scholarly journals Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

2021 ◽  
Author(s):  
Renato Salazar ◽  
Barbara Arbeithuber ◽  
Maja Ivankovic ◽  
Monika Heinzl ◽  
Sofia Moura ◽  
...  
Keyword(s):  
De Novo ◽  
Age Groups ◽  
Low Frequency ◽  
Congenital Disorders ◽  
Sequence Information ◽  
De Novo Mutations ◽  
Coding Region ◽  
Male Germline ◽  
Significant Difference ◽  
Duplex Sequencing

AbstractDe novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

scholarly journals Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 749 ◽  
Author(s):  
Melanie Hiltbrunner ◽  
Gerald Heckel
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Phylogenetic Analyses ◽  
Common Vole ◽  
Natural Host ◽  
Sequence Information ◽  
Coding Region ◽  
Sequence Capture ◽  
Genome Wide ◽  
The Impact

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

scholarly journals Mutational pressure drives differential genome conservation in two bacterial endosymbionts of sap feeding insects

2020 ◽  
Author(s):  
Gus Waneka ◽  
Yumary M. Vasquez ◽  
Gordon M. Bennett ◽  
Daniel B. Sloan
Keyword(s):  
De Novo ◽  
Evolutionary Rate ◽  
Direct Detection ◽  
Low Frequency ◽  
Rate Variation ◽  
Sequence Evolution ◽  
Single Nucleotide Variants ◽  
Genome Decay ◽  
Sap Feeding ◽  
Duplex Sequencing

ABSTRACTCompared to free-living bacteria, endosymbionts of sap-feeding insects have tiny and rapidly evolving genomes. Increased genetic drift, high mutation rates, and relaxed selection associated with host control of key cellular functions all likely contribute to genome decay. Phylogenetic comparisons have revealed massive variation in endosymbiont evolutionary rate, but such methods make it difficult to partition the effects of mutation vs. selection. For example, the ancestor of auchenorrhynchan insects contained two obligate endosymbionts, Sulcia and a betaproteobacterium (BetaSymb; called Nasuia in leafhoppers) that exhibit divergent rates of sequence evolution and different propensities for loss and replacement in the ensuing ∼300 Ma. Here, we use the auchenorrhynchan leafhopper Macrosteles sp. nr. severini, which retains both of the ancestral endosymbionts, to test the hypothesis that differences in evolutionary rate are driven by differential mutagenesis. We used a high-fidelity technique known as duplex sequencing to measure and compare low-frequency variants in each endosymbiont. Our direct detection of de novo mutations reveals that the rapidly evolving endosymbiont (Nasuia) has a much higher frequency of single-nucleotide variants than the more stable endosymbiont (Sulcia) and a mutation spectrum that is even more AT-biased than implied by the 83.1% AT content of its genome. We show that indels are common in both endosymbionts but differ substantially in length and distribution around repetitive regions. Our results suggest that differences in long-term rates of sequence evolution in Sulcia vs. BetaSymb, and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis.SIGNIFICANCE STATEMENTTwo ancient endosymbionts in the same host lineage display stark differences in genome conservation over phylogenetic scales. We show the rapidly evolving endosymbiont has a higher frequency of mutations, as measured with duplex sequencing. Therefore, differential mutagenesis likely drives evolutionary rate variation in these endosymbionts.

scholarly journals Mutation bias shapes gene evolution in Arabidopsis thaliana

2020 ◽  
Author(s):  
J. Grey Monroe ◽  
Thanvi Srikant ◽  
Pablo Carbonell-Bejerano ◽  
Moises Exposito-Alonso ◽  
Mao-Lun Weng ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
De Novo ◽  
Gene Evolution ◽  
Gc Content ◽  
Mutation Rates ◽  
Rate Variation ◽  
Recent Theory ◽  
De Novo Mutations ◽  
Coding Region ◽  
Mutational Hotspots

Classical evolutionary theory maintains that mutation rate variation between genes should be random with respect to fitness 1–4 and evolutionary optimization of genic mutation rates remains controversial 3,5. However, it has now become known that cytogenetic (DNA sequence + epigenomic) features influence local mutation probabilities 6, which is predicted by more recent theory to be a prerequisite for beneficial mutation rates between different classes of genes to readily evolve 7. To test this possibility, we used de novo mutations in Arabidopsis thaliana to create a high resolution predictive model of mutation rates as a function of cytogenetic features across the genome. As expected, mutation rates are significantly predicted by features such as GC content, histone modifications, and chromatin accessibility. Deeper analyses of predicted mutation rates reveal effects of introns and untranslated exon regions in distancing coding sequences from mutational hotspots at the start and end of transcribed regions in A. thaliana. Finally, predicted coding region mutation rates are significantly lower in genes where mutations are more likely to be deleterious, supported by numerous estimates of evolutionary and functional constraint. These findings contradict neutral expectations that mutation probabilities are independent of fitness consequences. Instead they are consistent with the evolution of lower mutation rates in functionally constrained loci due to cytogenetic features, with important implications for evolutionary biology8.

