Human Germline Mutations Research Articles

Mutational signature analyses in multi-child families reveal sources of age-related increases in human germline mutations.

Whole-genome sequencing studies of parent-offspring trios have provided valuable insights into the potential impact of de novo mutations (DNMs) on human health and disease. However, the molecular mechanisms that drive DNMs are unclear. Studies with multi-child families can provide important insight into the causes of inter-family variability in DNM rates but they are highly limited. We characterized 2479 de novo single nucleotide variants (SNVs) in 13 multi-child families of Mexican-American ethnicity. We observed a strong paternal age effect on validated de novo SNVs with extensive inter-family variability in the yearly rate of increase. Children of older fathers showed more C > T transitions at CpG sites than children from younger fathers. Validated SNVs were examined against one cancer (COSMIC) and two non-cancer (human germline and CRISPR-Cas 9 knockout of human DNA repair genes) mutational signature databases. These analyses suggest that inaccurate DNA mismatch repair during repair initiation and excision processes, along with DNA damage and replication errors, are major sources of human germline de novo SNVs. Our findings provide important information for understanding the potential sources of human germline de novo SNVs and the critical role of DNA mismatch repair in their genesis.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

ELAC2 is a functional prostate cancer risk allele.

Stentenbach and colleagues have unveiled a functional role of a human germline mutation found in the ribonuclease (RNase) Z enzyme, ELAC2, in prostate cancer. Here, we discuss the importance of these findings in enhancing our understanding of how risk variants enable prostate cancer progression and the post-transcriptional mechanisms underlying oncogenesis.

Limited role of generation time changes in driving the evolution of the mutation spectrum in humans.

Recent studies have suggested that the human germline mutation rate and spectrum evolve rapidly. Variation in generation time has been linked to these changes, though its contribution remains unclear. We develop a framework to characterize temporal changes in polymorphisms within and between populations, while controlling for the effects of natural selection and biased gene conversion. Application to the 1000 Genomes Project dataset reveals multiple independent changes that arose after the split of continental groups, including a previously reported, transient elevation in TCC>TTC mutations in Europeans and novel signals of divergence in C>Gand T>A mutation rates among population samples. We also find a significant difference between groups sampled in and outside of Africa in old T>C polymorphisms that predate the out-of-Africa migration. This surprising signal is driven by TpG>CpG mutations and stems in part from mis-polarized CpG transitions, which are more likely to undergo recurrent mutations. Finally, by relating the mutation spectrum of polymorphisms to parental age effects on de novo mutations, we show that plausible changes in the generation time cannot explain the patterns observed for different mutation types jointly. Thus, other factors - genetic modifiers or environmental exposures - must have had a non-negligible impact on the human mutation landscape.

P306: PAX5 DEFICIENCY AND GERMLINE SUSCEPTIBILITY TO PEDIATRIC LEUKEMIA

Background: Somatic mutations affecting the PAX5 gene are common in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, germline PAX5 mutations are extremely rare. While it is known that PAX5 acts as an indispensable master regulator of proliferation and differentiation in B-cells, the pre-leukemic setting mediated by reduced PAX5 levels is still poorly understood. Aims: To investigate the genetic and immunological effect of inherited PAX5 germline mutations in humans and to characterize the effect of Pax5 heterozygosity on B-cell development utilizing single-cell RNA Sequencing in mice. Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Sequencing, as well as whole exome Sequencing technology. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 and B-ALL development, including one family with a novel amino acid substitution p.Gly183Arg. Exome analysis of both families together with one family harboring PAX5 p.Gly183Ser previously reported by Auer et al., (Leukemia, 2014) yielded no additional common germline variants that could explain the incomplete penetrance in the observed leukemia development. A study of the respective B-cell differentiation comparing PBMCs from unrelated non-carriers, healthy carriers, and patients revealed a significant decrease of intermittent B-cells and memory B-cells in carriers and patients compared to non-carriers (Student’s T-test; p<0.05 and p<0.01, respectively). Finally, immunological studies performed using bone marrow samples from a patient and an unrelated control showed a reduction in the number of mature B cells, suggesting a block of the B-cell differentiation at the pre-B stage in the patient. In line, a comprehensive flow cytometry analysis of the B-cell development in the bone marrow of healthy Pax5 heterozygous (Pax5+/-) mice, showed a robust and age independent enrichment of the pre-BII population (B220+CD19+IgM-CD25+) compared to their Wildtype (WT) littermates (Student’s t-test; p=0.003). Bulk RNA-Sequencing of sorted preB-II cells (3 WT vs. 3 Pax5+/- mice) revealed deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs. Subsequent scRNA-Sequencing (4 WT vs. 4 Pax5+/- mice, pool of 20,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) confirmed a delayed BCR assembly and showed a skewing of light chain rearrangements towards the lambda isotype in Pax5+/- mice. Summary/Conclusion: Overall, our data provide evidence for B-cell dysregulations mediated by reduced Pax5 levels in the germline of mice and men. Ultimately these characterizations will help to advance the understanding of molecular mechanisms and susceptible populations associated with B-ALL. In turn, these findings are important to address the far more frequent somatic PAX5 mutations with the goal to prevent or treat a significant proportion of childhood leukemias.

