Thèses sur le sujet « Normal Karyotype »

The aim of this thesis was to examine the role that gene specific mutations play in leukaemogenesis of normal karyotype (NK) acute myeloid leukaemias (AMLs), by studying the number of genes involved, types of mutations, order by which they arise and their function. AML is a stem cell disease in which the bulk of the disease is perpetuated by a rare population of leukaemic stem cells (LSCs), which arise due to an accumulation of genetic events within the target cell. Eight genes known to be mutated in AML were screened from a panel of 88 NK AML patients. One hundred and twenty-seven mutations were detected multiple mutations were common and 44% of the AMLs contained mutations that affect both differentiation and proliferation, indicating a requirement for cooperating mutations. Regions of acquired uniparental disomy (aUPD) detected by SNP arrays were found to contain homozygous mutations in genes implicated in AML, such as homozygous FLT3-ITDs on 13q, indicating that mitotic recombination can act as a second genetic event in leukaemogenesis. Semi-quantitative real-time PCR mutation specific assays, were designed against mutations in the transcription factor CEBPA. These assays were used to test AML samples which had been sorted into populations corresponding to stem cell and early progenitors in normal haematopoiesis. CEBPA mutation was detected in all compartments tested, indicating it is an early event in leukaemogenesis, which is supported by their occurrence in familial AML. Functional studies of mutations in CEBPA indicated that the 30kDa isoform of the protein was able to direct differentiation of lineage depleted cord blood mononuclear cells towards the myeloid pathway, where the addition of an ITD within the C-terminal region promoted erythroid differentiation. The work presented here demonstrates that gene specific mutations play a key role in leukaemogenesis.

Loss of heterozygosity (LOH) is detectable in many forms of cancer including leukaemia. It contributes to tumorigenesis through the loss of one of the two alleles of one tumor suppressor gene at a given locus, caused by deletion or uniparental disomy (UPD). UPD can only be the result of homologous recombination. Little is known about the mechanisms of UPD and what connection this aberration has with the outcome of this disease. In this study, 146 patients with primary AML were analysed using a novel technique based on single nucleotide polymorphisms (SNPs). Leukaemic cells and healthy T-cells from each patient were obtained using FACS-Vantage cell sorting. In cases with very few sorted cells whole genome preamplification was done. Genome-wide SNP analysis was carried out according to the standard GeneChip Mapping Assay protocol (Affymetrix, USA) using the Human Mapping 10K Arrays. Moreover, the impact of the FLT3-ITD mutation on the homologous recombination using pmHPRT-DRGFP /pCbASce vectors system and yHA2x assay was investigated. Of 146 patients with normal karyotype LOH was found in 30 cases. The potential LOH regions, were confirmed by microsatellite analysis of short tandem repeat (STR) markers. In 21 of these cases STR-analysis of T-cells, representing the corresponding tumor-free material, confirmed the regions of partial UPD. This aberration affected different chromosomes, but most commonly chr. 2, 6, 11, 21, 13, and 7, and covered between 11.5 and 88 Mb. Interestingly, in 6 LOH cases, long stretches of homozygosity present at the same positions as in the healthy cells and in the blasts were found. The impact of this phenomenon is unknown. Additionally, chromosome losses were detected in 3 patients classified with normal karyotype according to current methods. These 9 cases were not included in the UPD positive group. No differences were observed regarding any clinical factors including age, WBC-counts and sex. The FAB M1 subtype was observed in 47.6% of the UPD positive patients, compared to only 19.2% of the UPD negative patients (P=0.04, n=146). In addition, no correlation between FLT3-ITD, MLL-PTD and NPM1 mutations in the UPD patients was found, but the data indicate that patients with UPD have a higher rate of treatment failure. Moreover, in this study the relationship between UPD and gene aberrations was able to be confirmed. In some cases, UPD found on chromosomes 21, 19 and 11 was correlated with mutations in the RUNX1, CEBPA and WT genes, respectively. Furthermore, AML cases with and without UPD showed different but specific gene expression profiles, revealing different expression levels for genes involved in double strand break repairs. Furthermore, it was found that different mutations could be responsible for the increase in efficiency of HR, such as FLT3-ITD or BCR-ABL. Moreover, cells with a FLT3-ITD mutation (without wt expression) rapidly increased the HR efficiency compared with heterozygous (FLT3-ITD/wt) cells. Preliminary results showed that the high repair efficiency was mainly dependent on the translocation of RAD51. In conclusion, SNP array technology allow the identification and mapping of LOH in AML patients with normal karyotype. The obtained data also point out the necessity of analysing tumour-free material to confirm the somatic origin of the alteration. Furthermore, the available results indicate that compared to patients without UPD, patients with UPD have a higher relapse rate, which might be used as a prognostic marker in the future. Also, it could be hypothesized that downregulation of RAD51 (for example by FLT3 inhibition) might be beneficial DNA damage occurs through the genotoxic agent by reducing the relapse risk of AML.

