X chromosome methylation-based chimerism assay for sex-mismatched hematopoietic stem cell transplantation

Analysis of hematopoietic chimerism is important for monitoring engraftment , graft failure, and disease recurrence. Although several techniques are no w available, their sensitivity is unsatisfactory. In sex-mismatched stem ce ll transplantation (SCT) with a female donor, Y chromosome-specific sequenc es have proven the most sensitive marker. However, in the case of a male do nor, no such reliable marker has been available to date. In this study, we report a novel method we developed to detect microchimerism in female recip ients who receive SCT from male donors. The X-linked human androgen recepto r gene (HUMARA) contains a highly polymorphic CAG trinucleotide repeat. Nea r this polymorphic site are methyl-sensitive HpaII restriction enzyme sites . After HpaII digestion, unmethylated male HUMARA sequences are completely digested, while methylated female ones remain intact among the male origin cells. This allows a highly efficient detection of a small number of female cells. Combined with the nested PCR technique, the X chromosome methylatio n-based chimerism assay could attain a 10(-4) level of sensitivity, which i s 1000-fold higher than that of conventional assays. The applicability of t he method was confirmed in two transplant cases. This highly sensitive meth od can also be applied to detect minimal residual disease or microchimerism in conditions other than hematopoietic SCT.