Pyrosequencing to identify homogeneous phenomenon when using recipients/donors with different CYP3A5*3 genotypes in living donor liver transplantation

Abstract

This study used pyrosequencing to determine the proportional distribution of CYP3A5*3 genotypes to further confirm the homogeneous phenomenon that is observed when recipients and donors in living donor liver transplantation (LDLT) have a different single nucleotide polymorphism (SNP) genotype. We enrolled 42 recipient/living donor pairs and the SNPs of CYP3A5*3 were identified by polymerase chain reaction-restriction fragment length polymorphism. We performed 120 liver graft biopsies as part of clinical investigations after LDLT. Pyrosequencing of the CYP3A5*3 SNPs revealed that among the 16 recipients with the G/G genotype, 94.68% had the G and 5.32% the A allele. Among the 14 recipients with the A/G genotype, 78.08% had the G and 21.92% the A allele, and among the 12 recipients with the A/A genotype, 18.45% had the G and 81.55% the A allele. Among the 12 donors with the G/G genotype, 93.85% had the G and 6.14% the A allele. Among the 26 donors with the A/G genotype, 75.73% had the G and 24.27% the A allele, and among the 4 donors with the A/A genotype, 11.09% had the G and 88.91% the A allele. There were a total of 120 liver graft biopsy samples; among the 37 recipients with the G/G genotype, 89.74% had the G and 10.26% the A allele, among the 70 recipients with the A/G genotype, 71.57% had the G and 28.43% the A allele, and among the 13 recipients with the A/A genotype, 48.25% had the G and 51.75% the A allele. The proportional distribution of G and A alleles of the CYP3A5*3 SNP between recipients/donors and liver grafts after LDLT was significantly different (p<0.001). Pyrosequencing was useful in identifying detailed proportional changes of the CYP3A5*3 SNP allele distribution, and to confirm the homogeneous phenomenon when recipients and donors in LDLT have a different genotype.

Figure 1. The single nucleotide polymorphism of CYP3A5*3 was identified by polymerase chain reaction-restriction fragment length polymorphism analysis.

Pyrosequencing was performed with peripheral blood mononuclear cells and liver tissues of graft biopsies.

Figure 2. The proportional changes of G and A alleles were determined by pyrosequencing of peripheral blood mononuclear cells of a recipient (#668) and a donor, and of a liver graft biopsy sample after living donor liver transplantation (recipient: A/A; donor: A/G, identified by restriction fragment length polymorphism analysis).

Figure 3. The proportional changes of G and A alleles were determined by pyrosequencing of peripheral blood mononuclear cells of a recipient (#532) and a donor, and of a liver graft biopsy sample after living donor liver transplantation (recipient: A/A; donor: G/G, identified by restriction fragment length polymorphism analysis).

Figure 4. The proportional changes of G and A alleles were determined by pyrosequencing of peripheral blood mononuclear cells of a recipient (#523), a donor, and a liver graft biopsy sample after living donor liver transplantation (recipient: G/G; donor: A/G, identified by restriction fragment length polymorphism analysis).