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Environment of human being usually contains a high number of environmental mutagens, which may modify chemically nucleic acid bases into promutagenic analogues. Hydroxylamine (NH2OH) is a strong mutagen which modifies cytosine and adenine to N4-hydroxycytosine and N6-hydroxyadenine, respectively. Once these analogues are present in DNA or RNA, they may cause transition point mutations by the exchange between two pairs C:G and A:T into T:A and G:C, respectively. The reason for these mutations is the change of preferences between intermolecular hydrogen bonds resulting from the shift of the tautomeric equilibrium from the preferred amino form into the imino one. In the case of the amino<-->imino tautomeric equilibrium of N6-hydroxyadenosine, it was also shown that preferential hydrogen bonding between its imino form and cytidine, or uridine and the amino form of this base leads to the shift of the tautomeric equilibrium in favour of these tautomers in solution. N4-hydroxy-dCMP analogues exhibited very interesting inhibitory properties versus the biosynthesis of dTMP catalyzed by thymidylate synthase. These properties help to further the knowledge on the molecular mechanism of the catalytic reaction of this enzyme as well as on the role of syn-anti photoisomerization of the N4-hydroxy group in this reaction. Examinations gathered in the article were conducted from 1979 to 1985 under supervision, and afterwards from 1986 to 2004 in collaboration with professor David Shugar.

Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in folate and homocysteine metabolism. Research performed during the past decade has clarified our understanding of MTHFR deficiencies that cause homocystinuria or mild hyperhomocysteinemia. Our cloning of the MTHFR coding sequence was initially followed by the identification of the first deleterious mutations in MTHFR, in patients with homocystinuria and marked hyperhomocysteinemia. Shortly thereafter, we identified the 677C-->T variant and showed that it encoded a thermolabile enzyme with reduced activity. Currently, a total of 41 rare but deleterious mutations in MTHFR, as well as about 60 polymorphisms have been reported. The 677C-->T (Ala222Val) variant has been particularly noteworthy since it has become recognized as the most common genetic cause of hyperhomocysteinemia. The disruption of homocysteine metabolism by this polymorphism influences risk for several complex disorders, including cardiovascular disease, neural tube defects and some cancers. We describe here the complex structure of the MTHFR gene, summarize the current state of knowledge on rare and common mutations in MTHFR and discuss some relevant findings in a mouse model for MTHFR deficiency.

Objectives To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. Methods We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. Results The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. Conclusions Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI. [ABSTRACT FROM AUTHOR]

Adrenoleukodystrophy is a rare neurogenetic disease, and adrenomyeloneuropathy is the most common phenotype in adults. The clinical data of a patient with adrenoleukodystrophy and spinal-peripheral neuropathy caused by a novel point mutation in exon 4 of the ABCD1 gene (c.1256T > G (p.Val419Gly)) were retrospectively analyzed. Furthermore, we constructed wild-type and mutant vectors of the ABCD1 (NM0000334) gene to validate the effect of this mutation on the expression of the ABCD1 gene and protein and to explore the mechanism of X-linked adrenoleukodystrophy occurrence and development to identify therapeutic targets for clinical treatment.

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14P124 or SRP14WT and SRP14P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14WT and SRP14P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

Single-point mutation diseases in which substitution of one nucleotide with another in a gene occurs include familial Alzheimer's disease (fAD), familial Parkinson's disease (fPD), and familial Creutzfeldt-Jacob disease (fCJD) as well as Huntington's disease (HD), sickle cell anemia, and hemophilia. Inevitability of occurrence of these diseases is certain. However, the time of appearance of symptoms could be influenced by the diet, environment, and possibly other genetic factors. There are no effective approaches to delay the onset or progression of symptoms of these diseases. The fact that increased oxidative stress and inflammation significantly contribute to the initiation and progression of these point mutation diseases shows that antioxidants could be useful. The major objectives are (a) to present evidence that increased oxidative stress and chronic inflammation are associated with selected single-point mutation diseases, such as fAD, fPD, and fCJD, HD, sickle cell anemia, and hemophilia; (b) to describe limited studies on the role of individual antioxidants in experimental models of some of these diseases; and (c) to discuss a rationale for utilizing a comprehensive mixture of micronutrients, which may delay the development and progression of symptoms of above diseases by simultaneously reducing oxidative and inflammatory damages.Key teaching pointsSelected single-point mutation diseases and their pattern of inheritanceCharacteristics of each selected single-point mutation diseaseEvidence for increased oxidative stress and inflammation in each diseasePotential reasons for failure of single antioxidants in human studiesRationale for using a comprehensive mixture of micronutrients in delaying the onset and progression of single-point mutation diseases.

