PCR-SSOP molecular typing of HLA-C alleles in an Iranian population.

HLA-C alleles were characterized by a polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) hybridization protocol in a sample of 120 Iranians from Tehran. A total of 23 alleles were identified with the four most predominant--Cw*0401, Cw*0602, Cw*1202, and Cw*0701/06--accounting for almost 50% of HLA-C alleles. A comparison of HLA-C diversity among several populations indicates that Iranians stand at an intermediate genetic position between Europeans and Africans, an observation that may be related to their geographical location at a continental crossroads. The results also reveal a very high correlation between genetic and geographic distances on a global scale. A total of 30 HLA-C-DRB1 haplotypes were found in the Iranians, with the highest frequencies of 6.6% and 6.04 % being for Cw*0602-DRB1*0701 and Cw*1202-DRB1*1502, respectively.

Relation between the heavy chain complementarity region 3 characteristics and rheumatoid factor binding properties.

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecificity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3's (15.3+/-2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3's of smaller length (11.3+/-2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3's and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.

Comparison of rheumatoid factors of rheumatoid arthritis patients, of individuals with mycobacterial infections and of normal controls: evidence for maturation in the absence of an autoimmune response.

We analyzed the rheumatoid factors (RF) produced by Epstein-Barr virus-transformed monoclonal B cells established from four patients with rheumatoid arthritis (RA), three individuals with a history of Mycobacterium tuberculosis (TB) and four normal controls (NI). Fifty-eight RF were analyzed for specific activity (international units-RF/microgram) for the Fc part of IgG and their interaction with tetanus toxoid (TT) and DNA (polyspecificity). Furthermore, we sequenced the V-D-J heavy chain region of 16 (9TB-/7RA-) RF. Significant differences were observed between the NI-RF and the TB- and RA-RF. While the RF repertoire of normal individuals comprised of low-avidity RF of which the majority (15/17) were polyspecific, more than half of the TB- and RA-RF were monoreactive. Furthermore, the monospecific TB- and RA-RF were of significantly higher avidity than the NI-RF (RA > TB > > NI). With respect to polyspecificity specificity, the RF in the three groups were comparable: the interaction with DNA, TT as well as with Fc was inhibited either by an increase of the ionic strength to 0.3-0.5 M NaCl or by addition of the polyanion dextran sulfate, indicating that the antibodies interacted with similar anionic epitopes shared by the three antigens. Analysis of the V-D-J heavy chain regions showed significant differences between the respective RF. The salt-sensitive binding was highly correlated with the presence of arginine in the complementarity-determining region 3 (CDR3). Furthermore, whereas the polyspecific RF consisted predominantly of germ-line encoded antibodies, the genes of the monospecific RA/TB-RF were somatically mutated (RA > TB). It is therefore likely that maturation of RF can be initiated by chronic infections and that monospecific, somatically mutated RF are not a unique characteristic of autoimmune diseases.

Oligotyping of HLA-A2, -A3, and -B44 subtypes. Detection of subtype incompatibilities between patients and their serologically matched unrelated bone marrow donors.

We have set up a simple PCR-SSO oligotyping procedure that is able to discriminate ten HLA-A2 (2 PCR/11 probes), two HLA-A3 (1 PCR/1 probe), and two HLA-B44 subtypes (1 PCR/2 probes). The frequency of these subtypes has been determined in a large panel of local blood donors and leukemic patients in combination with their unrelated potential donors. A*0201 and A*0301 were the predominant subtypes (> 95%) for A2 and A3, respectively. B*4402 occurred twice as frequently as B*4403. A2 and B44 subtype mismatches were analyzed in a group of 30 patients and their 116 unrelated potential donors who were matched serologically (low-stringency matching: AB without splits, DR1-10). For seven patients (23%) at least one A2- or B44-subtype-mismatched donor was found. For two of these patients (7%), the subtype-mismatched donor would have been considered as compatible on the basis of high stringency matching (AB splits, DRB1 subtypes, DRB3/B5). For one patient of Mediterranean origin, all five donors recruited from a north European registry (matched with high stringency) appeared to be subtype incompatible (A*0201/A*0205). The rather low percentage of A2- and B4-subtype mismatches in DRB1/B3/B5 matched combinations confirms the significance of linkage disequilibria of HLA antigens. Because unrelated donor selection is done through international registries, however, class I subtyping might be necessary when individuals originate from different geographic areas.