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.

Homogeneous phenomenon of the graft when using different genotype characteristic of recipients/donors in living donor liver transplantation

“…In the early stage within 1 mo after LDLT, this proportional change of the alleles was not stabilized. After a longitudinal followed up liver graft biopsy, the proportional change of the alleles was going to constant more than 1 mo after LDLT [4] . Followed up liver graft biopsy sampling more than 5 times per recipient was 24.4% (10/41) in CYP3A5*3, 17.1% (7/41) in MDR1-3435 and 66.7% (2/3) in CYP3A4*18.…”

“…Interestingly, the liver graft still presented the original CYP2C19 genotype characteristics of the donor when liver graft biopsy for a part of the clinical investigation at that moment. This clinical phenomenon could not be demonstrated used with Western blotting analysis [10] but it represented from the traditional sequencing so-called homogeneous phenomenon [3,4] . In the present study, pyrosequencing goes forward to identify the proportional change only requires a standard biotinylation of one of the PCR primers.…”

“…Followed up liver graft biopsy sampling more than 5 times per recipient was 24.4% (10/41) in CYP3A5*3, 17.1% (7/41) in MDR1-3435 and 66.7% (2/3) in CYP3A4*18. It was also suggested that the acute rejection is easily occurred within one month after liver transplantation [2,5] and the anti-rejection agent concentration in the serum was higher in one month later after LDLT [4] . As we know, the effect of MDR1-3435, CYP3A5*3, and CYP3A4*18 SNPs on cyclosporine A pharmacokinetics is very important [16][17][18][19][20][21][22] .…”

“…The PyroMark Q24 Plate containing the samples was heated at 80 ℃ for 2 min using a PyroMark Q24 Plate Holder and a heating block. The plate was then removed from the plate holder and the samples were allowed to cool to room temperature (15-25 ℃) for at least 5 min, and the reagents, including enzyme and substrate mixtures, and nucleotides were added to the cartridge (PyroMark Q24, Qiagen) [4,9] . The samples were analyzed using a PyroMark Q24 system (Qiagen) according to standard protocols.…”

“…The different genotype of the drug metabolic isoenzyme represented with an uncertain phenomenon when the new liver graft continuously circulated with the original recipient peripheral blood [3] . Recently, we successfully identified the homogeneous phenomenon of the single nucleotide polymorphism (SNP) from the liver graft biopsy specimen using by the pyrosequencing method [4] . Herein, we would like to expose all of the polymorphisms of CYP3A5*3, MDR1-3435 and CYP3A4*18 because of the importance for the immunosuppressive agent metabolism in the liver transplantation setting.…”