Carmen Botella

Association analysis of MICA gene polymorphism and MICA-129 dimorphism with inflammatory bowel disease susceptibility in a Spanish population

MICA is located at 46 kb centromeric of HLA-B, is highly polymorphic and interactions with NKG2D, its receptor on the surface of NK, Tgammadelta, and T CD8 lymphocytes. A variation at amino acid position 129 of the alpha2-heavy chain domain seems to categorize MICA alleles into strong and weak binder of NKG2D receptor, and thereby to influence effector cell function. Our aim was to study allele polymorphism of MICA and the functionally relevant dimorphism (129val/met) of MICA gene in inflammatory bowel disease (IBD) patients in our population. DNA was obtained from IBD patients (n = 88) and unrelated healthy Murcians (n = 154) and used to MICA genotyping using polymerase chain reaction-sequence-specific oligonucleotides. We did not find statistical differences in the distribution of MICA alleles between the IBD and control groups. However, we found a higher frequency of MICA-129met/met and a lower frequency of MICA-129val/met genotypes in IBD patients (mainly in ulcerative colitis) than in controls (pc = 0.02). These preliminary data could suggest a relevant role of MICA-129-val/met SNP (weak/strong binders of NKG2D receptor) in the pathogenesis of IBD.

Allelic diversity of MICA gene and MICA/HLA-B haplotypic variation in a population of the Murcia region in southeastern Spain

Major histocompatibility complex class I-related chain A (MICA) is located at 46 kb centromeric of HLA-B. It is highly polymorphic and interacts with NKG2D, its receptor on the surface of NK, Tgammadelta and T CD8 lymphocytes. Data on MICA polymorphism in different populations are still limited. Our aim was to establish allelic diversity of MICA gene and linkage disequilibrium with HLA-B in our population. DNA was obtained from 154 unrelated healthy individuals from the Murcia region in southeastern Spain. HLA-B genotyping was performed using polymerase chain reaction (PCR)-sequence-specific oligonucleotide probes and allele-specific PCR-sequence-specific primers, and MICA genotyping by using PCR-sequence-specific oligonucleotide probes. A total of 19 MICA alleles were detected on this study. MICA*008 was the most frequent allele (25.3%), followed by MICA*002 (16.1%), MICA*004 (14.9%), MICA*001 (7.8%), MICA*009 and MICA*016 (7.1%), and MICA*010 (4.6%). Eleven alleles had frequencies of <1%. In the haplotype analysis, MICA*008-B*0702 was found to be the most common, followed by MICA*004-B*4403 and MICA*001-B*1801, MICA*002-B*3501, MICA*008-B*4402, MICA*004-B*4901, MICA*008-B*0801, and MICA*002-B*3801. The frequency of MICA*010-B*1501, MICA*008-B*1302, MICA*015-B*4501, and MICA*008-B*4001 was remarkable inasmuch as these two last haplotypes have not been reported in Spanish population. Indeed, MICA*016 linked to B*1402 has also not been reported in the literature. In conclusion, the allelic diversity in our population is similar to other Caucasian populations; however we found a series of less frequent alleles, in addition to as-yet-undescribed haplotypic associations in other populations of Caucasian origin.

Specific "intra-allele" and "intra-broad antigen" human leukocyte antigen alloantibodies in kidney graft transplantation.

Human leukocyte antigen (HLA) antibodies are epitope specific and not antigen specific. This work presents a case of intra-allele (IA) sensitization. A 40-year-old-man underwent transplantion with identical broad DR. He was apparently not sensitized to HLA antigens by complement-dependent cytotoxicity (CDC), with one previous transplantation 15 years previously. In post-transplantation monitoring, we detected an intra-broad antigen (IBA) anti-DRB1*13 DSA by Luminex. We performed post-transplantation B-cell cross-matching (CM) by CDC, this being completely negative. We detected allele-specific antibodies by single antigens (SA), anti-DRB1*1303 (IBA), -DQB1*0301 (IA), -DRB1*1101, -DRB3*0101, anti-DPB1*0202, and anti-DRB1*0103. These antibodies originated from the first transplantation, HLA-DR6+ homozygous and serologically broad matched, but retrospectively typed as DRB1*1401, *1303; DRB3*0101, *0202; DQB1*0301, *0503; DPB1*0401, *0202 (mismatches in italics). However the second donor was DRB1*1301, *1401 (DR6+ homozygous); DRB3*0202; DQB1*0603, *0503; DPB1*0401 (mismatches in italic). Therefore, the stronger antibodies generated in the first transplantion (anti-DRB1*1303 and -DQB1*0301) were not specific for the specific subtypes (DRB1*1301 and -DQB1*0603) on the second transplantation. Finally, it was possible to exactly define the potential immunizing epitopes the recognition of which determined antibody production. Therefore, our patient had low titers of pretransplantation IBA and IA antibodies that were not prospectively detected by CDC. Post-transplantation with Luminex, we detected these alloantibodies, but as they were not IA and IBA DSA, they did not cause allograft injury.

Influence of human leukocyte antigen mismatching on rejection development and allograft survival in liver transplantation: is the relevance of HLA-A locus matching being underestimated?

