Feasibility of a Cost-Effective Approach to Evaluate Short Tandem Repeat Markers Suitable for Chimerism Follow-Up
Department of Genetics, Institute of Hematological Research, Mariano R Castex, National Medicine Academy of Buenos Aires, Argentina. Molecular Diagnosis
02/2004; 8(2):87-91. DOI: 10.2165/00066982-200408020-00002
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.
Available from: PubMed Central
- "identity and paternity testing). In addition, they are used in medical applications, including identification of fetal cells in maternal blood (12) and monitoring of allogeneic bone marrow transplants (13). "
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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.
Available from: Milena Velizarova
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ABSTRACT: The chimerism status after allogeneic bone marrow transplantation (BMT) provides substantial information about the replacement of host cells with donor cells after the transplantation. We studied six Bulgarian patients after sex-mismatched allogeneic BMT by fluorescent in situ hybridization (FISH) with alternatively labeled X and Y probes and three male-to-male donor-recipient pairs by genotyping of microsattellite loci. Complete hematopoietic chimerism (CC) was found in one patient who did not experience graft failure. Near-complete chimerism (NCC) was detected in three patients who experienced an autologous recovery over several months followed by relapse and graft failure. In one patient, low-level mixed chimerism (MC) that progressed to high-level MC, accompanied by full remission, was found. One patient showed low-level MC followed by complete loss of chimerism. In all patients the percentage of chimeric cells correlated with clinico-hematological data for remission or relapse. Of the three sex-matched BMT patients, one showed chimerism 3 months after BMT followed by recurrence, one died before the first monitoring, and in another patient, the markers produced no informative results. We conclude that dynamic changes in chimerism have clinical and predictive value for the hematological status of patients after BMT.
Key words: Bone marrow transplantation (BMT), Chimerism, Monitoring
Available from: Biju George
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ABSTRACT: Analysis of chimerism by polymerase chain reaction amplification of STR or VNTR has become a routine procedure for the evaluation of engraftment after allogeneic stem cell transplantation. Knowledge of the frequency of different STR or VNTR alleles in unrelated individuals in a population is useful for forensic work. In the context of HLA identical sibling bone marrow transplantation the informativeness of these markers needs to be evaluated. We evaluated five STRs (THO1, VWA, FES, ACTBP2, and F13A1) and 1 VNTR (APOB) for informativeness in stem cell transplants from HLA identical sibling donors. All four markers used individually allowed us to discriminate 20-56% of the patient donor pairs. Using a combination of all these markers along with a polymorphic marker in the beta-globin gene and the sex chromosome specific amelogenin marker, we were able to discriminate 99% of the patient donor pairs. We have established an algorithm for evaluating chimerism following HLA identical sibling donor transplants in the Indian population using molecular markers in 310 patients. Analysis of heterozygote frequencies in different populations is similar suggesting that this algorithm can be used universally for transplant centers to evaluate chimerism following allogeneic bone marrow transplantation.
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