Dissertations / Theses on the topic 'Cytogenetic abnormalities'

SCC cell lines were karyotyped to identify recurrent aberrations and those providing a selective advantage in individual cases. Karyotypic heterogeneity provided evidence for ongoing chromosomal instability (CIN) in all cell lines but revealed discordance between the levels of numerical CIN (N-CIN) and structural CIN (S-CIN), supporting the notion that different mechanisms underlie these processes. This molecular cytogenetic analysis identified a novel reciprocal translocation t(8:12)(p21.3;p13.31) present in all cells of the SCC cell line MS751, indicative of a selective advantage. The rearrangement resulted in two novel fusion genes, PEX5-LPL on der(8) and LPL-PEX5 on der(12). LPL-PEX5 encodes a truncated transcript but PEX5-LPL encodes a full length chimeric protein, comprising the first exon of PEX5 followed by the majority of LPL coding region, and is the most likely candidate for having driven selection of the translocation. Reverse transcription PCR was used to show that LPL is generally expressed at negligible levels in the cervix whereas PEX5 is expressed constitutively. In concordance, PEX5-LPL was expressed at substantially higher levels than LPL-PEX5. The function of PEX5-LPL might be to drive aberrant expression of the 3’ partner or the chimeric protein might have a modified or novel function. Overexpression of LPL relative to normal cervix was found in over one third of cervical SCC cell lines and primary tumour samples, suggesting it is common in cervical SCC. Findings suggest that PEX5-LPL and LPL overexpression have similar roles in cervical carcinogenesis; functional studies of overexpression elucidated a role for both proteins in increased cellular invasion. The functionally important domain might be shared between these proteins and is most likely to be in the C terminal region of LPL. This C terminal domain may be considered as a novel candidate for targeted therapy of cervical SCC.

A study was undertaken to determine whether cytogenetic abnormalities can be identified in an invasive melanoma cell population that has been selected in vitro out of a larger cell population of low invasive potential. The selecting agent was a denuded human amniotic membrane situated within Mega-Membrane Invasion Culture System chambers. Invasive cells were collected, grown, and harvested for cytogenetic analysis. Metaphases of these cells were examined for chromosomal abnormalities and for evidence of gene amplification in the form of double minute chromosomes. Invasive cell lines evinced changes in their degree of aneuploidy which were not seen in parental control lines of the same passage number. Significant karyotypic abnormalities identified in invasive cell lines were an increased dosage of chromosome 7 and multiple 1q translocation marker chromosomes. Double minute chromosomes were found in up to 18% of invasive cell metaphases examined and in 3% of parental controls. The incidence of double minutes was found to decrease as a function of passage number.

Chromosome banding analysis of human glial tumors was performed using G- and Q-banding techniques in an attempt to establish recurring sites of chromosome change. Results revealed a nonrandom karyotypic profile including aneuploidy and considerable variation in chromosome number (range 40 → 200). All tumors examined displayed numerical abnormalities, with the most common numeric change being a gain of chromosome 7. Chromosomes most frequently involved in structural abnormalities included #1, #3, #7, and #11. Double minutes, reported to be frequently associated with human glial tumors, were observed in only one of ten tumors examined. These results (taken in conjunction with previously published reports) suggest that the single most frequently altered chromosome in human glial tumors is chromosome 7. An attempt was then made to correlate the observed chromosome 7 changes with activation of the cellular proto-oncogene c-erb-B, whose product is the epidermal growth factor receptor (EGFR). Six human glial tumors were analyzed for ¹²⁵I-EGF binding, EGFR gene copy number, EGFR gene rearrangement, mRNA expression, and karyotypic profile. Saturation analysis at 4°C revealed significant numbers of EGFR's in all 6 tumors. Southern blotting analysis utilizing cDNA probes for the EGFR failed to demonstrate significant amplification or structural rearrangement of the EFGR gene. Analysis of EGFR mRNA revealed significant levels in 3 of the tumors studied as compared to the A341 cell line. Karyotypic analysis revealed that all six cell lines displayed extra copies of both whole and structurally altered chromosome 7. These results may suggest that EGFR overexpression is associated with alterations of chromosome 7 (the locus for the EGFR gene). In contrast to previous reports, EGFR mRNA levels did not directly parallel EGF receptor numbers. These results suggest that overexpression of the EGFR may be related to an alternative mechanism, other than gene amplification and elevated mRNA levels, such as the regulation of receptor biosynthesis and degradation. In summary, findings indicate that alterations of chromosome 7 are the most prevalent chromosomal change in human glial tumors, and that these alterations may lead to overexpression of the proto-oncogene c-erb-B.

Chromosome abnormalities are observed very frequently in humans. Several types of structural chromosome abnormalities have been identified, with chromosome translocations, both reciprocal and Robertsonian, being the most common in the population. Balanced carriers of such rearrangements could be at risk of generating abnormal offspring due to the meiotic segregation of the translocation. Preimplantation Genetic Diagnosis (PGD) has allowed the extensive cytogenetic investigation of embryos from such patients with the application of Fluorescent in situ hybridisation (FISH). The first part of this work involved the development of robust three-colour FISH protocols for their clinical application for the PGD for three reciprocal translocations, two different Robertsonian translocations and two cases of suspected gonadal mosaicism. Five of these patients underwent 1-2 cycles of treatment, and 21 normal/balanced embryos were detected and transferred to the maternal uterus. One clinical pregnancy was established with a subsequent live birth of a healthy male infant in a case of a female reciprocal translocation carrier. Extensive FISH examination of the non-transferred embryos showed evidence of post-zygotic mosaicism in 73.4% of them, with chaotic embryos predominating. Both meiotic and mitotic mechanisms leading to chromosome gain and/or loss were identified in this group of embryos.

Introduction: Advances in diagnosis and screening of preimplantation embryos or oocytes for chromosomal abnormalities have helped many couples achieve a normal pregnancy. They also pointed to the fact that numerical and structural chromosomal abnormalities are frequent in human preimplantation embryos and can arise at any point during gametogenesis and meiosis through to early embryonic development and mitotic division. However, information coming from studies in this area is far from complete and uniform.;Aim: To investigate aneuploidy and its mechanisms in human preimplantation embryos and oocytes. To develop protocols and improve on existing molecular cytogenetic techniques for the advance of preimplantation genetic diagnosis or screening (PGD/PGS) in routine clinical analysis. To evaluate the impact of PGD and PGS on the treatment of various types of infertility.;Methods: Fluorescent In situ Hybridisation (FISH) and Comparative genomic hybridisation (CGH) were the main methods used. I) Protocols were developed and implemented for the clinical PGD and PGS program. The PGD protocols included 2 couples with rare structural chromosomal abnormalities II) All untransferred embryos were studied and information was obtained for 101 PGS cycles (77 couples-935 embryos) and 18 PGD cycles for structural chromosomal abnormalities. III) Immature and undivided oocytes were studied using CGH from PGS, PGD and routine IVF couples.;Results and discussion: Specific and highly efficient methods and their clinical application to detect a variety of rare and common chromosomal abnormalities in PGD and PGS embryos were achieved. This study adds to the accumulating evidence showing the extent and mechanisms of genetic abnormalities in human oocytes and preimplantation embryos. It is one of the first studies to identify significant differences in the types of chromosomal abnormalities in embryos from couples with different reproductive history suggesting susceptibility to particular types of aneuploidy in these couples. The problems and effectiveness of PGS and PGD are also discussed.

