Loss Of Genetic Material Research Articles

Síndrome de Phelan McDermid: descripción de cinco pacientes e informe del primer caso descripto en gemelas siamesas

Phelan McDermid Syndrome is caused by the loss of genetic material in a chromosome from pair 22, at the band q13.3. We describe five patients with deletion 22q13.3 in order to establish a genotype-phenotype association, and report the first case described in conjoined twins. We analyzed the perinatal history, psychomotor behavior, language, and the presence of minor dysmorphism. Karyotypes and in situ hibridization (FISH) for critical region 22q13.3 were performed to all patients. There were hypotonia, developmental delay, and delay or absence of language. A 22q13.3 deletion was detected in all patients described, two cases had a deletion and the other three had a ring of chromosome 22, one in a pure cell line, while the twins presented mosaicism. Karyotype and FISH for 22q11.2 critical region should be performed, with 22q13.3 control probe to detect the deletion of Phelan McDermid syndrome in all patients with clinical phenotype suggestive and evocative of velo-cardio-facial syndrome.

Williams syndrome (syndrome of idiopathic hypercalcemia)

Williams syndrome was first described in 1961 by J.C.P. Williams et al. The population frequency is 1:7500-10 000 live births; the ratio of boys and girls is about 1:1. The nature of this genetic disease is the loss of genetic material (microdeletion) of the long arm of chromosome 7. During microdeletion the loss of elastin, LIM-kinase-1 and replication factor C2 (RFC) genes occurs, some of the «lost» genes have not yet been identified. A more rare form of the syndrome exhibits deletions on chromosomes 11 and 12 - 11q13-q14, and 22q. Presented was the authors’ clinical observation. A child was born from the second pregnancy (the first one ended in miscarriage at 12 weeks of the term), which had a physiological course, first childbirth was given at the term of 40 weeks, the newborns body weight at birth was 2600 g, body length 49 cm. At 1 year 8 months the baby’s height was 80 cm, body weight was 10 kg 800 g, head circumference - 50 cm, chest circumference - 51 cm. Multiple stigmas of dysembryogenesis were present. The head was irregularly shaped, the large fontanelle was closed. A marked face dysmorphism: a broad forehead, the unusual shape of the eyes with swelling around them, epicanthal fold, drooping full cheeks, the characteristic shape of the nose with a rounded blunt end and anteriorly open nostrils, a small pointed chin, a large open mouth, full lips, especially the lower one, progeny, small teeth, jagged, and affected by caries. The thorax was narrow, a depressed sternum in the form of a «сobbler’s chest». The muscle tone was moderately reduced. The psychomotor development at the level of a 8-9 months old. Cardiologist’s conclusion: a congenital heart defect, hypoplasia of the isthmus of the aortic arch. Conclusion of the geneticist: multiple stigmas of dysembryogenesis, a characteristic phenotype. Karyological analysis 46,XY - without karyotype abnormalities. Chromatography of the amino acids - without any pathology. Diagnosis: Williams syndrome. Diagnosis of Williams syndrome sometimes possesses known difficulties. The described clinical case illustrates the possibilities of diagnosing the syndrome, based primarily on a careful analysis of the medical history, of the clinical picture and physical examination.

Abstract 1164: MTUS1 as a potential progression marker at chromosome 8p in bladder cancer