scholarly journals Marfan syndrome: whole-exome sequencing reveals de novo mutations, second gene and genotype–phenotype correlations in the Chinese population

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Yuduo Wu ◽  
Hairui Sun ◽  
Jianbin Wang ◽  
Xin Wang ◽  
Ming Gong ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Marfan Syndrome ◽  
Whole Exome Sequencing ◽  
Chinese Population ◽  
De Novo ◽  
Low Frequency ◽  
Monogenic Disease ◽  
De Novo Mutations ◽  
Fbn1 Gene ◽  
Whole Exome

Abstract Marfan syndrome (MFS) is a dominant monogenic disease caused by mutations in fibrillin 1 (FBN1). Cardiovascular complications are the leading causes of mortality among MFS. In the present study, a whole-exome sequencing of MFS in the Chinese population was conducted to investigate the correlation between FBNI gene mutation and MFS. Forty-four low-frequency harmful loci were identified for the FBN1 gene in HGMD database. In addition, 38 loci were identified in the same database that have not been related to MFS before. A strict filtering and screening protocol revealed two patients of the studied group have double mutations in the FBN1 gene. The two patients harboring the double mutations expressed a prominent, highly pathological phenotype in the affected family. In addition to the FBN1 gene, we also found that 27 patients had mutations in the PKD1 gene, however these patients did not have kidney disease, and 16 of the 27 patients expressed aortic related complications. Genotype-phenotype analysis showed that patients with aortic complications are older in the family, aged between 20 and 40 years.

scholarly journals Mutational Disruption ofcis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions

2015 ◽  
Vol 89 (23) ◽  
pp. 11761-11772 ◽  
Author(s):  
S. Smithee ◽  
S. Tracy ◽  
N. M. Chapman
Keyword(s):  
Rna Structure ◽  
De Novo ◽  
Virus Genome ◽  
Viral Rna ◽  
Sequence Information ◽  
Virus Population ◽  
Wild Type ◽  
Coding Region ◽  
Free System ◽  
Genomic Deletions

ABSTRACTFollowing natural human or experimental murine infections and in cell culture, coxsackievirus B (CVB) RNA can persist for weeks in the absence of a cytopathic effect, yet viral RNA remains detectable. Our earlier studies demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the genomic 5′ terminus which partially degraded the cloverleaf (or domain I), an RNA structure required for efficient viral replication. Acis-acting replication element (CRE) in the 2C protein-coding region [CRE(2C)] templates the addition of two uridine residues to the virus genome-encoded RNA replication primer VPg prior to positive-strand synthesis. Because our previous work also demonstrated that the genomes of CVB with a 5′-terminal deletion (CVB-TD) have VPg covalently linked, even though they rarely terminate in the canonical UU donated by CRE(2C)-mediated uridylylation of VPg, we hypothesized that a functional (uridylylating) CRE(2C) would be unnecessary for CVB-TD replication. Using the same 16 mutations in the CVB3 CRE(2C) structure that were considered lethal for this virus by others, we demonstrate here both in infected cell cultures and in mice that wild-type (wt) and CVB3-TD strains carrying these mutations with a nonuridylylating CRE(2C) are viable. While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to those observed in a CVB3-TD strain, mutation of the CRE(2C) function in a CVB3-TD strain does not further decrease replication. Finally, we show that replication of the parental CVB3 strain containing the mutated CRE(2C) drives thede novogeneration of genomic deletions at the 5′ terminus.IMPORTANCEIn this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), the replication rate suffers significantly. Further, deletions at the 5′ terminus of the genome are generated in this virus population, with this virus population supplanting the wild-type population. This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in the loss of sequence information from the 5′ terminus. These findings have significance when considering the replication of human enteroviruses, and we believe that these data are unattainable in a cell-free system due to the poor replication of these CRE-deficient viruses.

scholarly journals Germline and somatic mutations in STXBP1 with diverse neurodevelopmental phenotypes