The Mutationathon highlights the importance of reaching standardization in estimates of pedigree-based germline mutation rates.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various nonhuman species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and appropriately accounting for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a 'Mutationathon,' a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a twofold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria, and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.

Gene expression levels modulate germline mutation rates through the compound effects of transcription-coupled repair and damage.

Of all mammalian organs, the testis has long been observed to have the most diverse gene expression profile. To account for this widespread gene expression, we have proposed a mechanism termed 'transcriptional scanning', which reduces germline mutation rates through transcription-coupled repair (TCR). Our hypothesis contrasts with an earlier observation that mutation rates are overall positively correlated with gene expression levels in yeast, implying that transcription is mutagenic due to effects dominated by transcription-coupled damage (TCD). Here we report evidence that the compound effects of both TCR and TCD during spermatogenesis modulate human germline mutation rates, with TCR dominating in most genes, thus supporting the transcriptional scanning hypothesis. Our analyses address potentially confounding factors, distinguish the differential mutagenic effects acting on the highly expressed genes and the low-to-moderately expressed genes, and resolve concerns relating to the validation of the results using a de novo mutation dataset. We also discuss the theoretical possibility of transcriptional scanning hypothesis from an evolutionary perspective. Together, these analyses support a model by which the coupling of transcription-coupled repair and damage establishes the pattern of germline mutation rates and provide an evolutionary explanation for widespread gene expression during spermatogenesis.

Distinct Mutational Processes Influence Germline DNA Mutations.

Nine distinct mutational processes underlie human germline mutations.

Population sequencing data reveal a compendium of mutational processes in the human germ line.

Mechanistic processes underlying human germline mutations remain largely unknown.Variation in mutation rate and spectra along the genome is informative about the biological mechanisms. We statistically decompose this variation into separate processes using a blind source separation technique. The analysis of a large-scale whole genome sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. Seven of these processes lend themselves to a biological interpretation. One process is driven by bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes independently track direction of replication fork and replication timing. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions. We also demonstrate that a recently discovered mutagenic process specific to oocytes can be localized solely from population sequencing data. This process is spread across all chromosomes and is highly asymmetric with respect to the direction of transcription, suggesting a major role of DNA damage.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Targeting the tetraspanin CD81 reduces cancer invasion and metastasis

Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiology, including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biology. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathological conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-negative breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.

Heterogeneity of TP53 Mutations and P53 Protein Residual Function in Cancer: Does It Matter?

The human TP53 locus, located on the short arm of chromosome 17, encodes a tumour suppressor protein which functions as a tetrameric transcription factor capable of regulating the expression of a plethora of target genes involved in cell cycle arrest, apoptosis, DNA repair, autophagy, and metabolism regulation. TP53 is the most commonly mutated gene in human cancer cells and TP53 germ-line mutations are responsible for the cancer-prone Li-Fraumeni syndrome. When mutated, the TP53 gene generally presents missense mutations, which can be distributed throughout the coding sequence, although they are found most frequently in the central DNA binding domain of the protein. TP53 mutations represent an important prognostic and predictive marker in cancer. The presence of a TP53 mutation does not necessarily imply a complete P53 inactivation; in fact, mutant P53 proteins are classified based on the effects on P53 protein function. Different models have been used to explore these never-ending facets of TP53 mutations, generating abundant experimental data on their functional impact. Here, we briefly review the studies analysing the consequences of TP53 mutations on P53 protein function and their possible implications for clinical outcome. The focus shall be on Chronic Lymphocytic Leukemia (CLL), which also has generated considerable discussion on the role of TP53 mutations for therapy decisions.

Deviation from Mendelian transmission of autosomal SNPs can be used to estimate germline mutations in humans exposed to ionizing radiation.

We aimed to estimate the rate of germline mutations in the offspring of individuals accidentally exposed to Cesium-137 ionizing radiation. The study included two distinct groups: one of cases, consisting of males and females accidentally exposed to low doses of ionizing radiation of Cs137, and a control group of non-exposed participants. The cases included 37 people representing 11 families and 15 children conceived after the accident. Exposed families incurred radiation absorbed doses in the range of 0.2 to 0.5 Gray. The control group included 15 families and 15 children also conceived after 1987 in Goiânia with no history of radiation exposure. DNA samples from peripheral blood were analyzed with the Affymetrix GeneChip® CytoScanHD™ to estimate point mutations in autosomal SNPs. A set of scripts previously developed was used to detect de novo mutations by comparing parent and offspring genotypes at the level of each SNP marker. Overall numbers of observed Mendelian deviations were statistically significant between the exposed and control groups. Our retrospective transgenerational DNA analysis showed a 44.0% increase in the burden of SNP mutations in the offspring of cases when compared to controls, based on the average of MFMD for the two groups. Parent-of-origin and type of nucleotide substitution were also inferred. This proved useful in a retrospective estimation of the rate of de novo germline mutations in a human population accidentally exposed to low doses of radiation from Cesium-137. Our results suggested that observed burden of germline mutations identified in offspring was a potentially useful biomarker of effect to estimate parental exposure to low doses of IR and could become an important marker suitable for biomonitoring human population exposed to environmental mutagens.

Higher Germline Mutagenesis of Genes with Stronger Testis Expressions Refutes the Transcriptional Scanning Hypothesis.

Why are more genes expressed in the testis than in any other organ in mammals? The recently proposed transcriptional scanning hypothesis posits that transcription alleviates mutagenesis through transcription-coupled repair so has been selected in the testis to modulate the germline mutation rate in a gene-specific manner. Here, we show that this hypothesis is theoretically untenable because the selection would be too weak to have an effect in mammals. Furthermore, the analysis purported to support the hypothesis did not control known confounding factors and inappropriately excluded genes with no observed de novo mutations. After remedying these problems, we find the human germline mutation rate of a gene to rise with its testis expression level. This trend also exists for inferred coding strand-originated mutations, suggesting that it arises from transcription-associated mutagenesis. Furthermore, the testis expression level of a gene robustly correlates with its overall expression in other organs, nullifying the need to explain the testis silencing of a minority of genes by adaptive germline mutagenesis. Taken together, our results demonstrate that human testis transcription increases the germline mutation rate, rejecting the transcriptional scanning hypothesis of extensive gene expressions in the mammalian testis.

Extreme differences between human germline and tumor mutation densities are driven by ancestral human-specific deviations

Mutations do not accumulate uniformly across the genome. Human germline and tumor mutation density correlate poorly, and each is associated with different genomic features. Here, we use non-human great ape (NHGA) germlines to determine human germline- and tumor-specific deviations from an ancestral-like great ape genome-wide mutational landscape. Strikingly, we find that the distribution of mutation densities in tumors presents a stronger correlation with NHGA than with human germlines. This effect is driven by human-specific differences in the distribution of mutations at non-CpG sites. We propose that ancestral human demographic events, together with the human-specific mutation slowdown, disrupted the human genome-wide distribution of mutation densities. Tumors partially recover this distribution by accumulating preneoplastic-like somatic mutations. Our results highlight the potential utility of using NHGA population data, rather than human controls, to establish the expected mutational background of healthy somatic cells.

A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53

SUMMARYThe physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53178C/C mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and trans-activated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development.

Association of functional variants and protein-to-protein physical interactions of human MutY homolog linked with familial adenomatous polyposis and colorectal cancer syndrome.

The human gene MUTYH codes for a DNA glycosylase involved in the repair of oxidative DNA damage. Faulty MUTYH protein activity causes the accumulation of G→T transversions due to unrepaired 8-oxoG:A mismatches. MUTYH germ-line mutations in humans are linked with a recessive form of Familial Adenomatous Polyposis (FAP) and colorectal cancer predisposition. We studied the repair capacity of variants identified in MUTYH-associated polyposis (MAP) patients. MAP is inherited in an autosomal recessive type due to mutations in MUTYH (Y165C, G382D, P54S, A22V, Q63R, G45D, S136P and N43S), indicating that both copies of the gene become inactivated. However, the parents of an individual with an autosomal recessive condition may serve as carriers, each harboring one copy of the mutated gene without showing signs or symptoms of MAP. Six protein partners have been associated with MUTYH, four via direct physical interactions, namely, hMSH6, hPCNA, hRPA1, and hAPEX1. We examined, for the first time, specific interactions of these protein partners with MAP-associated MUTYH mutants using molecular dynamics simulations. The approach provided tools for exploration of the conformational energy landscape accessible to protein partners. The investigation also determined the impact before and after energy minimization of protein-protein interactions and binding affinities of MUTYH wild type and mutant forms, as well as the interactions with other proteins. Taken together, this study provided new insights into the role of MUTYH and its interacting proteins in MAP.

