1. Supported by Czech Govt. Funding MSM 6198959205
MADCAM1 GENE POLYMORPHISMS AND THE OUTCOME OF THE ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION
Z. Ambruzova1, F. Mrazek1, L. Raida2, A. Stahelova1, E. Faber2, K. Indrak2, M. Petrek1
1Laboratory of Immunogenomics and Proteomics, Department of Immunology, 2Department of Haematooncology
Palacky University and University Hospital Olomouc, Czech Republic
Supported by Czech Govt. Funding MSM
and IGA MZ CR NR 9099

2. INTRODUCTION
various non-HLA gene polymorphisms are intensively studied for their possible relevance for allogeneic haematopoietic stem cell transplantation (aHSCT) outcome
migration and distribution of activated donor T cells to the recipient mucosal sites and parenchymal target organs is important for development of graft-versus-host disease (GVHD)
organ-specific „homing“ of donor cells is mediated via interaction between adhesion molecules and their ligands

3. MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1)
glycoprotein expressed on the surface of the endothelial cells (high level of expression in Peyer´s patches and mesenteric lymph nodes)
ligands:
α4β7 integrins (LPAM-1, CD49d/β7)
L-selectin (CD62L)
function:
lymphocyte traffic to mucosal sites
adhesion of leucocytes on the endothelium
aHSCT - donor T cells „homing“ into the recipient mucosal tissue
Eksteen et al, Clin Med 2004; 4:173-80

4. MADCAM1 GENE POLYMORPHISM
many single nucleotide polymorphisms (SNPs) were described throughout the MADCAM1 gene (chromosome 19)
particular variants of the MADCAM1 gene may affect structure and/or expression of MAdCAM-1 molecule
SNPs selected to our study:
rs T/C promotor
rs G/A exon synonymous (Pro/Pro)
rs G/T exon non-synonymous (His/Pro)

5. AIM OF THE STUDY
to investigate whether the selected MADCAM1 gene SNPs are the risk factors for development of the serious complications after aHSCT
MADCAM1 gene SNPs
rs T/C
rs G/A
rs G/T
acute or chronic GVHD overall survival

6. INVESTIGATED SUBJECTS
aHSCT pairs 87
Age – median (range) Donor type
Patients (18-61) Related 70
Donors (18-69) Unrelated 17
Recipient gender Cell source
Female PBSC 86
Male Bone Marrow 1
Diagnosis Acute GVHD
Acute leukaemia (AML, ALL) 43 Grade 0-I 53
Chronic leukaemia (CML, CLL) 15 Grade II 23
Non-Hodgkin lymphoma 14 Grade III 4
Other Grade IV 8
Conditioning regimen Chronic GVHD
Non-myeloablative 48 None 56
Myeloablative Limited 17
Donor HLA compatibility Extensive 14
Identical 87
Mismatched 0

7. MADCAM1 genotyping (rs758502)
METHODS
Genotyping
Polymerase Chain Reaction with Sequence Specific Primers (PCR-SSP)
Primers designed according to
the reference MADCAM1 gene sequence:
rs T/C
rs G/A
rs G/T
2. Statistics
Conformity to the Hardy-Weinberg equilibrium: Chi-square test
Differences between allele and genotype frequencies: Pearson´s Chi-squared test, Bonferroni correction for multiple comparisons, binary logistic regression model
Overall survival analysis: Kaplan-Meier, log-rank test and Cox regression analysis (SPSS software, version 15.0)
TT TC CC
MADCAM1 genotyping (rs758502)

8. MADCAM1 SNPs GENOTYPE FREQUENCIES IN PATIENTS AND DONORS
Patients Donors
n = 87 n = 85
MADCAM1 T/C (rs758502)
Genotype CC (37) (44)
Genotype TC (41) (31)
Genotype TT (9) (8)
MADCAM1 G/A (rs )
Genotype AA (22) (29)
Genotype GA (43) (33)
Genotype GG (20) (20)
MADCAM1 G/T (rs )
Genotype GG (54) (63)*
Genotype GT (28) (15)
Genotype TT (3) (4)
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*MADCAM1 (rs ) GG genotype frequency: p = 0.03, pcorr >0.05 for comparison patients with donors

9. FREQUENCIES OF MADCAM1 GENOTYPES IN PATIENTS WITH AND WITHOUT ACUTE GVHD
Patients aGVHD+ Patients aGVHD p*
MADCAM1 T/C (rs758502) n = 35 n = 49
Genotype CC (13) (22)
Genotype TC (19) (21)
Genotype TT (3) (6)
MADCAM1 G/A (rs ) n = 34 n = 48
Genotype AA (5) (15)
Genotype GA (21) (22)
Genotype GG (8) (11)
MADCAM1 G/T (rs ) n = 34 n = 48
Genotype GG (20) (33)
Genotype GT (12) (14)
Genotype TT (2) (1)
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* comparison of particular homozygous genotype versus carriage of an alternative allele

10. FREQUENCY OF CHRONIC GVHD IN RECIPIENTS ACCORDING TO THEIR MADCAM1 rs2302217 GENOTYPES
pcorr = NS

11. MULTIVARIATE ANALYSIS OF RISK FACTORS FOR OVERALL SURVIVAL AFTER aHSCT
Factor p HR
aGVHD , ,35
cGVHD <0, ,7
MADCAM1 rs AA genotype 0,001 2,99
Factors included to the analysis:
Diagnosis (malignancy vs. non-malignancy), type of the donor (related vs. unrelated), stem cell
source, conditioning regimen, GVHD prophylaxis, donor-recipient gender combination (female
donor in male recipient vs. others), aGVHD, cGVHD, MADCAM1 gene variants

12. CONCLUSION
preliminary data suggest that MADCAM1 rs AA genotype in recipients may be associated with the risk of chronic GVHD and decreased overall survival after aHSCT
possible impact of MADCAM1 SNP variants for aHSCT outcome has to be confirmed in substantially larger cohorts of donor-recipient aHSCT pairs

13. Dept. of Immunology, Medical Faculty Palacky University and University Hospital Olomouc
Prof. Martin Petrek
Frantisek Mrazek
Anna Stahelova
Jana Onderkova
Silva Zachova
Dept. of Haematooncology, Medical Faculty Palacky University and University Hospital Olomouc
Prof. Karel Indrak
Ludek Raida
Edgar Faber
Thank you for your attention…