Allelic distribution of HLA-B*5 in HLA-B5-positive Israeli patients with Behçet's disease

In order to investigate the sub-typing of the B5 antigen in Israeli (Jewish and Arabic) patients with Beh?et's disease (BD) allele-specific genotyping of B51 and B52 alleles was performed in Israeli BD patients and healthy controls. Among the HLA-B51-positive BD patients, B*5101 was found to be the predominant allele, identified in 62% of all BD patients and 78% of Jewish BD patients. HLA-B*5101 was also the predominant allele in HLA-B51-positive healthy controls. HLA-B*5108 and B*5104 alleles were identified in 23% and 15% of B51-positive BD patients, respectively. The HLA-B*5201 allele was identified in all HLA-B52-positive patients and controls. Our study suggests that both HLA-B*5101 and HLA-B*5201 are the dominant alleles of HLA-B5 in Israeli BD patients.     

A strong association between HLA-B*5101 and Behçet's disease in Greek patients

Behçet's disease is known to be associated with HLA-B51, one of the split antigens of HLA-B5, among many different ethnic groups. In a Greek population, an increased incidence of HLA-B5 in the patient group also been reported. Because the B51 antigen has been recently identified to comprise seven alleles, B*5101-B*5107, we performed HLA-B51 allele genotyping by the PCR-SSP method as well as serological HLA-A and -B typing among 31 Greek patients with Behçet's disease to investigate whether there is any correlation between one particular B51-associated allele and Behçet's disease. The frequency of B51 was remarkably high (80.6%) in the patient group as compared to the ethnically matched control group (26.7%). In addition, HLA-A26 was also increased in the patients (29.0%) as compared with the healthy controls (3.3%). B51 allele genotyping revealed that all these B51-positive patients carried B*5101. This study revealed a strong association of Behçet's disease in Greeks with one of B51 subantigens, providing insight into the molecular mechanism underlying an HLA association with Behçet's disease.     

HLA-B*51 allele analysis by the PCR-SBT method and a strong association of HLA-B*5101 with Japanese patients with Behçet's disease

Abstract: Behçet's disease (BD) is known to be associated with human leukocyte antigen (HLA) B51 in many different ethnic groups. An increased incidence of HLA-B51 in the patient group has also been reported in a Japanese population. Recently, the B51 antigen has been identified to comprise 21 alleles, B*5101–B*5121. Further, not only HLA-B*5101 but also HLA-B*5108 were found to be relatively increased in the patient groups among Italian and Saudi Arabian populations. Therefore, we performed HLA-B*51 allele genotyping by the polymerase chain reaction-sequencing based typing (PCR-SBT) method in order to investigate whether there is any correlation of one particular B51-associated allele with Japanese BD. Ninety-six Japanese patients with BD and 132 healthy Japanese volunteers were enrolled in this study. As a result, the phenotype frequency of the B51 antigen was confirmed to be remarkably increased in the patient group as compared to the ethnically matched control group (59.4% in patients vs. 13.6% in controls; P c=0.0000000000098, R.R.=9.3). In the B*51 allele genotyping, 56 out of 57 B51-positive patients were defined as B*5101 and the remaining one was B*5102. In contrast, all of 18 B51-positive normal controls were B*5101. None of the Japanese patients and healthy controls carried the HLA-B*5108 allele. This study revealed that B*51 allelic distribution in Japanese was different from those in Italian and Saudi Arabian populations, and that the significantly high incidence of the HLA-B51 antigen in the Japanese BD patient group was mostly caused by the significant increase of the HLA-B*5101 allele.     

