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Cellular and Molecular Characterisation of Familial Haemophagocytic Lymphohistiocytosis Type 1

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Title: Cellular and Molecular Characterisation of Familial Haemophagocytic Lymphohistiocytosis Type 1
Authors: Chakravorty, Subarna
Item Type: Thesis or dissertation
Abstract: Haemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition in which absent or markedly reduced T cell and natural killer (NK) cell cytotoxicity results in uncontrolled proliferation of T cells, activation of macrophages, hypercytokinaemia, pancytopaenia, and hepatosplenomegaly. Familial HLH (FHL), an autosomal recessive disorder affecting young infants and children, is fatal unless treated with chemotherapy and allogeneic haemopoietic stem cell transplantation. Mutations in the gene encoding perforin, a lytic protein involved in cell cytotoxicity, account for 30% of cases of FHL (FHL II). Mutations in genes encoding hMunc 13-4, syntaxin 11, and syntaxin binding protein 2, all essential for perforin release and T/NK cell cytotoxicity, have also been identified in FHL (FHL III IV and V respectively). A previous report form our group showed that in the UK, in consanguineous families of Pakistani descent, FHL (designated FHL I) maps to 9q21.3-22 in an area spanning 5Mb and containing 14 genes. The aim of my project was to identify the gene responsible for FHL I and to study the function of the corresponding protein. Based on the premise that similar to other types of FHL, the protein product of FHL I candidate gene would function in the perforin-dependent cytotoxic pathway, 4 out of the 14 genes emerged as strong functional candidates: KIF27, RASEF, UBQLN1 and FRMD3. My mutation screening strategy aimed at amplifying and sequencing all exons and intronexon boundaries of the candidate genes, followed by screening the remaining genes of the FHL I locus. The material used was either genomic DNA extracted from 4 parental and 1 patient B cell line, or cDNA from the latter. In 2 newly identified patients, genomic DNA from granulocytes was used. PCR amplification of genomic DNA and direct sequencing or cloning and sequencing of individual clones or in some cases direct sequencing of the full length cDNA from the patient B cell line did not reveal any mutations in any of the known genes in the FHL I locus. For KIF27, mutations were excluded in gene regulatory areas by amplification and direct sequencing of the whole promoter region, up to 1kb upstream of the transcription start site. KIF27 protein expression was also studied by immunoblotting and immunostaining followed by confocal microscopy but no abnormality was identified at protein level. Promoter area mutation was also excluded in UBQLN1, the other strong candidate. The gene responsible for FHL I remains unidentified. Further refinement of the locus with high-density SNP arrays in newly diagnosed patients demonstrating linkage to the FHL I locus might help identify the gene responsible for FHL I.
Issue Date: 2011
Date Awarded: Mar-2011
URI: http://hdl.handle.net/10044/1/6433
DOI: https://doi.org/10.25560/6433
Supervisor: Layton, Mark
Karadimitris, Anastasios
Sponsor/Funder: Leukaemia and Lymphoma Research
Author: Chakravorty, Subarna
Department: Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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