Showing papers in "Tissue Antigens in 1999"

Behçet's disease, the Silk Road and HLA-B51: historical and geographical perspectives.

Abstract: Behcet’s disease (BD), also known as the Silk Road disease, is a blinding inflammatory disorder of young adults found predominantly between the Mediterranean basin and the Orient, and is strongly associated with the major histocompatibility complex (MHC) antigen HLA-B51. In this article we review the history of Behcet’s disease since its first description by Hippocrates, the development of the trading routes collectively known as the Silk Road and the effect of population movement on the distribution of HLA-B51. The global distribution of this antigen among healthy control populations bears a striking similarity both to the ancient trading routes and the distribution of Behcet’s disease, suggesting a genetic risk that migrated in parallel with population movement between the Mediterranean and Asia. However, certain indigenous Amerindian peoples have a high prevalence of HLA-B51 but no reported cases of BD. Furthermore, a clear genealogical relationship exists between eastern, but not central, Siberian populations with the Amerindians. Since a high level of recombination within the MHC is known to have occurred in these eastern populations before their migration into Beringia, we suggest that disruption of genetic loci in linkage disequilibria with HLA-B51 may be one reason for the absence of disease in these high HLA-B51-bearing populations. However, a contributory influence of environmental factors is not excluded by this data, and the wide variation that exists in relative risk of HLA-B51 even within Europe would support other non-genetic risk factors on the Silk Road which may be absent, or non-contributory to disease, in the Americas.

Nomenclature for factors of the HLA system, 1998.

Abstract: 1. The Naming of New Genes Within the HLA Region The Committee agreed in 1996 to defer the naming of new genes within the HLA region. However, in view of the great interest and work being carried out on the MIC (MHC Class I chain related) genes, already named by the Human Genome Nomenclature Committee as MICA, MICB, MICC, MICD and MICE in April and September 1994 [15, 16], it was agreed that they should be included within the official HLA nomenclature for the purpose of naming alleles of these genes. In addition the change of name of the gene HLADNA to HLA-DOA, as published in the monthly updates, was confirmed, following further results showing that the gene codes for the alpha chain associated with HLA-DOB. The updated list of genes in the HLA region is given in Table 1.

Detection of soluble HLA‐G molecules in plasma and amniotic fluid

Abstract: Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.

CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis.

Abstract: The human cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene may be a candidate susceptibility gene in multiple sclerosis (MS). In this study the distribution of the dimorphisms of exon 1 (+49 A/G) and promoter (-318 C/T) regions of the CTLA4 gene was analysed in 296 unrelated Norwegian MS patients and 271 matched controls by polymerase chain reaction and restriction fragment length polymorphism. The frequency of the exon 1 (+49) A-G genotype was increased in patients (57%) compared with controls (44%) (Pcorrected=0.01), and even more increased in patients with relapsing remitting MS (59%) (Pcorrected=0.006). No other significant differences were found between clinical subgroups of patients or between HLA-DRB1*1501, DQB1*0602-positive and negative patients and controls.

Molecular studies of trophoblast HLA-G: polymorphism, isoforms, imprinting and expression in preimplantation embryo.

Abstract: : There is considerable interest in human HLA-G arising from the observation that it is expressed selectively on the surface of extravillous trophoblast, the fetal cell population directly in contact with the mother. We investigated several aspects of the molecular biology of this unusual molecule. Limited polymorphism at the nucleotide level, and even more restricted variation at the amino acid level, was found in our Caucasian population. A further unusual aspect of HLA-G is the occurrence of alternatively spliced mRNAs. Spliced messages that could give rise to either membrane-bound or soluble proteins have been reported and six of these alternative forms were detected in all first trimester and term placentae, highly purified villous and extravillous trophoblast and the cell lines, JEG-3 and 221-G. An additional novel splice variant involving loss of part of the 3′-untranslated region was observed with two alleles. Using a sensitive RNase protection assay higher levels of the membrane-bound RNAs as compared to the soluble forms were detected in first trimester and term placentae as well as in JEG-3. Contrary to previous findings our term samples taken from the maternal aspect showed higher levels of both mRNA species when compared to first trimester placenta. The question of imprinting was addressed through the detection of heterozygotes both in placental tissue and, more tellingly, in the purified trophoblast cells. There was no evidence of imprinting. In addition we did not find mRNA for HLA-G in human two to eight-cell embryos or in blastocyst or in sperm samples1.

