The new phylogenesis of the genus Mycobacterium

Abstract Phylogenetic knowledge of the genus Mycobacterium is based on comparative analysis of their genetic sequences. The 16S rRNA has remained for many years the only target of such analyses, but in the last few years, other housekeeping genes have been investigated and the phylogeny based on their concatenated sequences become a standard. It is now clear that the robustness of the phylogenetic analysis is strictly related to the size of the genomic target used. Whole genome sequencing (WGS) is nowadays becoming widely accessible and comparatively cheap. It was decided, therefore, to use this approach to reconstruct the ultimate phylogeny of the genus Mycobacterium . Over 50 types of strains of the same number of species of Mycobacterium were sequenced using the Illumina HiSeq platform. The majority of the strains of which the whole sequence was already available in GenBank were excluded from this panel with the aim of maximizing the number of the species with genome available. Following assembling and annotation with proper software, the phylogenetic analysis was conducted with PhyloPhlAn and the pan-genome analysis pipeline. The phylogenetic three which emerged was characterized by a clear-cut distinction of slowly and rapidly growing species with the latter being more ancestral. The species of the Mycobacterium terrae complex occupied an intermediate position between rapid and slow growers. Most of the species revealed clearly related and occupied specific phylogenetic branches. Thanks to the WGS technology, the genus Mycobacterium is finally approaching its definitive location

Structured reporting for fibrosing lung disease: a model shared by radiologist and pulmonologist

Objectives: To apply the Delphi exercise with iterative involvement of radiologists and pulmonologists with the aim of defining a structured reporting template for high-resolution computed tomography (HRCT) of patients with fibrosing lung disease (FLD). Methods: The writing committee selected the HRCT criteria\ue2\u80\u94the Delphi items\ue2\u80\u94for rating from both radiology panelists (RP) and pulmonology panelists (PP). The Delphi items were first rated by RPs as \ue2\u80\u9cessential\ue2\u80\u9d, \ue2\u80\u9coptional\ue2\u80\u9d, or \ue2\u80\u9cnot relevant\ue2\u80\u9d. The items rated \ue2\u80\u9cessential\ue2\u80\u9d by < 80% of the RP were selected for the PP rating. The format of reporting was rated by both RP and PP. Results: A total of 42 RPs and 12 PPs participated to the survey. In both Delphi round 1 and 2, 10/27 (37.7%) items were rated \ue2\u80\u9cessential\ue2\u80\u9d by more than 80% of RP. The remaining 17/27 (63.3%) items were rated by the PP in round 3, with 2/17 items (11.7%) rated \ue2\u80\u9cessential\ue2\u80\u9d by the PP. PP proposed additional items for conclusion domain, which were rated by RPs in the fourth round. Poor consensus was observed for the format of reporting. Conclusions: This study provides a template for structured report of FLD that features essential items as agreed by expert thoracic radiologists and pulmonologists

Exome sequencing in a patient with Catele-Manzke-like syndrome excludes the involvement of the known genes and reveals a possible candidate

In the present study we describe the exome sequencing and analysis of a patient with Catel-Manzke-like phenotype showing bilateral hyperphalangism of the second finger and thumb clinodactyly due to a unilateral delta phalanx, associated with growth, cardiac and vertebral defects. The exome sequencing analysis excluded pathogenetic mutations in the genes known to cause syndromes with hyperphalangism and did not identify any alteration in the X-chromosome or de novo mutations in likely candidate genes. Under the assumption of an autosomal recessive mode of inheritance and based on the frequency of the single nucleotide variants found in homozygous or double heterozygous states and the results of computer prediction programs, only one gene, DNAH10, emerged as a candidate in the pathogenesis of the disease in our patient. However, the differences among the known biological functions of DNAH10 and the genes involved in the other syndromes with hyperphalangism, suggest caution in the interpretation of the results

First-trimester prenatal screening for the common 35delG GJB2 mutation causing prelingual deafness

