Identification of a new HLA-A null allele, A*2436 N.

We describe a new HLA-A null allele in a donor. This null allele resulted from the deletion of two nucleotides in exon 2, which effects a frameshift as well as a premature stop codon. This new null allele has been officially named HLA-A*2436 N.

Identification of a new HLA-B null allele, B*1817 N, in a family.

We describe a new HLA-B null allele found in a daughter and her mother. This null allele was due to a mutation at position 41 of exon 1 which resulted in a premature stop codon. This new null allele was officially named HLA-B*1817N*.

Microsatellite polymorphism of the MICA gene and susceptibility to rheumatoid arthritis.

We examined the role of MICA gene alleles in susceptibility to rheumatoid arthritis (RA). Ninety adult Caucasian patients with classical seropositive RA and 85 normal healthy Caucasian subjects from the same geographical area were typed for microsatellite repeat polymorphism in the transmembrane region of the MICA gene by the polymerase chain reaction. The results show that the MICA allele 6 may confer protection from the development of RA.

Polymorphism in the Y box controls level of cytokine-mediated expression of HLA-DRB1 genes.

The HLA class II molecules play an important role in immune response. The quality of immune response is dependent not only on the polymorphisms in the class II molecules, but also on the level of their cell-surface expression. In fact, it has been demonstrated that differences in the level of expression of DRB1 and DRB3 genes restricted and activated distinct CD4+ T lymphocytes. We and others have previously described allelic polymorphisms in the upstream regulatory regions of DRB genes, which affected DNA-protein interactions and resulted in significantly different promoter strengths. We showed that polymorphisms in both the X1 and Y box motifs affect level of constitutive expression of DRB1 genes in the DR1, DR51 and DR53 haplotype groups. In the present study, we examined the effect polymorphisms in the X1 box and the Y box on the cytokine (interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha) and granulocyte macrophage-colony-stimulating factor (GM-CSF))-mediated transcriptional activities of DRB1 promoters in these, i.e. DR1, DR51 and DR53, haplotype groups. The results demonstrate that the polymorphism in the X1 box does not affect cytokine-mediated strength of DRB1 gene promoters. In contrast, the polymorphism in the Y box, which affects the inverted CCAAT sequence, plays a dominant role on the cytokine-mediated transcriptional activity of DRB1 promoters.

HFE gene mutations in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the role of C282Y and H63D mutations in HFE gene in susceptibility to rheumatoid arthritis (RA). METHODS: The distribution of C282Y and H63D mutations in patients with RA and in healthy subjects was examined by restriction endonuclease digestion of polymerase chain reaction amplified genomic DNA. RESULTS: The prevalence of C282Y mutation in patients with RA was the same as in healthy controls. In contrast, the distribution of H63D mutation was significantly higher in the total RA patient population and in DRB1 QKRAA/QRRAA epitope positive patients compared to respective groups of controls. Analysis of data showed that (1) both H63D mutation and QKRAA/QRRAA DRB1 epitope are individually associated with RA susceptibility; (2) there is interaction between these 2 factors in development of RA; and (3) both these factors combined have stronger association with RA susceptibility than with these factors individually. CONCLUSION: H63D mutation appears to play a role in pathogenesis of RA. This study is small and must be regarded as preliminary. These data therefore need confirmation from independent studies.

HLA class III region and susceptibility to rheumatoid arthritis.

OBJECTIVE: We examined the contribution of the HLA class III region in susceptibility to rheumatoid arthritis (RA). METHODS: Patients with RA, healthy subjects and homozygous typing cell (HTC) lines were typed for HLA class I (A, B, C), class II (DR, DQ) and class III (D6S273, Bat 2, and TNFa microsatellites, and HSP70 promoter region) alleles by molecular techniques. RESULTS: Based on the distribution of microsatellites D6S273, Bat2 and TNFa, and HSP70 promoter region alleles in HTCs and homozygous unrelated individuals, a class III region haplotype, D6S273 138-HSP70c-Bat2 138-TNFa2 was identified. This haplotype showed a significant primary association with susceptibility to RA in DRB 1 QKRAA/QRRAA epitope-negative patients. CONCLUSION: Since the QKRAA/QRRAA epitope does not provide any risk for disease susceptibility in RA-susceptibility DRB1 epitope-negative patients, the present data suggest that the class III region haplotype D6S273 138-HSP70c-Bat2 138-TNFa2 provides an additional risk for the development of RA. These results show that two regions in MHC, class II (DRB1) and class III (D6S273 138-HSP70c-Bat 2 138-TNFa2), contribute to susceptibility to RA and more completely define the risk for development of the disease.

NRAMP1 gene polymorphisms in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with HLA-DR genes that share amino acid sequence motif QKRAA/QRRAA from position 70 to 74 in the third hypervariable region of DR1 molecule. The contribution of HLA in RA is however about 37%, suggesting a role for other genes. One such candidate is the gene that encodes natural resistance-associated macrophage protein (NRAMP1), which plays a crucial role in inflammation and tissue destruction. In the present study, we examined the role of NRAMP1 gene polymorphisms in susceptibility to RA. The results show that variation at position 543 in exon 15, which involves substitution of negatively charged aspartic acid (D) by uncharged asparagine (N), and the deletion of TGTG in the 3' UTR may confer protection from development of RA.

Polymorphism in heat-shock protein 70-1 (HSP70-1) gene promoter region and susceptibility to tuberculoid leprosy and pulmonary tuberculosis in Asian Indians.

Heat shock proteins (HSP) act as immunological target structures either by themselves because of an unusual expression pattern, or they are carrier proteins for immunogenic peptides. A three-allele polymorphism of HSP70-1 promoter region was analysed in random patients with pulmonary tuberculosis (PTB), or with tuberculoid (TT) leprosy and healthy controls from North India. HSP70-1A and HSP70-1C occurred more frequently (> 60%) while HSP70-1B occurred infrequently in this population. Only HSP70-1A allele was significantly increased in TT leprosy as compared to healthy controls (91.8% Vs 71.1%, Pc < 0.03, RR = 4.58). Although a strong association of HLA-DR15 was observed with both of these patient groups in earlier studies, no correlation was found between HSP70-1 promoter alleles with any of the HLA allotypes. Amongst six possible genotype combinations of HSP70-1 promoter allele, only four (A/A, A/B, A/C, C/C) were encountered in Asian Indians. A significant increase of HSP70-1 A/C genotype was observed among DR15 negative PTB patients as compared to DR15 negative controls (87.5% Vs 35.7%, X2 = 8.6, Pc < 0.02) giving highest relative risk of 12.6. These findings suggest that HSP70-1 genes may play a secondary role to HLA-DR in governing susceptibility to mycobacterial infectious diseases.

Genetic basis for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common disabling disorder of unknown etiology. In the past 2 decades, a number of studies have examined the genetic basis for RA. One major focus of these studies has been to identify genes within the MHC class II (HLA-DR) chromosomal region, which confer susceptibility/resistance to RA. A strong association between HLA-DR4 and adult seropositive RA has been observed in majority of populations. In addition, there is evidence of a positive association between HLA-DR1 and RA. On the basis of prevalence of DR1 (B1*0101) and of subtypes of DR4 (B1*0401, B1*0404 and B1*0405), it has been suggested that a five amino acid sequence motif (QKRAA/QRRAA) from position 70 to 74 in the third hypervariable region of DRbeta1 molecules is associated with susceptibility to RA. These associations between RA and HLA-DR genes are however incomplete in that about 1/4 of patients do not carry RA-susceptibility DRB1 epitope. Since MHC class III region contains genes that are involved in immune response, we have recently examined the role of a number of microsatellites (D6S273, Bat2, TNFa) and HSP70 promoter region alleles in susceptibility to RA. The results demonstrate that two regions in MHC, class II (DRbeta1) and class III (D6S273, HSP70, Bat2, TNFa) more completely define the risk for development of RA.