Acute Myeloid Leukemia (AML) accounts for approximately 25% of all leukemias in adults in the Western world, and therefore is the most frequent form of blood neoplasia. Leukemic stem cells show abnormal proliferation, activation of antiapoptotic pathways and the impairment normal cell differentiation resulting in the dysregulated production of not functional blood cells, known as blast. AML is an aggressive disease, with a relative survival rate for all ages 5 years after diagnosis of 29.5%, the clinical manifestations of AML reflect the accumulation of malignant, poorly differentiated myeloid cells within the bone marrow, peripheral blood and in other organs. Diagnostic tests are mainly constituted by blood cells count and morphology, AML diagnosis is established by the presence of >=20% myeloid blasts in the bone marrow or peripheral blood. The prognostic assessment of AML patients is of capital importance for the management of the disease and to set up risk adapted therapies. Although clinical factors play an important role in disease development, karyotype is the most independent prognostic factor to forecast patients’ survival and it is adopted to provide the framework for risk-adapted treatment approach (Deschler and Lübbert, 2006; De Kouchkovsky and Abdul-Hay, 2016). The European Leukemia Net (ELN) guidelines aims to standardize risk stratification in adult AML patients by incorporating cytogenetic and known molecular abnormalities in hot spot genes. Accordingly, AML patients could be stratified into distinct prognostic risk groups (favorable, intermediate or adverse) based on their cytogenetic and molecular profile. Although this classification is the gold standard for the stratification of patients, it is fulfilled for only the 75% of AML whereas it is poorly satisfying for those patients resulted with normal karyotype (nk) at the conventional cytogenetic analysis. Normal karyotype AML (nkAML) patients mostly belong to the intermediate risk category but they experience an extremely heterogeneous outcome that represents an unmet needs in the clinical context of AML (De Kouchkovsky and Abdul-Hay, 2016; Döhner et al., 2017). In the last few years, large-scale tumour-sequencing studies have demonstrated that the majority of cancers, including hematologic neoplasia, are driven by Structural Variants (SVs) that are, for instance, genomic rearrange- ments larger than 50 bp. SVs include insertions, translocations, inversions and Copy Number Alterations (CNAs) (deletions and duplications). The recent development of high-throughput sequencing platforms provided impressive insights into leukemia pathogenesis and contributed to consider SVs as the hallmark of the genome instability leading to the establishment of the neoplasia. Beside karyotype, SVs detection is currently addressed by Next Generation Sequencing (NGS) technologies that allow the simultaneous and accurate detection of recurrent SVs breakpoints (Schütte et al., 2019), nothwithstanding, NGS faces inaccuracy and limitations when applied to resolve wide and structurally complex SVs due to the short length (100-500 bp) of the sequencing read employed (Norris et al., 2016). In this study, we exploited the long-reads Oxford Nanopore Sequencing technology to explore the genome of a cohort of 152 AML patient with normal cytogenetics, aiming to address the genomic analysis challenges and to identify new potential genomic biomarkers able to refine the prognostic forecasting for nkAML patients. Of 152 bone marrow samples collected at diagnosis, 85 referred to the hematology unit of the A.O.U.Careggi and 67 were prospectively collected for the AML #1310 study by the Italian Hematologic Network GIMEMA (Venditti et al., 2019). The DNA purified from nkAML samples was used to sequence the whole genome by the nanopore long-reads approach and further analysed by the bioinformatic pipeline specifically developed for SVs calling. Two SVs caller, Sniffles (Sedlazeck et al., 2018) and cuteSV (Jiang et al., 2020), were employed for the identification of an high-confidency callset of SVs that were further clustered and filtered before correlating them with patients’ outcome data. We employed an univariate Cox proportional-hazards analysis to weight the correlation between patients’ survival and each predictor variables. Further, to better estimate the cumulative impact of multiple genome and clinical variables, we developed a multi- variate Cox regression model including those SVs selected by Cox univariate model (pvalue <.05) and other predictors such as age, white blood cells count and the known molecular abnormalities in specific hotspot genes included in the ELN guidelines (Fms related Receptor Tyrosine Kinase 3 (FLT3)-ITD, Nucleophosmin 1 (NPM1), CCAAT Enhancer Binding Protein alpha (CEBPa)). Multivariate analysis allowed to select 12 