Genetic aspects of outcome in kidney transplantation : cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.Terminaalisen munuaissairauden paras hoitomuoto on munuaissiirto. Immuunivastetta heikentävää lääkitystä (immunosuppressiivit) annetaan estämään siirteen immunologinen hyljintä. Toisaalta immunosuppressiivit ovat haitallisia munuaisille ja lisäävät infektioiden ja syövän vaaraa. Tärkeimmät siirteen immunologiseen hyväksyntään vaikuttavat geenit ovat ihmisen valkosoluantigeeneja koodaavat geenit (HLA). Muut HLA:n ulkopuolisten geenien eri muodot voivat ennustaa ennen siirtoa hoidon sivuvaikutuksia ehkä mahdollistaen yksilöllisen immunosuppressiivisen lääkityksen. Siirron jälkeen siirteen toimintaa seurataan yleisluontoisten sairauden oireiden ja laboratoriokokeiden avulla. Siirteen hyljintä määritetään ottamalla neulakoepala siirteestä. Äkillisen hyljinnän määritykseen tarvitaan koepalan oton sijaan helposti toistettava ja yksinkertainen menetelmä säännölliseen seurantaan, mikä voi parantaa siirteen pitkäaikaistoimintaa. Väitöskirjassa tutkittiin sytokiinigeeneistä ja laskimotukokseen liittyvistä geeneistä eri geenimuotoja ja näiden vaikutusta munuaissiirteen ennusteeseen. Hyljinnän määrityksen avuksi tutkittiin myös soluille myrkyllisiä ja soluja ärsyttäviä T-imusolumolekyyligeenien ilmentymisbiomerkkejä. Sytokiinigeenien TNF ja IL10 tietyt muodot siirteen saajissa vaikuttivat hyljintäriskiin pienessä aineistossa. Lisäksi IL10 geenin tietyt muodot siirteen luovuttajissa vaikuttivat sytomegaloviruksen lisääntymiseen tietyissä siirteen saajissa. Toisaalta emme löytäneet laskimotukokselle, infarktille, hyljintään sairastuvuudelle tai siirteen elinikää lyhentäville altistavia sytokiini- tai tukosgeenimuotoja. Hyljinnän biomerkkejä tutkittaessa reaaliaikaisella kvantitatiivisella polymeraasiketjureaktiolla CD154- ja ICOS-geenien ilmentyminen oli erilaista hyljintäpotilaiden ja normaalien potilaiden välillä muttei muiden hyljinnänkaltaisia oireita ilmentäneiden potilaiden ja hyljintäpotilaiden välillä. Tutkittaessa kliinisesti oireettomien mutta koepalan perusteella hyljintätautimäärityksen saaneita lapsipotilaita hyljintäpotilaita ei pystytty erottamaan normaalipotilaista tutkimalla ehdokasgeenien ilmentymistä tai koko genomin ilmentymistä. Munuaissiirtoa voidaan pitää kompleksitautina, jossa useat ympäristö- ja geneettiset tekijät vaikuttavat siirron jälkeiseen aikaan. Suuri joukko tällä hetkellä tuntemattomia geneettisiä tekijöitä vaikuttavat mahdollisesti siirron onnistumiseen

Donor haplotype B of NK KIR receptor reduces the relapse risk in HLA-icentical sibling hematopoetic stem cell transplantation of AML patients

Creative Commons Attribution License (CC BY 4.0)Successful allogeneic hematopoietic stem cell transplantation (HSCT) depends not only on good HLA match but also on T-cell mediated graft-versus-leukemia (GyL) effect. Natural killer (NK) cells are able to kill malignant cells by receiving activation signal from the killer-cell immunoglobulin-like receptors (KIR) recognizing HLA molecules on a cancer cell. It has been recently reported that the risk of relapse in allogeneic hematopoietic stem cell transplantation (HSCT) is reduced in acute myeloid leukemia (AML) patients whose donors have several activating KIR genes or KIR B-motifs in unrelated donor setting, obviously due to enhanced GyL effect by NK cells. We studied the effect on relapse rate of donor KIR haplotypes in the HLA-identical adult sibling HSCT, done in a single center, in Helsinki University Central Hospital, Helsinki, Finland. Altogether, 134 patients with 6 different diagnoses were identified. Their donors were KIR genotyped using the Luminex and the SSP techniques. The clinical endpoint, that is, occurrence of relapse, was compared with the presence or absence of single KIR genes. Also, time from transplantation to relapse was analyzed. The patients with AML whose donors have KIR2DL2 or KIR2DS2 had statistically significantly longer relapse-free survival (P = 0.015). Our data support previous reports that donors with KIR B-haplotype defining genes have a lower occurrence of relapse in HSCT of AML patients. Determination of donor KIR haplotypes could be a useful addition for a risk assessment of HSCT especially in AML patients.Peer reviewe

Faecal Microbiota Composition in Adults Is Associated with the FUT2 Gene Determining the Secretor Status

Peer reviewe

Secretor Genotype (FUT2 gene) Is Strongly Associated with the Composition of Bifidobacteria in the Human Intestine

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa

Isolation of bifidobacteria for blood group secretor status targeted personalised nutrition

Background: Currently, there is a constant need to find microbial products for maintaining or even improving host microbiota balance that could be targeted to a selected consumer group. Blood group secretor status, determining the ABO status, could be used to stratify the consumer group. Objective: We have applied a validated upper intestinal tract model (TIM-1) and culturing methods to screen potential probiotic bacteria from faeces of blood secretor and non-secretor individuals. Design: Faecal samples from healthy volunteers were pooled to age- and sex-matched secretor and non-secretor pools. Faecal pools were run through separate TIM-1 simulations, and bacteria were cultivated from samples taken at different stages of simulations for characterisation. Results: Microbes in secretor pool survived the transit through TIM-1 system better than microbes of non-secretor pool, especially bifidobacteria and anaerobes were highly affected. The differences in numbers of bifidobacteria and lactobacilli isolates after plate cultivations and further the number of distinct RAPD-genotypes was clearly lower in non-secretor pool than in secretor pool. Conclusions: In the present study, we showed that microbiota of secretor and non-secretor individuals tolerate gastrointestinal conditions differently and that a combination of gastrointestinal simulations and cultivation methods proved to be a promising tool for isolating potentially probiotic bacteria

Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained

Low-medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay

Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained