Cytogenomic characterization of an unexpected 17.6 Mb 9p deletion associated to a 14.8 Mb 20p duplication in a dysmorphic patient with multiple congenital anomalies presenting a normal G-banding karyotype

We describe a female patient with developmental delay, dysmorphic features and multiple congenital anomalies who presented a normal G-banded karyotype at the 550-band resolution. Array and multiplex-ligation probe amplification (MLPA) techniques identified an unexpected large unbalanced genomic aberration: a 17.6 Mb deletion of 9p associated to a 14.8 Mb duplication of 20p. The deleted 9p genes, especially CER1 and FREM1, seem to be more relevant to the phenotype than the duplicated 20p genes. This study also shows the relevance of using molecular techniques to make an accurate diagnosis in patients with dysmorphic features and multiple anomalies suggestive of chromosome aberration, even if on G-banding their karyotype appears to be normal. Fluorescence in situ hybridization (FISH) was necessary to identify a masked balanced translocation in the patient's mother, indicating the importance of associating cytogenetic and molecular techniques in clinical genetics, given the implications for patient management and genetic counseling. (C) 2012 Elsevier B.V. All rights reserved.

Molecular Characterization and Physical Mapping of Two Classes of 5S rDNA in the Genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I. Copyright (C) 2012 S. Karger AG, Basel

Copy number variation in Williams-Beuren syndrome: suitable diagnostic strategy for developing countries

Abstract Background Williams-Beuren syndrome (WBS; OMIM 194050) is caused by a hemizygous contiguous gene microdeletion at 7q11.23. Supravalvular aortic stenosis (SVAS), mental retardation, and overfriendliness comprise typical symptoms of WBS. Although fluorescence in situ hybridization (FISH) is considered the gold standard technique, the microsatellite DNA markers and multiplex ligation-dependent probe amplification (MLPA) could be used for to confirm the diagnosis of WBS. Results We have evaluated a total cohort of 88 patients with a suspicion clinical diagnosis of WBS using a collection of five markers (D7S1870, D7S489, D7S613, D7S2476, and D7S489_A) and a commercial MLPA kit (P029). The microdeletion was present in 64 (72.7%) patients and absent in 24 (27.3%) patients. The parental origin of deletion was maternal in 36 of 64 patients (56.3%) paternal in 28 of 64 patients (43.7%). The deletion size was 1.55 Mb in 57 of 64 patients (89.1%) and 1.84 Mb in 7 of 64 patients (10.9%). The results were concordant using both techniques, except for four patients whose microsatellite markers were uninformative. There were no clinical differences in relation to either the size or parental origin of the deletion. Conclusion MLPA was considered a faster and more economical method in a single assay, whereas the microsatellite markers could determine both the size and parental origin of the deletion in WBS. The microsatellite marker and MLPA techniques are effective in deletion detection in WBS, and both methods provide a useful diagnostic strategy mainly for developing countries.

Analyse genomischer Imbalancen in hepatozellulären Karzinomen und präneoplastischen Leberläsionen

Das hepatozelluläre Karzinom (HCC) ist mit ungefähr 1,000,000 neuen Fällen pro Jahr einer der häufigsten malignen Tumore weltweit. Es ist hauptsächlich in Südost-Asien und im südlichen Afrika verbreitet. Risikofaktoren sind chronische Infektionen mit Hepatitis Viren (HBV, HCV), Aflatoxin B1-Belastung und chronischer Alkoholkonsum. Um Veränderungen auf genomischer Ebene in HCCs zu untersuchen, wurden in der vorliegenden Untersuchung Frischgewebeproben von 21 Patienten mit HCCs und formalin-fixiertes, paraffineingebettetes Material von 6 Dysplastischen Knoten mittels Comparativer genomischer Hybridisierung (CGH) analysiert. In den untersuchten HCCs konnte Zugewinne auf 1q (12/21), 6p ( 6/21), 8q (11/21), 17q (6/21), 20q (6/21), sowie Verluste auf 4q (7/21), 6q (4/21), 10q (3/21), 13q (4/21), 16q (3/21) identifiziert werden. Die Validität der mit diesem Ansatz erzielten Ergebnisse konnte anhand von unabhängigen Kontrollexperimenten mit Interphase-FISH-Analyse nachgewiesen werden. Die in Dysplastische Knoten identifizierten Veränderungen sind Gewinne auf 1q (50% ) sowie Verluste auf 8p und 17p. Daher stellt 1q eine Kandidatenregion für die Identifizierung jener Gene dar, die bereits im frühem Stadium der Hepatokarzinogenese aktiviert sind. Die Gen-Expressionsanalyse eines HCCs mit Gewinnen auf 1q, 8q, und Xq zeigte die Überexpression von einigen Genen, die in den amplifizierten Regionen liegen. Daher kann spekuliert werden, dass die DNA-Amplifikation in der Hepatokarzinogenese bei einigen Genen ein Mechanismus der Aktivierung sein kann. Zusammengefasst konnte somit durch CGH-Analyse charakteristische, genomische Imbalances des HCC ermittelt werden. Der Vergleich mit Veränderungen bei prämalignen Läsionen erlaubt die Unterscheidung früher (prämaligner) und später (progressionsassoziierter) Veränderungen

