Applicability and universality of formulae for chimerism monitoring considering stutter PCR products

Summary

Introduction

Analysis of genetic chimerism is one of the main methods to monitor the bone marrow engraftment after stem cells transplantation. PCR amplification of short tandem repeats (STR) utilizing the differences in the STRs length between donor and recipient is used as a routine test for chimerism. However, chimerism estimation is often complicated by “stutter” PCR peaks appearing due to irregular DNA polymerase activity. Generally, these sequences are 4 nucleotides shorter than a specific marker and may concur with a specific sequence of recipient’s DNA, thus hindering chimerism estimation based on that locus. In our previous work, we showed that percentage of the stutter peaks in total DNA product is a locus- and patient-specific constant. Therefore, we have derived a formula for the mixed chimerism calculation in the presence of stutter peaks. It was also shown that the results of chimerism estimation by means of the formulae proposed were similar for “stutter-complicated” and “stutter-free” markers (p<0.05). The aim of our study was to validate these formulas for different allelic statuses, including cases with heterozygous uninformative donor allele that overlay an informative allele of the recipient (Fig.1). The formulas should be considered valid if the ratios of the stutter 1 to both peak 1 and peak 2 of the heterozygous STR allele are constant. We expect this to be true, since the stutter peaks are caused by irregular DNA polymerase activity and depend on the product length and nucleotide sequence of the STR.

Materials and methods

The study was done with 20 DNA samples. Genomic DNAs of donors and patients were isolated from bone marrow specimens. Genetic chimerism was assessed by the STR-PCR analysis with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci. The fragment analysis was performed on a 3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software. The informative loci were chosen in advance by comparing pre-transplant patient DNA and donor DNA. The ratios stutter-peak 1/peak 1 and the stutter-1/peak 2 in heterozygotes were calculated using standard formulae. The variances of these ratios for each locus and patient were then compared with respect to contribution of stutter components to the total amount of the product.

Results

In twenty transplant cases at seven time points, we analyzed the variances for the locus and patient in repeated measurements (Fig. 2). The following mean variances were obtained: 0.01 for the ratio of a stutter-peak 1 to peak 1 in heterozygote, and 0.014 for the ratio of stutter-peak to another heterozygote peak (peak 2). The variance for the contribution of the stutters to the total DNA from the previous study is 0.01. Thus, not only contribution of the stutters to the total DNA amounts, but also the ratio of stutter to any heterozygous peak can be considered an individual constant for each locus and patient. Therefore, it can be used in the chimerism calculation. However, in case of using stutter 1 and peak 2, it is necessary to increase sensitivity to 1.5%. In cases of low mixed chimerism, these loci are not recommended for the chimerism calculation. The calculations for case A (Fig. 2) will be made according to the formula 2 (Fig. 1), and, for cases 2 and 3, according to the formula 1 as for the homozygote, since the coinciding peak in this case may not be taken into account.

Conclusion

The resulting formulae shown in Fig. 1 are valid for different allelic combinations, since the ratio of any stutter to any major peak in the heterozygote is a constant, which is associated with a definite probability of DNA polymerase error, constant for each particular sequence. However, due to random fluctuations in the product quantity for the stutter 1/peak 2 ratio, the error is larger than for the stutter 1/peak 1 ratio. Thus, the stutter-peaks are not an obstacle to a correct calculation of gene chimerism. The stutter contribution to the total amount of the product in a given locus may be considered constant for the chimerism calculations. Alternatively, one may use a ratio of stutter-to-allele product at which it was obtained due to DNA polymerase errors.

Keywords

Allogeneic hematopoietic stem cell transplantation, chimerism, short tandem repeats, stutter-band.