2017 ◽  
Vol 3 (6) ◽  
pp. e199 ◽  
Author(s):  
Mohammed Uddin ◽  
Marc Woodbury-Smith ◽  
Ada Chan ◽  
Ledia Brunga ◽  
Sylvia Lamoureux ◽  
...  
Keyword(s):  
Brain Tissue ◽  
De Novo ◽  
Focal Cortical Dysplasia ◽  
Autism Spectrum ◽  
Epileptic Encephalopathy ◽  
De Novo Mutation ◽  
Chromosomal Microarray ◽  
Published Data ◽  
De Novo Mutations ◽  
Coding Region

Objective:To expand the clinical phenotype associated with STXBP1 gene mutations and to understand the effect of STXBP1 mutations in the pathogenesis of focal cortical dysplasia (FCD).Methods:Patients with STXBP1 mutations were identified in various ways: as part of a retrospective cohort study of epileptic encephalopathy; through clinical referrals of individuals (10,619) with developmental delay (DD) for chromosomal microarray; and from a collection of 5,205 individuals with autism spectrum disorder (ASD) examined by whole-genome sequencing.Results:Seven patients with heterozygous de novo mutations affecting the coding region of STXBP1 were newly identified. Three cases had radiologic evidence suggestive of FCD. One male patient with early infantile epileptic encephalopathy, DD, and ASD achieved complete seizure remission following resection of dysplastic brain tissue. Examination of excised brain tissue identified mosaicism for STXBP1, providing evidence for a somatic mechanism. Cell-type expression analysis suggested neuron-specific expression. A comprehensive analysis of the published data revealed that 3.1% of severe epilepsy cases carry a pathogenic de novo mutation within STXBP1. By contrast, ASD was rarely associated with mutations in this gene in our large cohorts.Conclusions:STXBP1 mutations are an important cause of epilepsy and are also rarely associated with ASD. In a case with histologically proven FCD, an STXBP1 somatic mutation was identified, suggesting a role in its etiology. Removing such tissue may be curative for STXBP1-related epilepsy.

scholarly journals Genetic Mosaicism as a Cause of Inborn Errors of Immunity

2021 ◽  
Author(s):  
Jahnavi Aluri ◽  
Megan A. Cooper
Keyword(s):  
Immune Response ◽  
De Novo ◽  
Somatic Mosaicism ◽  
Genetic Diagnosis ◽  
Low Frequency ◽  
De Novo Mutations ◽  
Inborn Errors ◽  
Tissue Samples ◽  
Inborn Errors Of Immunity ◽  
Genetic Mosaicism

AbstractInborn errors of immunity (IEIs) are a heterogeneous group of disorders due to genetic defects in the immune response that have a broad clinical spectrum. Diagnosis of the precise genetic cause of IEI has led to improved care and treatment of patients; however, genetic diagnosis using standard approaches is only successful in ~40% of patients and is particularly challenging in “sporadic” cases without a family history. Standard genetic testing for IEI evaluates for germline changes in genes encoding proteins important for the immune response. It is now clear that IEI can also arise from de novo mutations leading to genetic variants present in germ cells and/or somatic cells. In particular, somatic mosaicism, i.e., post-zygotic genetic changes in DNA sequence, is emerging as a significant contributor to IEI. Testing for somatic mosaicism can be challenging, and both older sequencing techniques such as Sanger sequencing and newer next-generation sequencing may not be sensitive enough to detect variants depending on the platform and analysis tools used. Investigation of multiple tissue samples and specifically targeting sequence technologies to detect low frequency variants is important for detection of variants. This review examines the role and functional consequences of genetic mosaicism in IEI. We emphasize the need to refine the current exome and genome analysis pipeline to efficiently identify mosaic variants and recommend considering somatic mosaicism in disease discovery and in the first-tier of genetic analysis.

The Male Is Significantly Implicated as the Cause of Unexplained Infertility

2020 ◽  
Vol 38 (01) ◽  
pp. 003-020
Author(s):  
Robert John Aitken
Keyword(s):  
Oxidative Stress ◽  
Male Infertility ◽  
De Novo ◽  
Deep Understanding ◽  
Epigenetic Factors ◽  
De Novo Mutations ◽  
Male Factor ◽  
Male Germline ◽  
Broad Array ◽  
Ejaculation Frequency

