Development of a Fully Automated Image Analysis Method for High Density cDNA and array CGH Microarray Based Genomic Studies

In this thesis, different techniques for image analysis of high density microarrays have been investigated. Most of the existing image analysis techniques require prior knowledge of image specific parameters and direct user intervention for microarray image quantification. The objective of this research work was to develop of a fully automated image analysis method capable of accurately quantifying the intensity information from high density microarrays images. The method should be robust against noise and contaminations that commonly occur in different stages of microarray development.

Caracterização de rearranjos cromossômicos citogeneticamente equilibrados associados a quadros clínicos

Este estudo teve como objetivos (a) identificar mecanismos pelos quais rearranjos cromossômicos citogeneticamente equilibrados possam estar associados de maneira causal a determinados quadros clínicos e (b) contribuir para a compreensão dos mecanismos de formação desses rearranjos. Para isso, foram estudados 45 rearranjos cromossômicos citogeneticamente equilibrados (29 translocações, 10 inversões e seis rearranjos complexos), detectados em pacientes que apresentavam malformações congênitas, comprometimento do desenvolvimento neuropsicomotor ou déficit intelectual. Foram 31 rearranjos cromossômicos esporádicos, três familiais que segregavam com o quadro clínico e mais 11 rearranjos cromossômicos herdados de genitores fenotipicamente normais. Inicialmente os pontos de quebra desses rearranjos foram mapeados por hibridação in situ fluorescente (FISH). A busca por microdeleções e duplicações genômicas foi realizada por a-CGH. A investigação dos pontos de quebra prosseguiu com a aplicação da técnica de Mate-Pair Sequencing (MPS), que permite localizar as quebras em segmentos de 100 pb - 1 kb, na maioria dos casos. Para obter os segmentos de junção das quebras no nível de pares de bases, os segmentos delimitados por MPS foram sequenciados pelo método de Sanger. A análise por aCGH revelou microdeleções ou microduplicações localizadas nos cromossomos rearranjados, em 12 dos 45 pacientes investigados (27%). A análise de 27 rearranjos por MPS permitiu a caracterização dos pontos de junção das quebras. MPS expandiu o número de pontos de quebra, detectados por análise do cariótipo ou aCGH, de 114 para 156 (em resolução < 2kb, na maioria dos casos). O número de pontos de quebra/rearranjo variou de 2 a 20. Os 156 pontos de quebra resultaram em 86 variantes estruturais equilibradas e outras 32 variantes não equilibradas. Perdas e ganhos de segmentos submiscroscópicos nos cromossomos rearranjados constituíram a principal causa ou, provavelmente, contribuíram para o quadro clínico de 12 dos 45 pacientes. Em cinco desses 12 rearranjos foram detectadas por MPS a interrupção de genes já relacionados à doença, ou provável alteração de sua região reguladora, contribundo para o quadro clínico. Em quatro dos 33 rearranjos não associados a perdas ou ganhos de segmentos, a análise por MPS revelou a interrupção de genes que já foram anteriormente relacionados a doenças, explicando-se, assim, as características clínicas dos portadores; outro rearranjo pode ter levando alteração da expressão gênica de gene sensível a dosagem e ao quadro clínico. Um rearranjo cromossômico familial, identificado na análise após bandamento G como uma translocação equilibrada, t(2;22)(p14;q12), segregava com quadro de atraso do desenvolvimento neuropsicomotor e dificuldade de aprendizado associados a dismorfismos. A combinação das análises por FISH, aCGH e MPS revelou que se tratava, na verdade, de rearranjo complexo entre os cromossomos 2, 5 e 22, incluindo 10 quebras. A segregação de diferentes desequilíbrios submicroscópicos em indivíduos afetados e clinicamente normais permitiu a compreensão da variabilidade clínica observada na família. Rearranjos equilibrados detectados em indivíduos afetados, mas herdados de genitores clinicamente normais, são, em geral, considerados como não tendo relação com o quadro clínico, apesar da possibilidade de desequilíbrios cromossômicos gerados por permuta desigual na meiose do genitor portador do rearranjo. Neste trabalho, a investigação de 11 desses rearranjos por aCGH não revelou perdas ou ganhos de segmentos nos cromossomos rearranjados. No entanto, a análise por aCGH da portadora de um desses rearranjos - inv(12)mat - revelou deleção de 8,7 Mb no cromossomo 8, como causa de seu fenótipo clínico. Essa deleção estava relacionada com outro rearranjo equilibrado também presente em sua mãe, independente da inversão. Para compreender os mecanismos de formação de rearranjos citogeneticamente equilibrados, investigamos os segmentos de junção no nível de pares de base. A análise por MPS que levou, na maioria dos casos, ao mapeamento dos pontos de quebras em segmentos <1kb permitiu o sequenciamento pelo método de Sanger de 51 segmentos de junções de 17 rearranjos. A ocorrência de blunt fusions ou inserções e deleções <10 pb, e a ausência de homologia ou a presença de micro homologia de 2 pb a 4 pb de extensão indicaram o mecanismo de junção de extremidades não homólogas (non-homologous end joinging; NHEJ), na maioria das 51 junções caracterizadas. As características de três dos quatro rearranjos mais complexos, com 17-20 quebras, indicaram sua formação pelo mecanismo de chromothripsis. Este estudo mostra a importância da análise genômica de variações de número de cópias por microarray, juntamente com o mapeamento dos pontos de quebra por MPS, para determinar a estrutura de rearranjos cromossômicos citogeneticamente equilibrados e seu impacto clínico. O mapeamento dos segmentos de junção por MPS, permitindo o sequenciamento pelo método de Sanger, foi essencial para a compreensão de mecanismos de formação desses rearranjos

