Fluorescence In Situ Hybridization Hybridization Research Articles

PYGO2-MDR1 Axis in Multiple Myeloma Patients with 1q21 Amplification As Promising Target to Overcome Carfilzomib Resistance

Multiple myeloma (MM) is a hematological malignancy characterized by an accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Gain and/or amplification of 1q21 (1q21+) is one of the most frequent secondary cytogenetic abnormalities present in MM patients and in smoldering MM (SMM). The incidence and copy number alteration (CNA) of the 1q21 region increase during the progression and relapse of the disease; in fact, additional copies of 1q21 can be detected in around 40% of newly diagnosed MM (NDMM) and 70% of relapse-refractory MM (RRMM). Recently, 1q21+ has been identified as an adverse prognostic factor in MM patients and its presence has been correlated with a shorter duration of the response in MM patients treated with carfilzomib (CFZ)-based regimens. Thus, the identification of possible target genes in 1q21 region is an emerging unmet medical need in MM patients. PYGOPUS2 (PYGO2) is a gene located in the 1q21 chromosomal region and it is a downstream target of β-catenin, and it has been shown to promote the transcription of gene target of the Wnt-signaling, forming a nuclear complex with β-catenin/BCL9. Different studies have reported that upregulation of PYGO2 is involved in tumorigenesis in breast and lung cancers, showing that overexpression of PYGO2 leads to drug resistance by promoting the activation of the multidrug resistance 1 polypeptide ( MDR1). The expression profile of PYGO2 in1q21+ MM patients and its possible role in CFZ resistance is still unknown and have been investigated in this study. Firstly, we purified CD138 + PCs from BM samples from 18 NDMM and from 11 SMM patients. Fluorescent in situ hybridization (FISH) analysis was performed on purified CD138 + PCs for all patients to access CNA in the region 1q21. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. 14/29 (48%) patients presented CNA in the 1q21 region. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA) . The sam package was used to identify differentially expressed genes between 1q21+ and control samples. Our analysis identified PYGO2 to be significantly upregulated in patients with 1q21+ when compared with controls (p= 0.0008). Additionally, we found a positive correlation between gene expression and the 1q21 copy number (p= <0.0001, r= 0.6738). Secondly, we evaluated PYGO2 mRNA expression levels in several human myeloma cell lines (HMCLs) with 1q21 CNA. Our results showed that PYGO2 is overexpressed in HMCLs with 1q21 CNA. Previous studies in solid tumor have shown that PYGO2 is upregulated in drug resistant cancer cells, and this upregulation results in overexpression of MDR1; indeed, we evaluated the mRNA expression of PYGO2 in CFZ-resistant HMCLs. Interestingly our results showed that both PYGO2 and MDR1 expression are upregulated in CFZ-R cells. We next examined the relationship between PYGO2 and MDR1. We generated a PYGO2-knockdown in JJN3 MM cell line using short hairpin RNA (shRNA) lentivectors. Knockdown of PYGO2 resulted in a significant decrease in expression PYGO2, a decrease in cell viability, and a reduction in MDR1 expression. In conclusion, our results show that PYGO2 expression is significantly upregulated in MM patients carrying 1q21+ and that its expression is correlated with 1q21 copy number. Moreover, we found that the PYGO2-MDR1 axis is involved in the CFZ resistance in HMCLs MM cells suggesting that PYGO2 could be a possible future druggable target in 1q21+ MM patients.