BackgroundPrader-Willi syndrome (PWS) is an imprinting disorder caused by the absence of paternal expressed genes in the Prader-Willi critical region (PWCR) on chromosome 15q11.2-q13. Three molecular mechanisms have been known to cause PWS, including a deletion in the PWCR, uniparental disomy 15 and imprinting defects.ResultsWe report the first case of PWS associated with a single-nucleotide SNRPN variant in a 10-year-old girl presenting with clinical features consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay with cognitive impairment, excessive eating with central obesity, sleep disturbances, skin picking and related behaviour issues. Whole-exome sequencing revealed a de novo mosaic nonsense variant of the SNRPN gene (c.73C>T, p.R25X) in 10% of DNA isolated from buccal cells and 19% of DNA from patient-derived lymphoblast cells. DNA methylation study did not detect an abnormal methylation pattern in the SNRPN locus. Parental origin studies showed a paternal source of an intronic single-nucleotide polymorphism within the locus in proximity to the SNRPN variant.ConclusionsThis is the first report that provides evidence of a de novo point mutation of paternal origin in SNRPN as a new disease-causing mechanism for PWS. This finding suggests that gene sequencing should be considered as part of the diagnostic workup in patients with clinical suspicion of PWS.

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.

Each human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. Whereas the rate and patterns of point mutations in the human germline are now well understood, far less is known about the frequency and features that impact de novo structural variants (dnSVs). We report a family-based study of germline mutations among 9,599 human genomes from 33 multigenerational CEPH-Utah families and 2,384 families from the Simons Foundation Autism Research Initiative. We find that de novo structural mutations detected by alignment-based, short-read WGS occur at an overall rate of at least 0.160 events per genome in unaffected individuals, and we observe a significantly higher rate (0.206 per genome) in ASD-affected individuals. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and we predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction, we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations and that these mechanisms most likely have markedly different rates and selective pressures than those leading to point mutations.

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.

Base-editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome-editing methods that use DNA double-strand breaks, such as zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system. This allows the generation of single-nucleotide-variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time- and resource-effective manner. These single-nucleotide-variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype-to-phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base-editing constructs, transfect adherent cells, quantify base-editing efficiencies in bulk, and generate single-nucleotide-variant clonal cell lines. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and production of plasmids for base-editing experiments Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing Alternate Protocol 1: Next-generation sequencing to quantify base editing Basic Protocol 4: Single-cell isolation of base-edited cells using FACS Alternate Protocol 2: Single-cell isolation of base-edited cells using dilution plating Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.MethodsWe developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available.ResultsEM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

Glyceronephosphate O-acyltransferase (GNPAT) p.D519G (rs11558492) was identified as a genetic modifier correlated with more severe iron overload in hemochromatosis through whole-exome sequencing of HFE p.C282Y homozygotes with extreme iron phenotypes. We studied the prevalence of p.D519G in HFE p.C282Y/p.H63D compound heterozygotes, a genotype associated with iron overload in some patients. Cases were Australian participants with elevated serum ferritin (SF) levels ≥300μg/L (males) and ≥200μg/L (females); subjects whose SF levels were below these cut-offs were designated as controls. Samples were genotyped for GNPAT p.D519G. We compared the allele frequency of the present subjects, with/without elevated SF, to p.D519G frequency in public datasets. GNPAT p.D519G was more prevalent in our cohort of p.C282Y/p.H63D compound heterozygotes with elevated SF (37%) than European public datasets: 1000G 21%, gnomAD 20% and ESP 21%. We conclude that GNPAT p.D519G is associated with elevated SF in Australian HFE p.C282Y/p.H63D compound heterozygotes.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

