27 research outputs found
Analysis of hMLH1 germline mutations in three Turkish hereditary nonpolyposis colorectal cancer kindreds
Ankara : The Department of Molecular Biology and Genetics and Institute of Engineering and Science, Bilkent Univ., 1998.Thesis (Master's) -- Bilkent University, 1998.Includes bibliographical references leaves 84-92.Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common genetic diseases in Western world. It is a clinical syndrome characterized by an inherited predisposition to early onset colorectal and an increased incidence of other cancers. The disease is caused by a germline defect in one of five human DNA mismatch repair genes, hMLHl, hMSH2, hPMSl, hPMS2, and hMSH6. Defects in hMLHl and hMSH2 account for the majority of mutations found in HNPCC families. In this study, a variety of mutation detection methods were used to identify hMLHl germline mutations in three Turkish HNPCC kindreds.
Restriction enzyme analysis of genomic DNA was used to analyze five members of an HNPCC family with a previously described G884C mutation. The genotypes of all five individuals were determined by Dde\ digestion and the results were confirmed by DNA sequence analysis. Hph\ restriction enzyme analysis was used to analyze twenty-nine members of an unrelated HNPCC family for a previously identified A1652C mutation. Genotypes were determined for all individuals and the results were confirmed by DNA sequence analysis. Both of these restriction enzyme analyses are reliable, cost-effective methods that can be used in mutation screening programs for family members who request genetic counseling.
Single strand conformation polymorphism analysis (SSCP) was used to screen for unknown germline mutations. Nine DN A samples with defined mutations in the hMLHl gene were analyzed using several gel formulations and electrophoretic conditions to determine the most sensitive protocols. These protocols were then used for routine mutation detection.
In a third HNPCC family, for whom no mutation has yet been defined, the complete coding sequence of the hMLHl gene was screened by SSCP. Two exons, 7 and 15, showed an altered mobility compared to control sequences. The nucleotide sequence of these two exons was determined by automated fluorescence DNA sequence analysis. The differential mobility observed for exon 15 appears to be due to an intonic polymorphism in the control sample. Preliminary results for exon 7 show no difference between proband and control nucleotide sequences. Thus, the DNA mismatch repair defect in this kindred appears not to be in hMLHl. Further studies will focus on the analysis of hMSH2.Akyerli, CemaliyeM.S
Analysis of candidate molecular targets in adult (CML) and childhood (AML, ALL) Leukemias
Cataloged from PDF version of article.Candidate molecular targets were investigated in three different types of
leukemias, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and
acute lymphocytic leukemia (ALL). The first group of these molecular targets was
identified through a cDNA based gene expression profile analysis in sixty-seven
CML patients who were classified according to clinical parameters known as new
prognostic score (NPS). CML patients can be divided into three groups of low-risk,
intermediate-risk, and high-risk, based on NPS. Response of these risk groups to
treatment is not uniform and the gene expression profiles associated with each risk
group remain unknown. Seven genes were chosen from a cDNA microarray study in
which two high versus two low-risk patients were analyzed. Semi-quantitative and
real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of these
differentially expressed transcripts highly correlated with the microarray data.
Expression levels of all genes, except PTGS1, were significantly different between
the high (n=9) and low-risk (n=7) CML by semi-quantitative RT-PCR (IFITM1 and
CXCL3 p=0.001; CCNH p=0.012; RAB1A p=0.01, PRKAR2B p=0.016; UCP2
p=0.04; and PTGS1 p=0.315). Real-time RT-PCR analysis showed similar results
for IFITM1 expression in thirty-four low and eleven high-risk patients (p=9.7976 x
10-11). Higher IFITM1 or lower CXCL3 expression correlated with improved
survival (p=0.01 and p=0.059 respectively). Gene expression profiling is a valuable
tool to identify candidate risk group indicator genes for the development of a
molecular classification system for CML, which may also predict survival.
Although the connection between DNA-repair gene mutations and
hematological malignancies are now well established, germ-line mutations in the
base excision repair (BER) pathway was only recently documented in an inherited
cancer syndrome in human homologue of E. coli mut Y (MYH). Interestingly, the
cancer associated MYH missense mutations Tyr165Cys and Gly382Asp have been
documented with a high frequency (1 percent) in a control group of the British
population. Therefore, we screened the above mentioned missense variants in two
different childhood leukemias, AML (n=45) and ALL (n=140). Neither mutation was
present in any of the patient samples and controls, except for one patient diagnosed
with AML/M3. Tyr165Cys mutation in the heterozygous state was present in the
sample obtained at the time of initial diagnosis. Further sampling, at remission, and
the analysis of parental DNA, showed only the normal allele. Therefore, the
mutation was considered to be specific for the leukemic blasts. Based on these
results, an association between childhood leukemias and the MYH missense variants
Tyr165Cys and Gly382Asp was not observed. Also, these variants appear to be
absent -if not at a very low frequency- in the Turkish population, contrary to the
British population.Boylu, Cemaliye AkyerliPh.D
Endothelial progenitor cells display clonal restriction in multiple myeloma
BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V(H)). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V(H )primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM
Sensitive Detection of Molecular Targets in Cancer by Minisequencing
Purpose: Molecular alterations leading to specific mutations are essential for tumor development and survival. Accurate
analysis of these molecular targets is important for diagnosis, early detection, forecasting of prognosis and aiding in the
treatment of different cancer types. Therefore, for sensitive analysis of molecular markers, we aimed to optimize and use
minisequencing protocols besides Sanger sequencing.
Methods and Materials: Sanger sequencing and minisequencing were performed for IDH1 R132, IDH2 R140/R172
and TERT promoter C228/C250 mutations using genomic DNA isolated from glioma samples. Minisequencing reactions
were performed with detection primers using SnaPshot Multiplex Ready Reaction Mix and run on an automated capillary
electrophoresis. Multiplex peaks were analyzed with GeneMapper Software.
Results: In the multiplex minisequencing analyses, peaks corresponding to wild type alleles and different mutations
were detected. The presence of the peaks next to the wild type peaks points to the presence of variations in that location
and the nature of the mutation can be identified according to the color.
Conclusions: Identification of molecular markers in cancer is very important. Minisequencing is a reliable method for the
detection of molecular targets