated with the number of their component substitutions [Chen et al., 2013]. On the other hand, simultaneously generated multiple mutations occurring within a distance of ≥3–20 bp to each other comprise more non-TBS than TBS mutations. In the latter regard, it is worth mentioning that we identified 45 non-TBS double mutations (i.e., double mutations separated by 1–18 bp) in an early analysis [Chen et al., 2009] and 29 TBS mutations comprising ≥3 component mutations (i.e., triplets, 16; and quadruplets to octuplets, 13) in our more recent analysis [Chen et al., 2013]. Both analyses were based on data from the HGMD; the number