22. Reducing diagnostic FISH turn-around-time: A rapid pipeline for BCR::ABL1 and PML::RARA suspected leukemias

Successful long-term outcome of patients with BCR::ABL1 positive chronic myeloid leukemia (CML) and PML::RARA positive acute promyelocytic leukemia (APL) is critically dependent on rapid cytogenetic and molecular studies. Fluorescence in situ hybridization (FISH) serves as a standard-of-care diagnostic assay in the workup of suspected BCR::ABL1 and PML::RARA positive leukemias. Nonetheless, turn-around-time for FISH results can be upwards of days, delaying highly-effective personalized treatments. To reduce FISH turn-around-time for patients with a suspected BCR::ABL1 or PML::RARA positive leukemia, our cytogenetics laboratory developed a rapid specimen processing algorithm to consistently return diagnostic BCR::ABL1 and PML::RARA FISH results within 24-hours from sample receipt. Our algorithm, a time-dependent decision matrix, leverages modified sample culturing, harvesting, and FISH hybridization protocols to return diagnostic BCR::ABL1 and PML::RARA FISH results as quickly as 6 hours from sample receipt. This pipeline enables accelerated treatment for patients with BCR::ABL1 and PML::RARA positive leukemias.

The classification of tetraploid wheat by phylogenetic and cytogenetic analyses

Tetraploid wheat (Triticum turgidum L.) is an important species within the genus Triticum and harbors many desirable agronomic traits. The classification, origin, and evolution of tetraploid wheat remain confused and controversial, resulting in useless germplasm resources. Two classification systems for tetraploid wheat are widely used: 1) tetraploid wheat comprises two species; 2) all forms of tetraploid wheat are classified as one species. The present study aimed to reassess the classification of tetraploid wheat using phylogenetic analysis of nuclear rDNA internal transcribed spacer region (ITS) sequence data, fluorescence in situ hybridization (FISH) karyotyping, and observation of meiotic pairing behavior in F1 hybrids. Network analysis of ITS sequences indicates that tetraploid wheat was not closely related to other Triticeae species with the exception of Aegilops speltoides and Ae. sharonensis. Phylogenetic analysis of ITS sequences and FISH show that Triticum turgidum and T. timopheevii clustered on distinct branches, and meiotic pairing in F1 hybrids of these species showed a high frequency of univalents. Meiotic behavior of F1 hybrids among forms of T. turgidum revealed a low number of univalents (means < 2) except for T. turgidum ssp. dicoccoides. The significant variation on chromosomes 1A, 2A, 5A, 1B, 2B, 3B, and 6B in the FISH hybridization patterns were observed between T. turgidum ssp. dicoccoides and other T. turgidum accessions. Furthermore, the results of ITS phylogenetic analyses correspond closely with observations of meiotic behavior and FISH karyotyping. The present results indicate that T. turgidum and T. timopheevii are two distantly related species of different origins. Triticum turgidum ssp. dicoccoides should be maintained as a subspecies of T. turgidum whereas other forms of T. turgidum should be reclassified as varieties.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications

BackgroundClementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine.ResultsA graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin.ConclusionsThe composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Rapid and Efficient FISH using Pre-Labeled Oligomer Probes

Fluorescence in situ hybridization (FISH) is used to visualize the distribution of DNA elements within a genome. Conventional methods for FISH take 1–2 days. Here, we developed a simplified, rapid FISH technique using pre-labeled oligonucleotide probes (PLOPs) and tested the procedure using 18 PLOPs from 45S and 5S rDNA, Arabidopsis-type telomere, and newly-identified Panax ginseng-specific tandem repeats. The 16 developed rDNA PLOPs can be universally applied to plants and animals. The telomere PLOPs can be utilized in most plants with Arabidopsis-type telomeres. The ginseng-specific PLOP can be used to distinguish P. ginseng from related Panax species. Differential labeling of PLOPs allowed us to simultaneously visualize different target loci while reducing the FISH hybridization time from ~16 h to 5 min. PLOP-FISH is efficient, reliable, and rapid, making it ideal for routine analysis, especially of newly sequenced genomes using either universal or specific targets, such as novel tandem repeats identified from whole-genome sequencing data.