The bacterial cell wall surrounds the cytoplasmic membrane and protects the cell against osmolysis in addition to providing shape. The cell wall is comprised of peptidoglycan, repeating units of N-acetly glucosamine and N-acetyl muramic acid form glycan strands and are crosslinked by short peptides that contain both L- and D-amino acids. Owing to the unique nature of peptidoglycan, and its absence in eukaryotic organisms, the cell wall has become an important target for many antibiotics, including the β-lactams and glycopeptides. Newly synthesised peptidoglycan contains pentapeptides, which extend from the lactyl moiety of the MurNAc sugar. These chains consist of L-alanine-D-γ- glutamate/glutamine-L-lysine/meso-diaminopimelic acid-D-alanine-D-alanine. The terminal D-alanine is often lost during cell wall maturation, either as a result of the crosslinking reaction, in which the penultimate D-alanine is attached to the side-chain of a neighbouring L-lysine or meso-diaminopimelic acid by an isopeptide bond, or as a consequence of the activities of DD-carboxypeptidases, and results in a tetrapeptide. The tetrapeptide can then be trimmed further to form a tripeptide by the action of LD-carboxypeptidases. Although many DD-carboxypeptidases have been well characterised, the majority of LD-carboxypeptidases that have been studied are active only against peptidoglycan fragments and so cannot be responsible for producing the tripeptides found in the cell wall. Of the LD-carboxypeptidases active against the mature cell wall, DacB (Streptococcus pneumoniae), Csd6 (Helicobacter pylori) and Pgp2 (Campylobacter jejuni), each has been shown to be essential in maintaining cell morphology. It should be noted, however, that neither Csd6 nor Pgp2 share any sequence similarity with DacB and belong to different peptidase families. This thesis concerns the structural and biochemical characterisation of DacB, herein renamed to LdcB (LD-carboxypeptidase B). The crystal structures of the apo form of LdcB from both S. pneumoniae and Bacillus subtilis were solved, revealing a single domain, globular protein with 2 sub-domains forming a V-shaped cleft in which the active site is located. LdcB binds one zinc ion per monomer, located at the bottom of the active site, and is a member of the LAS (lysostaphin, D-Ala-D-Ala peptidases, sonic hedgehog) family of metalloproteins. Additionally, the activity of LdcB as an LDcarboxypeptidase was confirmed and the crystal structure of LdcB from S. pneumoniae ii was solved in complex with a product mimic, M-Tri-Lys(D-Asn), revealing the molecular basis for peptidoglycan recognition in this family of enzymes. Finally, the affinity of LdcB for zinc and copper has been determined and it has been shown that catalysis is not inhibited by the substitution of zinc by copper or cobalt.

Em estudos etiológicos sobre a deficiência mental (DM), as anomalias cromossômicas, tanto numéricas quanto estruturais, são fatores que apresentam frequência relativa significante. O objetivo deste trabalho foi estudar as frequências e os tipos de anomalias cromossômicas em afetados por DM nas APAEs (Associação de Pais e Amigos dos Deficientes) de Batatais, Altinópolis e Serrana, objetivando conhecer melhor a contribuição destas anomalias na DM nessa região, caracterizando os tipos e as freqüências das aberrações cromossômicas observadas e compará-las entre as APAEs. Pacientes com suspeita de anomalias cromossômicas foram selecionados para o estudo. O critério usado para a seleção da amostra foi a realização do cariótipo em todos os afetados por DM com anomalias estruturais maiores e/ou menores. A análise citogenética foi feita através de cultura de linfócitos do sangue periférico e a coloração utilizada foi banda G, sendo analisadas 20 metáfases por paciente. Dos 505 indivíduos avaliados nas três APAES, 265 realizaram estudo citogenético, sendo encontradas 61 alterações cromossômicas (12,1% do total e 23,0% dos selecionados para cariótipo). Na APAE de Batatais, dos 305 indivíduos avaliados, 174 realizaram cariótipo, sendo encontradas 33 (10,8% do total) anomalias cromossômicas. Em Altinópolis, dos 107 indivíduos avaliados, 54 realizaram cariótipo, sendo observados 16 cariótipos anômalos (14,9% do total). Na APAE de Serrana, dos 93 indivíduos avaliados, 37 realizaram cariótipo, sendo encontradas 12 (12,9% do total) anomalias cromossômicas. Esses resultados demonstram que anomalias cromossômicas contribuem significativamente para a etiologia da DM e que a citogenética clássica possui importantes implicações na prática médica para o diagnóstico dos indivíduos afetados, assim como, para o aconselhamento genético das famílias. Além disso, observa-se que a APAE de Batatais, por apresentar uma porcentagem menor de indivíduos afetados por DM grave, 60,7%, possui uma menor incidência de anomalias cromossômicas quando comparada as APAEs de Altinópolis e Serrana que apresentam uma frequência de 87,8% e 83,9% de indivíduos com DM grave, respectivamente, indicando que alterações cromossômicas são mais frequentes em indivíduos afetados por DM grave.
In etiological studies on mental retardation (MR), the chromosomal abnormalities, both numerical and structural, are factors that have significant relative frequencies. The objective was to study the frequencies and types of chromosomal abnormalities in patients affected by MR in APAEs (Associação de Pais e Amigos dos Deficientes) of Batatais, Altinópolis and Serrana. This aims to better understand the contribution of these abnormalities to MR in these regions, and thus characterizing the types and frequencies of chromosomal aberrations observed in order to compare them between APAEs. Patients suspected of chromosomal abnormalities were selected for the study. The criterion used for sample selection was the achievement of the karyotype of all patients affected by MR with major and/or minor structural abnormalities. Cytogenetic analysis was performed on cultures of peripheral blood lymphocytes, where the band G was used for staining. Twenty metaphases were analyzed per patient. Of the 505 individuals evaluated in three APAEs, a cytogenetic study was performed on 265 patients, and 61 chromosomal abnormalities were found (12.1% of the total and 23.0% of the selected karyotypes). In APAE of Batatais, karyotypes were performed on 174 of the 305 subjects studied, and we found 33 chromosomal abnormalities (10.8% of total). In Altinópolis, 54 karyotypes were performed out of the 107 subjects studied, and we observed 16 abnormal karyotypes (14.9% of total). In APAE Serrana, 37 karyotypes were performed out of the 93 subjects studied, and 12 chromosomal abnormalities (12.9% of total) were found. These results show that chromosomal abnormalities contribute significantly to the etiology of MR and that classical cytogenetics have important implications in medical practice for diagnosis of affected individuals as well as for genetic counseling of the families. Moreover, it is noted that in the APAE of Batatais, because of the smaller percentage of individuals affected by severe MR, 60.7% have a lower incidence of chromosomal abnormalities when compared to the APAEs of Altinópolis and Serrana which have frequencies of 87.8% and 83.9% of individuals with severe MR, respectively. This indicates that chromosomal abnormalities are more frequent in individuals affected by severe MR.