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.

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.

Microarray based real-time analysis of nucleic acid hybridization kinetics and thermodynamics

Ziel dieser Dissertation ist die experimentelle Charakterisierung und quantitative Beschreibung der Hybridisierung von komplementären Nukleinsäuresträngen mit oberflächengebundenen Fängermolekülen für die Entwicklung von integrierten Biosensoren. Im Gegensatz zu lösungsbasierten Verfahren ist mit Microarray Substraten die Untersuchung vieler Nukleinsäurekombinationen parallel möglich. Als biologisch relevantes Evaluierungssystem wurde das in Eukaryoten universell exprimierte Actin Gen aus unterschiedlichen Pflanzenspezies verwendet. Dieses Testsystem ermöglicht es, nahe verwandte Pflanzenarten auf Grund von geringen Unterschieden in der Gen-Sequenz (SNPs) zu charakterisieren. Aufbauend auf dieses gut studierte Modell eines House-Keeping Genes wurde ein umfassendes Microarray System, bestehend aus kurzen und langen Oligonukleotiden (mit eingebauten LNA-Molekülen), cDNAs sowie DNA und RNA Targets realisiert. Damit konnte ein für online Messung optimiertes Testsystem mit hohen Signalstärken entwickelt werden. Basierend auf den Ergebnissen wurde der gesamte Signalpfad von Nukleinsärekonzentration bis zum digitalen Wert modelliert. Die aus der Entwicklung und den Experimenten gewonnen Erkenntnisse über die Kinetik und Thermodynamik von Hybridisierung sind in drei Publikationen zusammengefasst die das Rückgrat dieser Dissertation bilden. Die erste Publikation beschreibt die Verbesserung der Reproduzierbarkeit und Spezifizität von Microarray Ergebnissen durch online Messung von Kinetik und Thermodynamik gegenüber endpunktbasierten Messungen mit Standard Microarrays. Für die Auswertung der riesigen Datenmengen wurden zwei Algorithmen entwickelt, eine reaktionskinetische Modellierung der Isothermen und ein auf der Fermi-Dirac Statistik beruhende Beschreibung des Schmelzüberganges. Diese Algorithmen werden in der zweiten Publikation beschrieben. Durch die Realisierung von gleichen Sequenzen in den chemisch unterschiedlichen Nukleinsäuren (DNA, RNA und LNA) ist es möglich, definierte Unterschiede in der Konformation des Riboserings und der C5-Methylgruppe der Pyrimidine zu untersuchen. Die kompetitive Wechselwirkung dieser unterschiedlichen Nukleinsäuren gleicher Sequenz und die Auswirkungen auf Kinetik und Thermodynamik ist das Thema der dritten Publikation. Neben der molekularbiologischen und technologischen Entwicklung im Bereich der Sensorik von Hybridisierungsreaktionen oberflächengebundener Nukleinsäuremolekülen, der automatisierten Auswertung und Modellierung der anfallenden Datenmengen und der damit verbundenen besseren quantitativen Beschreibung von Kinetik und Thermodynamik dieser Reaktionen tragen die Ergebnisse zum besseren Verständnis der physikalisch-chemischen Struktur des elementarsten biologischen Moleküls und seiner nach wie vor nicht vollständig verstandenen Spezifizität bei.