Alternative PDGFD rearrangements in dermatofibrosarcomas protuberans without PDGFB fusions

Dermatofibrosarcoma protuberans is underlined by recurrent collagen type I alpha 1 chain-platelet-derived growth factor B chain(COL1A1-PDGFB) fusions but ~ 4% of typical dermatofibrosarcoma protuberans remain negative for this translocation in routine molecular screening. We investigated a series of 21 cases not associated with the pathognomonic COL1A1-PDGFB fusion on routine fluorescence in situ hybridization (FISH) testing. All cases displayed morphological and clinical features consistent with the diagnosis of dermatofibrosarcoma protuberans. RNA-sequencing analysis was successful in 20 cases. The classical COL1A1-PDGFB fusion was present in 40% of cases (n = 8/20), and subsequently confirmed with a COL1A1 break-apart FISH probe in all but one case (n = 7/8). 55% of cases (n = 11/20) displayed novel PDGFD rearrangements; PDGFD being fused either to the 5′ part of COL6A3 (2q37.3) (n = 9/11) or EMILIN2 (18p11) (n = 2/11). All rearrangements led to in-frame fusion transcripts and were confirmed at genomic level by FISH and/or array-comparative genomic hybridization. PDGFD-rearranged dermatofibrosarcoma protuberans presented clinical outcomes similar to typical dermatofibrosarcoma protuberans. Notably, the two EMILIN2-PDGFD cases displayed fibrosarcomatous transformation and homozygous deletions of CDKN2A at genomic level. We report the first recurrent molecular variant of dermatofibrosarcoma protuberans involving PDGFD, which functionally mimic bona fide COL1A1-PDGFB fusions, leading presumably to a similar autocrine loop-stimulating PDGFRB. This study also emphasizes that COL1A1-PDGFB fusions can be cytogenetically cryptic on FISH testing in a subset of cases, thereby representing a diagnostic pitfall that pathologists should be aware of.

Wolf-Hirschhorn syndrome presenting with cardiac manifestations at birth

Wolf–Hirschhorn syndrome (WHS) is a condition of developmental delay and dysmorphology caused by a deletion of short arm of chromosome 4. The main characteristics of WHS are intra and extrauterine growth retardation, mental retardation with typical facial dysmorphism, microcephaly, and midline fusion defects. The diagnosis is based on clinical features and chromosomal analysis, e.g., microsatellite analysis or molecular cytogenetic techniques and fluorescence in situ hybridization (FISH) to detect the deletion. Prenatal diagnosis is possible by FISH. Though no specific treatment is available, supportive management can be fruitful. Here, we describe a female baby with a 4p deletion, who had the majority of the main phenotypic features of WHS and severe congenital heart disease manifesting at birth. The case emphasized that any fetus with intrauterine growth retardation, dysmorphic features on antenatal scan, and midline defects should raise a suspicion of WHS. Conventional cytogenetic studies can miss the diagnosis; hence, these cases should be further investigated using molecular cytogenetic techniques such as FISH or array-comparative genomic hybridization.

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.

Chromosomal organization of centromeric Ty3/gypsy retrotransposons in Allium cepa L. and Allium fistulosum L.

This is the first report on the presence of Ty3/gypsy-like retrotransposons in the centromeric region of Allium cepa and Allium fistulosum. The paper identifies the putative Ty3/gypsy centromeric retrotransposons (CR) among the DNA sequences of A. cepa present in the NCBI database and evaluates their copy number in the genomes of Allium cepa and Allium fistulosum. The putative copy number of Ty3/gypsy CR constituted about 26000 for A. cepa and about 7000 for A. fistulosum. The chromosomal organization of Ty3/gypsy CR was analyzed with the help of fluorescent in situ hybridization (FISH). The 300-bp PCR products synthesized with genomic DNA of Allium cepa and Allium fistulosum and primers designed for the sequence ET645811 of A. cepa (Genome Survey Sequence database), displaying similarity to the reverse transcriptase of the CR Ty3/gypsy family, served as FISH hybridization probes. On the chromosomes of A. cepa, hybridization signals were mainly localized in the centromeric region. On the chromosomes of A. fistulosum the signals were less expressed in the centromeric regions, though they were abundant in other chromosomal regions. The pathways of evolution in these closely related species are discussed.