A novel specific genetic translocation in epithelioid hemangioendotelioma, showing a fusion of the WWTR1 and CAMTA1 genes, supports the monoclonality of multifocal epithelioid hemangioendotelioma.

Like other vascular tumors, epithelioid hemangioendothelioma (EHE) is multifocal in approximately 50% of cases, and it is unclear whether the separate lesions represent multifocal disease or metastases. We hypothesized that the identification of an identical WWTR1-CAMTA1 rearrangement in different EHEs from the same patient supports the monoclonal origin of EHE. To test our hypothesis, we undertook a molecular analysis of two multicentric EHEs of the liver, including separate tumor samples from each patient. Matherial and Methods: We retrieved two cases of EHE with available tissue for molecular analysis. In both cases, fluorescence in situ hybridization (FISH) was performed to identify the presence of the WWTR1-CAMTA1 rearrangement to confirm the histologic diagnosis of EHE, as previously described. The reverse transcription-polymerase chain reaction (RT-PCR) products were analyzed by electrophoresis and the RT-PCR–amplified products were sequenced using the Sanger method. Results: FISH analysis revealed signal abnormalities in both WWTR1 and CAMTA1. Combined results confirmed the presence of the t(1;3)(1p36.23;3q25.1) translocation in both cases of EHE. Using RT-PCR analysis, we found that the size of the rearranged bands was identical in the different tumors from each patient. The sequence of the fusion gene confirmed a different WWTR1-CAMTA1 rearrangement in each patient, but an identical WWTR1-CAMTA1 rearrangement in the different lesions from each patient. Discussion: Because of its generally indolent clinical course, EHE is commonly classified as a multifocal, rather than metastatic, disease. In this study, we examined two cases of multifocal liver EHE and found an identical WWTR1-CAMTA1 rearrangement in each lesion from the same patient, but not between the two patients. These findings suggest that multifocal EHE arises from metastasis of the same neoplastic clone rather than from the simultaneous formation of multiple neoplastic clones, which supports the monoclonal origin of multifocal EHE.

Role of new molecular approaches for the early diagnosis of bladder cancer in clinical practice

There is an urgent need to improve the performance of urine cytology for the diagnosis of bladder cancer. In preliminary studies, telomerase activity evaluated by telomeric repeat amplification protocol (TRAP) assay and chromosomal aneuploidy detected by fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer have produced important results. Urine cell-free (UCF) DNA has also been proposed as a potential marker for early bladder cancer diagnosis. In the first study the diagnostic performance of TRAP assay and FISH analysis was assessed, while the second study evaluated the potential role of UCF DNA integrity in early bladder cancer diagnosis. In the first cross-sectional study, 289 consecutive patients who presented with urinary symptoms underwent cystoscopy and cytology evaluation. In the second study, UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. c-Myc, BCAS1 and HER2 gene sequences longer than 250 bp were quantified by real time PCR to verify UCF DNA integrity. In the first study, sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the cytology and TRAP combination; 0.78 and 0.78 for the cytology, TRAP and FISH combination; and 0.65 and 0.93 for the TRAP and FISH combination. In the second study, at the best cutoff of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 and a specificity of 0.84 in healthy individuals and 0.83 in symptomatic patients. The preliminary results suggest that these biomarkers could potentially be used for the early diagnosis of bladder cancer, especially in high-risk populations (e.g, symptomatic individuals exposed to occupational risk) who may benefit from the use of noninvasive diagnostic tests in terms of cost-benefit.