SVs, represented by genomic deletions or insertions, with high impact on patients’ leukemia free and Overall Survival (OS). Of those, 8 resulted with an HR >1 (also referred as High Risk SVs (hrSVs)), thus associated with an increased risk of death, the other with an Hazard Ratio (HR) <1 (also referred as Low-risk SVs (lrSVs)) were associated to a reduced risk of death. The following stratification of the study cohort based on the presence of hrSVs enabled the identification of a high risk group of patients (accounting for the 17% of the cohort) with an extremely poor survival (median OS time 8.27 months for the group harbouring the hrSVs compared to 62.7 month fo the other, LogRank pvalue <.0001) and a low rate of response to therapy (46% for the patients with hrSVs compared to the 80%, pvalue <.0001). Taking together, these data suggest that the employ of an emerging long-reads sequencing technology capable to detect wide SVs together with a dedicated analysis pipeline could represent a powerful tool to accurately screen the whole genome of AML patients and identify new genomic biomark- ers for the prognostic assessment of nkAML patients capable to refine the actual ELN prognostic assessment in our cohort. inversions and Copy Number Alterations (CNAs) (deletions and duplications). The recent development of high-throughput se- quencing platforms provided impressive insights into leukemia pathogenesis and contributed to consider SVs as the hallmark of the genome instability leading to the establishment of the neo- plasia. Beside karyotype, SVs detection is currently addressed by Next Generation Sequencing (NGS) technologies that allow the simultaneous and accurate detection of recurrent SVs breakpoints (Schütte et al., 2019), nothwithstanding, NGS faces inaccuracy and limitations when applied to resolve wide and structurally com- plex SVs due to the short length (100-500 bp) of the sequencing read employed (Norris et al., 2016). In this study, we exploited the long-reads Oxford Nanopore Se- quencing technology to explore the genome of a cohort of 152 AML patient with normal cytogenetics, aiming to address the genomic analysis challenges and to identify new potential genomic biomarkers able to refine the prognostic forecasting for nkAML patients. Of 152 bone marrow samples collected at diagnosis, 85 referred to the hematology unit of the A.O.U.Careggi and 67 were prospectively collected for the AML #1310 study by the Italian Hematologic Network GIMEMA (Venditti et al., 2019). The DNA purified from nkAML samples was used to sequence the whole genome by the nanopore long-reads approach and further analysed by the bioinformatic pipeline specifically developed for SVs calling. Two SVs caller, Sniffles (Sedlazeck et al., 2018) and cuteSV (Jiang et al., 2020), were employed for the identification of an high-confidency call-set of SVs that were further clustered and filtered before correlating them with patients’ outcome data. We employed an univariate Cox proportional-hazards analysis to weight the correlation between patients’ survival and each predic- tor variables. Further, to better estimate the cumulative impact of multiple genome and clinical variables, we developed a multi- variate Cox regression model including those SVs selected by Cox univariate model (pvalue <.05) and other predictors such as age, white blood cells count and the known molecular abnormalities in specific hotspot genes included in the ELN guidelines (Fms related Receptor Tyrosine Kinase 3 (FLT3)-ITD, Nucleophosmin 1 (NPM1), CCAAT Enhancer Binding Protein alpha (CEBPa)). Multivariate analysis allowed to select 12 SVs, represented by genomic deletions or insertions, with high impact on patients’ leukemia free and Overall Survival (OS). Of those, 8 resulted with an HR >1 (also referred as High Risk SVs (hrSVs)), thus associ- ated with an increased risk of death, the other with an Hazard Ratio (HR) <1 (also referred as Low-risk SVs (lrSVs)) were as- sociated to a reduced risk of death. The following stratification of the study cohort based on the presence of hrSVs enabled the identification of a high risk group of patients (accounting for the 17% of the cohort) with an extremely poor survival (median OS time 8.27 months for the group harbouring the hrSVs compared to 62.7 month fo the other, LogRank pvalue <.0001) and a low rate of response to therapy (46% for the patients with hrSVs compared to the 80%, pvalue <.0001). Taking together, these data suggest that the employ of an emerging long-reads sequencing technology capable to detect wide SVs together with a dedicated analysis pipeline could represent a powerful tool to accurately screen the whole genome of AML patients and identify new genomic biomark- ers for the prognostic assessment of nkAML patients capable to refine the actual ELN prognostic assessment in our cohort.

Normal karyotype pediatric B-cell precursor ALL patients are heterogeneous with respect to chemotherapy response, relapse rates and prognosis and the reason is unknown. These patients are treated with a standard protocol, and stratified using MRD methodology that shows they have variable responses and predicted outcomes. This study aims to determine the reasons behind such heterogeneity. This study shows that through miRNA profiling, miR-221/222 are differentially expressed in normal karyotype patients and are up regulated in Poor Responder patients. Through proliferation, apoptosis, viability assays and cell cycle analysis, proliferation advantage was identified as the main cause of resistance to glucocorticoid treatment in miR-221/222 over expressing cell lines. SMAD1 and CREBBP were down regulated in miR-221/222 over expressing cell lines implicating the dysregulation of TGFβ and glucocorticoid-receptor pathways, while NLK was identified as a novel target of miR-221/222 through which resistance to chemotherapy is mediated.

目前廣泛應用于胎兒醫學的唐氏綜合症篩查法，即結合早孕期胎兒頸項透明層的超聲檢查，及母體血清生化指標的綜合篩查法。頸項透明層是指在早孕期利用超聲檢測到的胎兒頸后的皮下積水，其作為預測胎兒異常的一項重要“軟指標，其臨床意義，尤其是與胎兒染色體異常及器官結構異常之間的關係，逐漸得到深入的認識，但其形成機制尚未明確。現在已知有一百餘種畸形及遺傳綜合征與胎兒頸項透明層增厚相關，但其染色體異常譜系，尤其是亞顯微的染色體異常仍有待明確。大部分頸項透明層增厚但核型正常的胎兒預後良好，但約3-10%的這部分胎兒會伴有畸形或出生后的神經智力發育缺陷。而傳統核型分析無法檢測到亞顯微的染色體異常，從而無法判斷這部分核型正常卻伴有缺陷的胎兒是否因為這類染色體異常而致病。
微陣列比較基因組雜交芯片作為檢測兒童發育遲緩者及器官結構異常原因的重要手段已廣泛應用于臨床。在染色體核型正常的胎兒中，若伴有器官結構異常的胎兒，5-12%被檢出與該畸形相關的微缺失及微重複；若僅伴有孕婦高齡或唐氏篩查高危，則微缺失及微重複檢出率約1%。
該課題旨在研究頸項透明層增厚但核型正常的胎兒中，染色體拷貝數變異發生的頻率及頻譜；評估微陣列比較基因組雜交芯片在協助臨床判斷胎兒預後中的作用。因此，我們開展該多中心隊列研究，通過納入449例頸項透明層厚度≧3.5 mm但正常核型胎兒的，檢測其染色體拷貝數變異，監測并記錄其圍產、產後及新生兒期情況。微陣列比較基因組雜交芯片總共檢出2.8%的異常拷貝數變異，其大小範圍為0.1 kb至18Mb。在伴有器官結構異常的胎兒組中，異常拷貝數變異檢出率達7.8%。對於頸項透明層厚度≧4.0 mm的胎兒，異常拷貝數變異檢出率可達7.3%。
對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜。但，該研究中發現重複的致病拷貝數變異，如22號染色體長臂1區1帶的微重複或微缺失，2號染色體長臂2區2帶的微缺失。未在3號、7號、12號、13號、18號、20號、21號或Y染色體上發現與胎兒頸項透明層增厚相關的致病拷貝數變異。
頸項透明層增厚的胎兒79.3%預後良好；若經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則81.2%預後良好。如果僅頸項透明層增厚不伴有結構異常的胎兒，經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則93.5%預後良好。
綜上所述，微陣列比較基因組雜交芯片顯著提高了致病拷貝數變異的檢出率。可考慮將微陣列比較基因組雜交芯片作為頸項透明層厚度≧4.0 mm的胎兒染色體異常檢查的首要方法。對於僅頸項透明層增厚不伴有結構異常的胎兒，且經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，絶大部分預後良好。對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜，但發現重複出現的致病拷貝數變異。通過初步的基因本體分析及基因通路分析，神經嵴細胞的分化遷徙功能異常可作為今後研究頸項透明層增厚的病理生理機制的方向。
Measurement of nuchal translucency (NT) has been recognized as a sensitive marker for fetal chromosomal disorders for more than a decade, and is presently used as a routine first-trimester screening test. Although over 100 abnormalities and genetic syndromes have been reported to be associated with increased NT, these associations have not been fully explored and the relevant spectrum of associated submicroscopic chromosomal abnormalities has not been sufficiently investigated. The majority of euploid fetuses with increased NT have a good outcome, but around 3-10% of fetuses present with structural or neurodevelopmental abnormalities postnatally. A range of genetic syndromes has been reported, many of which are linked to submicroscopic chromosomal abnormalities that are typically missed by conventional karyotyping.