Interleukin-10 (IL-10) genotypes in inflammatory bowel disease.

Abstract: Interleukin-10 (IL-10) is an anti-inflammatory cytokine. Its production in humans is under genetic control, and genotype defines high or low producers of this cytokine. This study addresses the hypothesis that idiopathic inflammatory bowel disease (IBD) patients are more likely to have the low IL-10 producer genotype and phenotype. DNA was extracted from blood cells of patients with Crohn's disease (CD) or with ulcerative colitis (UC) for IL-10 genotyping. The frequency of the high IL-10 producer allele (-1082*G) was decreased in the whole IBD group (41% vs. 51%, P = 0.03) and in the UC patients compared with normal controls (37% vs. 51%; P = 0.04). Hence, there appears to be an association between the IL-10 genotypes and IBD. This suggests that individuals genetically predisposed to produce less IL-10 are at a higher risk of developing IBD, in particular, UC.

HLA class II haplotypes in primary sclerosing cholangitis patients from five European populations

Abstract: The association of primary sclerosing cholangitis (PSC) to HLA class II genes was studied by comparing patients from five different European populations. Deduced HLA-DRB1, DQA1, DQB1 haplotypes of 256 PSC patients from England, Italy, Norway, Spain and Sweden were compared to those observed in 764 ethnically-matched controls. Increased frequencies of the DRB1*03, DQA1*0501, DQB1*02 (RR=3.0, P<0.00001) and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes (RR=2.4, P<0.0001) were observed in all five patient groups. A total of 16% of the PSC patients were homozygous for the DRB1*03, DQA1*0501, DQB1*02 haplotype compared to 1% of the controls (RR=20, P<0.0001). The DRB1*04, DQA1*03, DQB1*0302 haplotype was significantly reduced in frequency(RR=0.4, P<0.00001). Among Norwegian, Swedish and British patients that did not carry neither the DRB1*03, DQA1*0501, DQB1*02 nor the DRB1*13, DQA1*0103, DQB1*0603 haplotype, an increased frequency of the DRB1*15, DQA1*0102, DQB1*0602 haplotype was observed (RR=2.0, P<0.0001). Thus, PSC was found to be positively associated to three different HLA class II haplotypes (i.e. the DRB1*03, DQA1*0501, DQB1*02, the DRB1*15, DQA1*0102, DQB1*0602 and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes) and negatively associated to one HLA class II haplotype (i.e. the DRB1*04, DQB1*0302 haplotype).

Association of tumor necrosis factor receptor 2 (TNFR2) polymorphism with susceptibility to systemic lupus erythematosus.

Abstract: Multiple genetic as well as environmental factors are considered to be involved in the development of systemic lupus erythematosus (SLE). A number of previous studies have suggested a possible role for tumor necrosis factor (TNF) in the pathogenesis of SLE. In addition, one of the candidate loci suggested by the genome-wide linkage analysis corresponds to the chromosomal position encompassing the TNF receptor 2 gene (TNFR2). The purpose of this study was to analyze the polymorphism of TNFR2 and its possible association with the susceptibility to SLE, using the case-control association analysis. Polymorphism screening of the exons containing previously reported nonsynonymous base substitutions was carried out by the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method, using genomic DNA from 81 Japanese patients with SLE and 207 healthy individuals. Two alleles were present in exon 6, coding for methionine (196M) and arginine (196R) at position 196. 30 of 81 patients (37.0%) with SLE were positive for the 196R allele, which was significantly more frequent compared with 39 of 207 healthy individuals (18.8%) (chi2=10.6, df=l, P=0.001, odds ratio=2.53, 95% CI: 1.45-4.43). Genotype analysis revealed that the presence of one 196R allele was sufficient for rendering susceptibility. The association of 196R allele with SLE was independent from that of HLA-DRB1*1501. In conclusion, the TNFR2 196R allele was found to be significantly associated with the susceptibility to SLE in the Japanese population. Further population and functional studies will be of particular importance to establish TNFR2 as one of the susceptibility genes to SLE.

The origin of cretan populations as determined by characterization of hla alleles

Abstract: The Cretan HLA gene profile has been compared with those of other Mediterranean populations in order to provide additional information regarding the history of their origins. The allele frequencies, genetic distances between populations, relatedness dendrograms and correspondence analyses were calculated. Our results indicate that the Indoeuropean Greeks may be considered as a Mediterranean population of a more recent origin (after 2000 B.C.), while all other studied Mediterraneans (including Cretans) belong to an older substratum which was present in the area since pre-Neolithic times. A significant Turkish gene flow has not been detected in the Greek or Cretan populations, although Greeks and Turks have two high frequency HLA-DRB-DQB haplotypes in common. It is proposed that Imazighen (Caucasoid Berbers living at present in the North African coast and Saharan areas) are the remains of pre-Neolithic Saharan populations which could emigrate northwards between about 8000-6000 B.C., when desert desiccation began. They also could be part of the stock that gave rise to Sumerians, Cretans and Iberians; this is supported by both linguistic and HLA genetic data.

CD28/CTLA4 gene region on chromosome 2q33 confers genetic susceptibility to celiac disease. A linkage and family-based association study.

Abstract: Celiac disease (CD) is a common small intestinal injury caused by sensitivity to gliadin in genetically-predisposed individuals. The only susceptibility locus established is the HLA-DQ. We tested whether the chromosomal region of the CD28/CTLA4 genes on 2q33 is linked to CD. These genes encode receptors regulating the T-lymphocyte activation. Recently, this gene region was reported to be linked to the susceptibility to many autoimmune diseases, including insulin-dependent diabetes (IDDM12locus). It is thus an obvious candidate locus also for CD, since the intestinal injury is mediated by the immune system. Genetic linkage between seven marker loci in this gene region and CD was studied in 69 Finnish families. In the multipoint linkage analysis, the highest non-pararametric linkage score (NPL) was 1.75 (P=0.04) for D2S116, suggesting weak linkage for this candidate locus. To evaluate this finding, an additional 31 families were typed for all markers. In the combined set of 100 families the NPL score for marker D2S116 was 2.55 (P=0.006) and for other markers 1.90-2.47 (P=0.029-0.007), supporting genuine linkage at this region. Significantly, locus D2S116 also showed a clear allelic association in these 100 families (P=0.0001). The transmission/disequilibrium test (TDT) for locus D2S116 gave preliminary evidence for preferential maternal non-transmission of allele *136 to patients (TDTmax=8.3; P<0.05). No paternal deviation was found suggesting that the effect of the locus might be mediated by a sex-dependent factor protective against CD. Our results indicate that the CD28/CTLA4 gene region can contain a novel susceptibility locus for CD and support the hypothesis that CD has an immune system-mediated component. Like the HLA, the CD28/CTLA4 genes appear to be associated with genetic susceptibility to various autoimmune diseases.

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

Abstract: The role of HLA-B*51 and other major histocompatibility complex (MHC) genes in Behcet'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.

Nomenclature for factors of the dog major histocompatibility system (DLA), 2000: Second report of the ISAG DLA Nomenclature Committee

Abstract: A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles.

A gene telomeric of the HLA class I region is involved in predisposition to both type 1 diabetes and coeliac disease.