Mitochondrial dysfunction is frequently associated with impairment in metabolic homeostasis and insulin action, and is thought to underlie cellular aging. However, it is unclear whether mitochondrial dysfunction is a cause or consequence of insulin resistance in humans. To determine the impact of intrinsic mitochondrial dysfunction on metabolism and insulin action, we performed comprehensive metabolic phenotyping of the polymerase gamma (PolG) D257A "mutator" mouse, a model known to accumulate supraphysiological mitochondrial DNA (mtDNA) point mutations. We utilized the heterozygous PolG mutator mouse (PolG+/mut ) because it accumulates mtDNA point mutations ~ 500-fold > wild-type mice (WT), but fails to develop an overt progeria phenotype, unlike PolGmut/mut animals. To determine whether mtDNA point mutations induce metabolic dysfunction, we examined male PolG+/mut mice at 6 and 12 months of age during normal chow feeding, after 24-hr starvation, and following high-fat diet (HFD) feeding. No marked differences were observed in glucose homeostasis, adiposity, protein/gene markers of metabolism, or oxygen consumption in muscle between WT and PolG+/mut mice during any of the conditions or ages studied. However, proteomic analyses performed on isolated mitochondria from 12-month-old PolG+/mut mouse muscle revealed alterations in the expression of mitochondrial ribosomal proteins, electron transport chain components, and oxidative stress-related factors compared with WT. These findings suggest that mtDNA point mutations at levels observed in mammalian aging are insufficient to disrupt metabolic homeostasis and insulin action in male mice.

Memory B cells (MBCs) are long-lived and produce high-affinity, generally, class-switched antibodies. Here, we use a multiparameter approach involving CD27 to segregate naïve B cells (NBC), IgD+ unswitched (unsw)MBCs and IgG+ or IgA+ class-switched (sw)MBCs from humans of different age, sex and race. Conserved antibody variable gene expression indicates that MBCs emerge through unbiased selection from NBCs. Integrative analyses of mRNAs, miRNAs, lncRNAs, chromatin accessibility and cis-regulatory elements uncover a core mRNA-ncRNA transcriptional signature shared by IgG+ and IgA+ swMBCs and distinct from NBCs, while unswMBCs display a transitional transcriptome. Some swMBC transcriptional signature loci are accessible but not expressed in NBCs. Profiling miRNAs reveals downregulated MIR181, and concomitantly upregulated MIR181 target genes such as RASSF6, TOX, TRERF1, TRPV3 and RORα, in swMBCs. Finally, lncRNAs differentially expressed in swMBCs cluster proximal to the IgH chain locus on chromosome 14. Our findings thus provide new insights into MBC transcriptional programs and epigenetic regulation, opening new investigative avenues on these critical cell elements in human health and disease.

The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of Escherichia coli with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

The probability of point mutations is expected to be highly influenced by the flanking nucleotides that surround them, known as the sequence context. This phenomenon may be mainly attributed to the enzyme that modifies or mutates the genetic material, because most enzymes tend to have specific sequence contexts that dictate their activity. Here, we develop a statistical model that allows for the detection and evaluation of the effects of different sequence contexts on mutation rates from deep population sequencing data. This task is computationally challenging, as the complexity of the model increases exponentially as the context size increases. We established our novel Bayesian method based on sparse model selection methods, with the leading assumption that the number of actual sequence contexts that directly influence mutation rates is minuscule compared with the number of possible sequence contexts. We show that our method is highly accurate on simulated data using pentanucleotide contexts, even when accounting for noisy data. We next analyze empirical population sequencing data from polioviruses and HIV-1 and detect a significant enrichment in sequence contexts associated with deamination by the cellular deaminases ADAR 1/2 and APOBEC3G, respectively. In the current era, where next-generation sequencing data are highly abundant, our approach can be used on any population sequencing data to reveal context-dependent base alterations and may assist in the discovery of novel mutable sites or editing sites.