Comparative analysis of the association of HLA-B*51 suballeles with Behçet's disease in patients of German and Turkish origin

The distribution of the different HLA-B*51 suballeles among patients with Beh?et's disease (BD) of German (n=33) and Turkish (n=92) origin in comparison to their presence in the respective ethnically matched healthy control groups (German: n=325, Turkish: n=93) was studied. HLA-B*51x was significantly increased in both patient groups in comparison to the controls (Germans: 58% vs. 12%, OR 9.76, P<0.001; Turkish: 75% vs. 25%, OR 9.13, P<0.001). Molecular subtyping of B*51x revealed HLA-B*51011 and B*5108 as the predominant suballeles in both patient groups and controls although with a slightly increased frequency of HLA-B*5108 in the diseased individuals. HLA-B*5105 was the only further HLA-B*51x subtype detected in one Turkish patient heterozygous also for HLA-B*5101. HLA-B*5107 although present in a Turkish as well as German control was absent in the patient groups. There was also a tendency towards a higher degree of homozygosity for HLA-B*51x in both patient groups versus the matched controls (Germans: 10% in patients vs. 2,5% in controls; Turkish: 27% in patients vs. 13% in controls). Our study further supports previous hypothesis of an association of BD with B51 suballeles which share amino-acid residues at positions 63 and 67 as well as at positions 77-83 for specific peptide binding and natural killer (NK)-cell interactions. This applies to HLA-B*5101 and B*5108, but not to HLA-B*5107 different at position 67, which could be negatively associated with BD.     

HLA and tumour necrosis factor (TNF) polymorphisms in ocular Behçet's disease

The role of HLA-B*51 and other major histocompatibility complex (MHC) genes in Beh?et's disease (BD) remains unknown. We have performed HLA and tumour necrosis factor (TNF) polymorphism analysis in BD and evaluated their contribution to ocular disease. In this study, 102 patients and 115 controls of Middle Eastern descent were investigated by HLA and B*51 subtyping using novel primers, and by LT alpha NCo 1 and TNF 308 promoter polymorphism analysis. The frequency of the HLA-B*51 family of alleles was raised in patients compared to controls (66% vs. 15%, Pc=2.5x10(-12), OR=10.9). The odds ratio (OR) of this group of alleles for subgroups of patients was as follows: non-ocular patients 7.8, all ocular patients 12.6, blind patients >22. HLA-B*51 subtyping detected B*5101, 07, 08 and 09 alleles, with a similar frequency among patients and controls. HLA-Cw*1602 was associated with B*5108, but was not an independent risk factor for disease. The LT alpha (TNFB*2) allele was associated with HLA-B*51 among patients and the frequency of this allele was significantly higher among completely blind patients compared to both non-ocular patients (P=0.048, OR >3.6) and to healthy controls (P=0.022, OR >4.3). The rare TNF-2 polymorphism at the TNF -308 promoter position was associated with HLA-B*50 (not B*51), and was not associated with BD. Thus, in this population the HLA*B51 family of alleles is a strong risk factor for BD, and in particular the development of ocular disease. HLA-B*51 subtyping did not define new markers for BD. A primary role for TNF gne polymorphisms in BD was not identified, but co-expression of the TNFB*2 allele with HLA-B*51 may contribute to severity of ocular disease.     

HLA-Cw*1602: a new susceptibility marker of Behçet's disease in southern Spain

Abstract: Genotyping of the HLA-C locus by PCR-SSP in Behçet's disease patients from southern Spain reveals a statistically significant association with Cw*1602 (OR 20.15, corrected ρ<0.05). This is an uncommon allele absent from the healthy control group, which seems to confer higher relative risk than B51 in this study (OR 1.85). Stratified frequencies do not show statistically significant differences but suggest that the Cw*1602-B51 haplo-type could be the main HLA marker of Behçet's disease in the analyzed population.     

HLA class II sequence-based typing in normal Saudi individuals

We have adopted a system that combines low resolution PCR-SSP followed by sequence-based typing (SBT) to analyze HLA-DRB1, -DPB1 and -DQB1 alleles in the Saudi population. The SBT method was used to identify HLA class II alleles in Saudis for the first time. Nineteen HLA-DRB1 alleles in currently recognized subtypes of the DRB locus were detected. DR1 and DR9 were not encountered. SBT did not detect diversity within the DR7 and DR10 alleles. Sixteen HLA-DQB1 and 10 HLA-DPB1 alleles were identified. This study represents the first molecular report on the HLA class II allele frequency in the population of Saudi Arabia.     