DiGeorge syndrome (DGS) is a developmental defect associated with deletions in chromosomal region 22q11.2. Recently, other syndromes (Velo-Cardio-Facial syndrome, Conotruncal Anomaly Face syndrome, isolated conotruncal cardiopathy) with overlapping phenotypes have been found to be associated with deletions of a similar extent in this chromosomal region. All these syndromes have been grouped under the acronym CATCH 22 (Cardiac defect, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia, chromosome 22q11.2 deletions). In order to characterize genetically this group of syndromes, we have searched for deletions in the 22q11.2 chromosomal region by fluorescence in situ hybridization (FISH). A set of 6 cosmid probes dispersed within the whole length of the DGS deleted region was used to screen 23 patients. A 22q11.2 deletion was observed in 96% of the patients studied. Furthermore, there does not seem to exist any correlation between the size of the deletion and the phenotype observed, since the majority of patients studied, although widely divergent in their clinical manifestation of DGS, appeared to present the same extent of deletion in this genomic region.
There appears to be a predominance of deletion-bearing mothers in familial CATCH 22 when published pedigrees are examined. Furthermore, our own familial cases and the sporadic cases where the parental origin of the deletion could be deduced using a chromosome 22 short arm heteromorphisms seem to confirm this tendency. Because we had isolated a CA-repeat locus mapping within the DGS deleted region, the parental origin of the deletion in sporadic DGS/VCFS cases was studied by assessing the inheritance pattern of this microsatellite marker. The deleted portion of chromosome 22 was of maternal origin in 16 out of 22 cases (72%). When cases of sporadic, familial and unbalanced translocation inheritance reported in the literature were pooled with these results, there appears to be a net tendency for the deletions to be of maternal origin in CATCH 22 (70 deletions of maternal origin, 21 of paternal origin, X$ sp2$ = 26.4, p $<$ 0.0001).
In order to identify the molecular defect underlying DGS, we embarked on a positional cloning approach. A detailed physical map of the 22q11.2 region was made using one- and two-color FISH on metaphases and G$ sb0$ interphase nuclei, and by hybridization to a chromosome 22 hybrid panel. This permitted delineation of a critical region, within which the breakpoint of a balanced translocation carrier affected with DGS was mapping. This breakpoint was cloned by the construction of cosmid contigs, and a novel gene mapped to this region was isolated. The gene potentially encodes an adhesion receptor, and is not interrupted by the balanced translocation breakpoint. Possible mechanisms through which this gene can be involved in the pathogenesis of DGS are presented.
This research project has contributed toward the understanding of the genetics of DGS and related syndromes. Furthermore, a candidate gene for the CATCH 22 syndromes has been isolated and further work will confirm whether it plays a major role in the pathogenesis of these syndromes.

As síndromes mielodisplásicas (SMD) constituem um grupo heterogêneo de doenças hematológicas de origem clonal, caracterizado por hematopoese ineficaz, citopenia e risco de evolução para leucemia mieloide aguda (LMA). As anormalidades citogenéticas adquiridas são marcadores prognósticos bem estabelecidos em SMD. No entanto, a técnica de citogenética metafásica apresenta limitações, incluindo baixa resolução e necessidade de divisão celular, sendo que defeitos cromossômicos podem não ser detectados. Tecnologias baseadas em microarranjo (array) de DNA, como o Single Nucleotide Polymorphism Array (SNP-A), são importantes para avaliação do genoma normal e neoplásico. O SNP-A foi desenvolvido para o estudo de todo o genoma, apresenta uma resolução superior a citogenética metafásica convencional, pode ser realizado em células na interfase, e detecta alterações cromossômicas não visualizadas pela citogenética metafásica. Além disso, o SNPA fornece dados de genotipagem para detecção de perda neutra de heterozigose, também denominada de dissomia uniparental somática. Regiões cromossômicas com deleção, perda neutra de heterozigose ou ganho são comuns em pacientes com neoplasias hematológicas e sugeriu genes candidatos a supressores de tumor e oncogenes. O objetivo do presente estudo foi a caracterização da coorte de pacientes com suspeita clínica de SMD e o uso integrado do método de citogenética convencional e SNP-A no serviço de hematologia da nossa instituição na investigação de alterações citogenéticas em pacientes com SMD e doenças relacionadas. Durante o período do estudo, foram recebidas um total de 114 amostras de pacientes com suspeita clínica de SMD. A análise clínica, morfológica e citogenética permitiu confirmar o diagnóstico de SMD ou doenças relacionadas em 43 pacientes (SMD [n=34], SMD/NMP [n=5], LMA com alterações mielodisplásicas [n=4]). Vinte e um pacientes foram classificados como citopenia idiopática de significado indeterminado (CISI) e 50 indivíduos apresentaram outros diagnósticos. SNP-A foi realizado em 17 pacientes com SMD e doenças relacionadas. Dentre os pacientes selecionados para o SNP-A, anormalidades cromossômicas foram observadas em 6/17 (35%) casos pelo cariótipo convencional e em 8/17 (47%) casos pela técnica de SNP-A. SNP-A não detectou quatro alterações cromossômicas previamente identificadas pela citogenética convencional: duas translocações balanceadas e duas alterações numéricas. SNP-A confirmou os demais achados identificados pela citogenética convencional e detectou um total de 32 novas lesões (1 ganho, 19 perdas e 12 UPDs) em 6 pacientes com SMD ou doenças relacionadas. SNP-A pode complementar a citogenética convencional na detecção de anormalidades cromossômicas em neoplasias mieloides.
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic diseases, characterized by inefficient hematopoiesis, peripheral blood cytopenias and a risk to progress to acute myeloid leukemia (AML). Acquired chromosomal abnormalities have prognostic value in MDS. However, metaphase cytogenetics has some limitations including low resolution and the requirement of cell division, and chromosomal abnormalities may not be detected. New technologies based on array, the Single Nucleotide Polymorphism Array (SNP-A), are able to evaluate the whole genome. The SNP-A has superior resolution compared to metaphase cytogenetics, may be used in interphase cells, and may detect chromosomal abnormalities not detected by metaphase cytogenetics. In addition, the SNP-A read-out includes genotyping calls and hybridization signal strength, corresponding to gene copy number, allowing detecting copy neutral loss of heterozigosity (CN-LOH), also known as uniparental dissomy (UPD). Deletions, copy neutral loss of heterozigosity or gain are frequent in patients with haematopoietic neoplasms and has already suggested the location of tumor suppressor genes and oncogenes. The aim of this study was to characterize the cohort of patients with clinical suspicion of MDS and to establish the integrative use of the conventional cytogenetic and the SNP-A in the investigation of chromosomal abnormalities in patients with MDS and related diseases followed at our institution. The clinical, morphological and cytogenetic evaluation allowed us to confirm the diagnosis of MDS or related disease in 43 patients (MDS [n=34], MDS/MPN [n=5], AML with myelodysplastic changes [n=4]). Twenty-one patients were diagnosed with idiopathic cytopenia with undetermined significance (ICUS) and 50 patients had other diagnosis. SNP-A were performed in 17 patients with MDS and related disease. Chromosomal abnormalities were observed in 6/17 (35%) cases by metaphase cytogenetics, and in 8/17 (47%) of the cases by SNP-A. SNP-A did not detected two balanced translocations and two numerical alterations previously observed by metaphase cytogenetics. SNP-A confirmed all the other findings observed by metaphase cytogenetics and SNP-A detected a total of 32 new lesions (1 gain, 19 losses and 12 UPDs) in 6 MDS and related diseases. SNP-A may complement metaphase cytogenetics to improve the detection of chromosomal abnormalities in myeloid neoplasms.