Constitutional telomeric association (Y;7) in a patient with a female phenotype

Telomeric associations (TAs) are fusions between two telomeres of two different chromosomes without visible loss of chromosomal material. Constitutional telomeric associations are rare chromosomal anomalies. We report on the cytogenetic and molecular analyses of a TA involving chromosomes Y and 7 in a child with a female phenotype. Prenatal cytogenetic analysis showed a 45,X chromosome complement in all cells. No fetal abnormality was identified at ultrasound examinations and the pregnancy went to term. During childhood, the proband had gonadal dysgenesis but no other phenotypic manifestations of Turner syndrome. Molecular genetic analyses showed the presence of genomic DNA of the SRY gene without any mutation. Karyotyping and fluorescent in situ hybridization (FISH) analyses on blood showed two cell lines: one cell line with a TA involving chromosomes Y and 7 [46,X,tas(Y;7)(p11.32;q36.3)] and a second cell line with a 45,X pattern. A human pantelomeric repeat TTAGGG probe hybridized to the junction of the TA within the derivative chromosome. FISH and array comparative genomic hybridization (aCGH) analyses demonstrated that tas(Y;7) occurred without detectable loss of any sequence at the derivative chromosome. SNP array analysis excluded an uniparental isodisomy of chromosome 7. Knowing more about TAs will help geneticists to deliver accurate genetic counseling.

Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by FluorescenceIn SituHybridization: Simulation of the Detection of Fetal Cells in Maternal Blood

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Molecular cytogenetic characterization and SSR marker analysis of a leaf rust resistant wheat line carrying a 6G(6B) substitution from Triticum timopheevii (Zhuk.)

A disease (powdery mildew, leaf rust) resistant line was selected from the progenies of a Triticum aestivum × Triticum timopheevii amphiploid produced at Martonvasar. This line was previously identified with C-banding as a 6G(6B) substitution. In order to detect the 6G chromosome in a wheat background, fluorescence in situ hybridization (FISH) and microsatellite marker analysis were used. Ten microsatellite markers of the 43 tested generated PCR products that were polymorphic between chromosomes 6B and 6G, and four showed length-polymorphism. The FISH hybridization pattern of 6G from T. timopheevii was identified using a combination of four repetitive DNA probes (Afa-family, pSc119.2, pTa71, (GAA)7). Genomic in situ hybridization (GISH) technique, capable of labelling the At and G genomes separately, was used on the same slides to differentiate the At and G genomes in T. timopheevii. The At and G genomes of T. timopheevii were grouped on the basis of the GISH patterns and a cyclic intergenomic translocation involving 6At-1G-4G was detected in T. timopheevii accession TRI667. The presence of 6G in the substitution line was demonstrated using FISH with the four repetitive DNA probes. Chromosome 6G was clearly identified and its FISH pattern was different from that of 6B in the parental wheat cultivar Fleischmann-481. According to field tests, the 6G(6B) substitution line has resistance to leaf rust.

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.

“Mini Review: Probes for Detecting Prymnesium Parvum and Preliminary Results from Gene Expression Studies”2

Medlin, Linda K., Sonja Diercks, and Sara Beszteri, 2010. Mini Review: Probes for Detecting Prymnesium parvum and Preliminary Results From Gene Expression Studies. Journal of the American Water Resources Association (JAWRA) 46(1):144-152. DOI: 10.1111/j.1752-1688.2009.00398.x Abstract: Prymnesium parvum is common in brackish and marine coastal waters within temperate zones, world-wide. P. parvum forms recurrent blooms causing fish kills in many parts of the world. Harmful blooms are formed in nutrient rich, low salinity lakes, ponds, river systems, or estuaries. Probes made to this species and to the genus Prymnesium have been tested in dot blot and fluorescent in situ hybridization (FISH) and used with a solid-phase cytometer using a tyramide signal amplification (TSA) enhanced FISH hybridization to provide a means to identify the cells before blooms develop with automated counting. Field counts from light microscopy have been compared with solid-phase cytometer counts. Additional detection systems, such as biosensors and microarrays, have also been developed to identify rapidly this species without resorting to electron microscopy. Advantages and disadvantages of each detection system are discussed. A study of the genes expressed by P. parvum under environmental conditions that can induce blooms or stress was undertaken to try to understand the ecology behind toxic blooms.

