RESUMEN
Cats with a distinctive white hair pattern of unknown molecular cause have been discovered in the Finnish domestic cat population. Based on the unique appearance of these cats, we have named this phenotype salmiak ("salty licorice"). The use of a commercially available panel test to genotype four salmiak-colored cats revealed the absence of all known variants associated with white-haired phenotypic loci: full White (W), Spotting (Ws) and the Birman white Gloves associated (wg) allele of the KIT proto-oncogene (KIT) gene. Whole-genome sequencing on two salmiak-colored cats was conducted to search for candidate causal variants in the KIT gene. Despite a lack of coding variants, visual inspection of the short read alignments revealed a large ~95 kb deletion located ~65 kb downstream of the KIT gene in the salmiak cats. Additional PCR genotyping of 180 domestic cats and three salmiak-colored cats confirmed the homozygous derived variant genotype fully concordant with the salmiak phenotype. We suggest the newly identified variant be designated as wsal for "w salmiak".
Asunto(s)
Color del Cabello , Proteínas Proto-Oncogénicas c-kit , Animales , Gatos/genética , Color del Cabello/genética , Proteínas Proto-Oncogénicas c-kit/genética , Fenotipo , Eliminación de Secuencia , Finlandia , Genotipo , Secuenciación Completa del Genoma/veterinariaRESUMEN
Hundreds of genetic variants implicated in Mendelian disease have been characterized in dogs and commercial screening is being offered for most of them worldwide. There is typically limited information available regarding the broader population frequency of variants and uncertainty regarding their functional and clinical impact in ancestry backgrounds beyond the discovery breed. Genetic panel screening of disease-associated variants, commercially offered directly to the consumer or via a veterinary clinician, provides an opportunity to establish large-scale cohorts with phenotype data available to address open questions related to variant prevalence and relevance. We screened the largest canine cohort examined in a single study to date (1,054,293 representative dogs from our existing cohort of 3.5 million; a total of 811,628 mixed breed dogs and 242,665 purebreds from more than 150 countries) to examine the prevalence and distribution of a total of 250 genetic disease-associated variants in the general population. Electronic medical records from veterinary clinics were available for 43.5% of the genotyped dogs, enabling the clinical impact of variants to be investigated. We provide detailed frequencies for all tested variants across breeds and find that 57% of dogs carry at least one copy of a studied Mendelian disease-associated variant. Focusing on a subset of variants, we provide evidence of full penetrance for 10 variants, and plausible evidence for clinical significance of 22 variants, on diverse breed backgrounds. Specifically, we report that inherited hypocatalasia is a notable oral health condition, confirm that factor VII deficiency presents as subclinical bleeding propensity and verify two genetic causes of reduced leg length. We further assess genome-wide heterozygosity levels in over 100 breeds, and show that a reduction in genome-wide heterozygosity is associated with an increased Mendelian disease variant load. The accumulated knowledge represents a resource to guide discussions on genetic test relevance by breed.
Asunto(s)
Relevancia Clínica , Pruebas Genéticas , Perros , Humanos , Animales , Prevalencia , Frecuencia de los Genes , FenotipoRESUMEN
Cryptorchidism is the most common congenital sex development disorder in dogs. Despite this, little progress has been made in understanding its genetic background. Extensive genetic testing of dogs through consumer and veterinary channels using a high-density SNP genotyping microarray coupled with links to clinical records presents the opportunity for a large-scale genome-wide association study to elucidate the molecular risk factors associated with cryptorchidism in dogs. Using an inter-breed genome-wide association study approach, a significant statistical association on canine chromosome 10 was identified, with the top SNP pinpointing a variant of HMGA2 previously associated with adult weight variance. In further analysis we show that incidence of cryptorchidism is skewed towards smaller dogs in concordance with the identified variant's previous association with adult weight. This study represents the first putative variant to be associated with cryptorchidism in dogs.
Asunto(s)
Criptorquidismo , Enfermedades de los Perros , Proteína HMGA2/genética , Animales , Criptorquidismo/genética , Criptorquidismo/veterinaria , Enfermedades de los Perros/genética , Perros , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation.IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.
Asunto(s)
Infecciones Asintomáticas , Variación Genética , Mutación , Neisseria meningitidis/genética , Alelos , Variación Antigénica , Adhesión Bacteriana , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Transferencia de Gen Horizontal , Humanos , Estudios Longitudinales , Fenotipo , Secuenciación Completa del GenomaRESUMEN
The distribution of DNA damage and repair is considered to occur heterogeneously across the genome. However, commonly available techniques, such as the alkaline comet assay or HPLC-MS/MS, measure global genome levels of DNA damage, and do not reflect potentially significant events occurring at the gene/sequence-specific level, in the nuclear or mitochondrial genomes. We developed a method, which comprises a combination of Damaged DNA Immunoprecipitation and next generation sequencing (DDIP-seq), to assess the induction and repair of DNA damage induced by 0.1 J/cm2 solar-simulated radiation at the sequence-specific level, across both the entire nuclear and mitochondrial genomes. DDIP-seq generated a genome-wide, high-resolution map of cyclobutane thymine dimer (T<>T) location and intensity. In addition to being a straightforward approach, our results demonstrated a clear differential distribution of T<>T induction and loss, across both the nuclear and mitochondrial genomes. For nuclear DNA, this differential distribution existed at both the sequence and chromosome level. Levels of T<>T were much higher in the mitochondrial DNA, compared to nuclear DNA, and decreased with time, confirmed by qPCR, despite no reported mechanisms for their repair in this organelle. These data indicate the existence of regions of sensitivity and resistance to damage formation, together with regions that are fully repaired, and those for which > 90% of damage remains, after 24 h. This approach offers a simple, yet more detailed approach to studying cellular DNA damage and repair, which will aid our understanding of the link between DNA damage and disease.
Asunto(s)
Ciclobutanos/química , Heterogeneidad Genética , Genoma Mitocondrial , Estudio de Asociación del Genoma Completo , Genoma , Dímeros de Pirimidina/química , Supervivencia Celular/genética , Daño del ADN , Reparación del ADN , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Arginina/antagonistas & inhibidores , Argininosuccinato Sintasa/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Arginasa/farmacología , Arginasa/uso terapéutico , Arginina/metabolismo , Línea Celular Tumoral , Colon/patología , Neoplasias Colorrectales/mortalidad , Interacciones Farmacológicas , Sinergismo Farmacológico , Estudios de Factibilidad , Femenino , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Estudios de Seguimiento , Humanos , Hidrolasas/farmacología , Hidrolasas/uso terapéutico , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Masculino , Ratones , Ornitina Carbamoiltransferasa/metabolismo , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Polietilenglicoles/farmacología , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Estudios Retrospectivos , Resultado del Tratamiento , Urea/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.
Asunto(s)
Linfoma de Burkitt/tratamiento farmacológico , Carbazoles/farmacología , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-6/biosíntesis , Factor de Transcripción STAT3/metabolismo , Animales , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Furanos , Humanos , Subunidad alfa del Receptor de Interleucina-10/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-bcl-6/genética , Factor de Transcripción STAT3/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing.
Asunto(s)
Metabolismo de los Lípidos/genética , Dislipidemias/genética , Frecuencia de los Genes , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Mutación Missense , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
The bacterium Clostridium difficile is a significant cause of nosocomial infections worldwide. The pathogenic success of this organism can be attributed to its flexible genome which is characterized by the exchange of mobile genetic elements, and by ongoing genome evolution. Despite its pathogenic status, C. difficile can also be carried asymptomatically, and has been isolated from natural environments such as water and sediments where multiple strain types (ribotypes) are found in close proximity. These include ribotypes which are associated with disease, as well as those that are less commonly isolated from patients. Little is known about the genomic content of strains in such reservoirs in the natural environment. In this study, draft genomes have been generated for 13 C. difficile isolates from estuarine sediments including clinically relevant and environmental associated types. To identify the genetic diversity within this strain collection, whole-genome comparisons were performed using the assemblies. The strains are highly genetically diverse with regards to the C. difficile "mobilome," which includes transposons and prophage elements. We identified a novel transposon-like element in two R078 isolates. Multiple, related and unrelated, prophages were detected in isolates across ribotype groups, including two novel prophage elements and those related to the transducing phage φC2. The susceptibility of these isolates to lytic phage infection was tested using a panel of characterized phages found from the same locality. In conclusion, estuarine sediments are a source of genetically diverse C. difficile strains with a complex network of prophages, which could contribute to the emergence of new strains in clinics.
