Analysis of somatic mutations at human minisatellite loci in tumors and cell lines

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.     

Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis

Variation in internal minisatellite structure can be analyzed by mapping variant repeat units within amplified alleles. A system capable of distinguishing greater than 10(70) allelic states at the human hypervariable locus D1S8 has been developed. Population surveys of internal allelic structure indicate that D1S8 alleles evolve rapidly along haploid chromosome lineages. Internal mapping of deletion mutant alleles physically selected from genomic DNA provides further evidence that germline and somatic mutations altering the number of allelic repeat units seldom if ever arise by unequal exchange between alleles. The existence of low level germline mosaicism for new mutants further indicates that many germline mutation events are premeiotic. Physical selection of new mutants also allows minisatellite mutation rates to be estimated directly in human DNA.     

Factors affecting germline mutations in a hypervariable microsatellite: a comparative analysis of six species of swallows (Aves: Hirundinidae)

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes.     

PipeIT2: A tumor-only somatic variant calling workflow for molecular diagnostic Ion Torrent sequencing data

Precision oncology relies on the accurate identification of somatic mutations in cancer patients. While the sequencing of the tumoral tissue is frequently part of routine clinical care, the healthy counterparts are rarely sequenced. We previously published PipeIT, a somatic variant calling workflow specific for Ion Torrent sequencing data enclosed in a Singularity container. PipeIT combines user-friendly execution, reproducibility and reliable mutation identification, but relies on matched germline sequencing data to exclude germline variants. Expanding on the original PipeIT, here we describe PipeIT2 to address the clinical need to define somatic mutations in the absence of germline control. We show that PipeIT2 achieves a > 95% recall for variants with variant allele fraction >10%, reliably detects driver and actionable mutations and filters out most of the germline mutations and sequencing artifacts. With its performance, reproducibility, and ease of execution, PipeIT2 is a valuable addition to molecular diagnostics laboratories.     

A Dual Model for Prioritizing Cancer Mutations in the Non-coding Genome Based on Germline and Somatic Events

We address here the issue of prioritizing non-coding mutations in the tumoral genome. To this aim, we created two independent computational models. The first (germline) model estimates purifying selection based on population SNP data. The second (somatic) model estimates tumor mutation density based on whole genome tumor sequencing. We show that each model reflects a different set of constraints acting either on the normal or tumor genome, and we identify the specific genome features that most contribute to these constraints. Importantly, we show that the somatic mutation model carries independent functional information that can be used to narrow down the non-coding regions that may be relevant to cancer progression. On this basis, we identify positions in non-coding RNAs and the non-coding parts of mRNAs that are both under purifying selection in the germline and protected from mutation in tumors, thus introducing a new strategy for future detection of cancer driver elements in the expressed non-coding genome.     

Direct mutation analysis by high-throughput sequencing: from germline to low-abundant, somatic variants

DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.     

Somatic mutations in organisms with complex life histories.

A theoretical model is developed of the fate of mutations for organisms with such life-history characteristics as indeterminate growth and clonal reproduction. It focuses on how the fate of a particular mutant depends on whether it arises during mitotic cell division (somatic mutation) or during meiotic cell division (meiotic mutation). At gamete production, individuals carrying somatic mutations will produce some proportion of gametes reflecting the original, zygotic genotype and some proportion reflecting genotypes carrying the somatic mutation. Focusing on allele frequencies at gamete production allows the effects of growth and clonal reproduction to be summarized. The relative strengths of somatic and meiotic mutation can be determined, as well as the conditions under which the change in allele frequency due to one is greater than that due to the other. Examples from a published demographic study of clonal corals are used to compare somatic and meiotic mutation. When there is no selection acting on either type of mutation, only a few cell divisions per time unit on average are needed for the change in allele frequency due to somatic mutation to be greater, given empirically based mutation rates. When somatic selection is added, the most dramatic effect is seen with fairly strong negative selection acting against the somatic mutation within individuals. In this case, selection within organisms can effectively counteract the effects of somatic mutation, and the change in allele frequency due to somatic mutations will not be greater than that due to meiotic mutations for reasonable numbers of within-generation cell divisions. The majority of the mutation load, which would have been due to somatic mutation, is purged by selection within the individual organism.     

Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene

New germline mutations in the human retinoblastoma gene are known to arise preferentially on paternally derived chromosomes, but the magnitude of that bias has not been measured. We evaluated 49 cases with a new germline mutation and found that in 40 cases (82%) the mutation arose on the paternally derived allele. We also evaluated 48 cases likely to have a somatic initial mutation; in this group the initial mutation arose on paternal or maternal chromosomes with approximately equal frequency. There was no statistically significant difference in the average age of fathers of children with new paternal germline mutations from the average age of fathers of children with new maternal germline mutations or somatic initial mutations. Combining the data with that from previous reports from other groups, the proportion of new germline mutations arising on a paternally derived allele is 85% (based on 72 cases; 95% confidence interval = 76–93%). This number can be useful in the genetic counseling of some families with retinoblastoma. Received: 18 December 1996 / Accepted: 30 April 1997     

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.     

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.     

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.     

Trends in antibody sequence changes during the somatic hypermutation process

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are approximately 4% for core residues, approximately 5% for surface residues, and approximately 12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.     

Germline fitness-based scoring of cancer mutations

A key goal in cancer research is to find the genomic alterations that underlie malignant cells. Genomics has proved successful in identifying somatic variants at a large scale. However, it has become evident that a typical cancer exhibits a heterogenous mutation pattern across samples. Cases where the same alteration is observed repeatedly seem to be the exception rather than the norm. Thus, pinpointing the key alterations (driver mutations) from a background of variations with no direct causal link to cancer (passenger mutations) is difficult. Here we analyze somatic missense mutations from cancer samples and their healthy tissue counterparts (germline mutations) from the viewpoint of germline fitness. We calibrate a scoring system from protein domain alignments to score mutations and their target loci. We show first that this score predicts to a good degree the rate of polymorphism of the observed germline variation. The scoring is then applied to somatic mutations. We show that candidate cancer genes prone to copy number loss harbor mutations with germline fitness effects that are significantly more deleterious than expected by chance. This suggests that missense mutations play a driving role in tumor suppressor genes. Furthermore, these mutations fall preferably onto loci in sequence neighborhoods that are high scoring in terms of germline fitness. In contrast, for somatic mutations in candidate onco genes we do not observe a statistically significant effect. These results help to inform how to exploit germline fitness predictions in discovering new genes and mutations responsible for cancer.     

Evolution of mutation rates in bacteria

Evolutionary success of bacteria relies on the constant fine-tuning of their mutation rates, which optimizes their adaptability to constantly changing environmental conditions. When adaptation is limited by the mutation supply rate, under some conditions, natural selection favours increased mutation rates by acting on allelic variation of the genetic systems that control fidelity of DNA replication and repair. Mutator alleles are carried to high frequency through hitchhiking with the adaptive mutations they generate. However, when fitness gain no longer counterbalances the fitness loss due to continuous generation of deleterious mutations, natural selection favours reduction of mutation rates. Selection and counter-selection of high mutation rates depends on many factors: the number of mutations required for adaptation, the strength of mutator alleles, bacterial population size, competition with other strains, migration, and spatial and temporal environmental heterogeneity. Such modulations of mutation rates may also play a role in the evolution of antibiotic resistance.     

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds,purpurin and alizarin

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.     

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.