Abstract 2684: Telomere length of carcinoma-associated fibroblasts as a biomarker for breast cancer local recurrence

Abstract The use of breast-conservative surgery (BCS) for the treatment of early-stage breast cancers has been increasing and the ability to identify patients at high risk of local recurrence is highly desirable for guiding treatment decisions. Currently, relatively few breast cancer local recurrent risk factors have been identified; those known risk factors, which are based on clinical and histological features of tumor tissue, have limited predictive power. It is known that molecular genetic changes often precede morphologic evidence of malignant transformation. Thus, early molecular genetic alterations in breast tumor and adjacent normal tissues are likely to be more sensitive tools for the identification of patients at increased risk of local recurrence than current histological criteria. A nested case-control study was carried out to evaluate whether telomere length in morphologically normal tissues adjacent to the tumor is predictive of breast cancer local recurrence. One hundred forty-two women who are diagnosed with breast cancer at Lombardi Comprehensive Cancer Center between 1998 and 2005 were included in the study. Cases (N = 42) are patients who had local recurrences of breast cancer after BCS. Controls (N = 100) are patient who had no local recurrence and are matched to cases on year of surgery, age at diagnosis, disease stage and type of surgery. Quantitative telomere fluorescent in situ hybridization (FISH) was used to determine the telomere length of four cell types: cancer cells, carcinoma-associated fibroblast cells (CAFs), adjacent normal epithelial cells and lymphocytes, using 5-micron sections of formalin fixed paraffin-embedded (FFPE) breast tumor tissues. Telomere length of lymphocytes was used to normalize the FISH hybridization variation and relative telomere length (RTL) was defined as telomere length in cells of interest divided by the telomere length of lymphocytes. Multivariate logistic regression was used to assess the association between RTL and breast cancer local recurrence. We found that mean RTL of CAFs was significantly longer in patients with breast cancer local recurrence (mean = 0.99) than in patients without local recurrence (mean = 0.74, p=0.022). When RTL of CAFs were dichotomized using median value of control subjects, patients who had longer RTL in CAFs had 2.4-fold (95% CI=0.86-6.56) increased risk of breast cancer local recurrence than women who had shorter RTL in CAFs. We observed no statistically significant case-control difference in mean RTL of normal epithelial cells adjacent to the tumor (p = 0.712) or in mean RTL of cancer cells (p = 0.285). This study is the first to report that telomere length in tumor stromal cells (CAFs) is associated with breast cancer local recurrence. If confirmed by future studies, telomere length in CAFs has the potential to become a novel molecular tool for the predicting breast cancer local recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2684.

Array-Based Karyotyping for Prognostic Assessment in Chronic Lymphocytic Leukemia: Performance Comparison of Affymetrix 10K2.0, 250K Nsp, and SNP6.0 Arrays

Specific chromosomal alterations are recognized as important prognostic factors in chronic lymphocytic leukemia (CLL). Array-based karyotyping is gaining acceptance as an alternative to the standard fluorescence in situ hybridization (FISH) panel for detecting these aberrations. This study explores the optimum single nucleotide polymorphism (SNP) array probe density for routine clinical use, presents clinical validation results for the 250K Nsp Affymetrix SNP array, and highlights clinically actionable genetic lesions missed by FISH and conventional cytogenetics. CLL samples were processed on low (10K2.0), medium (250K Nsp), and high (SNP6.0) probe density Affymetrix SNP arrays. Break point definition and detection rates for clinically relevant genetic lesions were compared. The 250K Nsp array was subsequently validated for routine clinical use and demonstrated 98.5% concordance with the standard CLL FISH panel. SNP array karyotyping detected genomic complexity and/or acquired uniparental disomy not detected by the FISH panel. In particular, a region of acquired uniparental disomy on 17p was shown to harbor two mutated copies of TP53 that would have gone undetected by FISH, conventional cytogenetics, or array comparative genomic hybridization. SNP array karyotyping allows genome-wide, high resolution detection of copy number and uniparental disomy at genomic regions with established prognostic significance in CLL, detects lesions missed by FISH, and provides insight into gene dosage at these loci.

Mini Review: Probes for Detecting Prymnesium parvum and Preliminary Results From Gene Expression Studies1

Medlin, Linda K., Sonja Diercks, and Sara Beszteri, 2010. Mini Review: Probes for Detecting Prymnesium parvum and Preliminary Results From Gene Expression Studies. Journal of the American Water Resources Association (JAWRA) 46(1):144‐152. DOI: 10.1111/j.1752‐1688.2009.00398.xAbstract: Prymnesium parvum is common in brackish and marine coastal waters within temperate zones, world‐wide. P. parvum forms recurrent blooms causing fish kills in many parts of the world. Harmful blooms are formed in nutrient rich, low salinity lakes, ponds, river systems, or estuaries. Probes made to this species and to the genus Prymnesium have been tested in dot blot and fluorescent in situ hybridization (FISH) and used with a solid‐phase cytometer using a tyramide signal amplification (TSA) enhanced FISH hybridization to provide a means to identify the cells before blooms develop with automated counting. Field counts from light microscopy have been compared with solid‐phase cytometer counts. Additional detection systems, such as biosensors and microarrays, have also been developed to identify rapidly this species without resorting to electron microscopy. Advantages and disadvantages of each detection system are discussed. A study of the genes expressed by P. parvum under environmental conditions that can induce blooms or stress was undertaken to try to understand the ecology behind toxic blooms.

A Case of Severe Mental and Developmental Retardation Associated with 14q Terminal Monosomy/5q Terminal Trisomy

I previously described the case of a 19 year-old female with severe mental retardation, developmental retardation, microcephalus, short stature, bilateral microphthalmia, ptosis and blepharophimosis(1). Now, I present clinical descriptions of her half-siblings, who have a different father. Subtelomeric fluorescence in situ hybridization (FISH) analysis of the proband demonstrated 5q terminal trisomy and 14q terminal monosomy. I presume that her mother harbors a balanced translocation between the terminal of chromosome 5q and 14q. I suggest that familial cases of mental retardation and dysmorphic features should be screened for terminal chromosomal abnormalities by FISH or comparative genomic hybridization (CGH), even if G-banding analysis or high-resolution chromosome analysis is normal.

Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization

We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea (Paradiplozoon homoion) and Cestoda (Caryophyllaeus laticeps, Caryophyllaeides fennica, and Nippotaenia mogurndae) but not in Acanthocephala (Pomphorhynchus laevis and Pomphorhynchus tereticollis). In the Pomphorhynchus species, no hybridization signals were obtained with the "nematode" (TTAGGC)n, "arthropod" (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

Long-term assessment of nitrification in a full-scale wastewater treatment plant

In this study, long term nitrification performances and microbial composition in a full-scale oxidation ditch type wastewater treatment plant (WWTP) were monitored and evaluated using different molecular methods; fluorescent in situ hybridization (FISH), slot-blot hybridization and quantitative real-time polymerase chain reaction (PCR) method. In situ and membrane hybridization results indicated that Nitrosomonas species were identified as the dominant ammonia oxidizing bacteria and Nitrospira related species were detected as the prevailing nitrite oxidizing bacteria in a full-scale wastewater treatment plant throughout 2 years of operation. Real-time PCR using the LightCyclerinstrument has been developed for the quantification of ammonia monooxygenase (amoA) and 16S rRNA genes. Results suggest that real-time PCR analysis, amoA/16S rRNA ratio, is an alternative method to understand nitrifying bacterial population and activity in wastewater treatment plant compared with the FISH and slot-blot hybridization assays. The autotrophic/heterotrophic bacterial ratio and their influence on reactor performances were investigated using real-time PCR amoA/16S rRNA gene copy ratios and the results showed that this ratio varied from 3.6% to 8.3% during operational period.