The absence of disease-specific polymorphisms within the HLA-B51 gene that is the susceptible locus for Behçet's disease

Beh?et's disease is known to be associated with HLA-B51 in many different populations. Genetic evidence supports that the susceptible gene for Beh?et's disease is the HLA-B51 allele at the HLA-B locus. This study was aimed to determine the HLA-B51 nucleotide sequence variation in three Beh?et's disease patients and three healthy controls in order to elucidate if any disease specific mutations or polymorphisms may exist in the HLA-B51 gene of patients. Long-range polymerase chain reaction (PCR) was first carried out to give a PCR-amplified product of 9.5 kb which was then used as a template for nested PCR to give a final amplified product of 4.2 kb. This final product containing the 1.3-kb promoter/enhancer region and the entire HLA-B gene except for a 363-bp 3' terminal end segment encoding the 3' untranslated region was subcloned by the BP cloning technique and sequenced. The sequencing results showed that all the patients possessed the HLA-B*51011 allele, and there were no differences in the exonic nucleotide sequences between the three Beh?et's disease patients and the three healthy controls. The HLA-B*51011 intronic and promoter/enhancer nucleotide sequences from the three patients had 22 single nucleotide polymorphisms (SNPs), a single insertion of 6 bp and a single deletion of 2 bp. On the other hand, the three healthy controls had 24 SNPs in their intronic and promoter/enhancer regions. However, none of these polymorphisms in the patients were specific for the disease. Therefore, these results clearly demonstrate that the HLA-B exonic sequence that encodes the HLA-B51 allele is the real pathogenic factor in Beh?et's disease.     

Significant associations of HLA-B*5101 and B*5108, and lack of association of class II alleles with Behçet's disease in Italian patients

Beh?et's disease has been known to be strongly associated with human leukocyte antigen (HLA) B51, one of the split antigens of HLA-B5. An increased incidence of HLA-B51 in the patient group has also been reported in an Italian population. Since the B51 antigen has been recently identified to comprise nine alleles, B*5101-B*5109, we performed HLA-B51 allele genotyping by the polymerase chain reaction-sequencing based typing (PCR-SBT) method as well as serological HLA-A and -B typing among 21 Italian patients with Beh?et's disease in order to investigate whether there is any correlation of one particular B51-associated allele with Behcet's disease. In addition, HLA class II genotyping was performed by the PCR-restriction fragment length polymorphism (RFLP) method. As a result, only the phenotype frequency of the B51 antigen was found to be significantly increased in the patient group as compared to the ethnically matched control group by the corrected P-value analysis (71.4% in patients vs. 17.9% in controls; chi2 = 14.26, Pc = 0.0042, R.R. = 11.5). In the B51 allele genotyping, 11 out of 15 B51-positive patients were B*5101 and the remaining four were B*5108, whereas all of 5 normal controls were B*5101, showing significant association of each allele with Beh?et's disease. No significant difference was observed between the patient and control groups in the HLA class II allelic distribution. This study revealed a strong association of Beh?et's disease in Italian with B*5108 as well as B*5101, providing important insight into the molecular mechanism underlying an HLA association with Beh?et's disease.     

