Common Breakpoint Research Articles

Base-Pair Resolution Mapping of IGH Translocations in Multiple Myeloma Using Targeted Capture and Massively Parallel Sequencing

AbstractAbstract ▪3490▪This icon denotes a clinically relevant abstractIGH loci translocations in multiple myeloma are primary events in the aetiology of the disease. There are 5 main translocation partner chromosomes which result in the over-expression of key oncogenes. These translocations are t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) and result in the over-expression of MMSET and FGFR3, CCND3, CCND1, MAF and MAFB, respectively. The translocations have a major impact on response and survival with the t(4;14), t(14;16) and t(14;20) resulting in poor prognosis. It is therefore imperative that these chromosomal abnormalities be identified. Translocations have traditionally been identified by fluorescence in situ hybridisation (FISH). Using targeted capture techniques, similar to exome capture technology, followed by massively parallel sequencing it should be possible to identify the translocations and the specific breakpoints.We have developed a targeted capture using the SureSelect (Agilent) system by tiling RNA baits across the IGH locus. Baits covered the V, D and J segments as well as being tiled across the entire constant region, including the switch regions. DNA from samples (n=120) were assayed using 150 ng of DNA and a modified capture protocol. The translocation partner had previously been identified by FISH in 36 samples which comprised 11 t(4;14), 3 t(6;14), 11 t(11;14), 9 t(14;16), 2 t(14;20). The remaining 84 samples were assayed by RQ-PCR for over-expression of the partner oncogenes to determine the translocation. Several identified translocations were verified by PCR.In 90% of samples which had FISH performed the correct IGH translocation was detected using the capture technique. The number of paired reads detecting the translocation varied from 2 to 102. Breakpoints could be determined for all of these samples and were mapped for each translocation group. In the t(4;14) group the breakpoints were clustered around exons 1, 4 and 5, corresponding to the MB4-1, MB4-2 and MB4-3 IgH-MMSET hybrid transcripts. Of the 11 t(4;14) with FISH only 2 did not express FGFR3 and had deletion of der(14). In these samples the breakpoint was located between LETM1 and MMSET, confirming that loss of FGFR3 expression is due to deletion of der(14) and not due to the location of the breakpoint. The sample with the breakpoint furthest from MMSET was located 67 kbp upstream of the start of translation within LETM1, in a position similar to that found in the KMS-11 cell line. In the t(11;14) samples the breakpoints varied dramatically on chromosome 11 but were always centromeric to CCND1. Breakpoints varied from 1.1 kbp centromeric to the start of CCND1 transcription to 1.1 Mbp centromeric, within the PPP6R3 gene. However, most breakpoints (70%) were in the intergenic region between MYEOV and CCND1. The distance from the breakpoint to CCND1 did not inversely correlate with CCND1 expression, in fact the sample with the breakpoint furthest from CCND1, within PPP6R3, had the highest expression of CCND1 as determined by gene expression array. No samples had breakpoints within the mantle cell lymphoma major translocation cluster. However, 2 samples had their breakpoint within 100 bp of one another, indicating a possible common breakpoint. Of the t(6;14) samples 2 had breakpoints in the first intron of CCND3, upstream of the start of translation. The remaining sample had its breakpoint 550 kbp upstream of the transcription start site within UBR2. The t(14;16) samples all had their breakpoints within the last intron of WWOX, 0.48–1.03 Mbp centromeric of MAF and in the location of the common fragile site FRA16D. The breakpoints cluster into 2 groups on either side of the fragile site. The t(14;20) breakpoints were located in the 1.5 Mbp intergenic region centromeric of MAF. The breakpoint furthest from MAF was 1.2 Mbp centromeric of the gene.In conclusion, we have developed and validated a targeted capture and sequencing approach for identifying translocations into the IGH locus in myeloma. This approach is important because of its capacity for high throughput low cost testing strategies that can identify these important prognostic events making a myeloma specific diagnostic platform and personalised medicine a reality for patients with myeloma. Importantly sequence analysis of the peri-breakpoint regions gives insight into molecular mechanisms acting early in the process of myelomagenesis. Disclosures:No relevant conflicts of interest to declare.

MLL Gene Rearrangements in 174 Infants with Acute Leukemia.

AbstractAbstract 2537 Background.MLL gene rearrangements are the most common genetic events in infant leukemia. Up to date more than 100 various MLL rearrangements were described. Purpose.To evaluate the distribution of MLL rearrangements among infants (aged from 1 to 365 days) with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods.174 infants (117 ALL and 57 AML cases) were included in the current study. 11q23/MLL rearrangements were detected by chromosome banding analysis (CBA), fluorescence in-situ hybridization (FISH) and reverse-transcriptase PCR (RT-PCR). CBA was done according to standard procedure. FISH analysis using LSI MLL Dual Color, Break Apart Rearrangement Probe (Abbott Molecular, USA) was performed on at least 200 interphase nuclei and on all available metaphases. RT-PCR was performed as previously described (A. Borkhardt et al.,1994, N. Palisgaard et al., 1998, J. van Dongen et al., 1999). In 39 cases genomic DNA breakpoint was detected in MLL and translocation partner genes by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996. Results.11q23/MLL rearrangements were revealed in 74 ALL patients (63.2%). Among this group MLL-AF4 was detected in the majority of cases (53.5%), less frequently were found MLL-MLLT1, MLL-MLLT3, MLL-MLLT10 and others (fig. 1a). Children with ALL under 6 months of age had significantly higher incidence of MLL rearrangements in comparison with older infants (84.0% vs. 47.8%, p<0.001). MLL-positive patients more frequently had BI-ALL and less frequently BII-ALL than infants without these rearrangements (p<0.001 for both). Fusion gene transcripts were sequenced in 26 MLL-rearranged ALL cases. Depending on breakpoint position within MLL and partner genes we detected 7 different types of MLL-AF4 fusion gene transcript, 3 types of MLL-MLLT1, 2 types of MLL-EPS15. The most common fusion site within MLL gene in ALL patients was exon 11, detected in 14 cases (53.8%). It was confirmed by LDI-PCR, that in addition to common breakpoint location in MLL gene (18 out of 27 cases in intron 11, 4 cases in intron 9) allowed to reveal less frequent breakpoint sites, like intron 12 (1 case), intron 10 (3 cases) and intron 7 (1 case). Interestingly, in the last case where LDI-PCR showed presence of MLL-AF4, this fusion gene transcript was not initially found by RT-PCR, because applied primer set did not cover exon 7. Moreover, due to lack of metaphases this patient was primary misclassified as MLL-rearranged, but MLL-AF4-negative. MLL rearrangements were found in 28 AML cases (49.1%). In AML patients the most common MLL rearrangements were MLL-MLLT10 (32% of cases) and MLL-MLLT3 (28%). Other ones were detected less frequently (fig. 1b). In AML patients frequency of MLL rearrangements was similar in children younger and older than 6 months (p=0.904). Among MLL-positive cases AML M5 were detected significantly more often and AML M7 significantly less frequent than in MLL-negative patients (p=0.024 and p=0.001, correspondingly). The most common breakpoint location within MLL gene in AML patients was intron 9, detected in 6 out of 12 cases (50%). Additional chromosomal abnormalities were revealed in 7 out of 21 MLL-positive AML patients with known karyotype (33%), while complex karyotype was detected in 5 cases (24%). Application of LDI-PCR allowed to verify rare MLL rearrangements, including MLL-AFF3 (1 ALL case), MLL-MYO1F (2 AML cases), MLL-SEPT6 (1 AML case), MLL-SEPT9 (1 AML case) In 4 ALL and 3 AML patients MLL rearrangements with concurrent 3’-deletion of MLL gene were found. 3’-deletion of MLL was not associated with breakpoint position in MLL gene and type of translocation partner gene. None of the patients with 3’-deletions had reciprocal fusion gene. Based on LDI-PCR data we assessed several mechanisms of fusion gene formation. Reciprocal translocations were detected in 29 cases, 3-way translocations in 3 cases, inversions in 5 cases, combination of inversion and insertion in 2 cases. Conclusion.In the current study we precisely characterized large cohort of MLL-rearranged infant acute leukemia patients. Combination of all available techniques, including cytogenetics, FISH, RT-PCR and LDI-PCR can lead to detailed verification of every single MLL rearrangement. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Activation of the two microRNA clusters C19MC and miR-371-3 does not play prominent role in thyroid cancer

Chromosomal rearrangements of band 19q13.4 are frequent cytogenetic alterations in benign thyroid adenomas. Apparently, these alterations lead to the upregulation of genes encoding microRNAs of two clusters mapping to the breakpoint region, i.e. miR-371-3 and C19MC. Since members of both clusters have been associated with neoplastic growth in other tumor entities the question arises whether or not their upregulation predisposes to malignant transformation of follicular cells of the thyroid. To address this question we have quantified the expression of miR-372 and miR-520c-3p in samples of 114 thyroid cancers including eight anaplastic thyroid carcinomas, 25 follicular thyroid carcinomas, 78 papillary thyroid carcinomas (including 13 follicular variants thereof), two medullary thyroid carcinomas and one oncocytic thyroid carcinoma. Additionally, we quantified miR-371a-3p and miR-519a-3p in selected samples. While in neither of the cases miR-520c-3p and miR-519a-3p were found to be upregulated, one papillary and one anaplastic thyroid carcinoma, respectively, showed upregulation of miR-372 and miR-371a-3p. However, in these cases fluorescence in situ hybridization did not reveal rearrangements of the common breakpoint region as affected in adenomas. Thus, these rearrangements do apparently not play a major role as first steps in malignant transformation of the thyroid epithelium. Moreover, there is no evidence that 19q13.4 rearrangements characterize a subgroup of thyroid adenomas associated with a higher risk to undergo malignant transformation. Vice versa, the mechanisms by which 19q13.4 rearrangements contribute to benign tumorigenesis in the thyroid remain to be elucidated.

Early recurrence in standard-risk medulloblastoma patients with the common idic(17)(p11.2) rearrangement

Medulloblastoma is diagnosed histologically; treatment depends on staging and age of onset. Whereas clinical factors identify a standard- and a high-risk population, these findings cannot differentiate which standard-risk patients will relapse and die. Outcome is thought to be influenced by tumor subtype and molecular alterations. Poor prognosis has been associated with isochromosome (i)17q in some but not all studies. In most instances, molecular investigations document that i17q is not a true isochromosome but rather an isodicentric chromosome, idic(17)(p11.2), with rearrangement breakpoints mapping within the REPA/REPB region on 17p11.2. This study explores the clinical utility of testing for idic(17)(p11.2) rearrangements using an assay based on fluorescent in situ hybridization (FISH). This test was applied to 58 consecutive standard- and high-risk medulloblastomas with a 5-year minimum of clinical follow-up. The presence of i17q (ie, including cases not involving the common breakpoint), idic(17)(p11.2), and histologic subtype was correlated with clinical outcome. Overall survival (OS) and disease-free survival (DFS) were consistent with literature reports. Fourteen patients (25%) had i17q, with 10 (18%) involving the common isodicentric rearrangement. The presence of i17q was associated with a poor prognosis. OS and DFS were poor in all cases with anaplasia (4), unresectable disease (7), and metastases at presentation (10); however, patients with standard-risk tumors fared better. Of these 44 cases, tumors with idic(17)(p11.2) were associated with significantly worse patient outcomes and shorter mean DFS. FISH detection of idic(17)(p11.2) may be useful for risk stratification in standard-risk patients. The presence of this abnormal chromosome is associated with early recurrence of medulloblastoma.

Abstract 3117: Complex genomic regions associated with ERBB2 amplification in breast cancer

Abstract Repeated DNA segments (Low Copy Repeats, Segmental Duplications) are highly homologous sequences within the human genome. They are typically 10-300 kb in length, and bear &amp;gt;95% sequence identity. In germline, repeated DNA segments mediate recurrent DNA rearrangements and thus cause specific genetic disorders called genomic disorders. Repeated genomic segments could also be prone to rearrangements in somatic cells and thus could mediate recurrent rearrangements in cancer. However, the important role of repeated genomic segments in genome instability in cancer remains elusive, mainly because such regions are less accessible by current genomic technologies due to the difficulty in assembling and interrogating sequences. Gene amplification is a cellular process characterized by the production of multiple copies of a particular gene through DNA rearrangements. ERBB2, an epidermal growth factor receptor gene, is amplified in 15-20% of breast tumors. Due to excessive cell growth signaling, this recurrent amplification is associated with advanced stages and poor prognosis. Despite the clinical importance, the mechanisms giving rises to recurrent ERBB2 amplification remains unclear. We hypothesized that, similar to the recurrent rearrangements for genomic disorders, repeated genomic segments cause DNA rearrangements for ERBB2 amplification. To test the hypothesis and understand the mechanism of ERBB2 amplification, we mapped a common copy-number breakpoint for primary ERBB2-amplified (HER2-positive) breast tumors. Copy number breakpoints were examined at every 300-kilo bases (kb) from the location of ERBB2 to its telomeric region using real-time PCR based assays. The breakpoint that marked a region between normal copy number and low level amplification resided within a 300-kb complex genomic region at the 1.5 mega-bases telomeric side of ERBB2. The region contains many repeated segments from two gene families. Furthermore, the HapMap SNPs, a set of SNPs that tag haploblocks in the human genome, are less well-defined, indicating the difficulty in interrogating the genomic region by current genomic technologies. Normal copy-number variations seem common, as there is several deletion polymorphisms reported in the region. Two sub-regions, each covering 40-kb region, are particularly complex due to a number of direct and inverted repeats. The prevalence of this copy number breakpoint in HER2-positive breast tumors was also shown in a study by Sircoulomb et al.,(BMC Cancer 2010), in which common amplifications and deletions were determined for 54 HER-2 positive breast tumors. Defining (1) the copy-number breakpoint with very high resolution and (2) critical DNA sequences for ERBB2 amplification will lead to better understanding the amplification mechanism underlying this aggressive type of breast cancer. 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 3117. doi:1538-7445.AM2012-3117

Genomic vulnerability to LINE-1 hypomethylation is a potential determinant of the clinicogenetic features of multiple myeloma

BackgroundThe aim of this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM).MethodsWe assessed global methylation levels using four repetitive elements (long interspersed nuclear element-1 (LINE-1), Alu Ya5, Alu Yb8, and Satellite-α) in clinical samples comprising 74 MM samples and 11 benign control samples (7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPC)). We also evaluated copy-number alterations using array-based comparative genomic hybridization, and performed methyl-CpG binding domain sequencing (MBD-seq).ResultsGlobal levels of the repetitive-element methylation declined with the degree of malignancy of plasma cells (NPC>MGUS>MM), and there was a significant inverse correlation between the degree of genomic loss and the LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA-methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also declined during the development of MM. We confirmed that levels of methylation of the 5' untranslated region of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and poorer overall survival (hazard ratio 2.8, P = 0.015).ConclusionGlobal hypomethylation of LINE-1 is associated with the progression of and poorer prognosis for MM, possibly due to frequent copy-number loss.

Association between deletion size and important phenotypes expands the genomic region of interest in Phelan–McDermid syndrome (22q13 deletion syndrome)

BackgroundThe clinical features of Phelan–McDermid syndrome (also known as 22q13 deletion syndrome) are highly variable and include hypotonia, speech and other developmental delays, autistic traits and mildly dysmorphic features. Patient...

Delineation of Subtelomeric Deletion of the Long Arm of Chromosome 6

Pure subtelomeric deletion of the long arm of chromosome 6 is rare. The frequency of this deletion accounts for approximately 0.05% of subjects with intellectual disability and developmental delay with or without dysmorphic features. Common phenotypes associated with this deletion include intellectual disability, developmental delay, dysmorphic features, seizure, hypotonia, microcephaly and hypoplasia of the corpus callosum. The smallest overlapped region is approximately 0.4 Mb, and contains three known genes. Of these genes, TBP has been considered as a plausible candidate gene for the phenotype in patients with a subtelomeric 6q deletion. Analysis of the breakpoints in 14 cases revealed a potential common breakpoint interval 8.0-9.0 Mb from the chromosome 6q terminus where the FRA6E fragile site exists and the PARK2 gene is located. This suggests that breakage at the FRA6E fragile site may be the mechanism behind chromosome 6q subtelomeric deletion in some of the cases.

Role of Phosphoinositide-Specific Phospholipase C η2 in Isolated and Syndromic Mental Retardation

Deletions in the distal region of the short arm of chromosome 1 (1p36) are widely diffuse, both as somatic abnormalities in tumors and as constitutive in the congenital 1p36 deletion syndrome. The deletion size varies from 1.5 to 10 Mb, with common breakpoints located from 1p36.13 to 1p36.33. Patients bearing constitutional deletion of a smaller region, 1p36.3, present with a number of features, including mental retardation. The gene PLCH2, codifying for the phosphoinositide-specific phospholipase C (PI-PLC) η2, maps on the 1p36.32 region. PI-PLC η2, expressed in the brain after birth, is a key enzyme in cellular calcium mobilization. In the brain, calcium plays a role in axon growth and retraction, growth cone guidance, synapse formation, and responses to various neurotransmitters. For its role in the nervous system, PI-PLC η2 might be a putative candidate gene for the neurodevelopmental delay observed in patients bearing 1p36.3 deletions.

Undifferentiated Embryonal Sarcoma of the Liver is Associated with Mesenchymal Hamartoma and Multiple Chromosomal Abnormalities: A Review of Eleven Cases

Undifferentiated embryonal sarcoma (UES) of the liver is a primitive mesenchymal, malignant neoplasm occurring in children. The link between UES and mesenchymal hamartoma (MH) is controversial. Whether they share the same histiogenesis, representing 2 ends of a spectrum, or are distinct entities is unclear. The genetic aberrations of these neoplasms are not well understood, although a common breakpoint (19q13.4) was recently identified. The purpose of this study was to elucidate immunohistochemical markers that may establish a link between the 2 tumors by reviewing cases of UES and MH. Cases of UES from 1990 to 2008 were identified. Clinical demographics were reviewed. Hematoxylin and eosin staining and immunohistochemical staining for vimentin, alpha-1 antitrypsin, and alpha-fetoprotein were performed. Eleven children were diagnosed with UES. Five cases were seen arising in association with MH, and transitional zones were evident. The mean age at presentation was 10 years. To our knowledge, the 11-month-old patient is the youngest reported case of UES in concurrence with MH. All UES tumor cells were positive for vimentin, diastase-resistant periodic acid-Schiff stain, and alpha-1 antitrypsin. Chromosomal analysis of 3 UES cases, 2 arising with MH, showed complex karyotypes with no involvement of 19q13.4. We suggest a continuum between UES and MH. Although a chromosomal anomaly of 19q13.4 was not identified, a submicroscopic involvement of this locus cannot be excluded. Additionally, our analyses suggest that multiple chromosomal aberrations may be associated with the MH/UES spectrum.

CML with e6a2 BCR-ABL1 transcript: an aggressive entity?

Dear Editor, The BCR-ABL1 fusion gene is characteristic of CML. Four different breakpoint cluster regions (bcr) have been described in BCR. The most common BCR breakpoints, in 95% of CML cases, occur in M-BCR, i.e., introns 13 or 14, resulting in an e13a2 (b2a2) or an e14a2 (b3a2) BCR-ABL1 transcript. “Atypical” transcripts, e8a2, e19a2, e13a3, e14a3, e1a3, and e6a2 BCR-ABL1, account for less than 1% of CML cases [1, 2]. Only 11 CML patients have been reported with the e6a2 variant. A male predominance and a worse outcome have been suggested for this variant [3]. Here, we describe an additional CML case with an e6a2 fusion transcript. This 57-year-old male patient presented with hepatosplenomegaly, weight loss, cough, and fever. Laboratory examination showed a hemoglobin level of 12.1 g/dL, a white blood cell count of 43.5×10*9/L with 5% myeloblasts and 10% eosinophils, a platelet count of 129×10*9/L, and an LDH of 3,363 U/L (normal value, 313–618 U/L). An abdominal CT scan confirmed the hepatomegaly (craniocaudal diameter of 24 cm) and splenomegaly (bipolar diameter of 20 cm). Sokal score was 2.11, and Hasford score, 2,211.6 (both high risk). Bone marrow aspirate showed a hypocellular marrow with 10% myeloblasts and marked eosinophilia. On trephine biopsy, a myeloproliferative disease with striking fibrosis was seen. Taqman RQ-PCR was negative for e13a2/e14a2 transcripts and weakly positive for the e1a2 transcript in peripheral blood. A subsequent cytogenetic analysis showed the t(9;22)(q34;q11) translocation in all metaphases and trisomy of chromosome 8 in 6 out of 10 metaphases, indicating clonal evolution. Fluorescent in situ hybridization (FISH), using the Vysis LSI BCR/ABL1 extra signal dual color translocation probe, revealed, in addition to the expected fusion signal on der(22), a small fusion on der(9), further suggesting the presence of a variant breakpoint, located centromeric to M-BCR (Fig. 1). Qualitative reverse transcription polymerase chain reaction (RT-PCR), using forward primers in BCR exon 1 and reverse primers in ABL1 exon 2, confirmed the presence of a variant transcript of 502 bp. This was identified as e6a2 after Sanger sequencing. Based on these findings, a diagnosis of CML in an accelerated phase was made, and the patient was started on imatinib 600 mg daily. Three months later, while still on imatinib, he was readmitted with CML in blast crisis. He had cutaneous chloromas, severe abdominal pain, a productive cough, and life-threatening dyspnea, necessitating intubation and mechanical ventilation. Peripheral leukocyte count was 465× 10*9/L with 29% myeloblasts; he had severe anemia (Hb, 6.5 g/L), thrombocytopenia (75×10*9/L), and an LDH of 10,612 U/L. Chest CT scan revealed multiple pathologically enlarged mediastinal and hilar lymph nodes. Fundoscopic examination showed features of hyperviscosity. Peripheral blood karyotype and FISH now showed an extra derivative der(22)t(9;22) in ~50% of cells. Following leukapheresis and PV is a senior clinical investigator of the Fund for Scientific ResearchFlanders (Belgium) (FWO Vlaanderen). K. A. Beel : J. Maertens Department of Hematology, Internal Medicine, University Hospital Leuven, Leuven, Belgium

Genomic imbalances in esophageal carcinoma cell lines involve Wnt pathway genes

To identify molecular markers shared across South African esophageal squamous cell carcinoma (ESCC) cell lines using cytogenetics, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism (SNP) array copy number analysis. We used conventional cytogenetics, FISH, and multicolor FISH to characterize the chromosomal rearrangements of five ESCC cell lines established in South Africa. The whole genome copy number profile was established from 250K SNP arrays, and data was analyzed with the CNAT 4.0 and GISTIC software. We detected common translocation breakpoints involving chromosomes 1p11-12 and 3p11.2, the latter correlated with the deletion, or interruption of the EPHA3 gene. The most significant amplifications involved the following chromosomal regions and genes: 11q13.3 (CCND1, FGF3, FGF4, FGF19, MYEOV), 8q24.21(C-MYC, FAM84B), 11q22.1-q22.3 (BIRC2, BIRC3), 5p15.2 (CTNND2), 3q11.2-q12.2 (MINA) and 18p11.32 (TYMS, YES1). The significant deletions included 1p31.2-p31.1 (CTH, GADD45α, DIRAS3), 2q22.1 (LRP1B), 3p12.1-p14.2 (FHIT), 4q22.1-q32.1 (CASP6, SMAD1), 8p23.2-q11.1 (BNIP3L) and 18q21.1-q21.2 (SMAD4, DCC). The 3p11.2 translocation breakpoint was shared across four cell lines, supporting a role for genes involved at this site, in particular, the EPHA3 gene which has previously been reported to be deleted in ESCC. The finding that a significant number of genes that were amplified (FGF3, FGF4, FGF19, CCND1 and C-MYC) or deleted (SFRP2 gene) are involved in the Wnt and fibroblast growth factor signaling pathways, suggests that these pathways may be activated in these cell lines.

Longitudinal analysis of HIV-1 BF1 recombinant strains in vertically infected children from Argentina reveals a decrease in CRF12_BF pol gene mosaic patterns and high diversity of BF unique recombinant forms

The HIV-1 epidemic associated to BF1 recombinants in South America is both complex and intriguing, with an underestimated diversity of recombinant structures. Our aim was to explore the characteristics and temporal dynamics of the HIV-1 BF1 epidemic in Argentina, through the study of 172 HIV-1 pol BF1 recombinant sequences obtained from HIV-1 vertically infected patients born from 1986 to 2008. Recombination patterns were characterized by bootscanning, subtype signature analysis, and phylogenetic approaches. Proportion of sequences sharing common ancestry and recombination breakpoints with the Circulating Recombinant Form (CRF) CRF12_BF was compared against sequences with a non-CRF12_BF pattern in three study periods, and by fitting the data to a logistic model. Twenty-eight HIV-1 pol BF1 mosaic structures were identified, including four of the seven South-American CRF_BF-like patterns. However, common ancestry of these sequences with reference CRF strains only confirmed the presence of CRF12_BF (51.1%) and CRF17_BF (1.2%) among the Argentine BF pol sequences. Most non-CRF_BF-like recombinant patterns shared at least one common recombination breakpoint with CRF12_BF. The number of transmissions caused by CRF12_BF viruses decreased in a linear way over time, from 69% in the period 1986–1993 to 46% in 2001–2008. In conclusion, the diversity of HIV-1 pol BF1 recombinant structures in Argentina is much more complex than previously described, with at least two CRFs_BF and 26 BF1 unique recombinant forms. For the first time, we provide evidence of a decrease in the proportion of CRF12_BF viruses transmitted from mother-to-child since the start of the epidemic to the present time in Argentina.

Mapping of Genetic Abnormalities of Primary Tumours from Metastatic CRC by High-Resolution SNP Arrays

BackgroundFor years, the genetics of metastatic colorectal cancer (CRC) have been studied using a variety of techniques. However, most of the approaches employed so far have a relatively limited resolution which hampers detailed characterization of the common recurrent chromosomal breakpoints as well as the identification of small regions carrying genetic changes and the genes involved in them.Methodology/Principal FindingsHere we applied 500K SNP arrays to map the most common chromosomal lesions present at diagnosis in a series of 23 primary tumours from sporadic CRC patients who had developed liver metastasis. Overall our results confirm that the genetic profile of metastatic CRC is defined by imbalanced gains of chromosomes 7, 8q, 11q, 13q, 20q and X together with losses of the 1p, 8p, 17p and 18q chromosome regions. In addition, SNP-array studies allowed the identification of small (<1.3 Mb) and extensive/large (>1.5 Mb) altered DNA sequences, many of which contain cancer genes known to be involved in CRC and the metastatic process. Detailed characterization of the breakpoint regions for the altered chromosomes showed four recurrent breakpoints at chromosomes 1p12, 8p12, 17p11.2 and 20p12.1; interestingly, the most frequently observed recurrent chromosomal breakpoint was localized at 17p11.2 and systematically targeted the FAM27L gene, whose role in CRC deserves further investigations.Conclusions/SignificanceIn summary, in the present study we provide a detailed map of the genetic abnormalities of primary tumours from metastatic CRC patients, which confirm and extend on previous observations as regards the identification of genes potentially involved in development of CRC and the metastatic process.

RecDraw: A Software Package for the Representation of HIV-1 Recombinant Structures

The crucial role of recombination in HIV-1 biology is being increasingly recognized. In vitro studies have shown that up to 30 strand-transfer events may occur per viral replication cycle. Thus, recombination may surpass mutation as a major mechanism driving HIV-1 evolution. Currently, recombinant strains comprise 37% of the full-genome HIV-1 sequence database, including sequences representing 47 Circulating Recombinant Forms (CRFs) and more than 250 different Unique Recombinant Forms (URFs). Mapping of recombination breakpoints helps establish relationships among strains that are related by descent, such as CRF07_BC and CRF08_BC in China, and sheds light on their origin and epidemic spread. Additionally, unrelated recombinants sharing common breakpoints may reflect recombination hotspots within the viral genome. Here we present a software tool, RecDraw, for the graphical representation and efficient comparison of recombinant HIV-1 structures and breakpoints. RecDraw is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/research.php?ServiceID = 5&SubServiceID = 6 .

Mutation and homozygous deletion of ARHGEF10 in bladder cancer; a candidate tumour suppressor gene at 8p23.3

Loss of chromosome arm 8p is found in urothelial carcinoma (UC) and other epithelial cancers and is associated with a more advanced tumour stage. The pattern of complex copy number loss without clear common breakpoints may reflect the need to lose several regions on 8p. We have been examining genomic changes throughout 8p using array CGH on a panel of 33 UC cell lines and 174 bladder tumours, followed by a more detailed investigation of the changes in the cell lines. One of the emerging regions of interest is 8p23.3.

Time series analysis based on running Mann-Whitney Z Statistics

A time series analysis method based on the calculation of Mann–Whitney U statistics is described. This method samples data rankings over running time windows, converts those samples to Mann–Whitney U statistics, and then normalizes the U statistics to Z statistics using Monte-Carlo generated null parameters. Based on the Z statistics’ magnitudes this algorithm can identify time windows containing significant incidences of low or high data rankings, where the window length is determined by the sample size. By repeating this process with sampling windows of varying duration ranking regimes of arbitrary onset and duration can be objectively identified in a time series. The simplicity of the procedure's output – a time series’ most significant non-overlapping ranking sequences – makes it possible to graphically identify common temporal breakpoints and patterns of variability in the analyses of multiple time series. This approach is demonstrated using United States annual temperature data during 1896–2008.

