Renata Žunec

The study of the extended haplotypes of rare HLA-B*2730 allele using microsatellite loci

The aim of the present study was to compare haplotypes of the most frequent B*27 alleles among Croatians (B*2702 and *2705) and the rare B*2730 allele. For this purpose, 37 families with members carrying human leukocyte antigen (HLA)-B27 were selected. All individuals were analysed for eight microsatellites (Msats): D6S2927, short tandem repeat - MHC class I-related gene (STR_MICA), D6S2793, D6S2811, tumor necrosis factor a (TNFa), tumor necrosis factor d (TNFd), D6S273 and D6S1014, while individuals carrying the HLA-B27 specificity were subtyped. Of 39 analysed haplotypes, 20 individuals had B*2702, 15 subjects were positive for the B*2705 allele, the B*2730 allele was found in three haplotypes from different families, while one individual carried the B*2703 allele. HLA-A3 and -DRB1*16 were shared by all three B*2730 haplotypes. The DRB1*16 allele was also observed in the majority of B*2702 haplotypes (76.5%), while HLA-A3 was, after HLA-A2, the second most frequent HLA-A specificity in B*2702 haplotypes. No such correlation was found for the B*2705 haplotypes. Msat analysis showed that B*2730 haplotypes also share the same allele at all tested Msats. The D6S2927, D6S2793, MICA and TNFd Msats were not useful in distinguishing B*2702 and B*2705 alleles because D6S2927-213bp, STR_MICA-179bp, D6S2793-206bp, D6S2811-83bp and TNFd-130bp were detected in almost all cases. Conversely, for the TNFa, D6S273 and D6S1014 loci, haplotypes carrying B*2702 and B*2730 shared a single Msat allele in the majority of cases (TNFa-113bp, D6S1014-134bp and D6S273-134bp), which was not observed for B*2705 haplotypes. In conclusion, the similarity between B*2702 and B*2730 DNA sequences as well as their sharing of the same haplotypic combinations corroborates the proposed mechanism of B*2730 evolution from B*2702 by interallelic recombination.

HLA-B27 subtypes in Croatian patients with ankylosing spondylitis

Sir, The association between ankylosing spondylitis (AS) and HLA-B27 is one of the strongest known and it has been con® rmed in various populations. HLAB27 is found in about 96% of AS patients, while only 4 ± 12% of healthy Caucasians are HLA-B27 positive (1). The study of the association between B27 subtypes and AS gave different results in various populations. The results available indicate that alleles B*2701, *2702, *2704, *2705, and *2707 are occurring in AS patients (1,2). Negative association of B*2706 and *2709 subtypes has been described in patient groups of the Thai and Sardinian population, respectively (2). The frequency of the B27 antigen in healthy Croatians is about 12%, while in AS patients it is approximately 90%. The aims of the study was to analyse the distribution of HLA class I antigens and B27 haplotypes in a group of 119 B27+ AS patients and a group of 165 B27+ healthy individuals, and to examine the frequency of B27 subtypes in B27+ AS patients and B27+ healthy controls. A total of 119 unrelated B27+ AS patients and 165 B27+ healthy individuals were included in this study. For all individuals HLA genotyping was performed using segregation data of parental haplotypes. The AS patients were clinically diagnosed according to the New York criteria of 1986. All the included families had been living in the same area for three generations at least. HLA-A and -B typing was performed using the standard microlymphocytotoxity test (MLCT) on local trays (3). PCR-SSP typing for the B27 subtypes was performed using Dynal HLA-B27 high resolution kit (DYNAL, Oslo, Norway) (4). Comparison of HLA class I antigen frequencies between both B27+ groups (AS patients and B27+ healthy individuals) and random controls, revealed an increased frequency of HLA-B44 antigen in random healthy controls (p= 0.65 and p= 0.59, respectively). Results of HLA-A and -B antigen frequencies showed a quite similar distribution in both B27+ groups. An increased frequency of HLAA9 antigen, observed among Basque, Indian, and USA AS patients, was not found in Croatian AS patients (9.1% vs 8.8%, p40.05) (5). In this study we did not observe HLA-B60 antigen more often than expected in B27+ AS patients as was suggested by Robinson et al (2.8% vs 3.9%, p40.05). Namely, they reported that B60 increases susceptibility to AS in B27+ patients while it does not seem to be a predisposing factor in the absence of B27 antigen (6). Our results indicate that there are no additional HLA-B alleles that are involved in a synergistic effect with B27 in a population of Croatian AS patients. This lack of agreement about association between AS and other HLA class I alleles could be explained by genetic characteristic of each population reported, but also support the hypothesis that B27 itself is a major genetic susceptibility factor for AS. The ® nding of lower frequency of the HLA-A26, -B27 haplotype in the group of AS patients in comparison to B27+ healthy controls (2.0% vs 5.9%,p50.05) suggest the potentially protective role of this haplotype in the Croatian AS population. This hypothesis needs to be con® rmed on a larger group of patients bearing B27 haplotypes. Molecular typing of HLA-B27 subtypes was performed in a group of 45 unrelated AS patients and in a group of 38 B27+ healthy controls. The Croatian population showed quite similar distribution of B27 subtypes as in other Caucasians reported so far (1). AS patients possessed similar B27 subtypes as B27+ control individuals. The B*2705 allele was the most common allele in AS patients and B27+ controls (83.3% and 73.7%, respectively), while B*2702 was the second most common allele in both groups (12.5% and 23.7%, respectively). The data from different populations suggest that both B*2702 and B*2705 are positively associated with AS (1,2). The other two subtypes observed in Croatian AS patients, B*2701 and B*2704, are previously reported as alleles associated with AS. B*2704 subtype is positively associated with AS in Oriental populations but its presence is also reported in the Mary population, a Finno-Ugric population from Russia. Thus, it seems that this allele is not only present in Orientals, as was suggested by Lopez-Larrea and further supported by this study (2). At this moment it is not possible to make conclusion for positive or non-association between B*2701 and AS because the publishing data are based on a small number of AS patients. Present study did not demonstrate any of B27 alleles as susceptible or protective genetic marker for AS in the Croatian population and it ® ts into the frame of the oldness of the association between AS Zorana Grubic , National Referral Organ Transplantation and Tissue Typing Center, University Hospital Center Zagreb, KisÏ patic eva 12, HR-10000, Zagreb, CroatiaA total of 119 unrealted B27+ AS patients and 165 B27+ healthy controls were included in this study. For all individuals HLA genotyping was performed using segregation data of parental haplotypes. The AS patients were clinically diagnosed according to the New York criteria of 1986. HLA-A and -B typing was performed using the standard MLCT test on local trays. PCR-SSP typing for the B-27 subtypes was performed using Dynal HLA-B27 high resolution kit. Present study did not demonstarte any of B27 alleles as susceptible or protective genetic marker for AS in the Croatian population and it fits into the frame of the oldness of the association between AS and B27. AS appears to be older than the origin of the B27 subtypes.