Lack of association of MICA transmembrane region polymorphism and Behçet''s disease in Spain

We have analyzed the distribution of the major histocompatibility complex (MHC) class I chain-related gene A (MICA) transmembrane alleles among 58 Spanish patients with Beh?et's disease (BD) and in 194 ethnically matched healthy controls. The study included the characterization of A4, A5, A5.1, A6 "new" and "old" and A9 MICA-TM alleles using polymerase chain reaction. As previously reported, the serological B51 specificity was increased among this BD patient group (36.25% vs. 19.6% in controls; P=0.009; OR=2.33). The MICA-TM alleles A6 ("new" and "old"), in linkage disequilibrium with HLA-B51 and HLA-B14 respectively, were only slightly increased among patients (70.7% vs. 61.3% in controls; P=NS). We conclude that, in contrast to previous finding reporting a strong association of MICA-TM genes and Beh?et disease in Japanese patients, in our population HLA-B51 is more closely associated to Beh?et susceptibility than MICA-TM genes. Finally, our data show that in Spain, as occurs in other populations, some MICA-TM alleles exhibit strong linkage disequilibrium with certain alleles of the HLA-B locus.     

Neuronal Hyaline Inclusions Observed in an Autopsy Case of Behçet's Disease

An autopsy case of Behçet's disease is reported. The patient, a 59 year old Japanese woman, died of intestinal bleeding after a 34 year clinical course of Behçet's disease. She also suffered from recurrent oral aphthous ulcers, erythema nodosum like cutaneous lesions and genital ulcerated lesions. Autopsy revealed marked atherosclerosis of the aorta and multiple deep ulcerations in the terminal ileum with no significant vascular lesions. Lewy bodies and globular hyaline inclusions in the neurons of the central nervous system were noted, although there were no clinical symptoms of Parkinson's disease throughout the clinical course. These findings appear to suggest that the patient was probably in the preclinical or early stage of Parkinson's disease. However, the presence of Lewy bodies in the 6th decade without any accompanying symptoms is very rare. This case seems to draw attention to the presence of these neuronal inclusions in Behçet's disease.     

Improvement of HLA class I and class II PCR-SSP typing by using timed-release activity of DNA polymerase

Abstract: Variable amounts of non-specific amplification may occur in HLA PCR-SSP typing, and this can be significantly reduced by the use of AmpliTaq Gold. In an effort to achieve an optimal balance between specificity and efficiency of the PCR amplification for both HLA alleles and the internal control, we designed a system of timed-release activity by combining two different Taq DNA polymerases. The reaction was started at a relatively low level of enzyme activity and as thermal cycling progressed more and more Ampli-Taq Gold was slowly activated for the reaction to continue. We applied this system to all routine HLA PCR-SSP typing. The number of repeat typings due to non-specific amplification and/or amplification failure of the internal control was remarkably reduced.     

Identification of a novel HLA-B allele (B*4202) in a Saudi Arabian family with Behçet's disease

A new HLA-B antigen, tentatively called HLA-B42AND, was identified as a B42 serologic variant in a Saudi Arabian family. DNA sequencing analysis of the second and third exon of this new B allele revealed that B42AND was identical to B*4201 except for a single T to C substitution at position 97 of exon 2. This substitution results in histidine (CAC) at codon 9 in B42AND instead of tyrosine (TAC) in B*4201. The antigen frequency of B42AND in a Saudi Arabian population was around 10%. This novel B42AND has officially been named HLA-B*4202.     

Intercellular adhesion molecule 1 K469E gene polymorphism is associated with presence of skin lesions in Tunisian Behçet's disease patients

Abstract
Intercellular adhesion molecule 1 ( ICAM1 ) gene polymorphisms have been implicated in the susceptibility to inflammatory diseases. The expression of both soluble and tissue ICAM1 were increased in Behçet's disease (BD) but the contribution of ICAM1 gene polymorphisms to this disease remains unknown. We sought to establish the association of ICAM1 gene K469E polymorphism in exon 6 with susceptibility for BD. One hundred and thirty-five Tunisian patients who satisfied the International Study Group criteria for BD and 157 healthy blood donor controls from the same geographic area were genotyped by polymerase chain reaction method for the K469E ICAM1 gene polymorphisms in exon 6. There were no significant differences in the distribution of the K469E allele or genotype frequencies between the BD patients and healthy controls in the ICA1 gene. Among patients, significant association was found between the presence of skin lesions and the studied polymorphism in the distribution of the K469E allele ( P = 0.004; odds ratio = 1.26; 95% confidence interval = 2.13–3.62) and genotype frequencies ( P = 0.0028;χ² = 11.75). Our findings suggest that K469E ICAM1 gene polymorphism was associated with Tunisian BD patients with skin lesions.     