Cell lineage context and type of genomic alteration predict for the therapeutic relevance of tyrosine kinase inhibitors in human cancers

Abstract Background Genomic alterations of receptor tyrosine kinases (RTK) or ligands are biomarkers for some targeted therapies in a fraction of cancers, with heterogeneous responses across tumor types. A general model to predict the efficacy of a targeted treatment in a given tumor type bearing a RTK genomic alteration is lacking. Through database and literature review, we investigated whether the role of a RTK in the normal lineage could predict the response to RTK inhibitors in cancers arising from the same lineage. Methods An analysis of the literature and query of public databases on RTK function in normal cell lineages and neoplastic cells was conducted, for 16 selected RTK (EGFR, HER2, KIT, FLT3, TRKA-B-C, ROS1, ALK, RET, MET, and FGFR1-2-3-4). The physiological role of RTK in the development/maintenance of cell lineages was assessed by looking at the phenotypes resulting from the alteration of the RTK in animal models and/or known human germline mutations. The activity of TKI monotherapy in genomic-driven clinical trials was analyzed through a systematic review of published phase I-II-III studies. Efficacy was defined as a median progression-free survival > 6 months and of a response rate > 30%. A matrix was built to test the association between the physiological role and clinical activity of each RTK according to lineage contexts. Results This analysis reveals a relationship between the role in lineage development and the magnitude of clinical activity of a TKI in a cancer arising from the same lineage. For missense mutations only, the clinical activity was higher when the RTK is involved in the development or maintenance of the normal cell lineage. Conversely, in cancers harboring fusion genes and proteins involving an RTK, TKI were active regardless of cell lineage. Single agent TKI activity was consistently limited in cancers bearing RTK gene amplifications. Conclusions This analysis identifies two preferred genomic biomarkers to select candidate patient for single agent TKI, 1) fusions genes involving an RTK; 2) activating missense mutations in RTK involved in normal cell lineage physiology. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure P.A. Cassier: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche/Genentech; Honoraria (self), Research grant / Funding (institution): Blueprint; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck Sharp & Dohme; Research grant / Funding (institution): Tayo; Research grant / Funding (institution): Loxo; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Innate. I.L. Ray-Coquard: Honoraria (self), Research grant / Funding (self): Roche; Honoraria (self), Research grant / Funding (institution): Amgen; Honoraria (self), Research grant / Funding (institution): Genmab; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Novartis; Honoraria (self), Research grant / Funding (institution): Pfizer. D. Perol: Honoraria (self): AstraZeneca; Honoraria (self): Ipsen; Honoraria (self): Novartis; Honoraria (self): Lilly; Honoraria (self): Bristol-Myers Squibb. J-Y. Blay: Research grant / Funding (self): PharmaMar; Research grant / Funding (self): GlaxoSmithKline; Research grant / Funding (self): Novartis; Research grant / Funding (self): Eisai; Research grant / Funding (self): Lilly; Research grant / Funding (self): Merck Sharp and Dohme; Research grant / Funding (self): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Similarities and differences in patterns of germline mutation between mice and humans

Whole genome sequencing (WGS) studies have estimated the human germline mutation rate per basepair per generation (~1.2 × 10−8) to be higher than in mice (3.5–5.4 × 10−9). In humans, most germline mutations are paternal in origin and numbers of mutations per offspring increase with paternal and maternal age. Here we estimate germline mutation rates and spectra in six multi-sibling mouse pedigrees and compare to three multi-sibling human pedigrees. In both species we observe a paternal mutation bias, a parental age effect, and a highly mutagenic first cell division contributing to the embryo. We also observe differences between species in mutation spectra, in mutation rates per cell division, and in the parental bias of mutations in early embryogenesis. These differences between species likely result from both species-specific differences in cellular genealogies of the germline, as well as biological differences within the same stage of embryogenesis or gametogenesis.

Signatures of replication timing, recombination, and sex in the spectrum of rare variants on the human X chromosome and autosomes

The sources of human germline mutations are poorly understood. Part of the difficulty is that mutations occur very rarely, and so direct pedigree-based approaches remain limited in the numbers that they can examine. To address this problem, we consider the spectrum of low-frequency variants in a dataset (Genome Aggregation Database, gnomAD) of 13,860 human X chromosomes and autosomes. X-autosome differences are reflective of germline sex differences and have been used extensively to learn about male versus female mutational processes; what is less appreciated is that they also reflect chromosome-level biochemical features that differ between the X and autosomes. We tease these components apart by comparing the mutation spectrum in multiple genomic compartments on the autosomes and between the X and autosomes. In so doing, we are able to ascribe specific mutation patterns to replication timing and recombination and to identify differences in the types of mutations that accrue in males and females. In particular, we identify C > G as a mutagenic signature of male meiotic double-strand breaks on the X, which may result from late repair. Our results show how biochemical processes of damage and repair in the germline interact with sex-specific life history traits to shape mutation patterns on both the X chromosome and autosomes.