Multiple Gene Sequences Research Articles

Insight into the Taxonomic Resolution of the Pleosporalean Species Associated with Dead Woody Litter in Natural Forests from Yunnan, China.

In the course of investigating the systematics of woody litter micromycete associates in Yunnan Province, China, we found one new species in Phaeoseptaceae, one new genus and three new species in Sulcatisporaceae from 16 specimens collected (ten collections of ascomycetous teleomorphs, four collections of hyphomycetous and two collections of coelomycetes anamorphs) from Ailaoshan, Chuxiong, Diqing, Honghe, Kunming, Lancang, Mengla and Yuxi in Yunnan Province. These taxonomic novelties were recognized with the aid of morphological comparisons and phylogenetic analyses of multiple gene sequences (non-translated loci and protein-coding regions). Pleopunctum menglaense sp. nov. is accommodated in Phaeoseptaceae (Pleosporales) based on its hyphomycetous anamorph, which is characterized by superficial sporodochia on the host surface, macronematous, mononematous, cylindrical, unbranched, aseptate, hyaline and smooth-walled conidiophores, monoblastic, terminal, hyaline conidiogenous cells, hyaline, muriform α conidia, and brown, muriform β conidia with tri-lobed wing like basal cells. Kazuakitanaka gen. nov. (type: K. yuxiensis) is introduced in Sulcatisporaceae (Massarineae, Pleosporales) for a saprobic ascomycete with teleomorphic and anamorphic (coelomycetous) features. The teleomorph possesses globose to subglobose ascomata with acentric ostiole, a peridial wall of textura angularis to textura prismatica, cylindric-clavate, pedicellate asci with an ocular chamber, and 1–2-septate, hyaline, fusiform, guttulate ascospores with a distinct mucilaginous sheath. The anamorph features pycnidial conidiomata, phialidic, ampulliform to cylindrical, hyaline conidiogenous cells and ampulliform to cylindrical, one-to-three-septate, hyaline, guttulate conidia. Loculosulcatispora was known only from its anamorph of L. thailandica. We observed the teleomorph of Loculosulcatispora hongheensis sp. nov. and amended the generic description of Loculosulcatispora accordingly. Loculosulcatispora hongheensis is characterized by globose to subglobose ascomata with a central ostiole, a peridial wall of textura angularis to globosa, branched, septate, pseudoparaphyses, clavate asci with a short pedicel and a minute ocular chamber and hyaline, fusiform, 1-septate ascospores with a thick irregular mucilaginous sheath. This study provides some insights into the diversity of fungi on dead woody litter in terrestrial habitats.

Seroprevalence of anti-Leptospira antibodies in equines and associated workers-Isolation of Leptospira interrogans serogroup Canicola from equine urine.

To investigate seroprevalence of anti-Leptospira antibodies in equines and associated workers in Uruguay, 891 equine and 150 human sera were drawn; 212 equine urine samples were also taken for culture. Environmental conditions and equine raising or managing practices were recorded in all 72 visited establishments; epidemiological information was obtained from each worker. Microscopic agglutination technique (MAT) was performed with 10 Leptospira strains for equines and 18 for human sera, that were also studied with IgM indirect immunofluorescence (IgM-IIF). Equine titres ≥100 were considered positive, and human sera titres ≥200 suggested probable recent or past infection. Urines were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) media; local identification of one obtained isolate with lipL32 PCR, Multiple Locus Variable number tandem repeat Analysis and partial rrs gene sequencing, were completed at Institut Pasteur, Paris. Estimated reactivity was 61.3% for equines, which was higher than the studied bovine national levels (21%) and mainly observed with Icterohaemorrhagiae serogroup (40.3%), Sejroe, Canicola, Pomona or Ballum. Aged animals from slaughterhouses and cattle farms were the most frequently positive. Multiple regression analysis confirmed a significant association between seropositivity and equine age. Only one positive culture could be fully studied, and confirmed to be Leptospira interrogans serogroup Canicola; it was added to the MAT antigen panel and revealed fairly frequent reaction with equine and human sera. Three workers (2%) showed titres=200 with Icterohaemorrhagiae or Canicola serogroups, without recent clinical manifestations. Their attended equines reacted with the same serogroups, suggesting common source infections or infection transmitted by equines. Three other humans yielded titres=100, and none of the 150 showed an IgM-IIF-positive result. Equines seem not to be an important origin of regional human leptospirosis, except perhaps during acute animal infection. More culture work is required to study intensity and lapses of leptospiruria, as well as to further identify circulating strains.