Abstract: We have recently shown that an as yet unidentified gene within or in the vicinity of the HLA complex, in linkage disequilibrium with microsatellite D6S2223, modifies the risk to develop type 1 diabetes independently of HLA-DR and -DQ genes. This microsatellite is located 2.5 Mb telomeric to HLA-F and particular alleles at this microsatellite modifies the risk encoded by the high-risk DRB1*03-DQA1*0501-DQB1*0201 (hereafter called DR3) haplotype. Coeliac disease and type 1 diabetes share some susceptibility HLA class II haplotypes, in Scandinavia particularly the DR3 haplotype. We therefore investigated whether the marker D6S2223 might also be associated with coeliac disease. In order to keep the contributions from the DRB1-DQA1-DQB1 genes constant (i.e., eliminate the effects of linkage disequilibrium to disease associated DR and/or DQ alleles), we only used cases and controls being homozygous for DR3. We found the frequency of allele 3 at D6S2223 to be reduced among patients with coeliac disease compared to controls, to a similar extent as seen in type 1 diabetes, which could not be explained by a different distribution of HLA-B alleles (as ascertained by typing for the MIB microsatellite). This negatively associated allele 3 at D6S2223 occurred in a homozygous combination at a significantly lower frequency among patients than controls. Thus, allele 3 at D6S2223 on DR3 haplotypes is associated with reduced susceptibility for development of both type 1 diabetes and coeliac disease. This suggests that a gene(s) in the vicinity of D6S2223 is involved in the pathogenesis of both of these immune-mediated diseases.

Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4

Abstract: Natural killer (NK) cells spontaneously detect and kill cancerous and virally infected cells through receptors that transduce either activating or inhibiting signals. The majority of well studied NK receptors are involved in inhibitory signaling. However, we have previously described an activating receptor, 2B4, expressed on all murine NK cells and a subset of T cells that mediate non-major histocompatibility complex (MHC) restricted killing. Anti-2B4 monoclonal antibodies directed against IL-2-activated NK cells enhanced their destruction of tumor cells. Recently, we determined binding of 2B4 to CD48 with a much higher affinity than CD2 to CD48. Here we describe the molecular characterization of a cDNA clone homologous to mouse 2B4, isolated from a human NK cell library. The cDNA clone contained an open reading frame encoding a polypeptide chain of 365 amino acid residues. The predicted protein sequence showed 70% similarity to murine 2B4. Additionally, it has 48, 45, and 43% similarity to human CD84, CDw150 (SLAM), and CD48, respectively. RNA blot analysis indicates the presence of 3 kb and 5 kb transcripts in T- and NK-cell lines. A single transcript of 3 kb is identified in poly(A)+ RNA from human spleen, peripheral blood leukocytes, and lymph node, whereas, the level of expression in bone marrow and fetal liver was indeterminate. Preliminary functional data suggests that NK-cell interaction with target cells via 2B4 modulates human NK-cell cytolytic activity.

Significant association of a single nucleotide polymorphism in the tumor necrosis factor-alpha (TNF-alpha) gene promoter with human narcolepsy.

Abstract: Narcolepsy is a sleep disorder in which multiple factors, including environmental and genetic factors, are involved. A genetic factor strongly associated with the disorder has been found in the human leukocyte antigen (HLA) class II region: the haplotype, DRB1*1501–DQB1*0602, predisposes to narcolepsy. No susceptibility genes other than the HLA-haplotype have been found. In this paper, we performed an association study of the tumor necrosis factor-alpha (TNF-α) gene located in the HLA class III region with human narcolepsy, in which we examined the known single-nucleotide polymorphisms (SNPs) in the promoter region in 49 narcoleptic patients, who were all positive for DRB1*1501, and 111 healthy control individuals. The results indicated that the frequency of the genotype at position −857 (−857SNP) was significantly different between the patients and controls, and the allele frequencies of –857SNP revealed that the frequency of –857T was significantly increased in the patients as compared with that in the controls (P=0.0068). In addition, haplotypes presumed from HLA-DRB1, –857SNP and HLA-B loci suggested that –857T was mainly associated with DRB1 alleles other than DRB1*1501: the significant increase in frequency of –857T in the patients was not caused by allelic association with DRB1*1501. Therefore, it is conceivable that the TNF-α with –857T was associated with narcolepsy independently of the strong association of DRB1*1501 with the disorder. Altogether, the data presented here lead us to propose that TNF-α could be a new susceptibility gene in human narcolepsy.

