A novel Filobacterium sp can cause chronic bronchitis in cats.

BACKGROUND: Cilia-associated respiratory bacillus (CARB; now known as Filobacterium rodentium gen. nov., sp. nov.) is a primary pathogen of rodents. A CARB-like organism was reported in post-mortem lung samples of cats using light and electron microscopy. Here we explore by molecular procedures if a Filobacterium sp. is a part of the normal feline lower respiratory microbiome and whether it could in some cats contribute to the development of chronic bronchial disease. METHODOLOGY: A Filobacterium sp. was identified in three Czech cats clinically diagnosed as having chronic neutrophilic bronchitis. Bronchoalveolar lavage fluid (BALF) specimens obtained from these cats were subjected to panbacterial 16S rDNA PCR followed by Sanger sequencing of the V5 to V8 region. After these cats were treated with specific antimicrobials, their clinical signs resolved promptly, without recurrence. Next, BALF specimens from 13 Australian and 11 Italian cats with lower respiratory disease and an additional 16 lung samples of Italian cats who died of various causes were examined using next generation sequencing (NGS). Subsequently, a Filobacterium-specific qPCR assay was developed and used to re-test BALF specimens from the 11 Italian cats and lung tissue homogenates from the additional 16 deceased cats. PRINCIPAL FINDINGS: An amplicon of 548 bp with 91.24% sequence agreement with Filobacterium rodentium was obtained from all three patients, suggesting the novel Filobacterium sp. was the cause of their lower respiratory disease. The novel Filobacterium sp., which we propose to call F. felis, was detected in 3/3 Czech cats with chronic neutrophilic bronchitis, 13/13 Australian cats and 6/11 Italian cats with chronic lower respiratory disease, and 14/16 necropsy lung specimens from Italian cats. NGS and qPCR results all showed identical sequences. The Filobacterium sp. was sometimes the preponderant bacterial species in BALF specimens from cats with lower airway disease. There was an association between the presence of large numbers (greater than 105 organisms/mL) of Filobacterium and the presence of neutrophilic and/or histiocytic inflammation, although only a subset of inflammatory BALF specimens had F. felis as the preponderant organism. CONCLUSION: The novel Filobacterium sp. comprises a finite part of the normal feline lower respiratory microbiome. Under certain circumstances it can increase in absolute and relative abundance and give rise to neutrophilic and/or histiocytic bronchitis, bronchiolitis and bronchopneumonia. These findings strongly suggest that F. felis could be an underdiagnosed cause of feline bronchial disease.

Merle phenotypes in dogs - SILV SINE insertions from Mc to Mh.

It has been recognized that the Merle coat pattern in dogs is not only a visually interesting feature, but it also exerts an important biological role, in terms of hearing and vision impairments. In 2006, the Merle (M) locus was mapped to the SILV gene (aka PMEL) with a SINE element in it, and the inserted retroelement was proven causative to the Merle phenotype. Mapping of the M locus was a genetic breakthrough and many breeders started implementing SILV SINE testing in their breeding programs. Unfortunately, the situation turned out complicated as genotypes of Merle tested individuals did not always correspond to expected phenotypes, sometimes with undesired health consequences in the offspring. Two variants of SILV SINE, allelic to the wild type sequence, have been described so far-Mc and M. Here we report a significantly larger portfolio of existing Merle alleles (Mc, Mc+, Ma, Ma+, M, Mh) in Merle dogs, which are associated with unique coat color features and stratified health impairment risk. The refinement of allelic identification was made possible by systematic, detailed observation of Merle phenotypes in a cohort of 181 dogs from known Merle breeds, by many breeders worldwide, and the use of advanced molecular technology enabling the discrimination of individual Merle alleles with significantly higher precision than previously available. We also show that mosaicism of Merle alleles is an unexpectedly frequent phenomenon, which was identified in 30 out of 181 (16.6%) dogs in our study group. Importantly, not only major alleles, but also minor Merle alleles can be inherited by the offspring. Thus, mosaic findings cannot be neglected and must be reported to the breeder in their whole extent. Most importantly, sperm cells seem to be a significant source of germline Merle allelic variants which can be passed to the offspring on Mendelian basis and explain unusual genotype / phenotype findings in the offspring. In light of negative health consequences that may be attributed to certain Merle breeding strategies, we strongly advocate implementation of the refined Merle allele testing for all dogs of Merle breeds to help the breeders in selection of suitable mating partners and production of healthy offspring.