Asunto(s)
Clostridioides difficile/genética , Genoma Bacteriano , Profagos/genética , Toxinas Bacterianas/genética , Clostridioides difficile/aislamiento & purificación , Clostridioides difficile/patogenicidad , Clostridioides difficile/virología , Microbiología Ambiental , Estuarios , Variación Genética , Sedimentos Geológicos/microbiología , Virulencia/genéticaRESUMEN
Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.
Asunto(s)
Proteínas Bacterianas/genética , Epigénesis Genética , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Streptococcus pneumoniae/genética , VirulenciaRESUMEN
BACKGROUND: Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. RESULTS: Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. CONCLUSIONS: The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis.
Asunto(s)
ADN Bacteriano , ADN Intergénico , Sitios Genéticos , Variación Genética , Genoma Bacteriano , Secuencias Repetitivas de Ácidos Nucleicos , Staphylococcus/genética , Secuencia Conservada , Evolución Molecular , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Staphylococcus/clasificación , Staphylococcus/patogenicidadRESUMEN
UNLABELLED: Receiver operating characteristic (ROC) analysis is a powerful and widely used technique for assessing predictive methods, yet there are no generic, open-source software tools for this that are freely available. Our ROCPLOT program performs ROC analysis on one or more files of search results (hits) and generates the following: (i) ROC values, giving a convenient numerical measure of method sensitivity and specificity; (ii) ROC plots graphically displaying sensitivity and specificity; (iii) classification plots to aid interpretation of the ROC plots and values; and (iv) a bar chart of the distribution of ROC values. ROCPLOT is generic and flexible: data in multiple hits files can be processed in series or parallel, allowing the results of multiple predictions to be viewed side-by-side or combined. AVAILABILITY: ROCPLOT is freely available for download as part of the European Molecular Biology Open Software Suite, EMBOSS (http://emboss.sourceforge.net/apps/rocplot.html).
Asunto(s)
Gráficos por Computador , Interpretación Estadística de Datos , Modelos Biológicos , Curva ROC , Programas Informáticos , Interfaz Usuario-Computador , Modelos EstadísticosRESUMEN
We identified key residues from the structural alignment of families of protein domains from SCOP which we represented in the form of sparse protein signatures. A signature-generating algorithm (SigGen) was developed and used to automatically identify key residues based on several structural and sequence-based criteria. The capacity of the signatures to detect related sequences from the SWISSPROT database was assessed by receiver operator characteristic (ROC) analysis and jack-knife testing. Test signatures for families from each of the main SCOP classes are described in relation to the quality of the structural alignments, the SigGen parameters used, and their diagnostic performance. We show that automatically generated signatures are potently diagnostic for their family (ROC50 scores typically >0.8), consistently outperform random signatures, and can identify sequence relationships in the "twilight zone" of protein sequence similarity (<40%). Signatures based on 15%-30% of alignment positions occurred most frequently among the best-performing signatures. When alignment quality is poor, sparser signatures perform better, whereas signatures generated from higher-quality alignments of fewer structures require more positions to be diagnostic. Our validation of signatures from the Globin family shows that when sequences from the structural alignment are removed and new signatures generated, the omitted sequences are still detected. The positions highlighted by the signature often correspond (alignment specificity >0.7) to the key positions in the original (non-jack-knifed) alignment. We discuss potential applications of sparse signatures in sequence annotation and homology modeling.