Feasibility of a cost-effective approach to evaluate short tandem repeat markers suitable for chimerism follow-up
ABSTRACT Precise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients.
To set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT.
Six dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients.
Four markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67-0.88) under our experimental system. A sensitivity of 0.8-1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH.
To our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage of Taq polymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient.
The data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.
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ABSTRACT: Malignant complications of celiac disease (CD) include carcinomas and lymphomas. The genetic basis behind cancer development in CD is not known, but acquisition of genetic abnormalities and genomic instability has been involved. The aim of this study was to explore molecular characteristics of genomic instability in CD patients by analyzing microsatellite instability (MSI) and loss of heterozygosis (LOH) with carefully selected microsatellites. We genotyped small bowel biopsies and peripheral blood samples from 20 untreated CD patients using five microsatellites related to MMR genes (panel A), and five repeats associated with tumor suppressor genes, chromosome instability, inflammation, and cancer (panel B). Genomic instability was found in seven out of 20 (35%) cases at: D5S107, D18S58, GSTP, TP53 or DCC, being TP53 the most frequently affected (five out of seven cases; 71%). Microsatellite alterations were significantly found using panel B markers (P=0.04). No cases with high frequency of MSI and replication error phenotype were detected. Only one case displayed MSI-L alone. Three patients exhibited LOH and three other cases showed LOH with low level of MSI, being classified as having chromosome instability phenotype. Two novel observations were found in this study: first, the finding that non-neoplastic cells from a group of untreated CD patients present genomic instability at nucleotide level; and second, the advantage to use carefully selected microsatellites to identify celiac patients with molecular instability. Our data support the existence of chromosome instability phenotype in CD, suggesting that stable and unstable patients are genomically distinct subtypes that may follow a different evolution.European journal of gastroenterology & hepatology 11/2008; 20(12):1159-66. DOI:10.1097/MEG.0b013e3283094ee9 · 2.15 Impact Factor
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ABSTRACT: Hematopoietic stem cell transplantation (HSCT) is a very successful method of treatment for children with different aquired or inborn diseases. The main goal of post-transplantation chimerism monitoring in HSCT is to predict negative events (such as disease relapse and graft rejection), in order to intervene with appropriate therapy and improve the probability of long-term DFS (disease free survival). In this context, by quantifying the relative amounts of donor and recipient cells present in the peripheral blood sample, it can be determined if engraftment has taken place at all, or if full or mixed chimerism exists. In a group of patients who underwent hematopoietic stem cell transplantation at the Mother and Child Health Care Institute, we decided to use standard human identfication tests based on multiplex PCR analyses of short tandem repeats (STRs), as they are highly informative, sensitive, and fast and therefore represent an optimal methodological approach to engraftment analysis.Archives of Biological Sciences 01/2007; 59(1). DOI:10.2298/ABS0701023K · 0.61 Impact Factor
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ABSTRACT: We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.Nucleic Acids Research 09/2010; 38(17):e172. DOI:10.1093/nar/gkq657 · 8.81 Impact Factor