Small Supernumerary Marker Chromosome May Provide Information on Dosage-insensitive Pericentric Regions in Human.

BACKGROUND: Cytogenetically visible chromosomal imbalances in humans are deleterious and adverse in the majority of the cases. However, healthy persons living with chromosomal imbalances in the range of several megabasepairs (Mbps) in size, like carriers of small Supernumerary Marker Chromosomes (sSMCs) exist. MATERIALS & METHODS: The identification of healthy sSMC carriers with euchromatic centromere-near (ECN) imbalances led to the following proposal: ECN-regions do not contain any dosage sensitive genes. Due to own previous work, dosage-insensitive pericentric ECN-regions were already determined with an accuracy of 0.3 and 5 Mbp. Based on this data we established 43 new pericentromeric probe sets spanning about 3-5 Mbp of each euchromatic human chromosome arm starting from the known insensitive regions towards distal. Such so called pericentromeric-critical region fluorescence in situ hybridization (PeCR-FISH) probe sets were applied exemplarily and successful here in 15 sSMC cases as available from the Else Kröner-Fresenius-sSMC-cellbank . CONCLUSION: Most of the involved sSMC breakpoints could be characterized as a higher resolution than before. An unexpected result was that in 5/15 cases cryptic mosaicism was characterized. The latter is also to be considered to have potentially an influence on the clinical outcome in these so-called discontinuous sSMCs. Overall, the suitability of PeCR-FISH to characterize sSMCs was proven; the potential of this probe set to further delineate sizes of dosage insensitive pericentric regions is obvious but dependent on suited cases. Furthermore, discontinuous sSMCs can be identified by this approach and this new subtype of sSMC needs to be studied in more detail in future.

Antifungal effects of the biological agent Pythium oligandrum observed in vitro.

Objectives Dermatophytosis, commonly known as ringworm, is a superficial fungal skin disease and zoonosis. Pythium oligandrum is a micromycete with mycoparasitic properties that is used in agriculture to control fungal infections on plants. Formulations containing P oligandrum were also developed for the treatment of dermatophytoses, but only a small number of case studies have been published. In order to document the process in simplified conditions in vitro, we investigated the effectiveness of P oligandrum against three pathogenic dermatophytes common in domestic animals. Methods Cultures of the pathogens grown on nutrient media and experimentally infected cat hair were treated with P oligandrum preparations in therapeutic concentration and the changes were documented by microscopic videos and scanning electron microscopy. Results There was strong mycoparasitic activity of P oligandrum against Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. Conclusions and relevance P oligandrum was demonstrated to be effective against three common causes of dermatophytosis in vitro.

Mantle cell lymphoma-variant Richter syndrome: Detailed molecular-cytogenetic and backtracking analysis reveals slow evolution of a pre-MCL clone in parallel with CLL over several years.

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

Comprehensive Analyses of White-Handed Gibbon Chromosomes Enables Access to 92 Evolutionary Conserved Breakpoints Compared to the Human Genome.

Gibbon species (Hylobatidae) impress with an unusually high number of numerical and structural chromosomal changes within the family itself as well as compared to other Hominoidea including humans. In former studies applying molecular cytogenetic methods, 86 evolutionary conserved breakpoints (ECBs) were reported in the white-handed gibbon (Hylobates lar, HLA) with respect to the human genome. To analyze those ECBs in more detail and also to achieve a better understanding of the fast karyotype evolution in Hylobatidae, molecular data for these regions are indispensably necessary. In the present study, we obtained whole chromosome-specific probes by microdissection of all 21 HLA autosomes and prepared them for aCGH. Locus-specific DNA probes were also used for further molecular cytogenetic characterization of selected regions. Thus, we could map 6 yet unreported ECBs in HLA with respect to the human genome. Additionally, in 26 of the 86 previously reported ECBs, the present approach enabled a more precise breakpoint mapping. Interestingly, a preferred localization of ECBs within segmental duplications, copy number variant regions, and fragile sites was observed.