Abstract Aims and background: The search for sensitive and specific progression and recurrence markers in bladder cancer is a major focus of many urological research groups. The aim of this study was to identify biomarkers on chromosome 8p in papillary urothelial carcinoma that give evidence for progression from non-invasive pTa to invasive pT1 stage. Candidate genes should be analyzed functionally in cell lines and validated immunohistochemically using tissue micro arrays. Material and methods: To find new progression markers 19 papillary urothelial carcinomas (9 pTa and 10 pT1) were investigated with array comparative genomic hybridization (aCGH) on Affymetrix Genome-Wide Human SNP Array 6.0. Therefore, tumor slides were manually microdissected, genomic DNA was isolated and DNA was hybridized on Affymetrix Chip together with reference DNA from 167 tumor-free patients. Computational analysis was performed with free Software “Genotyping-Console” from Affymetrix. MTUS1 gene and protein expression were investigated in bladder cancer cell lines and tumor tissue using qRT-PCR, Western blot and immunohistochemistry. Results: aCGH revealed more chromosomal aberrations overall as well as more deletions on chromosome 8p in pT1 tumors (50%) compared to pTa tumors (11%). Several putative progression-associated genes could be identified on 8p, for example SLC7A2, PDGFL, MTUS1, FGL1 and PCM1. MTUS1 (microtubule associated tumor suppressor 1) was chosen for further analysis, as in other tumor types this gene is known to be associated with frequent loss of heterozygosity at 8p22. However, to our knowledge, not much is known about its role in bladder cancer. Western blot analysis revealed expression of different isoforms in cell lines (140, 80 and 60 kDa) and tumor tissue (mainly 80kDa). Immunohistochemical staining of 128 papillary and 236 advanced tumors showed conflicting results compared to the aCGH findings. Conclusion: Whereas loss of genetic material at 8p22 harboring the MTUS1 gene is significantly associated with a pT1 stage in papillary urothelial carcinoma, pT1 tumors showed a significantly higher MTUS1 expression than pTa tumors as was shown by immunohistochemistry. At least in the pT1 group, expression loss contributed to worse overall survival, suggesting MTUS1 as a prognostic marker. Functional effects of MTUS1 overexpression will be analyzed in the future in RT112 cell line. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1164. doi:1538-7445.AM2012-1164

Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking

Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains.In this work we evaluated the usefulness of three different hybridization methods: spore to spore mating, rare-mating and protoplast fusion for the generation of intra- and inter-specific stable hybrids, being the first report about the comparison of different methods to obtain artificial hybrids to be used in fermentations. Spore to spore mating is an easy but time-consuming method; hybrids generated with this technique could lack some of the industrially relevant traits present in the parental strains because of the segregation occurred during meiosis and spore generation prior to hybridization. Hybrids obtained by protoplast fusion get the complete information of both parents but they are currently considered as genetically modified organisms (GMOs). Finally, hybrids obtained by rare-mating are easily obtained by the optimized methodology described in this work, they originally contain a complete set of chromosomes of both parents and they are not considered as GMOs. Hybrids obtained by means of the three methodological approaches showed a high genetic variability; however, a loss of genetic material was detected in most of them. Based on these results, it became evident that a last crucial aspect to be considered in every hybridization program is the genetic stabilization of recently generated hybrids that guarantee its invariability during future industrial utilization. In this work, a wine yeast genetic stabilization process was developed and vegetatively stable hybrids were obtained.

Congenital Diaphragmatic Hernia in a Fetus with a De novo Terminal Deletion of Chromosome 15q26.1

De novo terminal deletions of chromosome 15q26.1 are rare occurrences. Deletions of this region have been previously linked to congenital diaphragmatic hernia (CDH) as well as congenital malformations and developmental delay. This article presents a prenatal case of this de novo terminal deletion, detected by cytogenetic analysis and confirmed by fluorescence in situ hybridization (FISH), in a fetus with CDH and intrauterine growth restriction (IUGR). Genetic evaluation of pre- and postnatal cases of CDH should include at least a close examination of the terminal region of chromosome 15q26. As with any de novo substantial loss of genetic material, the prognosis is likely to include additional neurologic impairment and other congenital malformations in comparison to CDH patients without genomic alterations.

BCG sub-strains induce variable protection against virulent pulmonary Mycobacterium tuberculosis infection, with the capacity to drive Th2 immunity