Orientadores: Vera Lucia Gil da Silva Lopes, Iscia Lopes Cendes
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-12T17:53:05Z (GMT). No. of bitstreams: 1 Freitas_ErikaCristinaLopesBurronede_D.pdf: 4235837 bytes, checksum: 014a02ceef9fbd93a09deb49df5f0a37 (MD5) Previous issue date: 2009
Resumo: A Síndrome Blefaroqueilodôntica (BCD) e os Defeitos de Linha Média Facial com Hipertelorismo (DLMFH) são defeitos craniofaciais raros. Por esse motivo, os estudos de grandes casuísticas têm sido limitados. Contribuição científica significativa neste assunto tem sido dada por nosso grupo, que delineou características clínicas e diretrizes para seguimento de longo prazo e evidenciou achados neuroradiológicos em ambas as anomalias. Embasado nesses achados preliminares e evidências recentes da literatura pertinente, foi possível estabelecer uma estratégia inicial para investigação etiológica da BCD e dos DLMFH, sendo os objetivos desse projeto: investigar a etiologia da BCD nos indivíduos afetados pela síndrome, por meio de estudo dos genes candidatos IRF6, P63, OSR2, TBX10, FOXE1, SHH, FGF8 e PAX3; pesquisar, nos indivíduos com DLMFH, a presença de mutações nos genes candidatos SHH, FGF8 e PAX3; identificar, em ambas as malformações, possíveis alterações cromossômicas; e, por último, associar os achados clínicos detectados nas investigações anteriores aos possíveis achados moleculares. Foram utilizadas técnicas de sequenciamento direto, array-CGH, genotipagem automática e hibridação in situ por fluorescência. Não foi possível relacionar nenhuma mutação pontual nos genes estudados associadas às malformações em questão, pois não foram encontradas alterações gênicas patogênicas. Entretanto foram detectadas em pacientes com DLFMH três aberrações cromossômicas em regiões distintas do genoma, tratando-se de um caso de duplicação no cromossomo 6 (paciente DLMFH7), um caso com deleção no cromossomo 9 e duplicação no cromossomo 20 (paciente DLMFH10) e um caso de deleção no cromossomo 2 (paciente DLMFH1). Nesse último, o gene PAX3 encontrava-se dentro da região deletada, confirmando a hipótese inicial de que alguns genes de desenvolvimento estão envolvidos na etiologia dos DLMFH. Conclui-se, portanto, que em indivíduos com DLMFH, a técnica de array-CGH pode ser útil para detecção de aberrações cromossômicas diversas e o aconselhamento genético deve ser individualizado
Abstract: The Blepharocheilodontic Syndrome (BCD) and Midline Facial Defects with Ocular Hypertelorism (MFDH) are rare craniofacial anomalies. Considering the rarity of these two groups of congenital defects, studies with large casuistry have been limited. Our group has significantly contributed to the scientific knowledge about both anomalies, delineating clinical characteristics, evidencing neurological findings and designing protocols for long term follow-up. Based in these preliminary findings and recent evidences of pertinent literature, it was possible to determine an initial etiologic investigation strategy for the BCD and the MFDH. The objectives of this project are to investigate the etiology of the BCD in affected individuals through IRF6, P63, OSR2, TBX10, FOXE1, SHH, FGF8 and PAX3 candidate genes study; to search MFDH patients for mutations in the SHH, FGF8 and PAX3 candidate genes; to identify, in both syndromes, possible chromosomal anomalies; and finally, to associate the detected clinical findings in previous investigations to the molecular results. The direct sequencing, array-CGH, automatic genotyping and FISH were the molecular techniques used in this study. It was not possible to relate any punctual mutation in the studied genes associated to the syndromes investigated since none pathogenetic alteration was found. Although, chromosomal anomalies in different locations of the genome were detected in three MFDH patients, a chromosome 6 duplication (in patient MFDH7), a chromosome 9 deletion and chromosome 20 duplication (in patient MFDH10), and a chromosome 2 deletion (in patient MFDH1). In the latter case, the PAX3 gene was inside the deleted region, supporting the initial hypothesis that some development genes are involved in the MFDH etiology. Therefore, it can be concluded that in MFDH patients, the array-CGH investigation can be useful to detect a wide range of chromosomal anomalies and the genetic counseling must be individualized
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas

Les alteracions cromosòmiques constitucionals representen una de les principals causes d’anomalies congènites a la població, i el seu diagnòstic durant l’etapa prenatal és el principal objectiu de la majoria de procediments invasius que es realitzen durant l’embaràs. Actualment, el cariotip convencional és el gold standard del diagnòstic citogenètic prenatal, però els avanços tecnològics dels últims anys han portat al desenvolupament de tècniques de citogenètica molecular que ofereixen característiques molt atractives, la principal de les quals és l’elevada resolució. Donat que el paper que han de tenir aquestes tècniques durant l’etapa prenatal encara no està ben definit, els objectius de la present tesi han estat: a) determinar el potencial diagnòstic i la utilitat del cariotip i les tècniques de citogenètica molecular Fluorescent In Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA) i Chromosomal Microarray-based Analysis (CMA) en diagnòstic prenatal, especialment per a l’estudi de gestacions amb troballes ecogràfiques; i b) valorar la necessitat de modificar els procediments actuals de diagnòstic citogenètic prenatal. En primer lloc, s’han revisat els resultats dels 29.883 cariotips en líquid amniòtic realitzats entre 1998 i 2009 a l’Hospital Clínic de Barcelona. Els resultats obtinguts corroboren que el cariotip és una eina efectiva i robusta per a l’obtenció del cariotip fetal, revelant alteracions cromosòmiques en el 2,9% de les gestacions que es sotmeten a un procediment invasiu. La translucidesa nucal incrementada i les anomalies ecogràfiques destaquen com a indicadors excel•lents d’anomalia cromosòmica. En segon lloc, s’ha avaluat la utilitat de l’MLPA subtelomèrica per a l’estudi de gestacions amb troballes ecogràfiques i cariotip normal. Dels 229 fetus analitzats, 3 presentaven desequilibris subtelomèrics críptics (1,3%). Donat que la freqüència d’aquests desequilibris és baixa, i a més la correlació genotip-fenotip observada és pobra, l’MLPA subtelomèrica no sembla ser una eina crucial per a l’estudi d’aquestes gestacions. D’altra banda, s’ha estudiat la idoneïtat de reemplaçar els estudis de FISH de la regió 22q11.2 per kits d’MLPA dissenyats per a l’estudi d’aquesta regió i altres regions genòmiques associades a cardiopaties. S’han analitzat mitjançant aquests kits 55 gestacions amb troballes cardíaques, cariotip normal i estudi de FISH negatiu per a la microdeleció típica de 22q11.2, i no s’ha detectat cap anomalia. Aquest fet, juntament amb que l’MLPA presenta una taxa de repetició i de no obtenció de resultats superiors a la FISH, i que generalment requereix cultiu cel•lular, indica que la FISH hauria de seguir sent la tècnica d’elecció per a l’estudi de fetus amb troballes cardíaques (enfront l’MLPA). També s’ha volgut determinar la freqüència dels diferents tipus d’alteracions cromosòmiques, tant microscòpiques com críptiques, en fetus amb troballes ecogràfiques cardíaques. L’anàlisi retrospectiu dels resultats obtinguts en el període 2009-2011 a l’Hospital Clínic de Barcelona en aquest grup de gestacions (N=276) ha revelat una freqüència d’anomalies cromosòmiques microscòpiques i casos de Síndrome de la deleció cromosòmica 22q11.2 del 15,9% i 6,4%, respectivament; uns resultats que corroboren la forta associació existent entre troballes ecogràfiques cardíaques i anomalies cromosòmiques. A més, aquesta associació varia significativament en funció del tipus de troballa ecogràfica cardíaca i de la presència d’anomalies extracardíaques. D’altra banda, s’han analitzat mitjançant array CGH 51 fetus amb troballes ecogràfiques cardíaques, cariotip normal, i sense estudi o resultat de FISH negatiu per a la Sd. de la deleció cromosòmica 22q11.2. En aquest grup de gestacions, la taxa de detecció de variants en número de còpia patogèniques ha estat del 2%, i no s’han detectat variants de significat clínic incert. Si en la nostra sèrie de 276 gestacions amb troballes ecogràfiques cardíaques s’hagués utilitzat l’estratègia QF-PCR + CMA enlloc de cariotip, totes les anomalies cromosòmiques amb repercussió clínica clara diagnosticades per citogenètica convencional s’haguessin identificat, s’haguessin detectat els casos de delecions de 22q11.2, i s’hagués incrementat un 2% el nombre de casos diagnosticats. Per tant, aquests resultats mostren el potencial de les plataformes de microarray per al diagnòstic prenatal de fetus amb troballes ecogràfiques cardíaques. Finalment, s’han utilitzat les tècniques de citogenètica molecular FISH, MLPA i/o CMA per a la caracterització de 6 alteracions cromosòmiques visibles al cariotip però de difícil caracterització per citogenètica convencional. En tots els casos, la utilització d’aquestes tècniques ha contribuït a la correcte descripció de l’alteració identificada, posant de manifest la seva utilitat quan s’utilitzen de forma complementària al cariotip. L’elaboració d’aquesta tesi i la literatura recentment publicada evidencien la necessitat d’un nou plantejament dels protocols clínics de diagnòstic citogenètic prenatal, especialment en relació a la utilització de plataformes de microarray en diagnòstic prenatal.
Strategies for genetic diagnosis of fetuses with congenital malformations. Genotype-phenotype correlations. Constitutional chromosomal abnormalities represent one of the main causes of congenital abnormalities in the population, and their diagnosis during pregnancy is the main objective of most invasive procedures. Nowadays, conventional karyotyping is the gold standard for prenatal cytogenetic diagnosis, but recent technological advances have led to the development of molecular cytogenetic techniques which offer much higher resolution. Since there is still not a consensus on the role of these techniques in prenatal diagnosis, the goals of the present PhD work have been: a) to determine the diagnostic potential and usefulness of karyotype and molecular cytogenetic techniques Fluorescent In Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA) and Chromosomal Microarray-based Analysis (CMA) in prenatal diagnosis, especially in pregnancies with ultrasound findings; and b) to establish the need for modifications in current prenatal cytogenetic diagnostic protocols. The results obtained: - Corroborate the effectiveness and robustness of conventional karyotyping. The observed detection rate of chromosomal abnormalities in 29,883 consecutive amniotic fluid samples has been 2.9%, with increased nuchal translucency and ultrasound abnormalities being excellent indicators for chromosomal abnormalities. - Suggest that subtelomeric screening in pregnancies with ultrasound findings and normal karyotype is not a crucial tool, as detection rate of subtelomeric imbalances is low (1.3%; 3/229), and genotype-phenotype correlations are poor. - Reveal that FISH of 22q11.2 region is preferred to MLPA kits specific for this chromosome region and other genomic regions previously associated with congenital heart defects. - Reveal a strong association between cardiac ultrasound findings and chromosomal abnormalities, both microscopic and cryptic. In pregnancies with cardiac findings, the observed frequency of microscopic chromosomal abnormalities and chromosome 22q11.2 deletion syndrome has been 15.9% (44/276) and 6.4% (5/78), respectively. Moreover, CMA has been performed in 51 fetuses with cardiac ultrasound findings, normal karyotype and negative or no FISH study for chromosome 22q11.2 deletion syndrome, and the detection rate of pathogenic copy number variants has been 2%, without detection of variants of unknown clinical significance. If in the initial series of 276 pregnancies the strategy QF-PCR + CMA would have been applied; all the chromosomal abnormalities with clear clinical repercussion diagnosed by conventional cytogenetics would have been identified, together with the deletions of 22q11.2 region and an extra 2% of chromosomal abnormalities. These results show the potential of CMA for the prenatal diagnosis of these pregnancies. - Show the usefulness of the molecular cytogenetic techniques FISH, MLPA and CMA when used as a complement to conventional karyotyping in cases with chromosomal abnormalities not accurately characterizable by conventional cytogenetics. Accordingly, the results obtained suggest the need for updating current prenatal cytogenetic diagnostic protocols.