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

The prognostic significance of cytogenetic aberrations in childhood acute myeloid leukaemia. A study of the Swiss Paediatric Oncology Group (SPOG)

In childhood-onset acute myeloid leukaemia (AML) the clinical value of karyotypic aberrations is now acknowledged, although there is still debate concerning the prognostic significance of some events. To add to this knowledge, cytogenetic analysis was performed on a consecutive series of 84 childhood AML patients diagnosed in Switzerland. A result was obtained for all patients, with 69 (82%) showing a clonal karyotypic aberration. In the remaining 15 (18%), no karyotypic aberration was seen by either conventional or fluorescence in situ hybridisation analyses. The most frequent aberrations observed were t(11q23) (19% of all patients), t(8;21) (12%) and +8 (11%). Except for cytogenetics, no clinical parameter was shown to be significantly associated with outcome. The analysis of individual cytogenetic subgroups demonstrated that aberrations involving chromosome 16q were the strongest predictor of a good prognosis, while +8 and complex karyotypes represented the strongest predictors of a poor prognosis. It was also noteworthy that patients with the rare aberrations of del(11q) (n = 4) and t(16;21)(p11;q22) (n = 3) had a poor outcome. The results support the importance of cytogenetic analysis in childhood AML, but show that further work is required in the classification of the poor prognosis aberrations.

A de novo 1.1-1.6 Mb subtelomeric deletion of chromosome 20q13.33 in a patient with learning difficulties but without obvious dysmorphic features

We report on a de novo submicroscopic deletion of 20q13.33 identified by subtelomeric fluorescence in situ hybridization (FISH) in a 4-year-old girl with learning difficulties, hyperlaxity and strabismus, but without obvious dysmorphic features. Further investigations by array-based comparative genomic hybridization (array-CGH) and FISH analysis allowed us to delineate the smallest reported subterminal deletion of chromosome 20q, spanning a 1.1-1.6 Mb with a breakpoint localized between BAC RP5-887L7 and RP11-261N11. The genes CHRNA4 and KCNQ2 implicated in autosomal dominant epilepsy are included in the deletion interval. Subterminal 20q deletions as found in the present patient have, to our knowledge, only been reported in three patients. We review the clinical and behavioral phenotype of such "pure" subterminal 20q deletions.

Losses of chromosome arms 4q, 8p, 13q and gain of 8q are correlated with increasing chromosomal instability in hepatocellular carcinoma

OBJECTIVE: Chromosomal instability is a key feature in hepatocellular carcinoma (HCC). Array comparative genomic hybridization (aCGH) revealed recurring structural aberrations, whereas fluorescence in situ hybridization (FISH) indicated an increasing number of numerical aberrations in dedifferentiating HCC. Therefore, we examined whether there was a correlation between structural and numerical aberrations of chromosomal instability in HCC. METHODS AND RESULTS: 27 HCC (5 well, 10 moderately, 12 lower differentiated) already cytogenetically characterized by aCGH were analyzed. FISH analysis using probes for chromosomes 1, 3, 7, 8 and 17 revealed 1.46-4.24 signals/nucleus, which correlated with the histological grade (well vs. moderately,p < 0.0003; moderately vs. lower, p < 0.004). The number of chromosomes to each other was stable with exceptions only seen for chromosome 8. Loss of 4q and 13q, respectively, were correlated with the number of aberrations detected by aCGH (p < 0.001, p < 0.005; Mann-Whitney test). Loss of 4q and gain of 8q were correlated with an increasing number of numerical aberrations detected by FISH (p < 0.020, p < 0.031). Loss of 8p was correlated with the number of structural imbalances seen in aCGH (p < 0.048), but not with the number of numerical changes seen in FISH. CONCLUSION: We found that losses of 4q, 8p and 13q were closely correlated with an increasing number of aberrations detected by aCGH, whereas a loss of 4q and a gain of 8q were also observed in the context of polyploidization, the cytogenetic correlate of morphological dedifferentiation.

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.