The influence of tumor necrosis factor microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation

Aim To investigate the influence of tumor necrosis factor (TNF) microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation. Methods We analyzed TNFa, TNFb, and TNFd microsatellites among 100 patients who underwent allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor at the Internal Clinic of the University Hospital Center Zagreb in the period 2001-2009. The analysis was performed using polymerase chain reaction amplification and electrophoresis on a polyacrylamide gel in an automated sequencer. Results There was no significant difference in patient survival with respect to the allele length at a given microsatellite. However, a significantly lower survival rate was noticed among patients who were positive for TNFa8 allele (P < 0.001) and a significantly higher survival rate among those who were positive for TNFa10 allele (P = 0.0220). Conclusion These results for the first time suggest an influence of TNFa microsatellite on patient survival following HSCT and indicate a need for further studies of this microsatellite.

Analysis of HLA Class II Allele Frequencies on the Island of Hvar - Croatia

The island of Hvar belongs to the group of Middle Dalmatian Islands, part of the territory of the Republic of Croatia. Geography, ethnohistory and demography of the island of Hvar indicate that analyses of genetic features needs to be based on west-east dichotomy. As part of the anthropology component of the 12th workshop we have studied 105 unrelated inhabitants of Hvar. The DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 gene polymorphism was analysed by PCR-SSOP method using 12th Workshop primers and probes. On the basis of association data together with knowledge of haplotypes previously defined we were able to determine which DR-DQ haplotypes were probably carried by each individual. Thirty-one DRB1, 4 DRB3, 2 DRB5, / DQA1, 16 DQB1 alleles have been identified. Analysis of DRB1-DQA1-DQB1 haplotypes revealed a great diversity. Thirty-four and 38 distinct haplotypes have been identified on the east and west part of the island, respectively. Significant differences were observed between those two parts of island for 1.DRB1*0701 allele (p?0.001) 2.DQA1*0201 allele (p?0.01) and 3.DRB1*0101-DQA*0101-DQB1*0501 haplotype (p?0.05). Unusual haplotype DRB1*0101-DQA*0101-DQB1*0501 was found in 2 individuals from the west and in 3 individuals from the east while unusual haplotype DRB1*1501-DQA1*0102-DQB1*0604 was observed only on the western part. These data support the previously observed west-east dichotomy of the island of Hvar.