Noora Alakulppi

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.

Cytokine Gene Polymorphisms and Risks of Acute Rejection and Delayed Graft Function after Kidney Transplantation

Background. Pretransplantation identification of patients at an increased risk for adverse events would allow more individualized treatment strategies possibly improving long-term outcome. We studied cytokine gene polymorphisms of kidney allograft recipients and their donors to identify factors predisposing for acute rejection (AR) and delayed graft function (DGF). Methods. A total of 291 adult cadaver kidney recipients transplanted at a single transplantation centre between 1999 and 2002 were investigated. Recipients and donors were typed for TNF-&agr;(-308G/A), TGF-&bgr;1(codon 10T/C, codon 25C/G), IL-10(-1082G/A, -819C/T, -592C/A), IL-6(-174C/G), and IFN-&ggr;(+874T/A) polymorphisms using a SSP-PCR kit. An AR episode was defined based on clinical and histological findings (Banff criteria). Results. The incidence of AR was 17%. In univariate statistical analyses recipients with TNF-&agr; -308AA-genotype were found to be at a significantly increased risk for rejection (odds ratio [OR] 5.0, 95% CI 3.0–8.3, P=0.003). The association was independent from the patient-donor HLA-mismatch status. In addition, patients with IL-10 ACCACC, ATAATA, GCCATA (-1082A/G, -819C/T, -592C/A, respectively) haplotypes were predisposed to rejection (OR 1.9, 95% CI 1.1–3.1, P=0.016). Further, the combination of recipient TGF-&bgr;1 25GG-genotype and donor IL-10 -819T-allele was associated with rejection (OR 1.8, 95% CI 1.1–3.0, P=0.027). These variables remained significant risk factors also in a multivariate logistic regression analysis. The incidence of DGF was 22%. The risk was increased by a donor TNF-&agr; -308GA-genotype (OR 1.6, 95% CI 1.1–2.6, P=0.040). Conclusions. Our results confirm that cytokine gene polymorphisms influence the outcome of kidney transplantation. Our data especially identify the TNF-&agr; -308AA-genotype as a factor predisposing for AR episodes.

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

The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota.

Association study of FUT2 (rs601338) with celiac disease and inflammatory bowel disease in the Finnish population

Homozygosity for a nonsense mutation in the fucosyltransferase 2 (FUT2) gene (rs601338G>A) leads to the absence of ABH blood groups (FUT2 non-secretor status) in body fluids. As the secretor status has been shown to be a major determinant for the gut microbial spectrum, assumed to be important in the gut immune homeostasis, we studied the association of rs601338-FUT2 with celiac disease (CelD) and inflammatory bowel disease (IBD) in the Finnish population. Rs601338 was genotyped in CelD (n = 909), dermatitis herpetiformis (DH) (n = 116), ulcerative colitis (UC) (n = 496) and Crohns disease (CD) (n = 280) patients and healthy controls (n = 2738). CelD showed significant genotypic [P = 0.0074, odds ratio (OR): 1.28] and recessive (P = 0.015, OR: 1.28) association with the rs601338-AA genotype. This was also found in the combined CelD+DH dataset (genotype association: P = 0.0060, OR: 1.28; recessive association: P < 0.011, OR: 1.28). The A allele of rs601338 showed nominal association with dominant protection from UC (P = 0.044, OR: 0.82) and UC+CD (P = 0.035, OR: 0.84). The frequency of non-secretors (rs601338-GG) in controls, CelD, DH, UC and CD datasets was 14.7%, 18%, 18.1%, 14.3% and 16.1%, respectively. No association was evident in the DH or CD datasets alone. In conclusion, FUT2 non-secretor status is associated with CelD susceptibility and FUT2 secretor status may also play a role in IBD in the Finnish population.

Association of Genetic Variation in Inducible Costimulator Gene With Outcome of Kidney Transplantation

Background. The closely-linked genes of CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS), and programmed cell death 1 on chromosome 2q encode costimulatory molecules, which are regulators of the T-cell activity. The T-cell mediated immune response has a major role in allograft rejection. Hence, the variation in these genes may have an effect on graft survival and the amount of immunosuppression needed, but so far the studies have restricted solely to the CTLA4 gene. Methods. We determined 13 single nucleotide polymorphisms in CD28, CTLA4, ICOS, and PPCD1 genes in 678 adult patients who received a kidney from deceased donor. The effect of genetic variation on the outcome of renal transplantation was analyzed. Results. Two markers on the ICOS gene, rs10183087 and rs4404254, were associated with delayed graft function (odds ratio=5.8; P=0.020 and odds ratio=5.8; P=0.019, respectively). Interestingly, the same ICOS variation has been shown to regulate the expression level of ICOS. We also demonstrated an association of the ICOS polymorphism rs10932037 with the graft survival (P=0.026). Conclusions. The present results indicate that potentially functional genetic variation in T-cell costimulatory molecule ICOS has an effect on the outcome of kidney transplantation.

Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules.

Background. Currently, the diagnosis of acute rejection after kidney transplantation is based on a kidney biopsy taken after clinical rejection suspicion. A robust, noninvasive diagnostic method would allow easier and more frequent monitoring of the patient and the graft. Potentially, a straightforward method would be the analysis of lymphocyte marker molecule expression from whole blood samples. Methods. Whole blood samples were collected prospectively in a single kidney transplantation center from 50 adult kidney recipients transplanted between 2001 and 2005. The mRNA expression of granzyme B, perforin, FasL, granulysin, CD154, ICOS, CTLA4 and PD-1 were analyzed with real-time quantitative polymerase chain reaction. Results. The expression of ICOS and CD154 were significantly lower in rejection patients than in control patients (P<0.001). Both genes gave statistically significant area under receiver operating characteristic curve (AUC; 0.87, 0.88) with 84% sensitivity and 100% specificity for CD154 and 76% and 86% for ICOS, respectively. In paired rejection and postrejection therapy samples, the expression of both genes significantly increased during rejection therapy (P<0.001). When rejection patients were compared to patients biopsied because of other reasons of graft dysfunction, both CD154 and ICOS were lower in rejection patients but only CD154 was statistically significant (P=0.028, AUC=0.740, sensitivity 52%, specificity 90%). The other studied genes gave no consistent statistically significant results. Conclusions. The whole blood gene expression quantities of costimulatory molecules CD154 and ICOS reasonably robustly differentiated rejection patients from control patients. The clinical use of the analysis is limited by poor capability to differentiate patients with rejection from patients with other causes of graft dysfunction.

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

Successful allogeneic hematopoietic stem cell transplantation (HSCT) depends not only on good HLA match but also on T-cell mediated graft-versus-leukemia (GvL) 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 GvL 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.

Feasibility of diagnosing subclinical renal allograft rejection in children by whole blood gene expression analysis.

Background. Protocol biopsies are used to monitor allograft histology after transplantation. However, biopsy is an invasive procedure with potential complications, requires special facilities, and is unpractical for repeated monitoring of the graft. A noninvasive, robust, and rapid diagnostic method would be welcomed. Monitoring gene expression from blood samples could provide such a means. Methods. Whole blood samples taken at the time of 3- or 6-month protocol biopsy in 31 pediatric renal transplant recipients, 13 of whom had biopsy-proven subclinical rejection (SCR), were studied. The samples were collected into tubes containing an RNA stabilization reagent enabling feasible collection during a normal ward schedule. In all patients, the gene expression of candidate genes CD154 and inducible T-cell co-stimulator (ICOS) was measured. A low-density array containing 90 immunologic-related genes were measured with real-time quantitative PCR (RT-QPCR) in 10 patients. In addition, a whole genome microarray analysis was performed in eight patients. Results. Neither CD154 nor ICOS gene expression was diagnostic for SCR (median expression level 1.25 vs. 1.16 and 1.95 vs. 1.61 for CD154 and ICOS, respectively). In addition, expression levels of none of the genes on the low-density array were associated with SCR. Finally, in the microarray analysis none of the found differences between SCR and normal patients’ gene expression could be validated with RT-QPCR in 17 genes. Conclusions. In our relatively small series no robust whole blood gene expression biomarker for SCR was found. Further studies are needed to determine whether small changes in expression may provide a supporting diagnostic method.

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.

Immune Gene Polymorphisms Associate with Outcome in Kidney Transplantation

A short cold ischemic time, an optimal HLA match and other pre-transplant factors are in a key role in the success of kidney transplantation. Over the past decades, the acute rejection rate of kidney transplants has fallen dramatically and the 1-year graft survival rate has increased to 90% in transplantations with deceased donors and 95% with living related donors. This increase in graft survival is largely due to advances in immunosuppressant medication (Yates & Nicholson, 2006). But, due to undesired side effects usually associated with immunosuppressive regimens, reduced immunosuppression is warranted whenever possible. Acute rejection has been the most common end point of genetic association studies. This is natural as acute rejection predicts decreased long-term allograft survival. Despite progress in immunosuppression, the long-term graft survival has not increased in patients suffering acute rejection episodes (Meier-Kriesche et al., 2004). In many genetic studies, chronic allograft nephropathy and subsequent graft loss have been the endpoints with which genetic variation has been compared.