The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF␣), lymphotoxin-alpha (LT␣) and interleukin-10 (IL-10) genes on stimulated TNF␣ and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn's disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF␣, LT␣ and IL-10 genes. Production of the TNF␣ and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF␣ gene, three haplotypes of LT␣ and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT␣-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT␣-2 haplotypes were associated with higher TNF␣ production in CD patients, and the TNF-4 haplotype was associated with lower TNF␣ production in UC patients. The A allele in the IL-10 promoter region at position −1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF␣, LT␣ and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls. Genes and Immunity (2000) 1, 185-190.

Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system using primers with mismatches at the 3 ′ ends was developed to determine polymorphisms in IL-10 promoter region. Three previously described biallelic polymorphisms in IL-10 were linked in a 12 reaction PCR-SSP system and the method used to provide genotype data on 233 UK and 166 Polish controls. There are eight possible polymorphic combinations in IL-10 promoter gene but only three were observed in both control groups. Population frequencies of IL-10 genotypes show, in contrast to HLA, that UK and Polish frequencies are remarkably similar. Genes and Immunity (2000) 1, 321-324.

The molecular basis for the antigenic variation and red cell expression of the Duffy antigen system has recently been elucidated. We have developed a simple one‐step method for genotyping the single nucleotide polymorphisms in the promoter and exon of the Duffy gene using the polymerase chain reaction and sequence‐specific primers (PCR‐SSP). This method is also capable of haplotyping alleles at the two polymorphisms as being in cis or trans orientation. Twenty‐four serologically typed Caucasoid and Afro‐Caribbean samples were examined to validate the method, with absolute correlation between phenotype and genotype. A further 30 Gamb‐ian samples were genotyped, confirming homozygosity for the FY*null‐FY*B haplotype. Allele, gene and haplotype frequencies were examined in 100 Caucasoid controls. This method permits the rapid genotyping of large numbers of samples and will prove useful as a clinical and research tool.

A subgroup of common variable immunodeficiency (CVID) patients have distinct clinical features, particularly granulomata splenomegaly, characteristic blood lymphocyte phenotype, and elevated circulating TNF levels. To investigate the genetic basis for this phenotype, 150 CVID patients and 200 controls were genotyped for six biallelic TNF and lymphotoxin-alpha (LT alpha) polymorphisms and eight class I and II HLA loci using PCR and sequence specific primers (PCR-SSP) sequence-specific primers. Clinical and immunophenotypic data were collected for 90 patients to examine associations with CVID patient subgroups. The presence of granulomata (22% of patients) was strongly associated with splenomegaly, T and B lymphopenia, reduced CD4+ CD45RA+ T cells, and CD8+ CD57+ lymphocytosis, confirming the concept of a subgroup of patients with distinct clinical and laboratory features. The uncommon TNF +488A allele was strongly associated with this subgroup (p = 0.0005). The association between "granulomatous" CVID and TNF +488A was independent of HLA class I and II associations. We postulate that the presence of the TNF +488A allele, or alleles in linkage disequilibrium with it, contributes to the high levels of TNF and granulomatous complications characteristic of this subgroup of patients.