Genetics of rheumatoid arthritis (RA): two separate regions in the major histocompatibility complex contribute to susceptibility to RA.

We analyzed HLA-DR antigens and microsatellite Bat2 alleles in 97 adult caucasian patients with classical seropositive rheumatoid arthritis (RA) and 95 normal healthy controls. The results demonstrate that the prevalence of microsatellite Bat2 138 allele was significantly higher in RA-susceptibility DRB1 QKRAA/QRRAA epitope-negative patients as compared with normal controls. Analysis of the data suggested that Bat2 138 allele has primary association with RA-susceptibility in QKRAA/QRRAA epitope-negative patients. The Bat2 138 allele thus provides an additional risk in RA-susceptibility. In addition, microsatellite Bat2 138 allele showed a highly significant positive association with microsatellite D6S273 138 allele, which has similar (identical) association with RA development in DRB1 QKRAA/QRRAA epitope-negative patients. The present data demonstrate that DRB1 QKRAA/QRRAA epitope and microsatellite Bat2 138/D6S273 138 alleles more completely define the risk for development of RA. The results in the present study therefore suggest that two regions in MHC, class II (DRB1) and class III (Bat2 and D6S273 in HSP70-Bat2 region), contribute to susceptibility to RA.

D6S273 microsatellite polymorphism and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic articular inflammatory disease associated with HLA-DR genes that share a five amino acid sequence motif, QKRAA or QRRAA, from position 70 to 74 in the third hypervariable region of the DRB1 molecule. Since these associations between DRB1 genes and susceptibility to RA are incomplete, we examined the role of a CA repeat polymorphic microsatellite marker, D6S273, located between HSP70 and Bat2 genes in the class III region of MHC, in susceptibility to RA. Ninety-seven adult patients with seropositive RA and 100 normal healthy subjects were studied. Two D6S273 alleles (132 and 138) showed significant differences in their prevalence in RA patients as compared to normal controls; allele 132 was significantly higher in total patients and in DRB1 QKRAA/QRRAA epitope-positive patients, and allele 138 was significantly higher in QKRAA/QRRAA-negative patients. Analysis of data suggested that the association of D6S273 132 allele with RA was secondary to that of DRB1 genes. On the other hand, D6S273 138 allele showed primary association with RA susceptibility in QKRAA/QRRAA epitope-negative patients. The D6S273 138 allele thus provides an additional risk in RA susceptibility. The results in the present study therefore suggest that two regions in MHC, DRB1 and D6S273, contribute to susceptibility to RA.

DQCAR microsatellite polymorphism and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with HLA-DR genes that share a five amino acid sequence motif, QKRAA or QRRAA, from position 70 to 74 in the third hypervariable region of the DRbeta1 molecule. Since the associations between DRB1 genes and susceptibility to RA are incomplete, in this study we examined the CA repeat polymorphic marker DQCAR, located between DQA1 and DQB1 genes, alleles in 98 adult patients with seropositive RA and 100 normal healthy controls. The prevalence of the DQCAR 117 allele was significantly higher in RA patients as compared to normal controls. On the other hand, the frequency of DQCAR 99 was lower in patients than in normal subjects. Analysis of the data suggested that DRB1 genes sharing the QKRAA/QRRAA epitope have the primary association with disease susceptibility and DQCAR alleles do not provide an additional risk for the development of RA.

HLA-DM polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the contribution of HLA-DMA and DMB genes, which play a crucial role in the HLA class II restricted antigen presentation pathway, in susceptibility to rheumatoid arthritis (RA). METHODS: The distribution of DMA and DMB alleles was examined in patients with RA and in healthy subjects by oligotyping of PCR amplified genomic DNA with sequence specific oligonucleotide probes. RESULTS: There were no significant differences in the prevalence of DMA and DMB alleles in patients with RA as compared to healthy controls. In addition, no significant differences in frequencies of DMA and DMB alleles were observed in RA susceptibility epitope positive RA patients and controls. CONCLUSION: DMA and DMB genes do not appear to play a role in susceptibility to RA.

