Genotypic analysis of engraftment in thalassemia following bone marrow transplantation using synthetic oligonucleotides.

ABSTRACT

DNA hybridization with synthetic oligonucleotide probes was used to assess engraftment in 19 thalassemic patients who received bone marrow grafts from their respective healthy HLA-identical siblings. Three oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed so as to produce simple RFLP (restriction fragment length polymorphism) patterns. Each probe hybridizes to one or two bands in HinfI-digested genomic DNA. The combined use of these three probes allowed a discrimination between all the HLA-identical siblings tested. Both donor-specific and recipient-specific DNA fragments existed in 18 out of the 19 sibling pairs studied. One pair possessed only a donor-specific fragment. DNA analysis at an early stage after the graft detected donor-specific fragments in 15 out of 19 patients, recipient-specific fragments in three patients and a mix of recipient and donor fragments in one patient. At a later stage this patient possessed donor-specific fragments only. Follow-up DNA analysis confirmed these findings. Thus 16 patients continued to display donor-specific fragments over 60 days post-transplant. These DNA data showed strong correlation with the clinical status of the patients as well as with other markers of engraftment including cytogenetics and hemoglobin synthesis. The patients who showed donor-specific fragments over 60 days have been free of thalassemic symptoms for over 300 days. Moreover, in 11 cases it was possible to predict the fate of the graft within 15 days after transplantation. In conclusion, the use of the three synthetic oligonucleotide probes provides a powerful tool in documenting engraftment in bone marrow transplantation.