A Síndrome de Martin-Bell é uma forma hereditária de retardo mental determinada pela perda da expressão do gene FMR1 que está associado com a expansão da repetição dos tri-nucleotídeos citosina-guanina¬-guanina (CGG). Os indivíduos com um alto grau dessa expansão apresentam o silenciamento da expressão desse gene, com conseqüente alteração no desenvolvimento do sistema nervoso do embrião, causando danos neurológicos irreparáveis. A citogenética é uma metodologia clássica que contribui para o diagnóstico da síndrome em casos sem esclarecimentos, porém sua sensibilidade isoladamente em muitos casos é insuficiente para um diagnóstico positivo mesmo diante de características clínicas evidentes. Na busca de uma alternativa para suprir esta necessidade de definir exatamente as alterações encontradas em cada caso propôs-se a otimização de uma PCR de alta fidelidade no diagnóstico diferencial da Síndrome e simultaneamente comparar com os dados da citogenética clássica. Para tanto, foram coletadas amostras de sangue periférico de 102 pacientes e avaliou-se 100 a 150 metáfases para cada indivíduo pela citogenética clássica e para a PCR duplex. Os resultados demonstram pelo teste de Kruskal-Wallis que a PCR com a citogenética não apresentou diferenças significativas (p>0,05). Porém quando avaliadados pelo índice de Kappa sugere-se que os dois critéiros de diagnósticos devem ser utilizados simultaneamente com caracteristicas clínicas do paciente. A PCR mostrou-se rápida e com custo relativamente menor, sendo portanto, aplicável no auxílio ao aconselhamento genético dos indivíduos e suas famílias, bem como para um acompanhamento psico-pedagógico adequado.
The Martin-Bell Syndrome is a heredity mental retard form determined by the loss of FMR1 gene expression which is associated with the tri-nucleotides cytosine-guanine-guanine (CGG) repetition expansion. The individuals showing a high degree of this expansion present the silencing of this gene expression, with a consequent alteration of the embryo nervous system evolution, causing irreparable neurological damage The cytogenetics is a classical methodology that contributes for diagnosing the syndrome in cases without clarification, thus its sensitivity isolated in many cases is insufficient for a positive diagnosis even in front of evident clinical characteristics. On searching for an alternative to fulfill this need to define the found alterations in each case, an optimization of a high fidelity PCR to Syndrome diagnosis and simultaneously to compare with classical cytogenetics. Peripheral blood samples were collected from 102 patients for evaluating 100 to 150 metaphases evaluation by classical cytogenetics for each sample and to duplex PCR. The results showed by the Kruskal-Wallis test that the PCR with the cytogenetics have not presented significant differences (p>0,05). However, when evaluated by the Kappa index it was suggested that the two diagnosis criteria should be used simultaneously with patient s clinical characteristics. The PCR showed itself quick and with a relatively lower cost , and in this way, applicable in helping genetically counseling individuals and their families, as well as sending them to a more adequate psycho-pedagogical treatment.

Chromosome abnormalities are one of the most important causes of congenital disorders. The main goal of this research is to give a broad view of the use and evolution of prenatal and postnatal cytogenetic diagnosis in Girona province between 1999 and 2009. It also aims at linking prenatal cytogenetic diagnosis with the existing screening methods. The main conclusions are the following: - A rise in the use of cytogenetic diagnosis has been detected. This has been caused by a growing preventive medicine trend. - Case studies match the literature consulted when the study population is similar in accordance with national health policies. - The overall sensitivity of obstetric ultrasound scan was 60,8%, and this result matches, and in many cases exceeds, those found in the literature consulted. - The moving from second-trimester to first-trimester aneuploidy prenatal screening has meant a significant increase in aneuploidy detections. - Prenatal cytogenetic diagnosis appears as an interdisciplinary field in which the extent of prenatal screening tests, like obstetric ultrasound scans and aneuploidy screening, is crucial.
Les anomalies cromosòmiques són una de les causes més importants dels defectes congènits. L’objectiu d’aquest treball és donar una visió global de l’ús i l’evolució del diagnòstic citogenètic prenatal i postnatal a les comarques de Girona en el període 1999-2009 i relacionar el diagnòstic citogenètic prenatal amb els diferents mètodes de cribratge. Les conclusions principals són: - Es constata un augment de l’ús del diagnòstic citogenètic com a resultat d’una tendència creixent en medicina preventiva. - La casuística coincideix amb la bibliografia consultada quan la població d’estudi és semblant, atenent a la política sanitària del país. - La sensibilitat global de l’ecografia obstètrica va ser del 60,8%, i és un resultat concordant i en molts casos millor en comparació amb la literatura consultada. - La substitució del cribratge del segon trimestre pel cribratge del primer trimestre ha suposat un increment important en la detecció d’aneuploïdies. - El diagnòstic citogenètic prenatal es mostra com a una àrea interdisciplinària en què la contribució de les proves de cribratge prenatal com l’ecografia obstètrica i el cribratge d’aneuploïdies són importants.

Las LAM que acontecen tras un TPH efectuado a pacientes con LAM siempre han sido consideradas recaídas de la LAM inicial. Sin embargo, estos pacientes han recibido tratamientos con regímenes que incluyen antraciclinas y citarabina y, posteriormente, se han consolidado con un TPH, previo acondicionamiento con agentes alquilantes, antimetabolitos e ICT. Dado que la quimioterapia previa recibida, principalmente la dosis total acumulada de agentes alquilantes, se considera el factor de riesgo más importante para desarrollar una t-NM, alguna de estas LAM post-TPH podrían tratarse en realidad de una t-LAM. En la presente tesis doctoral, con el objetivo de analizar las características genéticas de las LAM post-TPH, se han analizado los datos citogenéticos y moleculares, en el momento del diagnóstico, de 151 pacientes trasplantados por una LAM y se han comparado con las características citogenéticas y moleculares que posteriormente presentaron los 58 pacientes de la serie que desarrollaron una LAM post-TPH. Al mismo tiempo, se han analizado en estos pacientes las variables descritas en la bibliografía que pueden tener una posible influencia en el desarrollo de una LAM post-TPH, así como la incidencia de las deleciones de TP53 (mediante hibridación in situ fluorescente) y su relación con el cariotipo complejo, el cariotipo monosómico y la deleción del cromosoma 5q. En este trabajo, los pacientes que desarrollaron una LAM post-TPH recibieron con más frecuencia un auto-TPH y presentaron, en el momento del diagnóstico, más s-LAM, más cariotipos complejos, un mayor número de alteraciones citogenéticas y más alteraciones desequilibradas. Por otro lado, las LAM post-TPH presentaron alteraciones cromosómicas relacionadas con la exposición previa a inhibidores de la topoisomerasa II y a agentes alquilantes con poca frecuencia. En una tercera parte de los casos se observaron las mismas alteraciones cromosómicas, asociadas o no a otras alteraciones, presentes en la LAM inical. Además, las LAM post alo-TPH presentaron más alteraciones citogenéticas y de mayor complejidad que las LAM post auto-TPH. La edad y el estado mutacional del gen de la NMP1, FLT3-ITD y FLT3-D835 al diagnóstico, la fase de la enfermedad y el número de ciclos de quimioterapia administrados previamente al TPH, el tipo de acondicionamiento recibido (agentes alquilantes vs ciclofosfamida e ICT), la profilaxis de la EICH (tratamiento inmunodepresor vs tratamiento inmunodepresor y metotrexato) y la presencia de EICH no se relacionaron, en el presente estudio, con el desarrollo de una LAM post-TPH. Todo ello parece indicar que el daño acumulado en el ADN de las CPH infundidas y el tratamiento previo al trasplante (inducción, consolidación y acondicionamiento) tiene menos influencia que la complejidad citogenética en el momento del diagnóstico, en el desarrollo de las LAM post-TPH.
Acute myeloid leukemias (AML) appearing after hematopoietic stem cell transplantation (HSCT) performed in patients with AML have always been considered as relapse of the original AML. However, these patients have been treated before HSCT with regimens that include anthracyclines and cytarabine and subsequently have been consolidated with HSCT, with conditioning regimens including alkylating agents, antimetabolites and, for some patients, total body irradiation (TBI). As the cumulated dose of alkylating agents is considered the most important risk factor for developing a therapy-related myelodysplasia or AML, some of these AML developing after-HSCT could be in fact therapy-related AML In order to characterize the genetic features of AML after-HSCT we have analyzed the cytogenetic and molecular characteristics at diagnosis in 151 AML patients submitted to HSCT and we have performed a comparison with the cytogenetic and molecular characteristics of 58 patients who developed an AML after-HSCT. In addition, we have analyzed the variables described in the literature that may have a potential impact on the development of AML after-HSCT, as well as the incidence of TP53 deletions (assessed by fluorescent in situ hybridization) and their relationship with the presence of complex karyotype, monosomal karyotype and deletion of chromosome 5q. In this study, patients who developed AML after-HSCT were submitted more often to an autologous HSCT and showed more frequently secondary AML, complex karyotypes, unbalanced karyotypic abnormalities and a higher number of cytogenetic abnormalities at diagnosis. On the other hand, AML after-HSCT showed a low frequency of chromosomal alterations related with previous exposure to topoisomerase II inhibitors and alkylating agents. One third of patients with AML developed after HSCT showed the same chromosomal alterations (either associated or not with additional alterations) as those detected at AML diagnosis. In addition, AML developed after allo-HSCT showed a higher number of cytogenetic abnormalities and more complex karyotypes than the AML arising after auto-HSCT. Age, mutational status of NMP1, FLT3-ITD and FLT3-D835 at diagnosis, disease state, the number of cycles of chemotherapy given prior to HSCT, the type of conditioning regimens (alkylating agents or cyclophosphamide and TBI), GVHD prophylaxis (immunosuppressive therapy and methotrexate or immunosuppressive therapy only) and the presence of GVHD have not been significantly associated with the development of AML after-HSCT. The results of our study suggest that the pre-transplant treatment (induction, consolidation and conditioning regimen) and the cumulative damage in the DNA of the HSC infused (in cases of auto-HSCT) have less influence in the development of AML after-HSCT than the cytogenetic complexity of AML at the time of diagnosis.