Long term follow-up in a patient with a de novo microdeletion of 14q11.2 involving CHD8.

We identified a de novo deletion of 14q11.2 in a Czech patient with developmental delay, mild autistic features, macrosomy, macrocephaly, orthognathic deformities, and dysmorphic facial features. The clinical follow-up of the patient lasting 14 years documented changes in the facial dysmorphism from infancy to adolescence. The deletion affects approximately 200 kb of DNA with five protein-coding genes and two snoRNA genes. Two of the protein-coding genes, SUPT16H and CHD8, have been proposed as candidate genes for a new microdeletion syndrome. Our patient further supports the existence of this syndrome and extends its phenotypic spectrum, especially points to the possibility that orthognathic deformities may be associated with microdeletions of 14q11.2. CHD8 mutations have been found in patients with neurodevelopmental disorders and macrocephaly. The HNRNPC gene, repeatedly deleted in patients with developmental delay, is another candidate as its 5́ end is adjacent to the deletion, and the expression of this gene may be affected by position effect.

Molecular characterization of the rare translocation t(3;10)(q26;q21) in an acute myeloid leukemia patient.

BACKGROUND: In acute myeloid leukemia (AML), the MDS1 and EVI1 complex locus - MECOM, also known as the ecotropic virus integration site 1 - EVI1, located in band 3q26, can be rearranged with a variety of partner chromosomes and partner genes. Here we report on a 57-year-old female with AML who presented with the rare translocation t(3;10)(q26;q21) involving the MECOM gene. Our aim was to identify the fusion partner on chromosome 10q21 and to characterize the precise nucleotide sequence of the chromosomal breakpoint. METHODS: Cytogenetic and molecular-cytogenetic techniques, chromosome microdissection, next generation sequencing, long-range PCR and direct Sanger sequencing were used to map the chromosomal translocation. RESULTS: Using a combination of cytogenetic and molecular approaches, we mapped the t(3;10)(q26;q21) to the single nucleotide level, revealing a fusion of the MECOM gene (3q26.2) and C10orf107 (10q21.2). CONCLUSIONS: The approach described here opens up new possibilities in characterizing acquired as well as congenital chromosomal aberrations. In addition, DNA sequences of chromosomal breakpoints may be a useful tool for unique molecular minimal residual disease target identification in acute leukemia patients.

Infections with the tick-borne bacterium "Candidatus Neoehrlichia mikurensis" mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases.

BACKGROUND: Candidatus Neoehrlichia mikurensis is a newly discovered noncultivatable bacterium spread among ticks and rodents in Europe and Asia that can infect humans, particularly immunocompromised patients. METHODS: We compiled clinical and laboratory data from 11 patients with hematological malignances or autoimmune diseases who were diagnosed with Candidatus N. mikurensis infection in Europe 2010-2013. Both published (6) and unpublished cases (5) were included. RESULTS: The patients had a median age of 67, were mostly male (8/11), and resided in Sweden, Switzerland, Germany, and the Czech Republic. All but one had ongoing or recent immune suppressive treatment and a majority were splenectomized (8/11). Less than half of them recalled tick exposure. The most frequent symptoms were fever (11/11), localized pain afflicting muscles and/or joints (8/11), vascular and thromboembolic events (6/11), that is, deep vein thrombosis (4), transitory ischemic attacks (2), pulmonary embolism (1), and arterial aneurysm (1). Typical laboratory findings were elevated C-reactive protein, leukocytosis with neutrophilia, and anemia. Median time from onset of symptoms to correct diagnosis was 2 months. In at least 4 cases, the condition was interpreted to be due to the underlying disease, and immunosuppressive therapy was scheduled. All patients recovered completely when doxycycline was administered. CONCLUSIONS: Candidatus N. mikurensis is an emerging tick-borne pathogen that may give rise to a systemic inflammatory syndrome in persons with hematologic or autoimmune diseases that could be mistaken for recurrence of the underlying disease and/or unrelated arteriosclerotic vascular events. Awareness of this new pathogen is warranted among rheumatologists, hematologists, oncologists, and infectious disease specialists.

