SNP-chip Analysis Research Articles

How Fish Population Genomics Can Promote Sustainable Fisheries: A Road Map.

Maintenance of genetic diversity in marine fishes targeted by commercial fishing is a grand challenge for the future. Most of these species are abundant and therefore important for marine ecosystems and food security. Here, we present a road map of how population genomics can promote sustainable fisheries. In these species, the development of reference genomes and whole genome sequencing is key, because genetic differentiation at neutral loci is usually low due to large population sizes and gene flow. First, baseline allele frequencies representing genetically differentiated populations within species must be established. These can then be used to accurately determine the composition of mixed samples, forming the basis for population demographic analysis to inform sustainably set fish quotas. SNP-chip analysis is a cost-effective method for determining baseline allele frequencies and for population identification in mixed samples. Finally, we describe how genetic marker analysis can transform stock identification and management.

Identification of Selection Signatures and Loci Associated with Important Economic Traits in Yunan Black and Huainan Pigs.

Henan Province is located in central China and rich in domestic pig populations; Huainan (HN) pigs are one of three Henan indigenous breeds with great performance, including early maturation, strong disease resistance and high meat quality. Yunan (YN) black pigs are a typical, newly cultivated breed, synthesized between HN pigs and American Duroc, and are subjected to selection for important traits, such as fast growth and excellent meat quality. However, the genomic differences, selection signatures and loci associated with important economic traits in YN black pigs and HN pigs are still not well understood. In this study, based on high-density SNP chip analysis of 159 samples covering commercial DLY (Duroc × Landrace × Large White) pigs, HN pigs and YN black pigs, we performed a comprehensive analysis of phylogenetic relationships and genetic diversity among the three breeds. Furthermore, we used composite likelihood ratio tests (CLR) and F-statistics (Fst) to identify specific signatures of selection associated with important economic traits and potential candidate genes. We found 147 selected regions (top 1%) harboring 90 genes based on genetic differentiation (Fst) in the YN-DLY group. In the HN-DLY group, 169 selected regions harbored 58 genes. In the YN-HN group, 179 selected regions harbored 77 genes. In addition, the QTLs database with the most overlapping regions was associated with triglyceride level, number of mummified pigs, hemoglobin and loin muscle depth for YN black pigs, litter size and intramuscular fat content for HN pigs, and humerus length, linolenic acid content and feed conversion ratio mainly in DLY pigs. Of note, overlapping 14 tissue-specific promoters' annotation with the top Fst 1% selective regions systematically demonstrated the muscle-specific and hypothalamus-specific regulatory elements in YN black pigs. Taken together, these results contribute to an accurate knowledge of crossbreeding, thus benefitting the evaluation of production performance and improving the genome-assisted breeding of other important indigenous pig in the future.

Maternal karyogene and cytoplasmic genotype affect the induction efficiency of doubled haploid inducer in Brassica napus

BackgroundArtificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far.ResultsIn this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration.ConclusionThe induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.

Whole-genome resequencing of extreme phenotypes in collared flycatchers highlights the difficulty of detecting quantitative trait loci in natural populations.

Dissecting the genetic basis of phenotypic variation in natural populations is a long-standing goal in evolutionary biology. One open question is whether quantitative traits are determined only by large numbers of genes with small effects, or whether variation also exists in large-effect loci. We conducted genomewide association analyses of forehead patch size (a sexually selected trait) on 81 whole-genome-resequenced male collared flycatchers with extreme phenotypes, and on 415 males sampled independent of patch size and genotyped with a 50K SNP chip. No SNPs were genomewide statistically significantly associated with patch size. Simulation-based power analyses suggest that the power to detect large-effect loci responsible for 10% of phenotypic variance was <0.5 in the genome resequencing analysis, and <0.1 in the SNP chip analysis. Reducing the recombination by two-thirds relative to collared flycatchers modestly increased power. Tripling sample size increased power to >0.8 for resequencing of extreme phenotypes (N=243), but power remained <0.2 for the 50K SNP chip analysis (N=1245). At least 1 million SNPs were necessary to achieve power >0.8 when analysing 415 randomly sampled phenotypes. However, power of the 50K SNP chip to detect large-effect loci was nearly 0.8 in simulations with a small effective population size of 1500. These results suggest that reliably detecting large-effect trait loci in large natural populations will often require thousands of individuals and near complete sampling of the genome. Encouragingly, far fewer individuals and loci will often be sufficient to reliably detect large-effect loci in small populations with widespread strong linkage disequilibrium.