碩士
國立陽明大學
遺傳學研究所
94
Amniocentesis is the most common, although invasive, test for prenatal diagnosis of fetal chromosome abnormalities. Indications for fetal chromosome analysis include advanced maternal age, abnormal maternal serum screening results, abnormal ultrasound findings, a previous child with congenital anomaly, family history of chromosome aberrations, and other minor purposes. We collected 101,528 amniocentesis cases for fetal chromosome analysis performed in a five-year interval, from year 2000 to 2004, in Taiwan. Among these, 59.84% were referred for advanced maternal age, 24.92% for abnormal maternal serum screening results, 7.45% for abnormal ultrasound findings, 1.89% for a previous child with congenital anomaly, 0.90% for a family history of chromosome aberrations, and 5.01% for other purposes. Chromosome aberrations were detected in 2,670 cases (2.63%), among these 44.76% cases were autosomal aneuploidy (25.24% were Down syndrome), 19.89% were sex chromosome aneuploidy, and 35.36% were structural abnormalities. Abnormal maternal serum screening results have a higher positive rate to detect cases with Down syndrome and structural abnormalities. Abnormal ultrasound findings were the most informative for Trisomy 18 and Turner syndrome. Sex chromosome abnormalities (SCA) are less damaging to the appearance and intelligence. Couples faced with a very difficult decision-making process when informed about their fetus’ diagnosis of SCA. We reported parental decisions following the prenatal diagnosis of SCA during these five years and compared with the outcome of prenatal SCA in other countries. Parents’ notion and perception were retrospectively interviewed to investigate what kind of information they had obtained during genetic counseling and what additional resources would have been helpful during their decision making. In this retrospective study, we also used semi-structured interview and questionnaires to examine patterns of genetic counseling, parents’ immediate emotion, and their satisfaction with counseling based on 50 parents of children with Turner syndrome. From these results, we are able to know their experience and difficulty, and what additional help and information should have been provided. These information can be beneficial for further improvements in both prenatal as well as postnatal genetic counseling.

Dissertação mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas
Limonium algarvense is a halophyte species that grows in salthmarshes. There is a few knowledge about this species biology, specifically on cytogenetic variability and reproductive biology. This knowledge is fundamental to apply conservation methods since their habitat is being degraded by anthropic impacts. The present study aims were to collect data regarding characterization of cytogenetic and male function of L. algarvense. Geographical and demography data confirmed that this species ranges from Southwest Portugal, in Algarve, to southern Spain. Flow cytometric studies in L. algarvense populations revealed that triploid cytotypes, indicating a cytotype consistency and homogeny across a geographical gradient. Cytogenetic analysis in distinct plants originated from different natural populations confirmed that L. algarvense presents 2n=3x=25 chromosomes, as previously reported. Exploratory studies using combined GISH and FISH analysis didn’t allow identifying the putative progenitors of L. algarvense. Finally, micro-sporogenesis and gametogenesis analyses showed several abnormalities during male gametes formation like presence of chromosomes bridges and laggard chromosomes during meiosis, the formation of restitution nuclei and cytomixis. These anomalies led to the formation of nonreduced pollen grains with different morphology which showed low viability and low germination rate. The results present in this thesis provided useful data for a better understanding of this species reproductive biology which can contribute to design better strategies for this species conservation.
Limonium algarvense é uma espécie halófita que ocorre em sapais. A biologia desta espécie é pouco conhecida, especificamente a sua biologia reprodutiva.Este tipo de conhecimento é fundamental para aplicar métodos de conservação, dado que o habitat desta espécie se encontra degradado devido a impactos de origem antrópica. Os objetivos deste estudo foram recolher informação relativamente à caracterização citogenética e reprodutiva função masculina em L. algarvense. A informação geográfica e demográfica adquirida confirmaram que L. algarvense extende-se desde o sudoeste de Portugal, no Algarve, até ao sul de Espanha. Estudos de citometria de fluxo em populações de L. algarvense revelaram a presença de citotipos triploides, indicando uma consistência e homogeneidade de citotipos ao longo de um gradiente geográfico. Análises citogenéticas em diferentes plantas originárias de diferentes populações naturais confirmaram que que L. algarvense apresenta 2n=3x=25 cromossomas, tal como relatado em estudos prévios. Estudos exploratórios usando análises FISH e GISH combinadas não permitiram a identificação dos supostos progenitores de L. algarvense. L. algarvense. Finalmente, análises da micro-esporogénese e gametogénese mostraram várias anomalias durante a formação de gâmetas masculinos tais como a presença de pontes entre cromossomas e de cromossomas perdidos na placa equatorial durante a meiose, a formação de núcleos de restituição e citomixia. Estas anomalias originaram a formação de grãos de pólen não reduzidos com diferentes morfologias que apresentaram baixa viabilidade e taxa de germinação. Os resultados apresentados nesta tese providenciam informação útil para um melhor conhecimento da biologia reprodutiva desta espécie, que pode contribuir para desenhar melhores estratégias de conservação desta espécie.