The hallmark of a vaccine is to induce long-term protective immunity against the pathogen. The use of Mycobacterium bovis BCG as a vaccine against tuberculosis has been problematic in that immunity induced by BCG wanes over time and it may be less effective against more virulent strains of Mycobacterium tuberculosis. Thus it is important to determine what factors might be associated with waning or inefficient immunity. One such factor has been associated with the difference in many types of BCG that are used around the world, or more specifically due to the loss of genomic material in the various sub-strains used in vaccination programs. To address this issue we investigated the long-term immune response generated by 3 sub-strains BCG in the C57BL/6 mouse model of experimental tuberculosis. Mice vaccinated with these diverse strains of BCG were assessed at 6 and 12 months post-vaccination. All BCG sub-strain induced elevated levels of IFN-γ-producing cells at each time point as determined by ELISpot assay. However, when mice were challenged at 6 and 12 months with either M. tuberculosis H37Rv or HN878 the ability of the BCG sub-strains to protect vaccinated mice varied, depending on the time of challenge and on the strain used to infect mice. BCG Pasteur was then used to vaccinate guinea pigs, which were subsequently infected with either H37Rv or HN878. Data showed that BCG Pasteur prolonged the survival of guinea pigs against infection with both strains. Taken together these data suggest that longevity of the immune response generated by BCG is not related to the loss of genetic material and that BCG can induce a protective immune response to infection with a clinical strain of M. tuberculosis.

Prevalence of JC polyomavirus genomic sequences from the large t‐antigen and non‐coding control regions among bulgarian patients with primary brain tumors

A total of 111 fresh brain biopsies from patients with primary brain tumors were examined for JC polyomavirus sequences from the Large T antigen encoding region (LT) and the viral non-coding control region (NCCR). SYBR Green and TaqMan real-time polymerase chain reaction assays were used. In the glioblastoma group of 39 patients 48.7% were positive for LT sequences. Among the astrocytoma group (19 patients) and the oligodendroglioma group (12 patients) 31.6% and 33.3% were also positive. The prevalence of LT genomic sequences among the other groups was as follows: in 2 out of 3 oligoastrocytomas; in 3 out 5 gangliogliomas; in 2 out of 5 meduloblastomas; in 1 out 3 pineocytomas; and in none of the tested 5 ependimomas. All positive samples had a late threshold cycle that varied from 36 to 49, indicative of very low starting viral number. Only 21 of all the 111 samples were positive for NCCR. Low copy number in range of 10-1,000 was present. Notably, only 8 of all NCCR positive specimens were also LT positive. It might be suggested that the disproportion between the results for LT and NCCR is either due to clonally integrated LT fragments, with loss of genetic material, or changes in the NCCR. The latter would alter the productive course of the infection and may establish a premise for continuous interaction of viral regulatory proteins with cell molecules that are responsible for the control of the cell cycle. This may lead subsequently to malignant transformation.

Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.

Karyotypic studies on Campanumoea (Campanulaceae)—endemic to China

Radix Campanumoeae (C. javanica Bl.) has been used in Miao herbal medicine to treat neurasthenia and consumptive disease for hundreds of years. Though Radix Campanumoeae shows great potential for utilization in medical studies, this herb crop has not been cultivated industrially in China. Many species in the Campanumoea genus are similar in phenotype; therefore, a karyotypic study would prove useful for clearly distinguishing Radix Campanumoeae from related species within the same genus, for germplasm preservation and for breeding Radix Campanumoeae. 10 accessions of four species in this genus and 5 accessions in 5 relative genera from SCSB were used for karyotype determination. The results showed a karyotype of 2n = 16 = 2m + 12sm + 1st in Campanumoea, and a karyotype of 2n = 18 = 6 m + 12sm in Cyclocodon. Based on the chromosome number and the karyotypic formula, we suggest that the Campanumoea genus can be divided into two genera, which is in agreement with results observed from pollen morphology and from homological usage in Chinese herbal medicine. The chromosome length in C. javanica subsp. javanica and C. javanica subsp. japonica Makino, ranged from 2.24 to 1.38 μm and 2.04 to 1.31 μm, respectively; and their haploid sets were almost identical (13.15 and 13.16 μm, respectively). This indicates that chromosomal rearrangements occur within chromosomes IV, V and VII without a net gain or loss of genetic material.