Establishment and characterization of novel human primary and metastatic anaplastic thyroid cancer cell lines and their genomic evolution over a year as a primagraft.

Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1β, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3-22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft.

TCIRG1 Associated Congenital Neutropenia

▪Severe congenital neutropenia (SCN) is a rare autosomal dominant hematopoietic disorder with an estimated incidence of 1 in 200,000 for individuals of European descent. Although several causal genes have been identified, the genetic basis for >30% of cases remains unknown. We report finding a novel single nucleotide variant (SNV) R736S identified in TCIRG1, utilizing SNP chip analysis, whole exome sequencing and confirmatory Sanger sequencing, in a multi-generation family (median blood neutrophil counts 0.486 x 109/L, range 0.074 to 1.235 x 109/L for 11 affected members and 3.808 x 109/L, range 1.548 to 6.488 x 109/L for 7 unaffected members). There is a perfect cosegregation of the variant with neutropenia in this family; all 11 affected, but none of the unaffected individuals share this novel variant.TCIRG1 is located at 11q13 and encodes the vacuolar H+-ATPase a3 subunit, a 116-kD component of V-ATPases called OC116, and is essential for bone tissue maintenance and homeostasis. Homozygous or compound heterozygous mutations in this gene cause autosomal recessive malignant osteopetrosis (arOP) characterized by bone resorption defect due to osteoclast malfunction. Interestingly, the same gene, through an alternative splicing and usage of an alternative initiation codon in exon 7, gives rise to another protein, i.e., TIRC7, which is a T cell-specific membrane protein demonstrated to play an essential role for T-lymphocyte activation and immune response. Recent publications suggest the existence of additional splice variants expressed in various tissues in normal volunteers. SCN due to mutations in TCIRG1 have not been previously described.The arginine (R) to serine (S) substitution is a critical change because arginine at position 736 is essential for proton translocation and is evolutionarily highly conserved with a Genomic Evolutionary Rate Profiling (GERP) score = 3.78. Although direct structural data on TCIRG1's protein products are lacking, the effect of this mutation on the protein structure is predicted to be “probably damaging” by Polyphen (an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of the protein).Western blot analysis of protein products of this gene performed using peripheral blood mononuclear cells show reduced expression of TCIRG1 protein in the affected family members from this family utilizing a commercially available polyclonal antibody developed against the N-terminal cytoplasmic domain of human OC116. The ∼45kDa TCIRG1 product was down-regulated by 26%, 49%, and 35% in the 3 affected individuals, compared to 2 normal controls. Another antibody, raised against the TCIRG1 fragment 121aa-220aa, detects a ∼120 kDa protein band with similar expression levels in both affected individuals and healthy controls. These assays confirm the multi-product nature of TCIRG1 and suggest deregulatory involvement of this protein in the pathogenesis of the neutropenia in this family.Sanger sequencing of additional 20 unrelated SCN patients known to be negative for mutations in the known causal genes, revealed two different SNVs in TCIRG1 in two unrelated SCN patients, based on Minor Allele Frequency < 0.5% and high conservation among mammals (GERP score > 3). The first variant, g.16065 G>A, (IVS 14 -1G>A), is located in the acceptor splice site of the intron 14, and predicted to alter the splicing. The second variant, c.479 G>A (p.GLY160GLU), is located in exon 5 and predicted to be possibly damaging by Polyphen. No other SNV's predicted to alter protein structure have been found.We conclude that mutations in TCIRG1 are a rare but important cause of autosomal dominant congenital neutropenia. Further studies are in progress to define the pathophysiological mechanisms for neutropenia and the importance of the vATPases in maintaining the integrity of neutrophil production and deployment. Disclosures:No relevant conflicts of interest to declare.

Severe intellectual disability, West syndrome, Dandy–Walker malformation, and syndactyly in a patient with partial tetrasomy 17q25.3

We report on a de novo 0.5 Mb triplication (partial tetrasomy) of chromosome 17q25.3 in a 10-year-old girl with severe intellectual disability, infantile seizures (West syndrome), moderate hearing loss, Dandy-Walker malformation, microcephaly, craniofacial dysmorphism, striking cutaneous syndactyly (hands 3-4, feet 2-3), joint laxity, and short stature. The triplication resulted from the unusual combination of a terminal duplication at 17qter and a cryptic translocation of an extra copy of the same segment onto chromosome 10qter. The breakpoint at 17q25.3 was located within the FOXK2 gene. SNP chip analysis suggested that the rearrangement occurred during paternal meiosis involving both paternal chromosomes 17.

QTL Scan for Meat Quality Traits Using High-density SNP Chip Analysis in Cross between Korean Native Pig and Yorkshire

We attempted to generate a linkage map using Illumina Porcine 60K SNP Beadchip genotypes of the F2 offspring from Korean native pig (KNP) crossed with Yorkshire (YS) pig, and to identify quantitative trait loci (QTL) using the line-cross model. Among the genotype information of the 62,136 SNPs obtained from the high-density SNP analysis, 45,308 SNPs were used to select informative markers with allelic frequencies >0.7 between the KNP (n = 16) and YS (n = 8) F0 animals. Of the selected SNP markers, a final set of 500 SNPs with polymorphic information contents (PIC) values of >0.300 in the F2 groups (n = 252) was used for detection of thirty meat quality-related QTL on chromosomes at the 5% significance level and 10 QTL at the 1% significance level. The QTL for crude protein were detected on SSC2, SSC3, SSC6, SSC9 and SSC12; for intramuscular fat and marbling on SSC2, SSC8, SSC12, SSC14 and SSC18; meat color measurements on SSC1, SSC3, SSC4, SSC5, SSC6, SSC10, SSC11, SSC12, SSC16 and SSC18; water content related measurements in pork were detected on SSC4, SSC6, SSC7, SSC10, SSC12 and SSC14. Additional QTL of pork quality traits such as texture, tenderness and pH were detected on SSC6, SSC12, SSC13 and SSC16. The most important chromosomal region of superior pork quality in KNP compared to YS was identified on SSC12. Our results demonstrated that a QTL linkage map of the F2 design in the pig breed can be generated with a selected data set of high density SNP genotypes. The QTL regions detected in this study

Update on ophthalmic molecular genetics

Abstract Purpose To provide an overview of the recent technological advances in human molecular genetics that can be applied in ophthalmic genetics. Methods Since the finalization of the Human Genome Project many novel genomic technologies emerged that led to significant advances in gene identification and genetic testing of hereditary eye disorders: (1) genomewide copy number screening (array CGH); (2) genomewide SNP genotyping; (3) next‐generation sequencing. Results (1) Microarray comparative genomic hybridisation or array CGH allows genomewide discovery of submicroscopic deletions and duplications in a single experiment. This technique is applied in routine molecular cytogenetic testing. Using array CGH a causal genomic defect can be found in at least 10% of all cases with mental retardation and/or multiple congenital anomalies. In ophthalmic genetics array CGH is mainly useful in the context of developmental eye disorders, with chorioretinal coloboma and anterior segment dysgenesis as an example. (2) Genomewide chip‐based SNP genotyping can be used for homozygosity mapping in inbred and outbred pedigrees. Recent successes in gene identification using this approach are illustrated. (3) Next‐generation sequencing or NGS. The application of this technology in gene identification and genetic testing of genetically heterogeneous conditions (with LCA as a paradigm) is discussed. Conclusion The rapid progress of genomic technologies such as array CGH, SNP chip analysis and next‐generation sequencing lead to a boost in gene identification and genetic testing of both developmental and retinal eye disease.

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

BackgroundEmerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Methodology/Significant FindingsUsing methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.ConclusionsThese findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions.

Abstract 3540: Down-regulation of solute carrier family 17 member 1(SLC17A1) expression contributes to chemotherapy resistance in acute myeloid leukemia

Abstract Cytosine arabinoside (Ara-C), an structural analogue of deoxycytidine, is generally considered to be the major drug in the treatment of AML. In acute myeloblastic leukemia (AML) treatment, resistance to cytotoxic nucleoside analogues is a major problem. To investigate novel genes associating with becoming Ara-C resistant cells, whole genome SNP chip analysis was performed. However, there was no significant in Ara-C metabolite genes. In that analysis, 12 region of loss of heterozygote (LOH) was found in non-CR group. SLC17 family gene and HIST1H family gene located in major of LOH in non-CR group. Since SLC29A1 expression levels directly affected the Ara-C mediated Apoptosis, we selected SLC17A1 among them and functional analysis of SLC17A1 was performed. Its expression levels were examined in four different AML cell lines using RT-PCR. Its expression level was a little different but that gene seemed to be constitutively expressed. To detect the pattern change of SLC17A1 in survival AML cells against Ara-C treatments, four AML cell-lines underwent 10−8 M of Ara-C addition/ depletion for 2 and 4 cycles. The results showed that expression of SLC17A1 was decreased more than SLC29A1 expression in the same treated condition. To examine whether the expression levels of SLC17A1 was associated with Ara-C resistance, Ara-C resistant HEL cell was established in vitro and in vivo (HEL-R1 and HEL-R1-vivo). Our results indicated that SLC17A1 mRNA expression of both resistant cells is decreased or less increased than HEL cells in Ara-C treatment. And the reduction of SLC17A1 mRNA by shRNA of SLC17A1 effectively suppressed Ara-C mediated apoptosis. Our results suggested that the function of SLC17A1 gene was similar to that of SLC29A1. Taken together, the expression levels of SLC17A1 also might involve the cytotoxic effects of Ara-C treatment. SLC17A1 could be a novel gene controlling Ara-C mediated apoptosis. 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 3540.

Prevalence and Prognostic Impact of Allelic Imbalances Associated with Leukemic Transformation of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms.

Abstract 1280Poster Board I-302Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are defined as clonal hematopoietic stem cell disorders. These disorders show an inherent tendency for transformation into leukemia (MPN-blast phase) which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, obtained a comprehensive profile of genomic alterations associated with leukemic transformation by using single-nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters.A relatively high number of genomic alterations was found in MPN after leukemic transformation with 4.6 ± 0.6 abnormalities per sample compared to only 1.4 ± 0.2 changes per patient in chronic phase (p<0.001). Compared to the cytogenetic data, SNP-chip analysis detected about 47% additional chromosomal changes in the MPN samples, and 31% more in the MPN-blast phase cases, whereas SNP-array allelokaryotyping practically captured all cytogenetic abnormalities in our study population. Several additionally altered regions were detected in patients with MPN-blast phase compared to chronic phase, including both deletion and copy-number neutral-loss of heterozygosity (CNN-LOH) on chromosome 12p (9%) and 21q (9%), involving ETV6 and RUNX1. Notably, deletion and CNN-LOH on 17p involving TP53 were diagnosed in 18% of MPN-blast phase samples, which was highly associated with preceding treatment with alkylating agents (p=0.016). Moreover, trisomy 8, as well as amplification of 8q24.21 involving the MYC gene, were detected in 13% of patients with MPN-blast phase who were almost exclusively negative for the JAK2V617F mutation. Genome-wide inspection of further critical regions with promising new candidate genes involved in the evolution to the MPN-leukemic phase included deletion and CNN-LOH on 7q22.1 (SH2B2) in 18%, duplication/amplification on 19p13.2 (PIN1, ICAM1, CDC37) in 13% and 21q22.2 (ERG) in 9% of MPN patients with blast crisis. In contrast, we detected a decreased frequency of JAK2V617F in MPN-blast phase samples (52%) compared to chronic phase (71%). Also, the percentage of patients with homozygous mutant JAK2 as a result of CNN-LOH was lower in the MPN-blast phase (43%) compared to the chronic phase (53%). Taken together, the data suggest that gain-of-function mutation of JAK2 is not a perquisite for leukemic transformation. Remarkably, CNN-LOH on either 7q or 9p was related to decreased survival after leukemic transformation (p=0.02 and p=0.012, respectively). Given the variety of allelic imbalances, our data suggest that MPN-blast phase appears to be a heterogeneous disease prone to have evolved multiple mechanisms to provide a proliferative advantage to the abnormal leukemic clone. Our analysis of MPN genomes in the chronic compared to the leukemic stage provided new prognostic insights, as well as novel causative genes which might be involved in the transformation to MPN-blast phase. DisclosuresNo relevant conflicts of interest to declare.

PAX5/TEL Exhibits a Dominant-Negative Role by Inhibiting the Expression of Genes Involved in BCR Signalling and Suppressing the Binding of Wild Type PAX5 to Its Direct Target Gene CD79A (mb-1) in a human Pre-B ALL Cell Line.

Abstract 3455Poster Board III-343Acute lymphoblastic leukemia (ALL), one of the most common malignancies in childhood, is a heterogeneous disease with individual leukemia subtypes differing in their response to chemotherapy. Recent findings suggest that disruptions of B cell receptor (BCR) signalling pathways may be involved in the development of ALL. The transcription factor PAX5 is essential for the commitment of lymphoid progenitors to the B-lymphocytic lineage. In 30% of childhood B-ALL cases, PAX5 is a frequent target of aberrancies, showing monoallelic loss, point mutations, or chromosomal translocations, whereas the role of these aberrancies is still poorly understood. Using high resolution SNP-chip analysis, we have recently identified several candidate partner genes fused to PAX5 in pediatric ALL, ETV6 (TEL), FOXP1, AUTS2, C20orf112, which bind to PAX5 recognition sequences as strongly as wild-type PAX5 (wt PAX5) suppressing its transcriptional activity in a dominant-negative fashion. In order to study the role of PAX5/TEL in leukemic evolution of B-ALL, we transfected the leukemic BCP cell line Nalm 6, which endogenously expresses PAX5, with a retroviral vector encoding PAX5/TEL and confirmed its expression by Western blotting and RT-PCR. Previously, the fusion gene PAX5/TEL has been cloned into the retroviral vector pMSCV-IRES-GFP (MIGR) from a patient diagnosed with B-cell precursor ALL (BCP) with t(9;12)(q11;p13). This fusion product consists of the 5′-end NH2 terminal region of the PAX5 gene and the almost whole sequence of the TEL gene. PAX5/TEL-MIGR expressing cells were sorted for GFP and analyzed by gene expression profiling on Affymetrix HG-U133 plus 2.0 Array in comparison to cells transfected with vector control (MIGR) and a MIGR vector encoding wt PAX5 (wtPAX5/MIGR). The probes were normalized with the Affymetrix MAS5.0 software. Probes were considered to be differentially expressed with a fold change ≤ 2 or ≥ 2, respectively. We identified a set of about 200 genes that were differentially expressed in the PAX5/TEL expressing cells, most of which were downregulated, compared to the controls. A subset of these genes encodes proteins important for BCR signalling: RAG1, one of two key mediators in the process of V(D)J recombination, VPREB3, which is involved in the early phase of pre-BCR assembly, the Runt domain transcription factor Runx1 (AML1) and FOXP1. The latter two genes are fusion partners of PAX5 in pediatric B-ALL and loss of FOXP1 leads to impaired DH–JH and VH–DJH rearrangement. Additionally, we found BACH2, which plays an important role during B-cell development, as well as protein kinase C-epsilon (PKCe) to be downregulated. PKCe is highly expressed in B cells linking the BCR to the activation of mitogen-activated protein kinases (MAPK). We confirmed the downregulation of the affected genes by RT-PCR. Strikingly, VPREB3 expression showed a significant downregulation of up to 170-fold, and RAG1 up to 90-fold. Loss of the RAG1/2 locus has been found in four precursor B-cell ALL cases, which indicates that defects in this process might contribute to leukemogenesis. We also detected a significant decrease in the expression of wt PAX5 as well as its direct downstream target CD79A (mb-1). CD79A (mb-1) encodes the B cell receptor component Ig-a and its early B cell-specific mb-1 promoter is a target for regulation by early B cell-specific transcription factors like E2A, early B cell factor (EBF), and PAX5. The latter is important for the activation of the mb-1 promoter by recruiting Ets proteins through protein-protein interactions. We investigated the binding efficiency of wt PAX5 to the promoter region of CD79A by chromatin-immunoprecipitation (ChIP). For the ChIP assay, we used a PAX5 antibody detecting the C-terminal region of PAX5 so that the antibody can bind the wt PAX5 but not the fusion product PAX5/TEL of which the C-terminal side is fused to TEL. Binding of wt PAX5 to the promoter region of CD79A was diminished by expression of the PAX5/TEL-fusion protein compared to the controls, leading to repression of CD79A, which we also confirmed by RT-PCR.In conclusion, we show that the expression of PAX5/TEL in a leukemic cell line has a repressor function on the expression of wt PAX5 as well as other genes important in BCR signalling. Also, we demonstrated that PAX5/TEL has a negative impact on the binding affinity of one of the direct downstream target genes of wt PAX5. Our results indicate a repressor role of the fusion gene PAX5/TEL including BCR signalling and point towards its contribution to leukemic transformation. DisclosuresNo relevant conflicts of interest to declare.

Chromosomal abnormalities and novel disease‐related regions in progression from Barrett's esophagus to esophageal adenocarcinoma

Barrett's esophagus (BE) is a metaplastic condition caused by chronic gastroesophageal reflux which represents an early step in the development of esophageal adenocarcinoma (EAC). Single-nucleotide polymorphism microarray (SNP-chip) analysis is a novel, precise, high-throughput approach to examine genomic alterations in neoplasia. Using 250K SNP-chips, we examined the neoplastic progression of BE to EAC, studying 11 matched sample sets: 6 sets of normal esophagus (NE), BE and EAC, 4 of NE and BE and 1 of NE and EAC. Six (60%) of 10 total BE samples and 4 (57%) of 7 total EAC samples exhibited 1 or more genomic abnormalities comprising deletions, duplications, amplifications and copy-number-neutral loss of heterozygosity (CNN-LOH). Several shared abnormalities were identified, including chromosome 9p CNN-LOH [2 BE samples (20%)], deletion of CDKN2A [4 BE samples (40%)] and amplification of 17q12-21.2 involving the ERBB2, RARA and TOP2A genes [3.1 Mb, 2 EAC (29%)]. Interestingly, 1 BE sample contained a homozygous deletion spanning 9p22.3-p22.2 (1.2 Mb): this region harbors only 1 known gene, basonuclin 2 (BNC2). Real-time PCR analysis confirmed the deletion of this gene and decreased the expression of BNC2 mRNA in the BE sample. Furthermore, transfection and stable expression of BNC2 caused growth arrest of OE33 EAC cells, suggesting that BNC2 functions as a tumor suppressor gene in the esophagus and that deletion of this gene occurs during the development of EAC. Thus, this SNP-chip analysis has identified several early cytogenetic events and novel candidate cancer-related genes that are potentially involved in the evolution of BE to EAC.

Single nucleotide polymorphism genomic arrays analysis of t(8;21) acute myeloid leukemia cells

Translocation of chromosomes 8 and 21, t(8;21), resulting in the AML1-ETO fusion gene, is associated with acute myeloid leukemia. We searched for additional genomic abnormalities in this acute myeloid leukemia subtype by performing single nucleotide polymorphism genomic arrays (SNP-chip) analysis on 48 newly diagnosed cases. Thirty-two patients (67%) had a normal genome by SNP-chip analysis (Group A), and 16 patients (33%) had one or more genomic abnormalities including copy number changes or copy number neutral loss of heterozygosity (Group B). Two samples had copy number neutral loss of heterozygosity on chromosome 6p including the PIM1 gene; and one of these cases had E135K mutation of Pim1. Interestingly, 38% of Group B and only 13% of Group A samples had a KIT-D816 mutation, suggesting that genomic alterations are often associated with a KIT-D816 mutation. Importantly, prognostic analysis revealed that overall survival and event-free survival of individuals in Group B were significantly worse than those in Group A.

Identified hidden genomic changes in mantle cell lymphoma using high-resolution single nucleotide polymorphism genomic array

Mantle cell lymphoma (MCL) is a lymphoma characterized by aberrant activation of CCND1/cyclin D1 followed by sequential genetic abnormalities. Genomic abnormalities in MCL have been extensively examined by classical cytogenetics and microarray-based comparative genomic hybridization techniques, pointing out a number of alterations in genomic regions that correlate with the neoplastic phenotype and survival. Recently, single nucleotide polymorphism genomic microarrays (SNP-chip) have been developed and used for analysis of cancer genomics. This technique allows detection of genomic changes with higher resolution, including loss of heterozygosity without changes of gene dosage, so-called acquired uniparental disomy (aUPD). We have examined 33 samples of MCL (28 primary MCL and 5 cell lines) using the 250,000 SNP-chip from Affymetrix. Known alterations were confirmed by SNP arrays, including deletion of INK4A/ARF, duplication/amplification of MYC, deletion of ATM, and deletion of TP53. We also identified a duplication/amplification that occurred at 13q involving oncogenic microRNA, miR17-92. We found other genomic abnormalities, including duplication/amplification of cyclin D1, del(1p), del(6q), dup(3q) and dup(18q). Our SNP-chip analysis detected these abnormalities at high resolution, allowing us to narrow the size of the commonly deleted regions, including 1p and 6q. Our SNP-chip analysis detected a number of aUPD sites, including whole chromosome 9 aUPD and 9p aUPD. We also found an MCL case with 19p, leading to homozygous deletion of TNFSF genes. SNP-chip analysis detected in MCL very small genomic gains/losses, as well as aUPDs, which could not be detected by more conventional methods.