CTLA-4 +49A/G polymorphism is associated with Behçet's disease in a Tunisian population

The involvement of excessive T-helper cell functions in the pathogenesis of Behçet's disease (BD) has been reported. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays a role in T-cell downregulation. In this report, we investigated the possible association between BD patients and the CTLA-4 +49A/G polymorphism in Tunisian population. A total of 135 Tunisian BD patients and 151 healthy blood donors from the same geographic area were genotyped by polymerase chain reaction for the CTLA-4 +49 A/G polymorphism. A highly significant difference between Tunisian BD patients and healthy controls was found regarding the distribution of CTLA-4 +49 A allele [ P  < 10⁻⁷; χ² = 75.63; odds ratio (OR) = 4.63; 95% confidence interval (CI) = 3.20–6.72] and genotype frequencies ( P  < 10⁻⁷; χ² = 71.02). Furthermore, in the BD group, the A allele was predominant in males (76.3%) when compared with females (62%), ( P  = 0.014; χ² = 5.97; OR = 1.99; 95% CI = 1.10–3.59). No relationship was found between the studied genotype and clinical manifestations. Our results show a gene dose effect of the A allele on the BD. The A allele exerts a stronger effect on disease susceptibility in males compared with females.     

DNA typing of HLA in the patients with moyamoya disease

Summary Moyamoya disease is a clinical entity demonstrating a chronic occlusion of the cerebrovascular system. Although some possible etiological factors have been postulated, the etiology of this disease is still unknown. So far, some investigations have suggested the association between moyamoya disease and HLA in the serological typing. However, DNA typing of HLA have not been performed yet. Thus, we performed DNA-typing of HLA in the unrelated Japanese patients with definite moyamoya disease, using the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) technique. In the total patients,DQB1*0502 had a positive association with the disease. On the other hand,DRB1*0405 andDQB1*0401 showed a negative association. In comparing the early-onset and late-onset groups, two groups did not share the same disease associated alleles at all. Thus, the etiology of moyamoya disease seem to have a genetic background. Furthermore, different genetic factors might also be involved in the difference between the early-onset and late-onset groups.     

A DNA-based detection and screening system for identifying HLA class I expression variants by sequence-specific primers

Molecular methods are now commonplace for HLA typing and they have replaced traditional serological methods in many histocompatibility laboratories. A consequence of reliance on molecular methods using primers or probes based on existing sequence information is that unsequenced or partially-sequenced null, or low expressed variants are not discriminated from expressed alleles. Failure to identify null alleles might have deleterious implications for allogeneic transplants. Expression variants may be classified into two categories: unique mutations and repeat mutations. For example, the alleles A*0303N, A*2409N, and B*1526N have apparently unique mutations. In contrast, repeat mutations may occur frequently at points where unusual nucleotide sequences make accurate DNA replication by DNA polymerases difficult. One example is between nucleotide positions 621-627, where HLA class I alleles may exhibit between three and seven consecutive cytosine residues. Incorrect insertion of an extra cytosine in this region is the cause of expression failure in A*2411N and A*0104N alleles. We hypothesise that insertion of an extra cytosine into the cytosine island between nucleotide positions 621-627 is likely to recur not only in other HLA-A alleles but also in HLA-B and even HLA-C alleles. We describe here a polymerase chain reaction using sequence-specific primers (PCR-SSP) system that can not only detect all previously-sequenced HLA class I expression variants but can also screen for mutations between positions 621-627 in HLA-A, B or C alleles which may give rise to potentially null alleles. Overall, in this study HLA class I expression variants were identified in 5 of 931 tested samples (0.53%).