Diagnostic and prognostic implications of the PAX8-PPARγ translocation in thyroid carcinomas-a TMA-based study of 226 cases

AIMS Follicular thyroid carcinoma (FTC) has been a diagnostic challenge for decades. The PAX8-PPARγ rearrangement has been detected in FTC and classic papillary thyroid carcinomas (PTCs). The aims of this study were to assess the presence of PAX8-PPARγ by using tissue microarrays in a large cohort of different thyroid neoplasms, and to assess its diagnostic and prognostic implications. METHODS AND RESULTS Fluorescence in-situ hybridization (FISH) analysis for PAX8-PPARγ was performed on 226 thyroid tumours, comprising FTCs (n = 59), PTCs (n = 126), poorly differentiated thyroid carcinomas (PDs; n = 34), follicular thyroid adenomas (FTAs; n = 5), and follicular tumours of unknown malignant potential (FTUMPs; n = 2). PAX8-PPARγ was detected in 12% of FTCs, 1% of PTCs, 7% of PDs, and in both cases of FTUMP. There was no correlation between the extent of capsular or vascular invasion and PAX8-PPARγ, or between lymph node or haematogenous metastasis and PAX8-PPARγ. Overall survival (OS), tumour-specific survival (TSS) and relapse-free-survival (RFS) were not influenced by PAX8-PPARγ. CONCLUSIONS In this study, we demonstrate for the first time the presence of PAX8-PPARγ in PDs and FTUMPs, whereas in FTCs and PTCs the prevalence of PAX8-PPARγ is lower than previously reported. PAX8-PPARγ did not correlate with invasiveness or affect prognosis in any tumour type.

Prognostic role of FGFR1 amplification in early-stage non-small cell lung cancer.

Background:Recently, fibroblast growth factor receptor 1 (FGFR1) was discovered in squamous cell carcinomas (SCC) of the lung with FGFR1 amplification described as a promising predictive marker for anti-FGFR inhibitor treatment. Only few data are available regarding prevalence, prognostic significance and clinico-pathological characteristics of FGFR1-amplified and early-stage non-small cell lung carcinomas (NSCLC). We therefore investigated the FGFR1 gene status in a large number of well-characterised early-stage NSCLC.Methods:FGFR1 gene status was evaluated using a commercially available fluorescent in situ hybridisation (FISH) probe on a tissue microarray (TMA). This TMA harbours 329 resected, formalin-fixed and paraffin-embedded, nodal-negative NSCLC with a UICC stage I-II. The FISH results were correlated with clinico-pathological features and overall survival (OS).Results:The prevalence of an FGFR1 amplification was 12.5% (41/329) and was significantly (P<0.0001) higher in squamous cell carcinoma (SCC) (20.7%) than in adenocarcinoma (2.2%) and large cell carcinoma (13%). Multivariate analysis revealed significantly (P=0.0367) worse 5-year OS in patients with an FGFR1-amplified NSCLC.Conclusions:FGFR1 amplification is common in early-stage SCC of the lung and is an independent and adverse prognostic marker. Its potential role as a predictive marker for targeted therapies or adjuvant treatment needs further investigation.

Expression analysis of heat shock protein 90 (HSP90) and Her2 in colon carcinoma.

PURPOSE The molecular chaperone heat shock protein 90 (HSP90) plays an important role in several types of tumors also participating in the modulation of the activity of receptor tyrosine kinases activity such as members of the Her family. We evaluated the significance of HSP90 and Her2 expression in colon cancer. METHODS HSP90 and Her2 expression was determined by immunohistochemistry and by fluorescence in situ hybridization (FISH) on 355 primary resected colon carcinomas. Results were correlated with pathologic features (Union for International Cancer Control (UICC) pTNM category, tumor localisation, tumor differentiation), additional molecular genetic characteristics (BRAF, KRAS mutational status, mismatch repair genes (MMR)), and survival. RESULTS HSP90 immunoreactivity was observed in various degrees. Fifty-one cases (14 %) were positive for Her2 (score 2+ and 3+) with 16/43 cases with Her2 2+ staining pattern showing amplification of Her2 determined by FISH. There was a significant correlation between high HSP90 expression and Her2 overexpression (p = 0.011). High HSP90 expression was associated with earlier tumor stages (p = 0.019), absence of lymph node (p = 0.006), and absence of distant metastases (p = 0.001). Patients with high tumoral HSP90 levels had a better survival (p = 0.032), but this was not independent from other prognostic relevant pathologic parameters. Her2 expression was not associated with any of the investigated histopathological, molecular, or clinical parameters. CONCLUSIONS High HSP90 levels are reflecting lower malignant potential in colon cancer. Her2 positivity can be observed in a small number of cases. Targeting HSP90 and/or Her2 may be an alternative therapeutic approach in colon cancer in a subset of patients.

Soil characteristics and abundance of archaea in samples from the Ross Sea Region, Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (>99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.