Microarray-based comparative genomic hybridization (arrayCGH) has been applied as the first-tier diagnostic tool for the evaluation of developmental delay and structural malformations in children. In fetuses with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 5-12% with a structural anomaly and in about 1% of those whose indications were advanced maternal age or positive screening results.
The objectives of this study were to delineate the frequency and spectrum of pathogenic chromosome copy number variants (CNVs) among fetuses with increased NT and normal karyotype; to evaluate the role of arrayCGH to predict the prognosis of the high NT fetuses; to explore the genotype-phenotype correlations of increased NT. Therefore, a multi-centre cohort of 449 fetuses with NT ≧3.5 mm and normal karyotype were further investigated by arrayCGH. Antenatal surveillance, pregnancy outcome and paediatric follow up were documented. ArrayCGH detected abnormal CNVs in 2.8% (14 of 449) of the fetuses with high NT; the size of CNVs ranged from 0.1 kb to 18Mb. Among fetuses with major congenital abnormalities the incidence of abnormal CNV reached 7.8% (4 of 51). By adjusting the NT to ≧4.0 mm as the referral indication, 7.3% (14 of 192) of the fetuses would have abnormal arrayCGH results. The spectrum of pathogenic CNVs found associated with increased NT was diverse. However, there were recurrent ones such as the deletions or duplications at chromosomal region 22q11, and deletions in ZEB2. There was no pathogenic CNV related with increased NT found in chromosomes 3, 7, 12, 13, 18, 20, 21, or Y. The total normal outcome rate of euploid fetuses with an increased NT was 79.3%; for fetuses with normal arrayCGH results 81.2% had a normal outcome. In fetuses with isolated increased NT, normal arrayCGH results predict a favorable prognosis of 93.5%.
In conclusion, arrayCGH significantly increased the diagnostic yield of pathogenic CNVs. In clinical practice arrayCGH may be considered as the first tier investigation in fetuses with an increased NT more than 4.0 mm. In cases with an isolated increased NT with normal arrayCGH results the pregnancy outcome is likely to be favorable. The spectrum of abnormal CNVs found by arrayCGH is diverse but there are recurrent cases such as del/dup 22q11 and del ZEB2. Our preliminary gene ontology and pathway analysis showed that gene pathways related to neural crest cells may be considered as a future study for physiopathologic mechanisms of NT.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Huang, Jin.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 106-120).
Abstracts also in Chinese.

Bibliography: leaves 653-669
Before God created human beings, He devised a plan to save them in case they sinned. In this plan, the second Person of the Godhead would become human. Thus, the incarnation of the second Person of the Godhead was solely for the purpose of saving fallen, sinful human beings. There would have been no incarnation if human beings had not sinned. Thus, the nature of the mission that necessitated the incarnation determined what kind of human nature Jesus was to assume. It was sin that necessitated the incarnation – sin as a tendency and sin as an act of disobedience. In His incarnational life and later through His death on Calvary’s cross, Jesus needed to deal with this dual problem of sin. In order for Him to achieve this, He needed to identify Himself with the fallen humanity in such a way that He would qualify to be the substitute for the fallen humanity. In His role as fallen humanity’s substitute, He would die vicariously and at the same time have sin as a tendency rendered impotent. Jesus needed to assume a human nature that would qualify Him to be an understanding and sympathetic High Priest. He needed to assume a nature that would qualify Him to be an example in overcoming temptation and suffering. Thus, in this study, after comparing and critically evaluating the Christological views of Jack Sequeira, Millard J. Erickson and Norman R. Gulley, I propose that Jesus assumed a unique post-fall (postlapsarian) human nature. He assumed the very nature that all human beings since humankind’s fall have, with its tendency or leaning towards sin. However, unlike other human beings, who are sinners by nature and need a saviour, Jesus was not a sinner. I contend that Jesus was unique because, first and foremost, He was conceived in Mary’s womb by the power of the Holy Spirit and was filled with the Holy Spirit throughout His earthly life. Second; He was the God-Man; and third, He lived a sinless life. This study contributes to literature on Christology, and uniquely to Christological dialogue between Evangelical and Seventh-day Adventist theologians.
Philosophy, Practical and Systematic Theology
D. Phil. (Systematic Theology)