Methylation of the regulatory region of the elongation of very-long-chain fatty acids-like 2 (ELOVL2) gene, an enzyme involved in elongation of long-chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved. Here, we report that Elovl2 regulates age-associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age-related eye diseases. We show that an age-related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5-Aza-2'-deoxycytidine (5-Aza-dc) leads to increased Elovl2 expression and rescue of age-related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2-specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well-established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components found in human drusen, a pathologic hallmark of age related macular degeneration. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual function, and suggest novel therapeutic strategies for the treatment of age-related eye diseases.

Introduction: Severe Combined Immunodeficiency (SCID) is a life-threatening immunodeficiency caused by several pathogenic genetic variants, and it is characterized by profound defects in T-cell numbers and immune function. First performed in the late 1960's, hematopoietic stem cell transplantation remains the standard treatment for most cases of SCID. There is a growing number of post-transplant SCID patients, and it is imperative to assess the long-term outcomes of these patients. We have reported here the longest follow-up of a post-transplant SCID patient who, to our knowledge, bears the first gamma chain (γc) variant to show intact IL-21 signaling. Case Presentation: The patient presented at 5 months of age with recurrent thrush and Pneumocystis jiroveci pneumonia. In 1971, at the age of 11 months, he received an unconditioned, matched, related donor transplant comprising whole, unprocessed bone marrow. He is now 48 years old without significant illness and has never required immunoglobulin replacement. He exhibits T-dependent vaccine responses. He does suffer from chronic warts and bacterial infections that have worsened in recent years. We confirmed a known pathogenic variant in the IL2RG gene showing a hemizygous variant NM_000206.2:c.675C>A, resulting in p.Ser225Arg. His chimerism studies revealed donor T cells, host B cells, host myeloid cells, and mixed NK cells. Lymphocyte enumeration revealed normal numbers and distribution of B cells. The host B cells carry the pathogenic variant in IL2RG, but, when stimulated with IL-21, they demonstrated intact, γc-dependent signaling. Conclusions: Even with host B cells, reconstitution with donor T cells can be sufficient to allow over four decades of survival when B-cell function is intact. Our case demonstrates that satisfactory B-cell function can arise as a consequence of both intact IL-21 signaling due to a hypomorphic γc variant, and close HLA matching with the donor to allow for effective T-cell help.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

Warmblood fragile foal syndrome (WFFS) is an autosomal recessive disorder caused by a single nucleotide variant in the procollagen-lysine-2-oxoglutarate-5-dioxygenase 1 gene (PLOD1:c.2032G>A, p.Gly678Arg). Homozygosity for the PLOD1 variant causes an Ehler-Danlos-like syndrome, which has to date only been reported in warmblood breeds but the WFFS allele has been also detected in the Thoroughbred. To investigate the breed distribution of the WFFS allele, 4081 horses belonging to 38 different breeds were screened. In total, 4.9% of the horses representing 21 breeds carried the WFFS allele. The affected breeds were mainly warmbloods, with carrier frequency as high as 17% in the Hanoverian and Danish Warmblood. The WFFS allele was not detected in most non-warmblood breeds. Exceptions include WFFS carriers in the Thoroughbred (17/716), Haflinger (2/48), American Sport Pony (1/12), and Knabstrupper (3/46). The origin of the WFFS allele remains unknown. The Arabian breed and specifically the stallion Bairactar Or. Ar. (1813), whose offspring were reported to have a similar phenotype in the 19th century, were hypothesized as the origin. DNA from a museum sample of Bairactar Or. Ar. showed that he did not carry the mutated allele. This result, together with the genotypes of 302 Arabians, all homozygous for the reference allele, does not support an Arabian origin of the WFFS allele. Our extensive survey shows the WFFS allele to be of moderate frequency and concern in warmbloods and also in breeds where it may not be expected.