Specific and complete human genome amplification with improved yield achieved by phi29 DNA polymerase and a novel primer at elevated temperature

BackgroundsWhole genome amplification (WGA) is a practical solution to eliminate molecular analysis limitations associated with genomic DNA (gDNA) quantity. Different methods have been developed to amplify the whole genome, including primer extension preamplification (PEP), degenerate oligonucleotide primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Each of these methods has its own merits and limitations.FindingsEffects of primer length and composition on amplification quality and quantity were explored in this study at two different temperatures (30°C & 40°C). New primer designs combined with elevated amplification temperature has significantly improved MDA as measured by amplification yield, genome coverage, and allele drop out (ADO) analysis. A remarkable finding was the comprehensive amplification, at 30°C & 40°C, of the human whole genome via the use of GGGCAGGA*N*G hotspot recombination consensus primer. Amplification was characterized by Affymetrix 10K SNP chip analysis. Finally, the use of new primer designs has suppressed the template-independent DNA amplification (TIDA) both at 30°C and 40°C.ConclusionThe use of new primers in this study combined with elevated incubation temperatures in MDA has remarkably improved the specificity, amplification yield, and suppressed TIDA.

Shorter Telomeres Are Associated with the Unfavourable Chromosomal Aberrations Del 17p and Del 11q in Chronic Lymphocytic Leukemia (CLL)

BACKGROUND: In chronic lymphocytic leukemia (CLL) short telomeres were shown to be associated with mutational status, progression free survival (PFS) and overall survival (Damle et al., 2004). Chromosomal instability increases with shortening of telomeres. Recently, a relationship between telomere length and number of chromosomal aberrations has been shown if telomere length was investigated by quantitative real-time polymerase chain reaction (Tel-PCR) (Roos et al., 2008). The aim of the present study was to correlate average telomere length of individual cells measured by multicolor flow-FISH to established prognostic factors and genomic aberrations.PATIENTS and METHODS: Blood samples from 64 patients with CLL were analyzed. Flow cytometry was performed for quantification of ZAP-70 and CD 38 expression with a cut off at 20%. Immunoglobulin variable heavy chain (IGVH) genes were sequenced. An IGVH gene sequence with less than 98% homology with the corresponding germ-line sequence was considered to be mutated. Chromosomal alterations were investigated by fluorescence in situ hybridization (FISH) with the following gene probes: LSI 13q14, LSI 13q34, CEP 12, LSI 17p13, LSI 11q22–23. Copy number changes were also detected in 18 samples by SNP-chip analysis. The average length of telomere repeats at chromosome ends was measured by multicolor flow-FISH. Values of telomere length from CLL cells were correlated to values of telomere lengths of B lymphocytes from healthy age matched individuals (delta telomere length=Δtel).RESULTS: The average telomere length of the clonal B-cells was short. Patient samples from advanced Binet stages (B/C) had significantly shorter telomeres (Δtel −4.8 ± 1.0 kb) than patients samples from Binet A (Δtel −3.4 ± 1.2 kb, p=0.03). The average telomere length was significantly shorter for ZAP-70+ (Δtel −5.0 ± 0.5 kb) and CD38+ (Δtel −4.9 ± 0.7 kb) patient samples than for ZAP-70− (Δtel −2.4 ± 0.8 kb) and CD38− (Δtel −3.0 ± 1.0 kb) patient samples, respectively (p<0.005, p<0.005). IGVH unmutated CLL samples exhibited significant shorter telomere lengths (Δtel −4.8 ± 0.4 kb) than mutated samples (Δtel −2.8 ± 0.9 kb, p<0.005). Interestingly CLL samples harbouring del 17p and del 11q had significantly shorter average telomere length (Δtel −5.3 ± 0.2 kb, n= 8) than samples without these aberrations (Δtel −4.0 ± 1.2 kb; n= 56, p<0.005). Furthermore we found a tendency of an increase in the number of chromosomal aberrations detected by SNP-chip with shorter telomeres.DISCUSSION: We are able to confirm significant shorter telomeres in CLL samples with unfavourable prognostic factors like advanced Binet stage, positivity for ZAP-70 or CD38 and unmutated IGVH genes compared to their favourable counterparts. CLL samples with the chromosomal aberrations del 17p and del 11q are associated with bad clinical outcome. CLL with these aberrations of the present study demonstrated significantly shorter telomeres compared to cases without these abnormalities. Additional studies relating impact of telomere length on genomic instability detected by SNP-chip are ongoing.

Genomic Changes Associated with Leukemic Transformation of Myeloproliferative Disorders.