A Custom DNA-Based NGS Panel for the Molecular Characterization of Patients With Diffuse Gliomas: Diagnostic and Therapeutic Applications.

The management of patients with Central Nervous System (CNS) malignancies relies on the appropriate classification of these tumors. Recently, the World Health Organization (WHO) has published new criteria underlining the importance of an accurate molecular characterization of CNS malignancies, in order to integrate the information generated by histology. Next generation sequencing (NGS) allows single step sequencing of multiple genes, generating a comprehensive and specific mutational profile of the tumor tissue. We developed a custom NGS-based multi-gene panel (Glio-DNA panel) for the identification of the correct glioma oncotype and the detection of its essential molecular aberrations. Specifically, the Glio-DNA panel targets specific genetic and chromosomal alterations involving ATRX chromatin remodeler (ATRX), cyclin dependent kinase inhibitor 2A (CDKN2A), isocitrate dehydrogenase (NADP+) 1 (IDH1) and the telomerase reverse transcriptase (TERT) promoter while also recognizing the co-deletion of 1p/19q, loss of chromosome 10 and gain of chromosome 7. Furthermore, the Glio-DNA panel also evaluates the methylation level of the O-6-methylguanine-DNA methyltransferase (MGMT) gene promoter that predicts temozolomide efficacy. As knowledge of the mutational landscape of each glioma is mandatory to define a personalized therapeutic strategy, the Glio-DNA panel also identifies alterations involving “druggable” or “actionable” genes. To test the specificity of our panel, we used two reference mutated DNAs verifying that NGS allele frequency measurement was highly accurate and sensitive. Subsequently, we performed a comparative analysis between conventional techniques - such as immunohistochemistry or fluorescence in situ hybridization - and NGS on 60 diffuse glioma samples that had been previously characterized. The comparison between conventional testing and NGS showed high concordance, suggesting that the Glio-DNA panel may replace multiple time-consuming tests. Finally, the identification of alterations involving different actionable genes matches glioma patients with potential targeted therapies available through clinical trials. In conclusion, our analysis demonstrates NGS efficacy in simultaneously detecting different genetic alterations useful for the diagnosis, prognosis and treatment of adult patients with diffuse glioma.

Integrating Venom Peptide Libraries Into a Phylogenetic and Broader Biological Framework.

The venomous marine snails are conventionally divided into three groups, the cone snails (family Conidae), the auger snails (family Terebridae) and the turrids (formerly all assigned to a single family, Turridae). In this study, a library of venom peptides from species conventionally assigned to the genus Turris was correlated to a phylogenetic analysis. Nucleotide sequences of multiple genes from transcriptomes were used to assess the phylogenetic relationships across a diverse set of species. The resulting tree shows that as conventionally defined, the conoidean genus Turris, is polyphyletic. We describe a new genus, Purpuraturris gen. nov., that comprises the outlier species. In addition to morphological distinctions, molecular data reveal that this group is divergent from Turris sensu stricto. The correlation between phylogenetic information and a family of peptide sequences was used to highlight those peptides mostly likely to be unique and intimately associated with biological diversity. The plethora of peptide sequences available requires two prioritization decisions: which subset of peptides to initially characterize, and after these are characterized, which to comprehensively investigate for potential biomedical applications such as drug developments. Life Science Identifiers: urn:lsid:zoobank.org; pub: 60D46561-28F0-4C39-BAC4-66DC8B4EAEA4

Diverse methylotrophic methanogenic archaea cause high methane emissions from seagrass meadows