Ionising radiation (IR) is a recognised carcinogen responsible for cancer development in patients previously treated using radiotherapy, and in individuals exposed as a result of accidents at nuclear energy plants. However, the mutational signatures induced by distinct types and doses of radiation are unknown. Here, we analyse the genetic architecture of mammary tumours, lymphomas and sarcomas induced by high (56Fe-ions) or low (gamma) energy radiation in mice carrying Trp53 loss of function alleles. In mammary tumours, high-energy radiation is associated with induction of focal structural variants, leading to genomic instability and Met amplification. Gamma-radiation is linked to large-scale structural variants and a point mutation signature associated with oxidative stress. The genomic architecture of carcinomas, sarcomas and lymphomas arising in the same animals are significantly different. Our study illustrates the complex interactions between radiation quality, germline Trp53 deficiency and tissue/cell of origin in shaping the genomic landscape of IR-induced tumours.

Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus.IMPORTANCE Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation.

Dbp5 is an essential DEAD-box protein that mediates nuclear mRNP export. Dbp5 also shuttles between nuclear and cytoplasmic compartments with reported roles in transcription, ribosomal subunit export, and translation; however, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5 specifically contributes to each of these processes remains unclear. Towards understanding the functions and transport of Dbp5 in Saccharomyces cerevisiae, alanine scanning mutagenesis was used to generate point mutants at all possible residues within a GFP-Dbp5 reporter. Characterization of the 456 viable mutants led to the identification of an N-terminal Xpo1-dependent nuclear export signal in Dbp5, in addition to other separation-of-function alleles, which together provide evidence that Dbp5 nuclear shuttling is not essential for mRNP export. Rather, disruptions in Dbp5 nucleocytoplasmic transport result in tRNA export defects, including changes in tRNA shuttling dynamics during recovery from nutrient stress.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.

MUTYH-associated polyposis (MAP) is a hereditary cancer syndrome that is caused by biallelic pathogenic variants in the MUTYH gene and should be evaluated for in patients with an attenuated colonic polyposis phenotype. Monoallelic pathogenic variants in MUTYH are associated with a moderate increased risk of colorectal cancer but not with the polyposis phenotype. We present a case of a patient presenting with multiple colonic adenomatous polyps, whose germline testing revealed a heterozygous pathogenic variant in MUTYH in exon 13, c.1187G > A (p.Gly396Asp) as well as a heterozygous variant of unknown significance (VUS) in MUTYH in exon 14, c.1379T > C (p.Leu460Ser). We interpret the VUS as pathogenic in light of the patient's phenotype; the fact that the VUS was in trans with a known pathogenic variant; and because all the in silico predictors suggested, it was likely to be deleterious. This case highlights the importance of a gastroenterologist recognizing the indication for genetic testing in a patient with greater than ten adenomas, the importance of a genetic counselor in interpretation of results, and is the first report of the specific variant in the literature with clinical information to suggest that it is likely pathogenic.

BackgroundBase editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. Recently, adenine base editors (ABEs) have been developed to mediate the conversion of A•T to G•C in genomic DNA of human cells, mice, and plants. Here, we investigated the activity and efficiency of several adenine base editors in zebrafish and showed that base editing can be used to create new models of pathogenic diseases caused by point mutations.ResultsThe original ABE7.10 exhibits almost no activity in zebrafish. After codon optimization, we found that a zABE7.10 variant could induce targeted conversion of adenine to guanine in zebrafish at multiple tested genomic loci, and all the target sites showed a high rate of germline targeting efficiency. Furthermore, using this system, we established a zebrafish model of 5q-Syndrome that contained a new point mutation in rps14. The further modification of zABE7.10 by a bipartite nuclear localization signals (bpNLS) resulted in 1.96-fold average improvement in ABE-mediated editing efficiency at four sites.ConclusionsCollectively, this system, designated as zABE7.10, provides a strategy to perform A•T to G•C base editing in zebrafish and enhances its capacity to model human diseases.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.

Background: Diminished ovarian reserve is one of the most important causes of female infertility. In the etiology study of DOR, besides age, it is known that chromosomal abnormality, radiotherapy, chemotherapy and ovarian surgery can result in DOR. For young women without obvious risk factors, gene mutation should be considered as a possible cause. However, the specific molecular mechanism of DOR has not been fully elucidated.Methods: In order to explore the pathogenic variants related to DOR, twenty young women under 35 years old affected by DOR without definite factors damaging ovarian reserve were recruited as the research subjects, and five women with normal ovarian reserve were recruited as the control group. Whole exome sequencing was applied as the genomics research tool.Results: As a result, we obtained a set of mutated genes that may be related to DOR, where the missense variant on GPR84 was selected for further study. It is found that GPR84Y370H variant promotes the expression of proinflammatory cytokines (TNF-α, IL12B, IL-1β) and chemokines (CCL2, CCL5), as well as the activation of NF-κB signaling pathway.Conclusion: In conclusion, GPR84Y370H variant was identified though analysis for WES results of 20 DOR patients. The deleterious variant of GPR84 could be the potential molecular mechanism of non-age-related pathological DOR through its role in promoting inflammation. The findings of this study can be used as a preliminary research basis for the development of early molecular diagnosis and treatment target selection of DOR.

The co-occurrence of a copy number variant (CNV) and a functional variant on the other allele may be a relevant genetic mechanism in schizophrenia. We hypothesized that the cumulative burden of such double hits-in particular those composed of a deletion and a coding single-nucleotide variation (SNV)-is increased in patients with schizophrenia. We combined CNV data with coding variants data in 795 patients with schizophrenia and 474 controls. To limit false CNV-detection, only CNVs called by two algorithms were included. CNV-affected genes were subsequently examined for coding SNVs, which we termed "CNV-SNVs." Correcting for total queried sequence, we assessed the CNV-SNV-burden and the combined predicted deleterious effect. We estimated P-values by permutation of the phenotype. We detected 105 CNV-SNVs; 67 in duplicated and 38 in deleted genic sequence. Although the difference in CNV-SNVs rates was not significant, the combined deleteriousness inferred by CNV-SNVs in deleted sequence was almost 4-fold higher in cases compared with controls (nominal P = 0.009). This effect may be driven by a higher number of CNV-SNVs and/or by a higher degree of predicted deleteriousness of CNV-SNVs. No such effect was observed for duplications. We provide early evidence that deletions co-occurring with a functional variant may be relevant, albeit of modest impact, for the genetic etiology of schizophrenia. Large-scale consortium studies are required to validate our findings. Sequence-based analyses would provide the best resolution for detection of CNVs as well as coding variants genome-wide.

Background:PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level β-lactam resistance is unknown. Objectives:To determine whether enzymatic activity of PBP4 is required for high-level β-lactam resistance and to investigate how the pbp4-associated mutations provide β-lactam resistance. Methods:The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. β-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC. Results:PBP4 inactivation imparted complete β-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart β-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment. Conclusions:PBP4's activity and overexpression both contribute to high-level β-lactam resistance.

BackgroundAutosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis.ObjectiveWe sought to establish genotype-phenotype correlation in patients with AD EDA-ID.MethodsA disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts.ResultsDisease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin β receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations.ConclusionsIκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.In prostate cancer, the role of mutations in the maternally-inherited mitochondrial genome are not well known. Here, the authors demonstrate frequent, age-dependent mitochondrial mutation in prostate cancer. Strong links between mitochondrial and nuclear mutational profiles are associated with clinical aggressivity.

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered.IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in product loss, a health hazard to workers, and ultimately abandonment of wells. Identification of the required genes is a critical step for determining the mechanism of biofilm formation by sulfate reducers. Here, the transporter by which putative biofilm structural proteins are exported from sulfate-reducing Desulfovibrio vulgaris Hildenborough cells was discovered, and a single nucleotide change within the gene coding for this transporter was found to be sufficient to completely stop formation of biofilm.

Exfoliation syndrome (XFS) is the most common known risk factor for secondary glaucoma and a major cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A, have previously been associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results across populations, and to identify new variants associated with XFS. We identified a rare protective allele at LOXL1 (p.Phe407, odds ratio (OR) = 25, P = 2.9 × 10-14) through deep resequencing of XFS cases and controls from nine countries. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10-8). We identified association signals at 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity.