Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 with >or=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases with >or=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases with >or=5%TP53-deleted cells (por=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with >20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes

Background: Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity. Results: The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data. Conclusion: After carrying out the analysis and validation for three sequenced Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.

Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer.

Background The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors. Results This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors. Conclusion This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.

Chromosomal aberrations in squamous cell carcinoma and solar keratoses revealed by comparative genomic hybridization

OBJECTIVE: To identify chromosomal copy numbers of frequent genetic aberrations within squamous cell carcinomas (SCCs) and solar keratoses (SKs), and provide further evidence to support or challenge current dogma concerning the relationship between these lesions. DESIGN: Retrospective analysis of genetic aberrations in DNA from SK and SCC biopsy specimens by comparative genomic hybridization. SETTING: University-based research laboratory in Queensland, Australia. PATIENTS: Twenty-two biopsy specimens from patients with diagnosed SKs (n = 7), cutaneous SCCs (n = 10), or adjoining lesions (n = 5). MAIN OUTCOME MEASURES: Identification of frequent genetic aberrations both specific to SK and SCC and shared by these lesions to investigate their clonal relationship. RESULTS: Shared genomic imbalances were identified in SK and SCC. Frequent gains were located at chromosome arms 3q, 17q, 4p, 14q, Xq, 5p, 9q, 8q, 17p, and 20q, whereas shared regional losses were observed at 9p, 3p, 13q, 17p, 11p, 8q, and 18p. Significant loss of 18q was observed only in SCC lesions. CONCLUSIONS: Our results demonstrate that numerous chromosomal aberrations are shared by the 2 lesions, suggesting a clonal relationship between SK and SCC. Additionally, the genomic loss of 18q may be a significant event in SK progression to SCC. Finally, the type and frequency of aberrations suggests a common mode of tumorigenesis in SCC-derived tumors.

Molecular cytogenetic analysis of basal cell carcinoma DNA using comparative genomic hybridization

In an attempt to define genomic copy number changes associated with the development of basal cell carcinoma, we investigated 15 sporadic tumors by comparative genomic hybridization. With the incorporation of tissue microdissection and degenerate oligonucleotide primed-polymerase chain reaction we were able to isolate, and then universally amplify, DNA from the tumor type. This combined approach allows the investigation of chromosomal imbalances within a histologically distinct region of tissue. Using comparative genomic hybridization we have observed novel and recurrent chromosomal gains at 6p (47%), 6q (20%), 9p (20%), 7 (13%), and X (13%). In addition comparative genomic hybridization revealed regional loss on 9q in 33% of tested tumors encompassing 9q22.3 to which the putative tumor suppressor gene, Patched, has been mapped. The deletion of Patched has been indicated in the development of hereditary and sporadic basal cell carcinomas. The identification of these recurrent genetic aberrations suggests that basal cell carcinomas may not be as genetically stable as previously thought. Further investigation of these regions may lead to the identification of other genes responsible for basal cell carcinoma formation.

Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.

Array Comparative Genomic Hybridization in Sarcomas

Over the past years, much research on sarcomas based on low-resolution cytogenetic and molecular cytogenetic methods has been published, leading to the identification of genetic abnormalities partially underlying the tumourigenesis. Continued progress in the identification of genetic events such as copy number aberrations relies upon adapting the rapidly evolving high-resolution microarray technology, which will eventually provide novel insights into sarcoma biology, and targets for both diagnostics and drug development. The aim of this Thesis was to characterize DNA copy number changes that are involved in the pathogenesis of soft tissue leiomyosarcoma (LMS), dermatofibrosarcoma protuberans (DFSP), osteosarcoma (OS), malignant fibrous histiocytoma (MFH), and uterine leiomyosarcoma (ULMS) by applying fine resolution array comparative genomic hybridization (aCGH) technology. Both low- and high-grade LMS tumours showed distinct copy number patterns, in addition to sharing two minimal common regions of gains and losses. Small aberrations were detected by aCGH, which were beyond the resolution of chromosomal comparative genomic hybridization (cCGH). DFSP tumours analysed by aCGH showed gains in 17q, 22q, and 21 additional gained regions, but only one region (22q) with copy number loss. Recurrent amplicons identified in OS by aCGH were 12q11-q15, 8q, 6p12-p21, and 17p. Amplicons 12q and 17p were further characterized in detail. The amplicon at 17p was characterized by aCGH in low- and high-grade LMS, OS, and MFH. In all but one case this amplicon, with minimal common regions of gains at 17p11-p12, started with the distal loss of 17p13-pter. OS and high-grade LMS were grouped together as they showed a complex pattern of copy number gains and amplifications at 17p, whereas MFH and low-grade LMS showed a continuous pattern of copy number gains and amplification at 17p. In addition to the commonly gained and lost regions identified in ULMS by aCGH, various biological processes affected by these copy number changes were also indicated by pathway analysis. The three novel findings obtained in this work were: characterization of amplicon 17p in low- and high-grade LMS and MFH, profiles of DNA copy number changes in LMS, and detection of various pathways affected by copy number changes in ULMS. These studies have not been undertaken previously by aCGH technology, thus this Thesis adds new information regarding DNA copy number changes in sarcomas. In conclusion, the aCGH technique used in this Thesis has provided new insights into the genetics of sarcomas by detecting the precise regions affected by copy number changes and some potential candidate target genes within those regions, which had not been uncovered by previously applied low resolution techniques.

Comparative genomic analysis links karyotypic evolution with genomic evolution in the Indian Muntjac (Muntiacus muntjak vaginalis)

The karyotype of Indian muntjacs (Muntiacus muntjak vaginalis) has been greatly shaped by chromosomal fusion, which leads to its lowest diploid number among the extant known mammals. We present, here, comparative results based on draft sequences of 37 bac

Comparative genomic data of the Avian Phylogenomics Project.

BACKGROUND: The evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics. FINDINGS: The 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses. CONCLUSIONS: Here we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2?kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.

16q24.1 microdeletion in a premature newborn: Usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn.

OBJECTIVE:: Report of a 16q24.1 deletion in a premature newborn, demonstrating the usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn and multiple congenital malformations. DESIGN:: Descriptive case report. SETTING:: Genetic department and neonatal intensive care unit of a tertiary care children's hospital. INTERVENTIONS:: None. PATIENT:: We report the case of a preterm male infant, born at 26 wks of gestation. A cardiac malformation and bilateral hydronephrosis were diagnosed at 19 wks of gestation. Karyotype analysis was normal, and a 22q11.2 microdeletion was excluded by fluorescence in situ hybridization analysis. A cesarean section was performed due to fetal distress. The patient developed persistent pulmonary hypertension unresponsive to mechanical ventilation and nitric oxide treatment and expired at 16 hrs of life. MEASUREMENTS AND MAIN RESULTS:: An autopsy revealed partial atrioventricular canal malformation and showed bilateral dilation of the renal pelvocaliceal system with bilateral ureteral stenosis and annular pancreas. Array-based comparative genomic hybridization analysis (Agilent oligoNT 44K, Agilent Technologies, Santa Clara, CA) showed an interstitial microdeletion encompassing the forkhead box gene cluster in 16q24.1. Review of the pulmonary microscopic examination showed the characteristic features of alveolar capillary dysplasia with misalignment of pulmonary veins. Some features were less prominent due to the gestational age. CONCLUSIONS:: Our review of the literature shows that alveolar capillary dysplasia with misalignment of pulmonary veins is rare but probably underreported. Prematurity is not a usual presentation, and histologic features are difficult to interpret. In our case, array-based comparative genomic hybridization revealed a 16q24.1 deletion, leading to the final diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins. It emphasizes the usefulness of array-based comparative genomic hybridization analysis as a diagnostic tool with implications for both prognosis and management decisions in newborns with refractory persistent pulmonary hypertension and multiple congenital malformations.