The influence of HLA matching on liver transplant is still controversial, as studies have failed to demonstrate an adverse effect of HLA mismatching on transplant outcome. We examined the effect of HLA mismatching on transplant outcome in a series of 342 consecutive liver transplants (224 finally analyzed). HLA typing was performed by serological and molecular methods. HLA-A matching was associated with an increased chronic rejection incidence (P=0.04). Indeed, HLA-A match also demonstrated a significant impact on allograft survival (P=0.03), confirming previous observation concerning to rejection, as complete HLA-A mismatching favored a better liver transplant outcome. Analysis of HLA-A+B+DR matching also demonstrated a significant impact on graft survival (P<0.05). Multivariate Cox regression analysis confirmed the effect of HLA-A and DPB1 matching as independent risk factors for graft loss. Another independent factor was a positive pre-transplant crossmatch. In conclusion, liver transplant outcome has not been found to be improved by HLA matching, however a poorer HLA compatibility favored a better graft survival and decreased rejection incidence, with a special relevance for HLA-A matching.

Lack of association between the −403G/A promoter polymorphism in the human CCL5/RANTES chemokine gene in liver transplant outcome

Chemokines play a major role in the inflammatory and immune responses that mediate allograft outcome. CCL5/RANTES expansion chemokine is potent eosinophil, monocyte, basophils and lymphocyte chemoattractant and has recently been studied in transplantation with discrepant results, but systemic concentrations have been correlated to liver graft survival and incidence of rejection. Recent studies revealed that a functional mutation at −403 in the promoter may have a significance for inflammatory and infectious immune responses. Our objective was to investigate CCL5/RANTES promoter polymorphism in rejection and graft survival in liver transplant. We examined the CCL5/RANTES polymorphism in a series of 218 liver transplants and 101 healthy Caucasian subjects. CCL5/RANTES genotyping was performed by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). After comparing recipients (with acute rejection episodes versus without rejection) with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CCL5/RANTES dimorphism in any comparison (Pu2003>u20030.05). Indeed, 5u2003years allograft survival was 61.3% in recipients with the GG genotype against 58.8% in recipients with the GA and AA genotypes. These differences were also not statistically significant. In conclusion, human CCL5/RANTES gene promoter polymorphism does not seem to influence acute rejection development and allograft survival in liver recipients.

Genetic relationship between Murcia Region (SE Spain) and other populations in the Iberian Peninsula and Mediterranean area with respect to HFE gene mutations distribution.

Dear Editor, Hereditary hemochromatosis (HH) is a common hereditary disorder in populations of European descent, characterized by iron overload and a variety of clinical manifestations such as liver cirrhosis and arthropathy. Most patients are homozygous for the C282Y mutation, 5–7% are compound heterozygous for the C282Y allele and an H63D mutation, and less than 2% are H63D homozygous; S65C has been also implicated in mild form of HH [1]. Unlike the situation in northern European countries in which the prevalence of HH is rather homogeneous, in Spain, the frequency of HH seems to decrease from north to south [2]. Other studies provided evidence that the prevalence of C282Y was much lower in HH patients from central and southern Spain than in those originating from northern Spain and Portugal [3, 4]. Because these differences likely reflect regional differences in the frequency of these alleles, we aimed to test the hypothesis that the C282Y allele frequency is lower in a restricted region in southern Spain. This information is relevant to know whether screening programs for HH in Spain should be designed on regional-based criteria, limiting large-scale screening studies to specific (Celticrelated) areas of northern Spain. Murcia, a region from the southeastern Spain, has approximately 1,500,000 inhabitants. From January to December 2004, 370 unrelated Caucasoid blood donors were enrolled in this anonymous study from blood-donor center in our hospital including 202 men and 168 women. All study participants were of white Spanish origin and adults. Indeed, the study was approved by the local medical committee. C282Y, H63D, and S65C were detected in all samples using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), as previously described [3]. Briefly, the primers sequences were 5′-CTT TGG GCT ACG TGG ATG ACC-3′ (forward) and 5′-CTG GCT TGA AAT TCT ACT GGA AAC C-3′ (reverse), used to amplify exon 2 of the hemochromatosis (HFE) gene. To amplify exon 4, a second set of oligonucleotides was used: 5′-GGT GTC GGG GCT TGA ACT ACT ACC-3′ (forward) and 5′A CAT ACC CCA GAT CAC AAT GAG G-3′. PCR reaction mixtures were digested with BclI and HinfI (exon 2) and SnaBI (exon 4) for 3 h following the protocol recommended by the manufacturer (Roche Diagnostics, Mannheim, Germany). HFE genotype distributions and Hardy–Weinberg equilibrium test were calculated using SPSS v12.0 (SPSS, Illinois) and Arlequin V2.0 software (University of Geneva, Switzerland), as previously published [5]. The haplotype frequencies were computed using the expectation-maximization algorithm. On the other hand, principal components analysis (PCA) was performed by analyzing HFE allele frequencies for each population by using the Visual Statistic System (VISTA) v5.05 program, where a correlation matrix Ann Hematol (2007) 86:455–457 DOI 10.1007/s00277-006-0242-x

HLA-DR antibodies in transfusion-related acute lung injury (TRALI): A case report

Transfusion-related acute lung injury (TRALI) is a serious adverse consequence of blood product transfusion. Cases of TRALI have gone unrecognized or misdiagnosed, since the symptoms can be confused with other transfusion-related events or with non-transfusion related comorbidities. Suspected cases of TRALI may be insufficiently investigated, and mild or moderate cases may not be investigated or reported at all. We report here the case of a 73-year man who developed TRALI following a transfusion of packed red blood cells (pRBCs) mediated by HLA class II antibodies (HLA-DR) detected by luminex technology. A very few cases of TRALI have been described being caused by HLA class II antibodies without the simultaneous presence of anti-HLA class I antibodies. Technology for antibody detection has increased the power and the specificity, especially with the use of flow cytometry with a better definition of the antigen/antibody pairs that have resulted in TRALI episodes. In this sense, HLA class II antibodies can exactly be detected with these methods and have surely been underestimated until now.