Objective: This study evaluates the prevalence of 35delG GJB2 mutation, the most common genetic mutation causing prelingual deafness, and its screening feasibility and acceptability in pregnant women undergoing first-trimester CVS for chromosomal abnormality investigation. Methods: Samples were taken from 5786 pregnant women who requested CVS for chromosomal analysis. The samples were split into two aliquots for chromosome and DNA analysis, respectively. The results of foetal karyotyping were provided 7 days after sampling, at which time the fully informed couple decided whether or not to undergo DNA testing. Results: Of the 5449 eligible candidates, 2997 (55.0%) chose to undergo 35delG testing. Among them, 67 proved to be carriers of the mutation, resulting in a prevalence rate of 1: 44.7 (2.23%). There were no homozygous foetuses, but two double heterozygous foetuses were found, and in one case the couple chose to terminate the pregnancy. Conclusions: The results confirm the high frequency of 35delG mutation in the Italian population. The study shows that prenatal screening for GJB2 mutations in pregnant women with no retrospective risk for deafness appears to be feasible and highly acceptable. Consequently, given evidence that early evaluation and treatment significantly improve speech and language skills, as well as social and emotional well-being in affected children, 35delG mutation analysis in pregnant women booking CVS primarily for chromosomal investigation could be considered a useful addition to more comprehensive population screening strategies. Copyright © 2004 John Wiley & Sons, Ltd

Next Generation Sequencing Analysis of MODY-X Patients: A Case Report Series

Background: Classic criteria for a maturity-onset diabetes of the young (MODY) diagnosis are often unable to identify all subjects, and traditional Sanger sequencing, using a candidate gene approach, leads to a high prevalence of missed genetic diagnosis, classified as MODY-X. Next generation sequencing (NGS) panels provide a highly sensitive method even for rare forms. Methods: We investigated 28 pediatric subjects suspected for MODY-X, utilizing a 15-gene NGS panel for monogenic diabetes (MD). Results: NGS detected variants of uncertain significance (VUS), likely pathogenic or pathogenic for rarer subtypes of MODY, in six patients. We found variants in the wolframin gene (WFS1), traditionally not considered in MD genetic screening panels, in three patients; KCNJ11 gene mutation, typically responsible for neonatal diabetes and rarely causing isolated diabetes in adolescents; INS gene mutation; a variant in the HNF1B gene in a young male with diabetes on sulfonylurea treatment. Conclusion: In our cohort, the availability of an NGS panel for MD was determined for the correct identification of MD subtypes in six patients with MODY-X. Our study underlines how a precise diagnosis utilizing NGS may have an impact on the management of different forms of MODY and, thus, lead to a tailored treatment and enable genetic counselling of other family members

ddSeeker: a tool for processing Bio-Rad ddSEQ single cell RNA-seq data

Abstract Background New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing. Results We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker. Conclusions ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments

Unexplored diversity and strain-level structure of the skin microbiome associated with psoriasis

Psoriasis is an immune-mediated inflammatory skin disease that has been associated with cutaneous microbial dysbiosis by culture-dependent investigations and rRNA community profiling. We applied, for the first time, high-resolution shotgun metagenomics to characterise the microbiome of psoriatic and unaffected skin from 28 individuals. We demonstrate psoriatic ear sites have a decreased diversity and psoriasis is associated with an increase in Staphylococcus, but overall the microbiomes of psoriatic and unaffected sites display few discriminative features at the species level. Finer strain-level analysis reveals strain heterogeneity colonisation and functional variability providing the intriguing hypothesis of psoriatic niche-specific strain adaptation or selection. Furthermore, we accessed the poorly characterised, but abundant, clades with limited sequence information in public databases, including uncharacterised Malassezia spp. These results highlight the skins hidden diversity and suggests strain-level variations could be key determinants of the psoriatic microbiome. This illustrates the need for high-resolution analyses, particularly when identifying therapeutic targets. This work provides a baseline for microbiome studies in relation to the pathogenesis of psoriasis

<i>JAK2</i> Exon 14 Skipping in Patients with Primary Myelofibrosis: A Minor Splice Variant Modulated by the JAK2-V617F Allele Burden

<div><p>Background</p><p>Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the <i>JAK2</i> gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of <i>JAK2</i>, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases.</p><p>Materials and Methods</p><p>By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the <i>JAK2</i> full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors.</p><p>Results</p><p>We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence.</p><p>Conclusions</p><p>Increased levels of <i>JAK2</i> full-length transcript and a small but significant increase in <i>JAK2</i> exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF.</p></div

In PMF patients, levels of mRNA isoform JAK2Δ14 correlate with the percentage of JAK2-V617F mutated alleles.

<p><i>R</i>² = 0.43, <i>p</i> < 0.001.</p