Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

The prognostic value of cytology and fluorescence in situ hybridization in the follow-up of nonmuscle-invasive bladder cancer after intravesical Bacillus Calmette-Guérin therapy

Molecular markers reliably predicting failure or success of Bacillus Calmette-Guérin (BCG) in the treatment of nonmuscle-invasive urothelial bladder cancer (NMIBC) are lacking. The aim of our study was to evaluate the value of cytology and chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in predicting failure to BCG therapy. Sixty-eight patients with NMIBC were prospectively recruited. Bladder washings collected before and after BCG instillation were analyzed by conventional cytology and by multitarget FISH assay (UroVysion, Abbott/Vysis, Des Plaines, IL) for aberrations of chromosomes 3, 7, 17 and 9p21. Persistent and recurrent bladder cancers were defined as positive events during follow-up. Twenty-six of 68 (38%) NMIBC failed to BCG. Both positive post-BCG cytology and positive post-BCG FISH were significantly associated with failure of BCG (hazard ratio (HR)= 5.1 and HR= 5.6, respectively; p < 0.001 each) when compared to those with negative results. In the subgroup of nondefinitive cytology (all except those with unequivocally positive cytology), FISH was superior to cytology as a marker of relapse (HR= 6.2 and 1.4, respectively). Cytology and FISH in post-BCG bladder washings are highly interrelated and a positive result predicts failure to BCG therapy in patients with NMIBC equally well. FISH is most useful in the diagnostically less certain cytology categories but does not provide additional information in clearly malignant cytology.

Prediction of outcome in patients with low-grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC

BACKGROUND Cytology is an excellent method with which to diagnose preinvasive lesions of the uterine cervix, but it suffers from limited specificity for clinically significant lesions. Supplementary methods might predict the natural course of the detected lesions. The objective of the current study was to test whether a multicolor fluorescence in situ hybridization (FISH) assay might help to stratify abnormal results of Papanicolaou tests. METHODS A total of 219 liquid-based cytology specimens of low-grade squamous intraepithelial lesions (LSIL), 49 atypical squamous cells of undetermined significance (ASCUS) specimens, 52 high-grade squamous intraepithelial lesion (HSIL) specimens, and 50 normal samples were assessed by FISH with probes for the human papillomavirus (HPV), MYC, and telomerase RNA component (TERC). Subtyping of HPV by polymerase chain reaction (PCR) was performed in a subset of cases (n=206). RESULTS There was a significant correlation found between HPV detection by FISH and PCR (P<.0001). In patients with LSILs, the presence of HPV detected by FISH was significantly associated with disease progression (P<.0001). An increased MYC and/or TERC gene copy number (>2 signals in>10% of cells) prevailed in 43% of ASCUS specimens and was more frequent in HSIL (85%) than in LSIL (33%) (HSIL vs LSIL: P<.0001). Increased TERC gene copy number was significantly correlated with progression of LSIL (P<.01; odds ratio, 7.44; area under the receiver operating characteristic curve, 0.73; positive predictive value, 0.30; negative predictive value, 0.94) CONCLUSIONS: The detection of HPV by FISH analysis is feasible in liquid-based cytology and is significantly correlated with HPV analysis by PCR. The analysis of TERC gene copy number may be useful for risk stratification in patients with LSIL.

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue

BACKGROUND: Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. METHODS: Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). RESULTS: Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P<.001). Histologic analysis identified M. catarrhalis in crypts, intraepithelially, subepithelially, and (using CLSM) intracellularly. M. catarrhalis colocalized with macrophages and B cells in lymphoid follicles. CONCLUSIONS: Colonization by M. catarrhalis is more frequent than is determined by surface culture, because the organism resides both within and beneath the epithelium and invades host cells.

The NF-{kappa}B negative regulator TNFAIP3 (A20) is inactivated by somatic mutations and genomic deletions in marginal zone lymphomas

Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-kappaB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-kappaB activation.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

EcPV2 DNA in equine papillomas and in situ and invasive squamous cell carcinomas supports papillomavirus etiology

Equine penile papillomas, in situ carcinomas, and invasive carcinomas are hypothesized to belong to a continuum of papillomavirus-induced diseases. The former ones clinically present as small grey papules, while the latter 2 lesions are more hyperplasic or alternatively ulcerated. To test the hypothesis that these lesions are papillomavirus-induced, samples of 24 horses with characteristic clinical and histologic findings of penile papillomas or in situ or invasive squamous cell carcinomas were collected. As controls, 11 horses with various lesions--namely, Balanoposthitis (6 cases), melanoma (3 cases), follicular cyst (1 case), and amyloidosis (1 case)--were included. DNA was extracted and polymerase chain reaction applied to amplify papillomavirus DNA. The respective primers were designed to amplify DNA of the recently discovered equine papillomavirus EcPV2. All tested papilloma and squamous cell carcinoma samples were found to contain DNA of either of 2 previously published EcPV2 variants. Among the other samples 6 of 11 were found to contain EcPV2 DNA. To further support the findings and to determine where the papillomavirus DNA was located within the lesions, an in situ hybridization for the detection of EcPV2 DNA was established. The samples tested by this technique were found to clearly contain papillomavirus nucleic acid concentrated in the nucleus of the koilocytes. The findings of this study support previous data and the hypothesis that papillomaviruses induce the described penile lesions in horses.

Albinism in the American mink (Neovison vison) is associated with a tyrosinase nonsense mutation

Albino phenotypes are documented in various species including the American mink. In other species the albino phenotypes are associated with tyrosinase (TYR) gene mutations; therefore TYR was considered the candidate gene for albinism in mink. Four microsatellite markers were chosen in the predicted region of the TYR gene. Genotypes at the markers Mvi6025 and Mvi6034 were found to be associated with the albino phenotype within an extended half-sib family. A BAC clone containing Mvi6034 was mapped to chromosome 7q1.1-q1.3 by fluorescent in situ hybridization. Subsequent analysis of genomic TYR sequences from wild-type and albino mink samples identified a nonsense mutation in exon 1, which converts a TGT codon encoding cysteine to a TGA stop codon (c.138T>A, p.C46X; EU627590). The mutation truncates more than 90% of the normal gene product including the putative catalytic domains. The results indicate that the nonsense mutation is responsible for the albino phenotype in the American mink.

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.