A method to identify new molecular markers for assessing minimal residual disease in acute leukemia patients.

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients. Here we demonstrate a technical approach for the identification and mapping of novel clone-specific chromosomal abnormalities down to the nucleotide level. We used molecular cytogenetics, chromosome microdissection, amplification of the microdissected material, and next-generation sequencing to develop PCR-based MRD assays based on unique breakpoint sequences.

Severe intellectual disability, omphalocele, hypospadia and high blood pressure associated to a deletion at 2q22.1q22.3: case report.

BACKGROUND: Recently, array-comparative genomic hybridization (aCGH) platforms have significantly improved the resolution of chromosomal analysis allowing the identification of genomic copy number gains and losses smaller than 5 Mb. Here we report on a young man with unexplained severe mental retardation, autism spectrum disorder, congenital malformations comprising hypospadia and omphalocele, and episodes of high blood pressure. An ~ 6 Mb interstitial deletion that includes the causative genes is identified by oligonucleotide-based aCGH. RESULTS: Our index case exhibited a de novo chromosomal abnormality at 2q22 [del(2)(q22.1q22.3)dn] which was not visible at the 550 haploid band level. The deleted region includes eight genes: HNMT, SPOPL, NXPH2, LOC64702, LRP1B, KYNU, ARHGAP15 and GTDC1. DISCUSSION: aCGH revealed an ~ 6 Mb deletion in 2q22.1 to 2q22.3 in an as-yet unique clinical case associated with intellectual disability, congenital malformations and autism spectrum disorder. Interestingly, the deletion is co-localized with a fragile site (FRA2K), which could be involved in the formation of this chromosomal aberration. Further studies are needed to determine if deletions of 2q22.1 to 2q22.3 define a new microdeletion syndrome.

How to narrow down chromosomal breakpoints in small and large derivative chromosomes--a new probe set.

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ∼10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.

Microdeletion and microduplication syndromes.

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted "genomic disorders" or "contiguous gene syndromes" is given.

The effects of siRNA-mediated RGS4 gene silencing on the whole genome transcription profile: implications for schizophrenia.

OBJECTIVE: The regulator of G-protein signaling (RGS) molecules represent a class of proteins that modulate the signaling activity of G-protein coupled receptors. Regulator of G-protein signaling 4 (RGS4) is of particular interest in schizophrenia due to reported downregulation of RGS4 transcripts in schizophrenia as well as a connection between RGS4 and a number of receptors implicated in schizophrenia. The mechanism of RGS4 involvement in the pathophysiology of this illness is not clear. METHODS: To elucidate thise role of RGS4 in pathophysiology of schizophrenia, we silenced RGS4 using siRNAs in human neuroblastoma cell lines and we studied the effects of differential RGS4 expression by microarray. RESULTS AND CONCLUSION: The cell lines with downregulated expression of RGS4 showed 67 genes with changed expression (30 underexpressed and 37 overexpressed). We have detected three subgroups of genes which might be implicated in schizophrenia pathophysiology: histone genes, which suggest epigenetic mechanisms of the disease; genes for transcription factors associated with other genes relevant to schizophrenia pathology (BDNF and DISCI1) and a heterogeneous group containing genes for G-proteins (GPR50 and GPR64) and calcium binding proteins.

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection.

Hematooncologic patients often host rare or fastidious pathogens. Using 16S rDNA sequencing and transmission electron microscopy, we have identified 2 lymphoma patients infected with Candidatus Neoehrlichia mikurensis. In both individuals, the clinical presentation suggested ehrlichiosis-like syndrome. We believe that molecular techniques open new vistas in the field of pathogen detection.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples.

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10-20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.