Constructing Defined Chromosome Segmental Duplications in Maize

We describe the production of chromosome segmental duplications and deletions of defined length in maize. The method utilizes a collection of previously isolated transposon-induced reciprocal translocations with sequence-defined breakpoints. These balanced translocations involve a first common breakpoint at the site of transposon Ac/fAc insertions on chromosome 1S, and a second variable endpoint elsewhere in the genome. Stocks containing translocations with different endpoints on the same chromosome arms were crossed together to generate heterozygous combinations. Subsequent meiotic segregation generates gametes that contain the original balanced translocations, or pairs of translocation chromosomes that are genetically unbalanced; i.e., they contain a duplication or deletion of the affected segment. Self-pollination produces progeny plants that are genetically balanced, or that contain 0 to 4 copies of the affected segment. The chromosome constitution of progeny plants can be determined using PCR primers flanking the translocation breakpoints. In addition, plants segregating genetically unbalanced gametes exhibit significant pollen grain abnormalities and thus can be easily detected in the field. In this way, we constructed 4 different segmental tetrasomic stocks involving genomic regions of approximately 7.8, 18.7, 21, and 42.6 Mb on chromosomes 5S, 5L, 5L, and 4L, respectively. FISH analysis using specific probes located within the affected segments confirms their aneuploid composition. These and similar lines containing precisely defined segmental duplications and deletions may be useful to study the genetic impact of gene dosage.

Characterization of Two Human Lung Adenocarcinoma Cell Lines by Reciprocal Chromosome Painting

Lung cancer is a leading cause of cancer death worldwide. Some lung cancer patients correlate with a gas of radon besides smoking. To search for common chromosomal aberrations in lung cancer cell lines established from patients induced by different factors, a combined approach of chromosome sorting, forward and reverse chromosome painting was used to characterize karyotypes of two lung adenocarcinoma cell lines: A549 and GLC-82 with the latter line derived from a patient who has suffered long-term exposure to environmental radon gas pollution. The chromosome painting results revealed that complex chromosomal rearrangements occurred in these two lung adenocarcinoma cell lines. Thirteen and twenty-four abnormal chromosomes were identified in A549 and GLC-82 cell lines, respectively. Almost half of abnormal chromosomes in these two cell lines were formed by non-reciprocal translocations, the others were derived from deletions and duplication/or amplification in some chromosomal regions. Furthermore, two apparently common breakpoints, HSA8q24 and 12q14 were found in these two lung cancer cell lines.

Genomic aberrations in borderline ovarian tumors

BackgroundAccording to the scientific literature, less than 30 borderline ovarian tumors have been karyotyped and less than 100 analyzed for genomic imbalances by CGH.MethodsWe report a series of borderline ovarian tumors (n = 23) analyzed by G-banding and karyotyping as well as high resolution CGH; in addition, the tumors were analyzed for microsatellite stability status and by FISH for possible 6q deletion.ResultsAll informative tumors were microsatellite stable and none had a deletion in 6q27. All cases with an abnormal karyotype had simple chromosomal aberrations with +7 and +12 as the most common. In three tumors with single structural rearrangements, a common breakpoint in 3q13 was detected. The major copy number changes detected in the borderline tumors were gains from chromosome arms 2q, 6q, 8q, 9p, and 13q and losses from 1p, 12q, 14q, 15q, 16p, 17p, 17q, 19p, 19q, and 22q. The series included five pairs of bilateral tumors and, in two of these pairs, informative data were obtained as to their clonal relationship. In both pairs, similarities were found between the tumors from the right and left side, strongly indicating that bilaterality had occurred via a metastatic process. The bilateral tumors as a group showed more aberrations than did the unilateral ones, consistent with the view that bilaterality is a sign of more advanced disease.ConclusionBecause some of the imbalances found in borderline ovarian tumors seem to be similar to imbalances already known from the more extensively studied overt ovarian carcinomas, we speculate that the subset of borderline tumors with detectable imbalances or karyotypic aberrations may contain a smaller subset of tumors with a tendency to develop a more malignant phenotype. The group of borderline tumors with no imbalances would, in this line of thinking, have less or no propensity for clonal evolution and development to full-blown carcinomas.