CTLA4 polymorphisms in Spanish patients with rheumatoid arthritis

Abstract: Cytotoxic T-lymphocyte antigen 4 (CTLA4) polymorphisms located in the promotor region at positions -318 (C/T) and in exon 1 (49 A/ G) were investigated in 138 Spanish patients (37 men and 101 women) with rheumatoid arthritis and in 305 ethnically-matched healthy controls. When the allelic and genotypic frequencies corresponding to the CTLA4 -318 position were compared, no significant differences between patients and controls were found. However, when the CTLA4 49 A/G polymorphism was analysed, a significant increase of A/G heterozygous individuals among female patients (48.5% vs. 33.8% in controls; P=0.008; OR=2.0) was observed. This increase was absent among males (37.8%, P=NS). Analysis of the CTLA4 49 polymorphism with respect to HLA-DRB1 typing demonstrated a significant increase of A/G heterozygosity in the HLA-DR3-positive patient group compared with HLA-DR3-negative patient group (14/19, 74% vs. 49/119, 41%; P=0.009, OR=4.0). The increase of A/G genotype among HLA-DR3-positive patients was found in both males (4/6, 67%) and females' (10/13, 77%), although statistical differences were only reached in the female group. These results provide new insight into this complex association, confirm previous data from other studies, and suggest that the CTLA4 gene could be involved in the pathogenesis of rheumatoid arthritis.

HLA class I expression and chromosomal deletions at 6p and 15q in head and neck squamous cell carcinomas.

Abstract: Loss at the chromosomal region 6p21.3 is a frequent event in head and neck squamous cell carcinomas (HNSCC). Since the human leukocyte antigen (HLA) complex is located at 6p21.3, loss of heterozygosity (LOH) of this region may provide tumour cells with an immune-escape tumour phenotype. In the present study, we have studied the correlation of HLA class I, TAP1 and TAP2 expression and LOH at 6p21.3. HLA class I and TAP1 and TAP2 protein expression was analysed by immunohistochemical procedures. A panel of 41 HNSCC with downregulated HLA class I expression was selected for LOH studies using 5 microsatellite markers located at 6p21.3 (D6S105, D6S265, D6S276, D6S273, D6S291) and 2 markers located at the chromosome 6 centromere (D6S473) and the 6p telomere (D6S277). In addition, LOH of the beta-2-nmicroglobulin (beta2m) gene was studied using 2 microsatellite markers flanking the beta2m gene (D15S126 and D15S153) and was correlated with beta2m and HLA class I expression. In 20/41 (49%) of the HNSCC, allelic loss for at least one locus at 6p21.3 was found. Loss at 15q was found in 4/10 (40%) HNSCC with downregulated beta2m expression and in 12/41 (29%) HNSCC with downregulated HLA class I expression. Our data show that downregulation of HLA class I expression is correlated with loss of chromosomal regions at 6p21.3 in HNSCC. In addition, LOH at 6p21.3 and 15q in 10 paired samples of DNA derived from the primary HNSCC, the lymph node metastases and from peripheral blood lymphocytes (PBLs) was studied. Five (5/10) primary tumours contained the same deletion as the corresponding lymph node metastases. The other cases contained deletions either in the primary tumour (3 cases) or in the lymph node metastases (1 case) or no deletions at all (1 case).

Polymorphism of human CD1 genes.

Abstract: Human CD1 genes have been reported to be invariant or to show limited polymorphism. Recently, certain functions of CD1 antigens have been described to include the presentation lipid and glycolipid antigens. These observations prompted a thorough survey of the genetic polymorphism in the five human CD1 genes (CD1a–CD1e). Using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) combined with sequence analyses, exons 2 and 3 from CD1a–CD1e were characterized from a total of 110 unrelated healthy donors. Results showed that all five genes (CD1a–CD1e) are polymorphic in exon 2. Substitutions in CD1b and CD1c are silent, whereas, substitutions in CD1a, CD1d and CD1e result in amino acid replacements in the deduced protein products. CD1a and CD1e polymorphisms are prevalent in the population. The substitutions in CD1a have characteristics that may influence interactions with β2-microglobulin (β2-m) or accessory molecules. The substitution in CD1e is located in the region predicted to interact with ligands and may differentially impact the ability of CD1e alleles to bind antigen.

