EXPERIMENTAL AND CLINICAL TRANSPLANTATION

After renal transplant, immunosuppression therapy is used to reduce the risk of rejection. Here, we describe the case of an adult living related donor renal transplant recipient with rare natural chimerism, as discovered by short tandem repeat sequence analysis. In our process of matching transplant patients, we perform human leukocyte antigen testing and short tandem repeat chimerism testing to decide posto-perative immunosuppression strategy for transplant patients. We analyzed the short tandem repeat chimerism status before renal transplant and determined that this patient represented a rare case of natural chimerism. Assessment of organ recipient chimerism can inform physicians regarding a dosage reduction of immunosuppressive agents. Short tandem repeat sequence analysis provides substantial information regarding existing polymor-phisms and can identify chimerism, if present, and thereby guide immunosuppression strategies after renal transplant, which may improve the long-term immunosup-pression-free survival of renal transplant recipients.

Key words : Chimera status, Kidney transplantation

In kidney transplant studies, research has focused on microchimerism in order to induce the post-transplant immune tolerance.¹ Microchimerism assessment can be used as a diagnostic method for various diseases.^2-4 We suggest that microchimerism assessment can be used as a reference biomarker to monitor the status of the organ recipient’s immune tolerance after kidney transplant. Here, we report our application of this method for a renal transplant patient. We performed blood type and human leukocyte antigen (HLA) tests before transplant. In this process, the dispersed chimerism was inadver-tently discovered by short tandem repeat (STR) sequence analysis, which is generally used in genetic disease detection and forensic medicine. Here, we report the STR sequence analysis in a living related donor adult renal transplant recipient with rare natural chimerism.

Materials and Methods

This study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The patient provided written informed consent.

A 23-year old male patient had been previously treated for uremia and had received hemodialysis for 3 months. He was admitted to our hospital in November 2021 with complaint of itching and fatigue. At that time, creatinine was 1700 ?mol/L, and renal biopsy indicated immunoglobin A nephro-pathy (grade V, Oxford classification: M1E0S1T2C2). The patient was diagnosed with uremia and underwent hemodialysis 3 times per week. To determine eligibility for renal transplant, the patient underwent chromosomal and HLA high-resolution typing tests. The patient and his parents collected the whole blood and throat swab samples.

Human leukocyte antigen typing

Whole blood and throat swab samples from the patient were sent for assessment of ABO blood group genotype, chromosome karyotype, chromosomal microdeletions, and microrepeated variations for STR, as detected by a blood DNA purification kit (Maxwell 16, Promega) and a buccal swab DNA purification kit (Maxwell 16 LEV, Promega). Sequence-specific oligonucleotide probes (LABType SSO HLA DNA typing trays; One Lambda) were used for the DNA analyses. The ABO blood group genotyping kit for human erythrocytes was from Xiaoping.

Genomic microdeletion and microduplication detection

Genomic microdeletion and microduplication detec-tion procedures were performed according to the high-throughput ligation probe amplification technique by Shanghai Tianhao Biotechnology (Shanghai, China). The assay provides 1602 probes covering more than 200 core regions and a certain range of regions on both sides of the core regions. The basic principle of this technique is the use of the ligase-linked reaction with high specificity to cross-link the target region, then by introduction of nonspecific sequences of different lengths at the end of the ligation probe and by ligase addition reaction, different-length ligation products corresponding to the sites are obtained. The polymerase chain reaction products are then amplified by universal primers labeled with fluorescent capillary electrophoresis, and the products are separated by electrophoresis. Finally, the peaks of each site are obtained by analysis of the electrophoretic patterns.

The HLA subtypes of the patient’s family are shown in Table 1. The patient inherited 2 genes from his father and 1 from his mother. The results showed the HLA A locus of the patient (Figure 1). The patient was identified to have a standard ABO blood type (A01 type) (Figure 2). The patient was identified to have a standard chromosome karyotype without sequence variation (Figure 3). Figure 4 shows that STR loci D7S820, G2S0002, and D18S51 all have 3 peaks, which represent 3 alleles.

Figure 5 shows the number of copies detected by each probe on all chromosomes. The probes on the autosome show 2 copies, and the probes on the sex chromosome show 1 copy. If there are 3 copies or 1 copy of multiple probes in a row, then this indicates that there is duplication or absence in the area where the probe is located (the experimental results showed no duplication or absence).

With regard to STR chimerism detection, the STR peak plot showed 2 sequence variants inherited from the father and 1 standard (invariant) sequence inherited from the mother (Figure 6). Moreover, the peak map of STR showed that the peak map area of the 2 sequence variants inherited from the father was basically the same as that of the mother. The 3 isogene peaks were observed at loci D7S820, CSF1PO, and D18S51.

After transplant, the induction immunosup-pression therapies included tacrolimus (2 mg twice daily), mycophenolate mofetil (0.5 g twice daily), and prednisone (5 mg/d); then mycophenolate mofetil was prescribed as the maintenance immunosup-pression therapy. The patient recovered from the transplant within 3 days. The laboratory results returned to baseline after a week. The follow-up laboratory reports showed healthy function of the kidney (Figure 7).

Our patient underwent HLA-matched living related kidney transplant; however, further follow-up was required to determine whether the patient was able to continue to tolerate kidney transplant in the long-term after cessation of the immunosuppressive agents. Here, we report a rare case of natural chimerism.

There have been previously reported cases for which donor origin was from the transplanted organs.^5,6 However, our case is quite different in that our living related kidney transplant recipient expressed rare natural chimerism..

The father provided the donor kidney for his son. Both of the father’s 2 haploids were passed to the son. The HLA test results showed that the son who received the transplanted kidney and his donor father were fully compatible at 6 sites, which represents a rare case of natural chimerism. We observed D7S820, G2S0002, and D18S51 by detection of chromosome microdeletion and microrepeated sequence variation, and we observed D7S820, CSF1PO, and D18S51 by STR chimerism detection; all had 3 allelic peaks, and 2 of the loci (D7S820 and D18S51) were mutually verified.

We followed up this patient after kidney transplant for 2 years, and we expected to reduce immunosuppressive agents in the later stage. We targeted this patient for further long-term follow-up observation.

The preoperative detection of this patient sug-gests that biomarker monitoring can guide operant immune tolerance and facilitate a better under-standing of the immune status of grafts during the process of immunosuppression reduction. Short tandem repeat sequence analysis provides substantial information regarding existing polymorphisms and can identify chimerism, if present, and thereby guide immunosuppression strategies after renal transplant, which may improve the long-term immunosup-pression-free survival of renal transplant recipients.

Volume: 21 Issue: 11 November 2023

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