Myeloproliferative disorders (MPD) are a group of heterogeneous diseases that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They are characterized by increased hematopoiesis leading to elevated numbers of non-lymphoid cells and/or platelets in the peripheral blood. Beside thrombotic and hemorrhagic complications, MPD may evolve into secondary acute myeloid leukemia (sAML). Recently characterized markers suggest an opportunity to diagnose and identify subpopulations of MPD patients. In particular, altered expression and point mutations of PRV-1, MPL and JAK2 were commonly found in MPD, as well as deletions on chromosome 20q (del20q). Acquired uniparental disomy (UPD) on chromosome 1p (1pUPD) and 9p (9pUPD) leading to copy-neutral loss of heterozygosity (LOH) is a further mechanism found in MPD which often leads to homozygous activated mutations of MPL and JAK2, respectively. However, the molecular mechanisms involved in the transformation process to sAML remains unclear. Using standard metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with MPD. We hypothesized that with new precise methods more genomic lesions can be uncovered that may be associated with leukemic transformation. To address this issue, we used 250K single nucleotide polymorphisms (SNP) Chip arrays to study chromosomal lesions in 40 sAML samples from patients who evolved from MPD; 7 had preexisting PV, 25 PMF, and 8 ET. Moreover, 43 additional samples of MPD (10 PV, 17 ET, and 16 PMF) were included in this study. SNP-chip analysis showed major chromosomal changes in almost all the sAML samples including monosomy 16, deletions of 1q-, 3p-, 6p-, 5q-, 7q-, 9q-, 12p-, 6q-, 3q-, 17p-, 19q-, and 20q- as well as trisomy 2, 3, 8, 9, 12, 15, 19, 21, and 22. We validated these data by MC. However, numerous new genomic alterations which contained potentially interesting genes that might contribute to leukemic transformation were detected by SNP Chip Array in patient samples with normal karyotype. Moreover, UPD was very frequent: 44% (19/43) of MPD and 53 % (21/40) sAML samples. 1pUPD occurred in 5 patients with MPD (1PV, 4 PMF; 12 %) compared to 5 patients with sAML (1 PV, 4 PMF; 13 %). 9pUPD was found in 16 MPD patients (8 PV, 7 PMF, 1 ET; 37%) and 6 sAML patients (3 PV, 2 PMF, 1ET; 15 %). All patients with 9pUPD proved to be positive for the JAK2 V617F mutation seen by allele specific PCR. Interestingly, the MPD samples only had UPD on 1p, 9p, and 12q. In contrast, sAML samples showed additional UPD regions on 7q, 11q, 12q, 16p, 17p, 19q, and 21q. Beside the evaluation of the non-matching groups of patients with MPD and sAML, we also evaluated 4 patients during their PMF and sAML stages by SNP Chip. The sAML samples acquired additional genomic changes including trisomy 8, 10, 14, 19, duplication on 3q and 6p, and heterozygote deletion on 18q. In contrast, 1pUPD, 9pUPD, and 12qUPD were detected in both MPD and sAML matching samples, suggesting that these changes do not play an immediate role in causing transformation. In conclusion, we detected chromosomal regions possessing genes which may be involved in the leukemic transformation of MPD patients.

Signal transduction pathways of mantle cell lymphoma: A phosphoproteome‐based study

Mantle cell lymphoma (MCL) is an incurable hematologic malignancy whose pathogenesis is only partly understood. The aim of the present study was to define a "core phosphoproteome" in MCL cell lines that is representative of primary MCL in order to improve knowledge of the signal transduction pathways involved in its tumorigenesis. We have analyzed phosphorylated proteins in several MCL cell lines by immobilized metal affinity chromatography and separation by 2-D PAGE, followed by RP-HPLC coupled with MS/MS identification. These data were correlated with information on copy number gains obtained by SNP-chip analysis. Several of the proteins identified could be linked to a specific signal transduction pathway, and have been recently recognized as important players in MCL pathogenesis, such as nuclear factor-kappaB (NF-kappaB) and phosphoinositide-3 kinase-mammalian target of rapamycin (PI3K-mTOR). However, our data also implicate a number of novel proteins and pathways in the pathobiology of MCL, one of which is mitochondrial signaling. A second-level analysis identified MAPK1, CK2, CK1, PKCzeta, and PKCepsilon as candidate upstream molecules. Our study provides new insights in MCL pathogenesis and helps to form the basis for testing new target-specific therapeutics.