Marine coastlines colonized by seagrasses are a net source of methane to the atmosphere. However, methane emissions from these environments are still poorly constrained, and the underlying processes and responsible microorganisms remain largely unknown. Here, we investigated methane turnover in seagrass meadows of Posidonia oceanica in the Mediterranean Sea. The underlying sediments exhibited median net fluxes of methane into the water column of ca. 106 µmol CH4 ⋅ m-2 ⋅ d-1 Our data show that this methane production was sustained by methylated compounds produced by the plant, rather than by fermentation of buried organic carbon. Interestingly, methane production was maintained long after the living plant died off, likely due to the persistence of methylated compounds, such as choline, betaines, and dimethylsulfoniopropionate, in detached plant leaves and rhizomes. We recovered multiple mcrA gene sequences, encoding for methyl-coenzyme M reductase (Mcr), the key methanogenic enzyme, from the seagrass sediments. Most retrieved mcrA gene sequences were affiliated with a clade of divergent Mcr and belonged to the uncultured Candidatus Helarchaeota of the Asgard superphylum, suggesting a possible involvement of these divergent Mcr in methane metabolism. Taken together, our findings identify the mechanisms controlling methane emissions from these important blue carbon ecosystems.

Detection of genetic mutations in patients with breast cancer from Saudi Arabia using Ion AmpliSeq™ Cancer Hotspot Panel v.2.0.

Next-Generation Sequencing allows for quick and precise sequencing of multiple genes concurrently. Recently, this technology has been employed for the identification of novel gene mutations responsible for disease manifestation among breast cancer (BC) patients, the most common type of cancer amongst Arabian women, and the major cause of disease-associated death in women worldwide. Genomic DNA was extracted from the peripheral blood of 32 Saudi Arabian BC patients with histologically confirmed invasive BC stages I-III and IV, as well from 32 healthy Saudi Arabian women using a QIAamp® DNA Mini Kit. The isolated DNA was quantified using a Qubit™ dsDNA BR Assay Kit with a Qubit 2.0 Fluorometer. Ion semiconductor sequencing technology with an Ion S5 System and AmpliSeq™ Cancer Hotspot Panel v2 were utilized to analyze ~2,800 mutations described in the Catalogue of Somatic Mutations in Cancer from 50 oncogenes and tumor suppressor genes. Ion Reporter Software v.5.6 was used to evaluate the genomic alterations in all the samples after alignment to the hg19 human reference genome. The results showed that out of the 50 genes, 26 mutations, including 17 (65%) missense point mutations (single nucleotide variants), and 9 (35%) frameshift (insertion/deletion) mutations, were identified in 11 genes across the cohort in 61 samples (95%). Mutations were predominantly focused on two genes, PIK3CA and TP53, in the BC genomes of the sample set. PIK3CA mutation, c.1173A>G located in exon 9, was identified in 15 patients (46.9%). The TP53 mutations detected were a missense mutation (c.215C>G) in 26 patients (86.70%) and 1 frameshift mutation (c.215_216insG) in 1 patient (3.33%), located within exon 3 and 5, respectively. This study revealed specific mutation profiles for every BC patient, Thus, the results showed that Ion Torrent DNA Sequencing technology may be a possible diagnostic and prognostic method for developing personalized therapy based on the patient's individual BC genome.

Genetic markers for inherited thrombophilia related pregnancy loss and implantation failure in Indian population – implications for diagnosis and clinical management

Aim The biology of recurrent pregnancy loss and recurrent implantation failure (RPL-RIF) is complex with multi-factorial etiology, with defective thrombosis being one of the most important and highly prevalent causes. The role of several thrombophilia related genes and variants associated with RPL-RIF is widely reported, and this study aimed to identify the risk associated with these genes in the Indian population. Methods Next generation sequencing (NGS) was employed for the current study. NGS enables sequencing of multiple genes, identification of new variants, and establishment of genetic correlations with reproductive failure in diverse population groups. The present NGS based study evaluates association of twenty-nine genotypes of ten coagulation pathway genes (F2, F5, F13, MTR, MTRR, MTHFR, ANXA5, PROZ, SERPINE1 and VEGFA) with RPL-RIF in 540 female subjects − 474 patients with early recurrent pregnancy loss, late pregnancy loss, pregnancy complications in late gestation and recurrent implantation failure, with 66 controls. Results The results emphasize inclusion of genotypes of seven thrombophilia genes (MTHFR, MTRR, MTR, ANXA5, PROZ, SERPINE1, VEGFA) for diagnosis of inherited thrombophilia risk for RPL-RIF in Indian population, as against the common practice of testing limited to F2, F5 and MTHFR genes. Conclusion Deriving risk magnitude from Combined Risk Analysis and interpretation of high-risk haplotypes are crucial components for evidence based personalized management such as selection of drugs and dosage, and prenatal or pre-implantation recommendations, for high-risk patients in fertility and obstetric clinics.

Impact of evolution of molecular technologies on evaluation of Neuromuscular Disorders in India at a Centre in Mumbai.

Abstract:
 Advances in molecular methods has made genetic testing as an imperative parameter, to be used in research studies and clinical practice. The present article includes the evolution of Molecular Diagnostic techniques over a period of two decades leading to the corresponding expansion in identification & classification of various Neuromuscular disorders in the population of mainly Western India.
 Initially conventional testing was done by mPCR, RFLP-PCR for single gene disorders like DMD/BMD & SMA respectively; followed by MLPA technique, for deletion duplication analysis. Under a research study, immuno-specific Western Blot was used for protein analysis as an initial screening tool for LGMD2B. Sangers sequencing was done for single gene (for a few LGMD2B cases) & known familial variants of LGMD2B & LGMD2A and GNE myopathy. Availability of Next generation sequencing technique involving massive parallel sequencing of multiple genes, improved the possibility of identifying the disease causing/ pathogenic mutations in phenotypically overlapping NMDs like LGMDs and Hereditary Neuropathies.
 Since last 12 years, over 2,600 patients from mainly western India have been analysed for various neuromuscular disorders. Carrier status of relatives of the probands was also determined in many of the cases. These molecular technologies have enabled to attained definitive diagnosis of different neuromuscular disorders in India. The diagnostic and predictive data generated helps in better management of the disease. It also has immense relevance in carrier and prenatal testing for the families at risk, thereby limiting the occurrence of the diseases in the population.

High Incidence of COL4A Genetic Variants Among a Cohort of Children With Steroid-Resistant Nephrotic Syndrome From Eastern India

High Incidence of COL4A Genetic Variants Among a Cohort of Children With Steroid-Resistant Nephrotic Syndrome From Eastern India

Functional leaf traits indicate phylogenetic signals in forests across an elevational gradient in the central Himalaya.

Traits are the primary attributes that distinguish a species niche. Species and higher taxa are part of a structured phylogeny, and variation in plant traits depends on lineage as well as on environmental conditions. Therefore, it is crucial to investigate linkages between taxonomic identity, shared ancestry, and environment for understanding the variation in leaf traits. We investigated the evolutionary relationships, based on multiple gene sequences among 26 plant species sampled along an elevational gradient from 650 to 3600m a.s.l. in the central Himalaya. We tested for the phylogenetic signal based on three different measures in 10 leaf traits having a significant association with the resource acquisition-conservation trade-offs axis and influencing plant growth, development, and ecological performance. We further assessed the role of elevation and growth forms as the potential drivers of leaf traits variation while controlling for phylogeny. 5 out of 10 leaf traits showed significant phylogenetic signal. Plant species clustered more often by growth forms at the tips of the phylogeny indicating multiple instances of independent evolution. Evergreen taxa showed niche separation with deciduous and incorporated larger trait variation. Trait variations were guided by both growth forms and elevation when accounted for phylogeny. Growth form has a higher contribution to trait variation compared to elevation. Trade-offs were detected between resource conservation and resource acquisition machinery traits (that would maximise carbon gain), differing between growth forms and along elevation.

Morpho-Phylo Taxonomy of Novel Dothideomycetous Fungi Associated With Dead Woody Twigs in Yunnan Province, China.

Within the field of mycology, macrofungi have been relatively well-studied when compared to microfungi. However, the diversity and distribution of microfungi inhabiting woody material have not received the same degree of research attention, especially in relatively unexplored regions, such as Yunnan Province, China. To help address this knowledge gap, we collected and examined fungal specimens from different plants at various locations across Yunnan Province. Our investigation led to the discovery of four species that are clearly distinct from extant ones. These taxonomic novelties were recognized based on morphological comparisons coupled with phylogenetic analyses of multiple gene sequences (non-translated loci and protein-coding regions). The monotypic genus Neoheleiosa gen. nov. (type: N. lincangensis) is introduced in Monoblastiaceae (Monoblastiales) for a woody-based saprobic ascomycete that possesses globose to subglobose or obpyriform ascomata with centric or eccentric, papillate ostioles, an ascomatal wall with thin-walled cells of textura globulosa, cylindric, pedicellate asci with an ocular chamber, and 1-septate, brown, guttulate, longitudinally striated, bicellular ascospores. Neoheleiosa has a close phylogenetic affinity to Heleiosa, nevertheless, it is morphologically dissimilar by its peridium cells and ornamented ascospores. Acrocalymma hongheense and A. yuxiense are described and illustrated as new species in Acrocalymmaceae. Acrocalymma hongheense is introduced with sexual and asexual (coelomycetous) features. The sexual morph is characterized by globose to subglobose, ostiolate ascomata, a peridium with textura angularis cells, cylindric-clavate asci with a furcate to truncate pedicel and an ocular chamber, hyaline, fusiform, 1-septate ascospores which are surrounded by a thick, distinct sheath, and the asexual morph is featured by pycnidial conidiomata, subcylindrical, hyaline, smooth, annelledic, conidiogenous cells, hyaline, guttulate, subcylindrical, aseptate conidia with mucoid ooze at the apex and with a rounded hilum at the base. Acrocalymma yuxiense is phylogenetically distinct from other extant species of Acrocalymma and differs from other taxa in Acrocalymma in having conidia with three vertical eusepta. Magnibotryascoma kunmingense sp. nov. is accommodated in Teichosporaceae based on its coelomycetous asexual morph which is characterized by pycnidial, globose to subglobose, papillate conidiomata, enteroblastic, annelledic, discrete, cylindrical to oblong, hyaline conidiogenous cells arising from the inner layer of pycnidium wall, subglobose, oval, guttulate, pale brown and unicelled conidia.

Insight into the Systematics of Microfungi Colonizing Dead Woody Twigs of Dodonaea viscosa in Honghe (China)

Members of Dodonaea are broadly distributed across subtropical and tropical areas of southwest and southern China. This host provides multiple substrates that can be richly colonized by numerous undescribed fungal species. There is a severe lack of microfungal studies on Dodonaea in China, and consequently, the diversity, phylogeny and taxonomy of these microorganisms are all largely unknown. This paper presents two new genera and four new species in three orders of Dothideomycetes gathered from dead twigs of Dodonaea viscosa in Honghe, China. All new collections were made within a selected area in Honghe from a single Dodonaea sp. This suggests high fungal diversity in the region and the existence of numerous species awaiting discovery. Multiple gene sequences (non-translated loci and protein-coding regions) were analysed with maximum likelihood and Bayesian analyses. Results from the phylogenetic analyses supported placing Haniomyces dodonaeae gen. et sp. in the Teratosphaeriaceae family. Analysis of Rhytidhysteron sequences resulted in Rhytidhysteron hongheense sp. nov., while analysed Lophiostomataceae sequences revealed Lophiomurispora hongheensis gen. et sp. nov. Finally, phylogeny based on a combined dataset of pyrenochaeta-like sequences demonstrates strong statistical support for placing Quixadomyces hongheensis sp. nov. in Parapyrenochaetaceae. Morphological and updated phylogenetic circumscriptions of the new discoveries are also discussed.

Is there any place for novel agents in treating biliary tract cancer?

Biliary tract cancer is an uncommon cancer in developed countries. In localized stages, surgery is the cornerstone of treatment with curative purpose. Conversely in advanced stages, chemotherapy with platinum-gemcitabine combination is the standard of care. Biliary tract cancers are a biologically heterogeneous group of malignancies, which perhaps explains the failure of targeted therapies in unselected patient populations to demonstrate benefit in advanced disease, although there are promises in selected populations (e.g. PD1/PD-L1 positive, BRAFV600E-mutated or IDH1-mutant). In view of the limited benefit of second line therapies in metastatic biliary tract cancer, various targeted agents have been tested in progressive disease. Furthermore, several ongoing trials are using next-generation sequencing of multiple genes to identify molecular abnormalities in the tumors of patients with refractory cancers that may potentially be used in pretreated disease (e.g. FGFR or IDH genes). Immunotherapy with immune checkpoint inhibitors may be interesting for patients whose tumors have programmed cell death 1 ligand 1 (PD-L1) overexpression. Ongoing and future trials will further advance our knowledge toward the optimal treatment strategy for the management of biliary tract cancer in its different stages, starting from metastatic and then reaching early stages of disease. We here provided an overview of these novel treatment strategies for advanced biliary tract cancers not amenable of curative treatment modalities.

Characterization of the domestic goat γδ T cell receptor gene loci and gene usage

Goats and cattle diverged 30 million years ago but retain similarities in immune system genes. Here, the caprine T cell receptor (TCR) gene loci and transcription of its genes were examined and compared to cattle. We annotated the TCR loci using an improved genome assembly (ARS1) of a highly homozygous San Clemente goat. This assembly has already proven useful for describing other immune system genes including antibody and leucocyte receptors. Both the TCRγ (TRG) and TCRδ (TRD) loci were similarly organized in goats as in cattle and the gene sequences were highly conserved. However, the number of genes varied slightly as a result of duplications and differences occurred in mutations resulting in pseudogenes. WC1+ γδ T cells in cattle have been shown to use TCRγ genes from only one of the six available cassettes. The structure of that Cγ gene product is unique and may be necessary to interact with WC1 for signal transduction following antigen ligation. Using RT-PCR and PacBio sequencing, we observed the same restriction for goat WC1+ γδ T cells. In contrast, caprine WC1+ and WC1- γδ T cell populations had a diverse TCRδ gene usage although the propensity for particular gene usage differed between the two cell populations. Noncanonical recombination signal sequences (RSS) largely correlated with restricted expression of TCRγ and δ genes. Finally, caprine γδ T cells were found to incorporate multiple TRD diversity gene sequences in a single transcript, an unusual feature among mammals but also previously observed in cattle.

Восточно-азиатская – восточно-североамериканская дизъюнкция в видовом комплексе Onoclea sensibilis: викариантность или перипатрия?

Disjunction between East Asian and eastern North American plants has been recognized since the time of Darwin. Although there is considerable evidence for congruent vicariance associated with late Neogene/Quaternary cooling among angiosperms, similar studies among specific fern families (e.g., Osmundaceae Martinov) have appeared incongruous with this pattern. Pteridologists continue to debate whether long-distance dispersal of wind-blown spores could have produced intercontinental disjunctions among ferns. To date, however, state-of-the-art historical biogeographical approaches have not been applied to this problem. In this investigation, multiple chloroplast gene (rbcL, atpA, atpB, and matK) sequences for ferns in the family Onocleaceae Pic. Serm. are drawn from GenBank, including those from the recently sequenced chloroplast genome of the East Asian fern O. sensibilis L. var. interrupta Maxim., and used to create molecular phylogenies using Bayesian (BEAUTi and BEAST) techniques. Using contemporary approaches for relaxed molecular clock divergence time estimation with fossil calibration, divergence time estimates for East Asian and eastern North American populations in the Onoclea sensibilis species complex are consistently reconstructed as the Pliocene (ca. 5 – 3.4 Ma), and the best-fitting historical biogeographic model is a DIVA-like (exclusively vicariant) model using BIOGEOBEARS in RASP4, with a low probability of peripatry. Accordingly, these molecular and fossil data appear congruent with the pattern observed among angiosperms, despite the propensity for long-distance dispersal of wind-blown spores in pteridophytes. The lack of evidence for peripatry in this lineage may be related to the presence of short-lived green spores in onocleoid ferns; however, this hypothesis requires further investigation.