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.

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.

Genomic microarrays in chromosomal analysis of leukemia

Chromosomal alterations in leukemia have been shown to have prognostic and predictive significance and are also important minimal residual disease (MRD) markers in the follow-up of leukemia patients. Although specific oncogenes and tumor suppressors have been discovered in some of the chromosomal alterations, the role and target genes of many alterations in leukemia remain unknown. In addition, a number of leukemia patients have a normal karyotype by standard cytogenetics, but have variability in clinical course and are often molecularly heterogeneous. Cytogenetic methods traditionally used in leukemia analysis and diagnostics; G-banding, various fluorescence in situ hybridization (FISH) techniques, and chromosomal comparative genomic hybridization (cCGH), have enormously increased knowledge about the leukemia genome, but have limitations in resolution or in genomic coverage. In the last decade, the development of microarray comparative genomic hybridization (array-CGH, aCGH) for DNA copy number analysis and the SNP microarray (SNP-array) method for simultaneous copy number and loss of heterozygosity (LOH) analysis has enabled investigation of chromosomal and gene alterations genome-wide with high resolution and high throughput. In these studies, genetic alterations were analyzed in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The aim was to screen and characterize genomic alterations that could play role in leukemia pathogenesis by using aCGH and SNP-arrays. One of the most important goals was to screen cryptic alterations in karyotypically normal leukemia patients. In addition, chromosomal changes were evaluated to narrow the target regions, to find new markers, and to obtain tumor suppressor and oncogene candidates. The work presented here shows the capability of aCGH to detect submicroscopic copy number alterations in leukemia, with information about breakpoints and genes involved in the alterations, and that genome-wide microarray analyses with aCGH and SNP-array are advantageous methods in the research and diagnosis of leukemia. The most important findings were the cryptic changes detected with aCGH in karyotypically normal AML and CLL, characterization of amplified genes in 11q marker chromosomes, detection of deletion-based mechanisms of MLL-ARHGEF12 fusion gene formation, and detection of LOH without copy number alteration in karyotypically normal AML. These alterations harbor candidate oncogenes and tumor suppressors for further studies.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

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.

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.

Microarray-Based Study of Carbohydrate-Protein Binding by Gold Nanoparticle Probes

In order to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate or glycoprotein microarrays by immobilizing amino modified carbohydrates on aldehyde-derivatized glass slides or glycoprotein on epoxide-derivatized glass slides and carried out lectin binding experiments by using these microarrays, respectively. The interaction events are marked by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry.

Microarray based Raman spectroscopic detection with gold nanoparticle probes

microarray approach based on surface-enhanced Raman spectroscopic (SERS) was developed for detection of spotted peptide, peptide-protein or protein-antibody interaction. The procedure involves the attachment of peptide-capped gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry. The well-known biomolecular recognition pairs, IgG/protein A and biotin/avidin, were used to demonstrate proof-of-concept of the SERS assay.

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.