Peptide transport in human lymphoblastoid and tumor cells: effect of transporter associated with antigen presentation (TAP) polymorphism.

CD8+ T-cells recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. These peptides bind to MHC class I molecules in the endoplasmic reticulum (ER) lumen. Antigenic peptides are translocated from the cytosol to the lumen of ER by transporter associated with antigen presentation (TAP) proteins. In this study, it is shown that TAP1 polymorphism influences the peptide substrate specificity in human B-lymphoblastoid and tumor cell lines. TAP1A and 1C alleles specifically enhance translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1B allele does not show specificity for the peptide C-terminus. Human basophilic leukemia (Ku812), and hepatocellular carcinoma (PLC/PRF/5) cells express TAP1 molecules and exhibit TAP-mediated allele-specific peptide uptake after gamma-interferon (gamma-IFN) treatment. Ku812 cells express TAP1A and preferentially take up antigenic peptides with a basic C-terminus, however, PLC/PRF/5 cells with the TAP1B allele take up low but equivalent levels of peptides regardless of basic, acidic, or hydrophobic C-termini. Moreover, TAP2 polymorphisms have no influence on the peptide translocation in normal or tumor cell lines. In addition, Daudi, a beta2-microglobulin (beta2m) deficient human Burkitt lymphoma, cell line also showed TAP-dependent peptide uptake. Taken together, these results suggest that human TAP1 but not TAP2 polymorphisms influence the antigenic peptide transport and that this transport is independent of beta2m in this system.

Transfection of TAP 1 gene restores HLA class I expression in human small-cell lung carcinoma.

HLA class I antigens of the human major histocompatibility complex (MHC) play an important role in immune response. Consistent with their role in immune surveillance, these antigens are expressed on most cell types. However, a marked deficiency or lack of expression of these antigens has been observed in a variety of human neoplasms. We have shown that a number of class I-deficient human tumor cell lines, including small-cell lung carcinoma, lacked products of MHC-encoded TAP1 and LMP2 genes. Since a direct evidence for the role of these genes in class I expression in tumor cells is not available, in the present study we transfected class I-deficient human small-cell lung carcinoma cells with cDNAs corresponding to TAP1 gene and to LMP2 gene. Following transfection, tumor cells expressed products of the respective transfected gene. Cell-surface expression of class I molecules was, however, observed in cells transfected with TAP1, but not in tumor cells transfected with LMP2 gene. Our results provide conclusive evidence for a role of TAP1 gene in class I expression and suggest that transfection of TAP genes may be useful to upregulate class I expression in tumor cells. This strategy for restoration of class I expression by transfection of TAP genes is relevant for tumor rejection and/or abrogation of metastases formation.

Lack of HLA-DR2-associated DRB1 gene in a family.

HLA-DR51 haplotypes co-express one DRB1 (B1*15 or B1*16) gene and one DRB5 gene. These haplotypes also carry two pseudogenes (DRB6 and DRB9) During routine HLA typing for transplantation, we observed an unexpected DR51 haplotype in two subjects (mother and daughter) in a family. Serological typing showed that these subjects are positive for DR51 and DQ6 but negative for DR15 and DR16. Investigations of genomic DNA by molecular techniques showed that these individuals carry DRB5*0101, DRB6*02 and DQB1*0602 genes, whereas the DRB1 gene associated with either DR15 (B1*1501 to B1*1504) or DR16 (B1*1601 to B1*1606) was not detected in the mother and daughter. It is possible that the new haplotype, DQB1*0602, DRB6*02, DRB5*0101, arose by deletion of DR2-associated DRB1 gene.