Quality assessment of a clinical next-generation sequencing melanoma panel within the Italian Melanoma Intergroup (IMI)

BackgroundIdentification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. Conventional methods focus on few genes per run and, therefore, are unable to screen for multiple genes simultaneously. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity.MethodsWe designed a customized IMI somatic gene panel for targeted sequencing of actionable melanoma mutations; this panel was tested on three different NGS platforms using 11 metastatic melanoma tissue samples in blinded manner between two EMQN quality certificated laboratory.ResultsThe detection limit of our assay was set-up to a Variant Allele Frequency (VAF) of 10% with a coverage of at least 200x. All somatic variants detected by all NGS platforms with a VAF ≥ 10%, were also validated by an independent method. The IMI panel achieved a very good concordance among the three NGS platforms.ConclusionThis study demonstrated that, using the main sequencing platforms currently available in the diagnostic setting, the IMI panel can be adopted among different centers providing comparable results.

Multiple cancer susceptible genes sequencing in BRCA-negative breast cancer with high hereditary risk.

BackgroundHereditary factors contributed to breast cancer susceptibility. Low BRCA mutation prevalence was demonstrated in previous BRCA mutation screening in Chinese breast cancer patients. Multiple-gene sequencing may assist in discovering detrimental germline mutation inBRCAnegative breast cancers.MethodsA total of 384 Chinese subjects with any two of high-risk factors were recruited and screened by next-generation sequencing (NGS) for 30 cancer susceptible genes. Variants with a truncating, initiation codon or splice donor/acceptor effect, or with pathogenicity demonstrated in published literature were classified into pathogenic/likely-pathogenic mutations.ResultsIn total, we acquired 39 (10.2%) patients with pathogenic/likely-pathogenic germline mutations, including one carrying two distinct mutations. Major mutant non-BRCA genes were MUTYH (n=11, 2.9%), PTCH1 (n=7, 1.8%), RET (n=6, 1.6%) and PALB2 (n=5, 1.3%). Other mutant genes included TP53 (n=3, 0.8%), RAD51D (n=2, 0.5%), CHEK2 (n=1, 0.3%), BRIP1 (n=1, 0.3%), CDH1 (n=1, 0.3%), MRE11 (n=1, 0.3%), RAD50 (n=1, 0.3%) and PALLD (n=1, 0.3%). A splicing germline mutation, MUTYH c.934-2A>G, was a hotspot (9/384, 2.3%) in Chinese breast cancer.ConclusionsAmong BRCA-negative breast cancer patients with high hereditary risk in China, 10.2% carried mutations in cancer associated susceptibility genes. MUTYH and PTCH1 had relatively high mutation rates (2.9% and 1.8%). Multigene testing contributes to understand genetic background of BRCA-negative breast cancer patients with high hereditary risk.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

A phytoplasma survey reveals the presence of ‘CandidatusPhytoplasma fragariae’ inUlmusspp. andAcer pseudoplatanusin Belgium

AbstractA Belgium‐wide survey to determine the presence of ‘CandidatusPhytoplasma ulmi’ elm trees was performed on nearly 600 elm trees. The randomly sampled elms were screened for the presence of phytoplasmas using a standard generic nested PCR method. In five symptomless elm trees, phytoplasmas were detected. Multiple gene sequence analysis (16S rRNA, tuf, rplV‐rpsC‐rplP), virtual RFLP of the 16S rRNA gene fragment and phylogenetic analysis showed that the phytoplasma strains detected in all five elm trees could be classified into the 16SrXII‐E subgroup (‘CandidatusPhytoplasma fragariae’‐related phytoplasma). In addition, a maple tree (Acer pseudoplatanus) located in close proximity to one of these elm trees was also determined to be infected with ‘Ca. P. fragariae’.UlmusandAcerare new host plant genera for this phytoplasma. Further studies are needed to elucidate the epidemiological impact of these findings.

A single-gene approach for the subspecies classification of Mycobacteroides abscessus.

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecieswith high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.