Application of Real-Time PCR Technlogies for Analysis of JCV as a Human Cancerogen

ABSTRACTViral agents such as Polyomavirus JC (JCV) raise an increased interest and are discussed as factors for development of several human malignancies. Real time PCR techniques allow better characterization of the studied samples as they possess superior sensitivity when compared to conventional PCR. Also quantitative analysis of the experimental results can be performed and information about the dynamics of the process is determined.Real time PCR technologies were applied to investigate fresh frozen biopsies from 132 patients for two particular JCV genomic sequences (LT and NCCR) that might be related to malignant transformation. Patients were divided into groups according to their histological type, and included: gliobastoma patients (n=39); astrocytoma patients (n=19); oligodendrogliomas (n=12); colorectal cancer patients (n=44); and patients with polyps (n=18). Colon samples were paired with matched adjacent mucosa. Results showed that viral genomic sequences were present in 22.7% to 61.5% among the different groups. Positive samples had a low starting viral number, as a late threshold cycle that varied from 36 to 49 was observed. The prevalence of the non-coding control region (NCCR) was lower than the prevalence of LT and only 12 of all 70 brain samples were found positive. None of the tested premalignant colonic lesions and cancers was positive, whereas two normal tissues had viral load in the range of 10–1000 starting copies.The disproportion between the results for LT and NCCR might be suggested to be either due to clonally integrated LT fragments, with loss of genetic material, or changes in the NCCR. This might lead to disturbance in the productive course of the infection and a premise for continuous interaction of viral regulatory proteins with cell molecules that are responsible for the control of the cell cycle may be establish. This subsequently might lead to malignant transformation. As infectious diseases acquire relevance in this process, new tools for cancer management and prevention are gained.

Chromosome 22q11.2 Microdeletion Syndrome

Chromosome 22q11.2 microdeletion syndrome is the most common microdeletion syndrome in humans. It involves the loss of genetic material on the short arm of one of the chromosome 22 alleles. Until advanced testing was available, this syndrome was known by various names including DiGeorge syndrome and velo-cardio-facial syndrome. This syndrome has a varied presentation with significant abnormalities including congenital heart disease, hypocalcemia, immunologic deficiencies, learning disabilities, and behavioral problems. A multidisciplinary approach is required to diagnose and manage the varied manifestations.

Progressive but previously untreated CLL patients with greater array CGH complexity exhibit a less durable response to chemoimmunotherapy

To better understand the implications of genomic instability and outcome in B-cell chronic lymphocytic leukemia (CLL), we sought to address genomic complexity as a predictor of chemosensitivity and ultimately clinical outcome in this disease. We used array-based comparative genomic hybridization (aCGH) with a one-million probe array and identified gains and losses of genetic material in 48 patients treated on a chemoimmunotherapy clinical trial. We identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14, and 17. Higher genomic complexity, as a mechanism favoring clonal selection, was associated with shorter progression-free survival, and predicted a poor response to treatment. Of interest, CLL cases with loss of p53 surveillance showed more complex genomic features and were found both in patients with a 17p13.1 deletion and in the more favorable genetic subtype characterized by the presence of 13q14.1 deletion. This aCGH study adds information on the association between poor trial response and increasing genetic complexity as CLL progresses.

Disorders caused by chromosome abnormalities

Many human genetic disorders result from unbalanced chromosome abnormalities, in which there is a net gain or loss of genetic material. Such imbalances often disrupt large numbers of dosage-sensitive, developmentally important genes and result in specific and complex phenotypes. Alternately, some chromosomal syndromes may be caused by a deletion or duplication of a single gene with pleiotropic effects. Traditionally, chromosome abnormalities were identified by visual inspection of the chromosomes under a microscope. The use of molecular cytogenetic technologies, such as fluorescence in situ hybridization and microarrays, has allowed for the identification of cryptic or submicroscopic imbalances, which are not visible under the light microscope. Microarrays have allowed for the identification of numerous new syndromes through a genotype-first approach in which patients with the same or overlapping genomic alterations are identified and then the phenotypes are described. Because many chromosomal alterations are large and encompass numerous genes, the ascertainment of individuals with overlapping deletions and varying clinical features may allow researchers to narrow the region in which to search for candidate genes.

Analysis of Mitosis In Acute Myeloid Leukemia Using Live-Cell Imaging.

AbstractAbstract 3363Proper mitotic control is a prerequisite to guarantee the equal distribution of the genetic material onto the two developing daughter cells. A mitotic key regulator is cyclin B. High levels of cyclin B facilitate entry into mitosis whereas its controlled degradation coordinates chromosome separation and cytokinesis. The latter events are coordinated by the anaphase- promoting complex / cyclosome (APC/C), a ubiquitin ligase that couples ubiquitin chains to cyclin B, mediating its proteasomal degradation. The regulation of the APC/C-activity by complex protein networks, such as the spindle assembly checkpoint, therefore presents the basis for an accurate mitosis.Mitotic errors give rise to daughter cells with an aberrant set of chromosomes and contribute to genetic instability. Genetic instability is a hallmark of cancer cells and plays an important role in the onset and progression of acute myeloid leukemia (AML). In rare cases, de novo AMLs present with multiple cytogenetic aberrations (complex karyotype). However, a larger number of patients develop karyotype deviations in the course of the disease, sometimes even under therapy, which comes along with an adverse prognosis. Understanding the biology that drives the gain and loss of genetic material therefore bears the potential of identifying new therapeutic targets.We compared a number of lymphoblastic and myeloid cell lines and found AML cell lines to be deficient in arresting at metaphase in the presence of the microtubule-disrupting agent nocodazole. Cyclin B was expressed at much lower levels in the AML cell line Kasumi-1 and did not accumulate following spindle disruption as observed in the lymphoblastic cell line DG-75. We could show that Kasumi-1 cells, when challenged with nocodazole, were not capable of properly maintaining chromatid-cohesion and underwent premature sister chromatid separation. These findings suggest that mitotic control mechanisms do not work tightly enough in AML cells to prevent chromosome separation in the presence of spindle disruption. We applied live-cell imaging to exactly characterize mitotic timing in Kasumi-1 cells at a single cell level. The expression of a GFP-tagged derivative of histone H2 served to visualize the nuclear envelope breakdown and anaphase onset. Detection of the latter events allowed the faithful measurement of mitotic timing. We could find a significant shortening of mitosis in Kasumi-1 cells as compared to the lymphoblastic cell line DG-75. In both AML cell lines and primary AML blasts we identified the spindle assembly checkpoint components BubR1 and Bub1 to be downregulated. Interestingly, re-expression of BubR1 in Kasumi-1 cells led to a significant stabilization of cyclin B on western blots. To address the question whether an increased expression of cyclin B leads to a more pronounced mitotic delay in the presence of spindle-disruption in AML cells is subject of current experiments. It was reported that different cell types can escape from a mitotic block as a consequence of cyclin B degradation. In the literature, this phenomenon was referred to as mitotic slippage and is known to drive genetic instability. To monitor cyclin B turnover and localization at a single cell level, we generated a chimeric cyclin B-molecule, SNAP-cyclin B, which can couple to a suitable fluorochrome in a self-labeling reaction after addition to the growth medium. In this system, the fluorescence intensity reflects the amount of chimeric cyclin B and allows the monitoring of APC/C-dependent proteolysis. In our current approaches we aim at studying cyclin B-turnover at a single cell level in AML cell lines as well as primary leukemia cells by using live-cell imaging before and after BubR1- and Bub1-rescue.An aberrant cell cycle control is found in most human malignancies and might be an important driving force in leukemogenesis. We hypothesize that BubR1, in concert with different other regulators, might lead to inaccuracies in mitotic control. This hypothesis is underlined by the shortened time to anaphase in Kasumi-1 cells and a decreased expression of cyclin B, both of which are characteristics of BubR1-depletion. Mitotic regulators are already targets in AML therapy and a deeper understanding of mitotic processes in AML might lead to improved approaches. Disclosures:No relevant conflicts of interest to declare.

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit A Less Durable Response to Chemoimmunotherapy

AbstractAbstract 2406The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. [Display omitted] Disclosures:Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Induction of Micro RNA-143 and 145 In Pre-Treatment CD34+ Cells From Patients with Myelodysplastic Syndrome (MDS) After In Vitroexposure to Lenalidomide Correlates with Clinical Response In Patients Harboring the Del5q Abnormality

AbstractAbstract 123 Background:Lenalidomide is an effective agent in the treatment of MDS particularily in those possesing the deletion 5q (del5q) abnormality. The disease phenotype is partly related to loss of genetic material found in the commonly deleted region (CDR) on the long arm of chromosome 5. Recently implicated genes include miRNA-143, 145 and 146 all of which appear to play a role in innate immune signalling. In this study we examined whether upregulation of miRNAs in pretreatment CD34+ marrow cells from patients with MDS (both del5q and non-del5q) exposed to lenalidomide in vitro predicts for clinical response to the drug. Patients and Methods:In total 31 pre-treatment patient samples were obtained from three North American centres. 11 patients' samples were positive for del5q. All patients had received lenalidomide as per local protocols. Clinical response was defined as per the International Working Group 2000 criteria. Clinical data were available for 26 patients. Hematologic improvement (HI), specifically the eythroid response (HI-E), was examined as the main clinical endpoint. Changes in miR-143, miR-145, miR-146a and miR-146b expression in CD34+ cells after in vitro lenalidomide exposure (10uM for 48 hours) were examined. The CD34- fraction was also analyzed. dCT values were normalized to 5S. A ≥ 1.5 fold change in miRNA expression was considered significant. Results:For the entire cohort mean fold changes in miRNA-143 and 145 post lenalidomide exposure were 1.6 and 1.7, respectively. Little change was seen in miR-146a and miR-146b expression (mean fold change 1.1 and 1.2, respectively). No significant increase was seen in any of the examined miRNA in the CD34 - fraction (Figure 1a). When separated based on del5q status both del5q and non-del5q groups showed a > 1.5 fold increase in expression of miR-143 and miR-145. No significant change in either miR-146a or miR-146b expression was seen (Figure 1b). In the CD34- fraction no significant change was seen in any of the examined miRNAs, irrespective of del5q status (Figure 1c). Examining HI-E, major responses (MR) were seen in 42% of patients (87.5% in the the del 5q cohort (7/8 had a MR) and 22.2% for the non-del5q cohort (4/18 had a MR)). Correlation with clinical response and miRNA expression in the CD34+ fraction is shown in Table I. In the del5q cohort MR was associated with an increase in both miR-143 and miR-145. No correlation with miRNA expression and clinical response was seen in the non del5q cohort. Conclusion:Exposure of CD34+ cells from patients with MDS to lenalidomide results in up regulation of miR-143 and miR-145, irrespective of del5q status. Clinically, upregulation of miR-143 and miR-145 correlates with a HI-E in patients with del5q and may be predictive of response to therapy. Interestingly, the non-responder in the del5q group failed to upregulate miR-145. A similar gene induction pattern was seen in the non-del5q cohort, yet no correlation was seen with clinical outcome. Given that non-del5q patients do not harbor any abnormalities within the CDR of chromosome 5 these miRNAs are unlikely to play a role in their disease phenotype, nor in their response to lenalidomide. [Display omitted] Table I:Correlation between clinical response and miRNA expression.Hematologic Improvement - Erythroid (n)miR143 increased (n)miR145 increased (n)Del5qMR (7)45No MR (1)10Total (8)55Non Del5qMR (4)02No MR (14)610Total (18)612 Disclosures:Nevill:Celgene: Honoraria. Karsan:Celgene: Research Funding.

Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities

Laboratory evaluation of patients with developmental delay/intellectual disability, congenital anomalies, and dysmorphic features has changed significantly in the last several years with the introduction of microarray technologies. Using these techniques, a patient's genome can be examined for gains or losses of genetic material too small to be detected by standard G-banded chromosome studies. This increased resolution of microarray technology over conventional cytogenetic analysis allows for identification of chromosomal imbalances with greater precision, accuracy, and technical sensitivity. A variety of array-based platforms are now available for use in clinical practice, and utilization strategies are evolving. Thus, a review of the utility and limitations of these techniques and recommendations regarding present and future application in the clinical setting are presented in this study.

Array comparative genome hybridization in patients with developmental delay: two example cases

Developmental delay is often a predictor of mental retardation (MR) or autism, two relatively frequent developmental disorders severely affecting intellectual and social functioning. The causes of these conditions remain unknown in most patients. They have a strong genetic component, but the specific genetic defects can only be identified in a fraction of patients. Recent developments in genomics supported the establishment of the causal link between copy number variants in the genomes of some patients and their affection. One of the techniques suitable for this analysis is array comparative genome hybridization, which can be used both for detailed mapping of chromosome rearrangements identified by classical cytogenetics and for the identification of novel submicroscopic gains or losses of genetic material. We illustrate the power of this approach in two patients. Patient 1 had a cytogenetically visible deletion of chromosome X and the molecular analysis was used to specify the gene content of the deletion and the prognosis of the child. Patient 2 had a seemingly normal karyotype and the analysis revealed a small recurrent deletion of chromosome 1 likely to be responsible for his phenotype. However, the genetic dissection of MR and autism is complicated by high heterogeneity of the genetic aberrations among patients and by broad variability of phenotypic effects of individual genetic defects.

Nanoparticle-enabled highly sensitive detection of DNA methylation in single tube

Abstract Background: One of the often occurred epigenetic events associated to cancer is inheritable transcriptional silencing of the tumor suppressor genes by aberrant methylation of cytosines at the promoter regions. Current approaches to detect promoter DNA methylation commonly involve three separate independent processes, including DNA extraction, bisulfite conversion and methylation detection through PCR amplification, such as methylation specific PCR (MSP). This method includes many disconnected steps with loss of genetic materials, potentially reducing the sensitivity required for analysis of challenging clinical samples. Methods and Results: To address this problem, we developed a new technique named Methylation-on-Beads (MOB). MOB uses the silica superparamagnetic nanobeads (SSB) to combine DNA extraction, bisulfite treatment (Bst) and PCR into a single tube. The single-tube scheme minimizes the DNA loss during the tube transfers and improves the analytical sensitivity for methylation detection. Further enhancement in sensitivity is achieved by combining MOB with quantum dot-enhanced MSP detection. The pre-PCR DNA yields using MOB were compared with those carried out with conventional organic solvent extraction and ethanol precipitation (PC). 15 serum samples from lung cancer patients (7 Stage I, 3 Stage II, 5 Stage III) were analyzed using both MOB and PC. The DNA recovery using MOB was higher than PC for each patient serum sample with a median increase of 6.61 fold. To demonstrate the versatile applicability of the new method, we further analyzed 10 samples to include fresh tissue and paraffin embedded tissue from normal patients, fresh tumors from cancer patients and sputum samples. Median DNA yield increase of 7.8, 5.3, 6.4 and 7.5 respectively was determined using MOB when compared to the conventional method. We examined the outcome of bisulfite conversion in the presence of SSBs using real-time methylation specific PCR (MSP) to analyze p16INK4a promoter methylation. A set of triplicate reactions were examined with bisulfite-treated DNA of varying treatment durations (0h, 1h, 3h, 4h, 8h), and compared to the control using 16 hrs of conventional bisulfite treatment. Results indicated that 4h of bisulfite treatment was sufficient for conversion, and that the presence of beads did not alter the conversion process. Assessment of methylation in serum or plasma can be a useful tool for early detection of cancer. However, extending the DNA methylation analysis to clinically usable serum/ blood-based tests has been limited by the lack of sensitivity of conventional methods. In order to address whether improving DNA yields could enhance methylation detection, we compared methylation of p16INK4a promoter in 49 patient serum samples (18 normal and 31 cancer) in a blinded study using both MOB and PC/Bst/MSP. The 31 tumor samples were pre-selected from patients diagnosed with lung cancer who were also methylated for p16INK4a promoter in corresponding tumors. While p16INK4a methylation was detected in 14/31 patients with lung cancer using conventional approach; using MOB, we were able to detect p16INK4a methylation in 23/31of these patients. When samples used for methylation analysis contained large amounts of DNA (cell lines, tumors etc), a single-tube analysis of entire input amount may be unnecessary. Instead, multiple reactions in parallel were feasible by directly splitting the SSB into several different tubes. Conclusion: MOB successfully combined three processes required for DNA methylation analysis into a single-tube using SSB thereby allowing for ease in handling and increased throughput in detection. Increased pre-PCR yield in MOB allowed for efficient, diagnostically sensitive methylation detection.

X-autosomal translocation — a distraction or a cause of primary ovarian insufficiency?

OBJECTIVE: To characterize the genetic basis of primary ovarian insufficiency (POI) in a patient with a balanced X-autosomal translocation involving one X chromosome and a deletion at Xq27.2 on the other X chromosome. X-autosomal translocations have been associated with POI, possibly due to aberrations in pairing or X inactivation during folliculogenesis. Additionally, the putative genes leading to POI are located at one of two loci on Xq: POF1 (Xq26-q28) or POF2 (Xq13.3-q22), with the minimal POF1 deletion associated with POI narrowed to a 10.5 Mb region at Xq27.2/Xq27.3-q28.DESIGN: Case report.MATERIALS AND METHODS: 23 year-old with oligomenorrhea and primary infertility. She reported menarche at age 13. After discontinuation of oral contraceptive pills at age 22, she experienced oligomenorrhea and infertility. There was no family history of POI. Her physical exam was unremarkable and showed no stigmata of Turner Syndrome. Relevant laboratory data included: day 3 FSH=61.2 mIU/mL, day 3 estradiol <20 pg/mL and normal FMR1 premutation testing.RESULTS: Standard karyotype testing (GTG band resolution 550) revealed 46,X,t(X;1)(q22.1;q42.1), indicating a balanced translocation involving chromosomes X and 1, with no apparent macroscopic loss of genetic material from the translocated chromosomes. Subsequent microarray comparative genomic hybridization (NimbleGen CGX-3 array) revealed a cryptic 382-482 kb deletion at Xq27.2 (arr Xq27.2(140,201,306-140,583,606)x1). In order to confirm the microarray findings and localize the deletion, fluorescence in situ hybridization (FISH) analysis was performed with a BAC probe (RP11-1082H4) mapping to the deleted region. The deletion was present on the X chromosome that was not involved in the translocation.CONCLUSION: Our patient exhibits a balanced X-autosomal translocation, which has been associated with POI, but appears to be an incidental finding in this patient. This case further narrows the minimal POF1 deletion associated with POI to a 382-482 kb deletion at Xq27.2. OBJECTIVE: To characterize the genetic basis of primary ovarian insufficiency (POI) in a patient with a balanced X-autosomal translocation involving one X chromosome and a deletion at Xq27.2 on the other X chromosome. X-autosomal translocations have been associated with POI, possibly due to aberrations in pairing or X inactivation during folliculogenesis. Additionally, the putative genes leading to POI are located at one of two loci on Xq: POF1 (Xq26-q28) or POF2 (Xq13.3-q22), with the minimal POF1 deletion associated with POI narrowed to a 10.5 Mb region at Xq27.2/Xq27.3-q28. DESIGN: Case report. MATERIALS AND METHODS: 23 year-old with oligomenorrhea and primary infertility. She reported menarche at age 13. After discontinuation of oral contraceptive pills at age 22, she experienced oligomenorrhea and infertility. There was no family history of POI. Her physical exam was unremarkable and showed no stigmata of Turner Syndrome. Relevant laboratory data included: day 3 FSH=61.2 mIU/mL, day 3 estradiol <20 pg/mL and normal FMR1 premutation testing. RESULTS: Standard karyotype testing (GTG band resolution 550) revealed 46,X,t(X;1)(q22.1;q42.1), indicating a balanced translocation involving chromosomes X and 1, with no apparent macroscopic loss of genetic material from the translocated chromosomes. Subsequent microarray comparative genomic hybridization (NimbleGen CGX-3 array) revealed a cryptic 382-482 kb deletion at Xq27.2 (arr Xq27.2(140,201,306-140,583,606)x1). In order to confirm the microarray findings and localize the deletion, fluorescence in situ hybridization (FISH) analysis was performed with a BAC probe (RP11-1082H4) mapping to the deleted region. The deletion was present on the X chromosome that was not involved in the translocation. CONCLUSION: Our patient exhibits a balanced X-autosomal translocation, which has been associated with POI, but appears to be an incidental finding in this patient. This case further narrows the minimal POF1 deletion associated with POI to a 382-482 kb deletion at Xq27.2.