Rapid screening of T‐cell receptor (TCR) variable gene usage by multiplex PCR: Application for assessment of clonal composition

Abstract: The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.

Corneodesmosin gene polymorphism demonstrates strong linkage disequilibrium with HLA and association with psoriasis vulgaris.

Abstract: Corneodesmosin (CD) is thought to play a key role in corneocyte cohesion, and its proteolysis appears to be a major event in the process of desquamation. Recently it was shown that CD is encoded by the S-gene, which is located approximately 160 kb telomeric of HLA-C. In the present study, the role of CD in the genetics of psoriasis vulgaris was studied in greater detail. The second exon of the CD gene was sequenced in 86 HLA-typed individuals from 13 psoriasis multiplex families. A total of 11 silent dimorphisms and 7 variants resulting in amino acid substitutions were found. Pedigree analysis showed that these variants could be grouped into 7 alleles, encoding 6 different amino acid sequences. These alleles are in strong linkage disequilibrium with HLA-B and -C, indicating that the polymorphism of the CD gene is ancient and well conserved rather than sporadic. One allele at the CD locus, designated CD2, displayed strong linkage disequilibrium with HLA-Cw6, the HLA allele most prominently associated with psoriasis. CD2 demonstrated a greater relative risk than Cw6 (3.4 vs. 2.5, not significant) and higher significant transmission disequilibrium with psoriasis than any of the investigated HLA-alleles. Due to its biologic function, cellular location and disease association, the CD gene appears to be an excellent candidate gene for PSORS1, the HLA-linked determinant of psoriasis vulgaris.

Distribution of HLA gene and haplotype frequencies in Taiwan: a comparative study among Min‐nan, Hakka, Aborigines and Mainland Chinese

Abstract: A total of 8,497 blood samples were typed for HLA-A, B, DR and DQ. Of these, 7,137 Min-nan, 714 Hakka, 535 Mainland Chinese (152 from North China, 211 from Middle China, and 172 from South China) and 111 Aborigines were randomly selected from Tzu Chi Taiwan Marrow Donor Registry (TCTMDR). Differences in HLA gene and antigen frequencies have been observed between various ethnic groups of the Chinese population in Taiwan. The phylogenic tree shows Taiwan Aborigines and Javanese cluster together; Min-nan shares a common cluster with Hakka, Southern Hans and Thai; and Northern Hans shares a cluster with Middle Hans. The separation between Northern/Middle and Southern Chinese Hans support the idea that Northern and Southern Chinese have different genetic background. Aborigines appeared to be quite distinct in the distribution of a majority of the class I and class II antigens. High frequency of HLA-A24 (60.4%) and relatively restricted HLA polymorphisms are noted in Aborigines. The HLA haplotypes with high frequency in Aborigines included A24-B60-DRB1*04, A24-B60-DRB1*14, A24-B48-DRB1*04, and A24-B48-DRB1*14, which are different from the other ethnic groups. Although the phylogenic tree separates Aborigines and Han Chinese populations, 4 out of 20 most common HLA-A, -B, and -DR haplotypes presented in both Aborigines and Han Chinese may reflect an ancient common origin or intermixture between early settlers of Han Chinese and Taiwan Aborigines. The results in this study are essentially a summary of the observed gene/haplotype frequencies and differences among various ethnic groups in Taiwan.

DQB1*0602 confers genetic susceptibility to multiple sclerosis in Afro-Brazilians.