AbstractMale infertility is recognized as a relatively common, complex condition, generated by a broad array of environmental and genetic factors. Historical reliance on the conventional semen profile has tended to underestimate the true contribution of “the male factor” to human infertility. This review highlights the importance of genetic and epigenetic factors in the etiology of male infertility, identifying a range of mutations responsible for primary testicular failure and impaired fertilizing potential. More than three quarters of all de novo mutations arise in the male germline via mechanisms that involve the inefficient or defective repair of DNA damage. Understanding the range of factors capable of creating genetic turmoil in the paternal germline is essential, if we are to gain a deep understanding of the causes of male infertility, rather than just the symptoms that characterize its presence. High levels of DNA fragmentation induced by oxidative stress are part of this equation. Oxidative stress is, in turn, driven by biological (age, ejaculation frequency, varicocele, infection), lifestyle (smoking, obesity), and environmental factors (heat, other forms of electromagnetic radiation, and toxins) that can impair the fertilizing potential of the spermatozoa and influence the incidence of spontaneous mutations that may cause infertility in the offspring.

scholarly journals The mutation spectrum in familial versus sporadic congenital cataract based on next-generation sequencing

2020 ◽  
Author(s):  
Fan Fan ◽  
Yi Luo ◽  
Jihong Wu ◽  
Chao Gao ◽  
Xin Liu ◽  
...  
Keyword(s):  
Congenital Cataract ◽  
De Novo ◽  
Genetic Diagnosis ◽  
Mutation Spectrum ◽  
Next Generation ◽  
De Novo Mutations ◽  
Additional Information ◽  
Significant Difference ◽  
Targeted Ngs ◽  
Sporadic Cases

Abstract Purpose Congenital cataract (CC) is a significant cause of lifelong visual loss. Its genetic diagnosis is challenging due to marked genetic heterogeneity. The purpose of this article is to report the genetic findings in sporadic and familial CC patients.Methods Patients (n=54) who were clinically diagnosed with CC and their parents were recruited. Blood samples were collected in our hospital. Mutations were detected by high-throughput, next-generation DNA sequencing (NGS) targeting 792 genes frequently involved in common inherited eye diseases.Results We identified variants in 11/38 cases (28.95%) of sporadic CC and 14/16 cases (87.5%) of familial CC, indicating a significant difference (P=0.000). Of the 14 variants identified in sporadic cases, 9 were previously reported mutations, and 5 were novel mutations, including 2 de novo mutations (CRYBB2 c.487C>T, FYCO1c.215A>T). The most frequent variants in our cohort were in crystallins and cytoskeletal genes (7/30, 23.33%), followed by X-linked syndromic proteins (13.33%) and transcriptional factors (10.00%). Additional information on the possibility of complications with inherited ocular or systemic diseases other than CC was provided in 20/30 (66.67%) variants.Conclusions These results contribute to expanding the mutation spectrum and frequency. Targeted NGS in CC provided significant diagnostic information and enabled more accurate genetic counseling. This study reports the different distributions of mutation genes in familial and sporadic CC cases.

scholarly journals Mutational pressure drives differential genome conservation in two bacterial endosymbionts of sap feeding insects

2020 ◽  
Author(s):  
Gus Waneka ◽  
Yumary M Vasquez ◽  
Gordon M Bennett ◽  
Daniel B Sloan
Keyword(s):  
De Novo ◽  
Evolutionary Rate ◽  
Direct Detection ◽  
Low Frequency ◽  
Sequence Evolution ◽  
De Novo Mutations ◽  
Cellular Functions ◽  
Single Nucleotide Variants ◽  
Genome Decay ◽  
Sap Feeding

Abstract Compared to free-living bacteria, endosymbionts of sap-feeding insects have tiny and rapidly evolving genomes. Increased genetic drift, high mutation rates, and relaxed selection associated with host control of key cellular functions all likely contribute to genome decay. Phylogenetic comparisons have revealed massive variation in endosymbiont evolutionary rate, but such methods make it difficult to partition the effects of mutation vs. selection. For example, the ancestor of Auchenorrhynchan insects contained two obligate endosymbionts, Sulcia and a betaproteobacterium (BetaSymb; called Nasuia in leafhoppers) that exhibit divergent rates of sequence evolution and different propensities for loss and replacement in the ensuing ~300 Ma. Here, we use the auchenorrhynchan leafhopper Macrosteles sp. nr. severini, which retains both of the ancestral endosymbionts, to test the hypothesis that differences in evolutionary rate are driven by differential mutagenesis. We used a high-fidelity technique known as duplex sequencing to measure and compare low-frequency variants in each endosymbiont. Our direct detection of de novo mutations reveals that the rapidly evolving endosymbiont (Nasuia) has a much higher frequency of single-nucleotide variants than the more stable endosymbiont (Sulcia) and a mutation spectrum that is potentially even more AT-biased than implied by the 83.1% AT content of its genome. We show that indels are common in both endosymbionts but differ substantially in length and distribution around repetitive regions. Our results suggest that differences in long-term rates of sequence evolution in Sulcia vs. BetaSymb, and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis.

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