The Importance of New Generation Sequencing (NGS) HLA Typing in Renal Transplantation-Preliminary Report.

INTRODUCTION: Thanks to new generation sequencing (NGS) and expansion of HLA typing with additional loci, it will be possible to increase the effectiveness of graft survival and to avoid complications related to the immune system. New pharmacogenetic factors are still being researched to develop better immunosuppressive treatment. MATERIAL AND METHODS: The incidence of polymorphic HLA loci variants was established, based on a high-resolution NGS method in kidney graft recipients. Furthermore, haplotypic analysis between examined loci was conducted to type additional loci that may influence the transplantation result. A total of 120 kidney recipients were enrolled in the study. A commercial DNA extraction kit in Tubes (QIAamp DNA Blood Mini Kit Qiagen, Germany) was used to isolate DNA from the blood. Sequencing library preparation was done with TruSight HLA set. The Conexio computer program was used to analyse the results of HLA typing. RESULTS: The patients with alleles A*02:01:01, B*44:02:01, C*03:03:01, C*01:02:01, C*05:01:01, C*07:02:01, DQB1*03:03:02, DQB1*06:04:01, or with haplotypic variation A*25:01:01-B*18:01:01- C*15:01:01 were taking the highest doses of cyclosporine (CsA), in contrast to patients with allele B*18:01:01, DQB1*06:02:01, DQB1*02:02:01, or haplotypic variation A*02:01:01- B*44:02:01-C*01:01:01, who were taking the lowest doses. The highest dose of tacrolimus (TAC) was administered to patients with alleles A*68:01:02, A*29:01:01, B*07:02:01, B*35:02:01, B*38:01:01, DRB1*12:01:01, DQB1*05:03:01, or haplotypic variations A*02:01:01-B*57:01:01-C*07:01:01, A*03:01:01-B*07:02:01-C*13:01:01, A*29:02:01-B*44:03:01- C*07:01:01, and A*01:01:01-B*08:01:01-C*03:01:01. Additionally, it was established that HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, and HLA-DQA1 show very slight polymorphism, which suggests that there is no need for their typing for transplantation purposes. Moreover, loci HLA-C, HLA-DQB1, and HLA-DPB1, which are not routinely examined in recipient-donor matching, show genetic variability that may increase the risk of transplant rejection or shortened graft life. CONCLUSIONS: Expanding the qualification procedure to include allele genotyping could allow clinicians to establish immunosuppressive treatment schemes that would be optimally suited for recipients' phenotype.

The influence of the tumor necrosis factor-alpa-308G>A polymorphism on the efficacy of immunosuppressive therapy in patients after kidney transplantation.

Cytokines play an important role in the immune response. The calcineurin inhibitors (cyclosporine CsA, tacrolimus TAC) widely used after renal transplantation to prevent allograft rejection are immunosuppressive drugs suppressing the production of cytokines. These drugs are characterized by interindividual variability and require monitoring their blood concentrations to predict their optimal dosage. Therefore, the aim of the study was to determine the correlation between therapeutic effects of immunosuppressants and the tumor necrosis factor-α (TNF-α)-308G>A polymorphism in renal transplant patients. A total of 412 patients receiving TAC and CsA were included in the study. Genotype frequencies were determined using the real-time PCR method. Patients with the GG genotype received higher doses of TAC as compared to carriers of the GA genotype (5.24 mg versus 3.35 mg) and had lower mean drug concentration in blood (5.86 ng/ml versus 6.92 ng/ml). Similar results were also obtained for CsA (GG: 185.33 mg versus GA: 153.30 mg, P < 0.05). The comparison of the TNF-α-308G>A polymorphism with the biochemical parameters did not reveal a potential risk for transplant rejection. These results indicate that the TNF-α-308G>A polymorphism may influence the dosage of immunosuppressive drugs in patients after transplantation as far as individualization of drug therapy is concerned.

Polymorphisms of OPG and their relation to the mineral density of bones in pre- and postmenopausal women.

OBJECTIVES: The aim of the study was to evaluate the frequency of gene polymorphisms OPG -163A/G, -950T/C and 1181G/C, assessing their relations with the clinical parameters of osseous turnover and the degree of postmenopausal osteoporosis. STUDY DESIGN: The study included 800 women of postmenopausal (505) and reproductive (295) age from Poland. The postmenopausal group included women with osteoporosis and osteopenia, as well as healthy individuals. All the women of reproductive age were healthy. The frequency of the tested gene polymorphisms was evaluated within the group where BMD (bone mineral density) was marked and also in the control group. MAIN OUTCOME MEASURES: The frequencies of the polymorphisms of OPG genotypes in the women were characteristic of the population. RESULTS: OPG -950T/C polymorphism has been associated with body weight and birth weight. OPG 1181G/C and OPG -163A/G polymorphisms have been associated not only with body weight and birth weight, but also with reduced bone density and an increased risk of postmenopausal osteoporosis. CONCLUSIONS: Evaluation of the polymorphism -950T/C of the OPG gene showed that the CC genotype may appear as an increased risk factor for the faster loss of bone mass and the onset of osteoporosis in Polish postmenopausal women. This polymorphism may be a genetic marker that is responsible for the development of osteoporosis. The homozygous genotypes of polymorphisms 1181G/C and -163A/G of the OPG gene may play a role in increased risks of osteoporosis and may be linked to the birth weights of women.