Abstract: We studied the distribution of the HLA-DRB1, -DQA1 and -DQB1 alleles in 44 Afro-Brazilian patients with multiple sclerosis and 88 controls. Although no significant differences were found between the patients and controls for the DRB1 and DQA1 alleles, the HLA-DQB1*0602 allele was positively associated with multiple sclerosis (45.0% vs. 17.0%, Pc=0.024, RR=3.31). The positive extended haplotypes for DQB1*0602 were more frequent in patients than controls, although the differences were not statistically significant in any of them. These results in Afro-Brazilians are in line with other studies which have found DQB1*0602 to be associated with the disease in the absence of the DRB1*1501 allele. We therefore think that the association with the disease in this ethnic group is more allelic than haplotypic.

DNA typing of HLA class II genes (HLA‐DR, ‐DQ and ‐DP) in Japanese patients with histiocytic necrotizing lymphadenitis (Kikuchi’s disease)

Abstract: : The pathogenesis of histiocytic necrotizing lymphadenitis (HNL), which was reported first by Kikuchi et al. and Fujimoto et al. in 1972, is as yet unknown. HNL is frequently reported in Asian countries including Japan, however it is rare in Europe and North America. To elucidate whether the human leukocyte antigen (HLA) alleles and haplotypes are associated with HNL, we performed DNA typing of HLA class II genes (HLA-DR, -DQ, and -DP) in 86 patients with HNL and 525 unrelated healthy Japanese controls with polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). In this study, we found DPA1*01 and DPB1*0202 allele frequencies in HLA class II genes are significantly higher in HNL patients than in normal controls. It is known that the frequency of DPB1*0202 alleles is extremely low or absent in Caucasians (e.g., French 0.4%, Italian 0.8%) and Negroid (e.g., South African 0%, Hottentot 0%), but relatively frequent in Asians (e.g., Korean 9.9%, Japanese 4.5%). Previous reports have said the incidence of HNL is frequent in Asians but rare in other races. In light of this background, HLA class II genes of HNL and the incidence of HNL in Asian countries, including Japan, might have a positive relationship to DPA1*01 and DPB1*0202 allele.

Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop.

Abstract: Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.

Genetic polymorphisms at the tumour necrosis factor loci (TNFA and TNFB) in cardiac sarcoidosis

Abstract: Many studies have previously confirmed high TNFalpha (tumor necrosis factor-alpha) production in sarcoidosis, and have shown that TNFalpha plays an important role in granuloma formation. We investigated TNFA and TNFB (lymphotoxin-alpha) gene polymorphisms in 26 cardiac sarcoidosis patients of Japanese origin. These studies revealed a significant increase in the more uncommon TNFA2 allele in the patient group, suggesting that the TNFA gene controls the genetic susceptibility to cardiac sarcoidosis.

HLA class II alleles in Japanese patients with inflammatory bowel disease

Abstract: The association of various HLA class II loci with Crohn's disease (CD) and ulcerative colitis (UC) has yet to be fully elucidated. To determine whether there is an association of HLA class II genes (DR, DQ and DP alleles) with inflammatory bowel disease (IBD), HLA class II genes for polymorphisms were analyzed at the DNA level in 111 patients with CD, 81 with UC and 525 healthy controls by the polymerase chain reaction-sequence specific oligonucleotide probe method. Allele and haplotype frequencies were compared between these populations. Results were as follows: 1) the presence of DQB1*0402 (RR=3.90, Pc=0.0001) was positively associated with CD; 2) the presence of DRB1*1502 (RR=4.51, P<1X10(-8)), DRB5*0102 (RR=4.70, Pc<1x10(-8)), DQA1*0103 (RR=3.72, Pc=1x10(-5)), DQB1*06011 (RR=3.78, PC=1x10(-5)), DPA1*0201 (RR=3.23, Pc=0.0001) and DPB1*0901 (RR=4.83, PC<1x10(8)) was positively associated and that of DRB4*0101 (RR=0.20, Pc<1X10(-8)) and DQA1*0302 (RR=0.34, Pc=0.001) negatively associated with UC; 3) haplotype analysis showed a positive association between the presence of DRB1*0410-DQA1*0302-DQB1*0402 and DRB1*0802-DQA1*0401-DQB1*0402 with CD, and a negative association between the presence of DRB1*1502-DQA1*0103-DQB1*06011 and CD, there was no association of DRB1*08032-DQA1*0103-DQB1*06011 with CD; and 4) in UC, a positive association with the presence of DRB1*1502-DQA1*0103-DQB1*06011 was found, but DRB1*08032-DQA1*0103-DQB1*06011 was not associated with it. In conclusion, in CD in the Japanese population, HLA-linked disease susceptibility alleles appear to be DQB1*0402 and DRB1*1502, a disease resistance allele. In UC, DRB1*1502 appears to be a disease susceptibility allele.

Susceptibility to ankylosing spondylitis is independent of the Bw4 and Bw6 epitopes of HLA-B27 alleles.

Abstract: We have characterized HLA-B27 alleles in a sample of the population from the Azores (n=46) with the aim of investigating the contribution of different subtypes to ankylosing spondylitis (AS). The study was carried out using PCR-SSOP and in some samples genomic sequencing was conducted. Some significant new finding have arisen from this study. First, B*2705,B*2702,B*2703,B*2707 and B*2708 alleles were found to be represented in this population. The polymorphism of B27 alleles found in a sample of the population from the Azores is higher than the Caucasian groups described. B*2703 and B*2707 have not previously been described to be represented in Caucasians and this could indicate admixtures with different populations of the world. In addition, the B*2708 allele was found to be associated with AS in a large family from the Azores. This association has not been previously reported in either ethnic group and needs to be confirmed in other population studies. This is of considerable interest since has only been described as a rare subtype underrepresented in the British population and has not been previously found to be associated with AS. B*2708 carries the sequence specifying the Bw6 epitope in contrast to most B27 alleles which carry a Bw4 sequence. Differences in this region (residues 77-83) can alter the F-pocket and affect T-cell recognition. The importance that these molecular changes can play in the pathogenesis of AS is discussed.

HLA‐B locus sequence‐based typing

Abstract: We describe an approach for HLA-B high-resolution typing. A single locus-specific amplification generates a 1-kb fragment useful for direct sequencing. Four internal primers are necessary for exon 2 and 3 cycle-sequencing in both directions. Fluorescent dye-labelled nucleotides are incorporated during cycle-sequencing and reaction products are analyzed in an automated DNA sequencer. At present, software programs allow automatic assignment of exon 2 only; analysis of exon 3 is not automatic. In the future, the development of more sophisticated software will improve allele assignment. The approach described in this work offers a precise and efficient identification of known allele sequences and at the same time can differentiate new alleles. Furthermore, it may be applied as a model for the development of similar molecular typing approaches for other polymorphic HLA loci.

Lack of a strong association of CTLA-4 exon 1 polymorphism with the susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese: an association study using a novel variation screening method.

Abstract: CTLA-4 is considered to be one of the attractive candidates for the susceptibility genes to rheumatic diseases. In the present study, the association of CTLA-4 polymorphism with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was examined in the Japanese population using the case-control association analysis. Polymerase chain reaction-preferential homoduplex formation assay (PCR-PHFA) was applied for the screening of genetic variations and for the genotyping of a large number of samples. A greater proportion of Japanese patients with RA (44%) and SLE (44%) compared with healthy individuals (37%) had exon 1 49 G/G genotype, but the difference did not reach statistical significance. However, when the patients with RA and healthy individuals were stratified according to HLA-DRB1 alleles, a weakly significant increase of the positivity of CTLA-4 49G allele was observed in HLA-DRB1*0405-positive patients (87%) compared with DRB1*0405-positive healthy individuals (71%) (P = 0.014, odds ratio = 2.77). These results indicate that CTLA-4 exon 1 polymorphism does not contribute greatly to the susceptibility to RA and SLE, at least in Japanese, although the presence of CTLA4 49G allele could be a minor predisposing factor for RA in HLA-DRB1*0405-positive individuals. In addition, PCR-PHFA was shown to be useful for a mass screening of gene variations.

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

Abstract: Behcet'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 Behcet'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 Behcet'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 Behcet's disease in Italian with B*5108 as well as B*5101, providing important insight into the molecular mechanism underlying an HLA association with Behcet's disease.