L’analyse des anomalies génomiques récurrentes est importante pour établir le diagnostic, le pronostic et pour orienter la thérapie des leucémies aiguës pédiatriques. L’objectif de notre étude est d’élaborer une stratégie optimale pour détecter les anomalies chromosomiques dans les leucémies aiguës lymphoblastiques (LAL) et myéloïdes (LAM) des enfants. Pour ce faire, nous avons caractérisé au caryotype, avec des panels d’hybridation in situ en fluorescence (FISH), par RT-PCR et par l’index d’ADN 253 leucémies de novo reçues au CHU Sainte-Justine entre 2005 et 2011 (186 LAL-B, 27 LAL-T et 40 LAM). Nous avons réussi à optimiser la détection des anomalies chromosomiques dans les trois types de leucémies, avec des fréquences de 93,5% dans les LAL-B (174/186), 66,7% dans les LAL-T (18/27) et 90% dans les LAM (36/40). Nos résultats suggèrent d’utiliser plusieurs tests génétiques concomitants afin d’optimiser la détection des anomalies génomiques dans les LAL et les LAM de novo pédiatriques.
Analysis of recurrent genomic abnormalities is important for the diagnosis, prognosis and therapeutic choices in paediatric acute leukemia. The aim of our study is to establish a strategy optimizing the detection of cytogenetic abnormalities in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To do so, we have characterized childhood AML and ALL cases received in cytogenetic laboratory of CHU Sainte-Justine (Montreal, Canada) between 2005 and 2011. Overall, 253 de novo cases have been analyzed (186 B-ALL, 27 T-ALL and 40 AML) by karyotyping, fluorescence in situ hybridization (FISH) panels, RT-PCR and DNA index. Chromosomal abnormalities detection rates achieved 93,5% in B-ALL (174/186), 66,7% in T-ALL (18/27) and 90% in AML (36/40). Our results suggest the analysis of both molecular and cytogenetic tests to optimize the detection of genomic abnormalities in new cases of childhood AML and ALL.

Fan Chung-sze.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references.
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
List of Tables --- p.vii
List of Figures --- p.viii
List of Abbreviations --- p.x
Table of Contents --- p.xi
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Nasopharyngeal Carcinoma --- p.1-1
Chapter 1.1.1 --- Histology of NPC --- p.1-1
Chapter 1.1.2 --- Etiological Factors --- p.1-2
Chapter 1.1.3 --- Genetic Changes in NPC --- p.1-5
Chapter 1.2 --- Background of Present Study --- p.1-14
Chapter 1.3 --- Aims of Study --- p.1-15
Chapter Chapter 2 --- Comparative Genomic Hybridization and Fluorescence In- Situ Hybridization
Chapter 2.1 --- Introduction --- p.2-1
Chapter 2.2 --- Fluorescence In-Situ Hybridization (FISH) --- p.2-2
Chapter 2.2.1 --- Principle of FISH --- p.2-2
Chapter 2.2.2 --- Applications of FISH --- p.2-2
Chapter 2.2.3 --- Advantages and Limitations --- p.2-5
Chapter 2.3 --- Comparative Genomic Hybridization (CGH) --- p.2-7
Chapter 2.3.1 --- Principle of CGH --- p.2-7
Chapter 2.3.2 --- Applications of CGH --- p.2-8
Chapter 2.3.3 --- Advantages and Limitations --- p.2-10
Chapter 2.4 --- Method of CGH --- p.2-13
Chapter 2.4.1 --- CGH Probe Preparation --- p.2-13
Chapter 2.4.2 --- CGH Template Preparation --- p.2-21
Chapter 2.4.3 --- Hybridization --- p.2-23
Chapter 2.4.4 --- Post-hybridization --- p.2-23
Chapter 2.4.5 --- Fluorescence Detection --- p.2-24
Chapter 2.4.6 --- Image Acquisition and Analysis --- p.2-24
Chapter 2.5 --- Method of Interphase FISH --- p.2-29
Chapter 2.5.1 --- FISH Probe Preparation --- p.2-29
Chapter 2.5.2 --- FISH Template Preparation --- p.2-29
Chapter 2.5.3 --- Hybridization --- p.2-30
Chapter 2.5.4 --- Post-hybridization --- p.2-30
Chapter 2.5.5 --- Fluorescence Detection --- p.2-30
Chapter 2.5.6 --- Scoring of FISH Signals --- p.2-31
Chapter 2.5.7 --- Threshold Determination --- p.2-31
Chapter Chapter 3 --- FISH Studies on NPC Biopsies Guided by CGH Information Derived from Cell Lines and Xenografts
Chapter 3.1 --- Introduction --- p.3-1
Chapter 3.2 --- Materials and Methods --- p.3-3
Chapter 3.2.1 --- CGH Analysis --- p.3-3
Chapter 3.2.2 --- Interphase FISH Analysis --- p.3-4
Chapter 3.2.3 --- Statistical Analysis --- p.3-7
Chapter 3.3 --- Results
Chapter 3.3.1 --- CGH --- p.3-9
Chapter 3.3.2 --- Interphase FISH Analysis --- p.3-10
Chapter 3.3.3 --- Statistical Analysis --- p.3-11
Chapter 3.4 --- Discussion --- p.3-27
Chapter 3.4.1 --- CGH --- p.3-27
Chapter 3.4.2 --- Interphase FISH Analysis --- p.3-31
Chapter 3.5 --- Summary of This Chapter --- p.3-36
Chapter Chapter 4 --- CGH Studies on Universally Amplified DNA from Microdissected NPC Biopsies and Interphase FISH Analysis
Chapter 4.1 --- Introduction --- p.4-1
Chapter 4.2 --- Materials and Methods --- p.4-4
Chapter 4.2.1 --- CGH on Universally Amplified DNA --- p.4-4
Chapter 4.2.2 --- Interphase FISH Analysis --- p.4-6
Chapter 4.2.3 --- Statistical Analysis --- p.4-8
Chapter 4.3 --- Results --- p.4-9
Chapter 4.3.1 --- CGH on Universally Amplified DNA --- p.4-9
Chapter 4.3.2 --- Interphase FISH Analysis --- p.4-10
Chapter 4.3.3 --- Statistical Analysis --- p.4-11
Chapter 4.3.4 --- Comparison of CGH and FISH Findings --- p.4-11
Chapter 4.4 --- Discussions --- p.4-30
Chapter 4.4.1 --- CGH Findings --- p.4-30
Chapter 4.4.2 --- Interphase FISH Analysis --- p.4-41
Chapter 4.4.3 --- Comparison of CGH and FISH Findings --- p.4-43
Chapter 4.5 --- Summary of This Chapter --- p.4-45
Chapter Chapter 5 --- Conclusion and Further Studies
Chapter 5.1 --- Conclusion --- p.5-1
Chapter 5.2 --- Further Studies --- p.5-3
Chapter 5.2.1 --- Combination of Microdissection --- p.5-3
Chapter 5.2.2 --- Multicolor Karyotyping --- p.5-3
Chapter 5.2.3 --- Microarrays --- p.5-4
References --- p.R-1

Lam, Man Ting.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 128-138).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.