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Inclusion body myositis (IBM) is a form of idiopathic inflammatory myopathy of unknown aetiology. A strong association with HLA class II (HLA-DR3) suggested a role for genes in the human major histocompatibility complex (MHC) in the predisposition to this disease. In this study, we have taken advantage of the ancestral haplotype (AH) concept and historical recombinations to map for a possible susceptibility gene(s) in the MHC. We performed detailed typing of three MHC-related HSP70 genes and defined allelic combinations in the context of MHC AH. We also modified existing methods to give a simple and accurate method for typing two TNF microsatellites. Using the HSP70 and TNF markers and HLA-DR, -B, and C4 typing of our patients with IBM, we defined a potential site for the MHC-associated susceptibility gene(s) in the region between HLA-DR and C4.

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.

Many new Major Histocompatibility Complex (MHC) genes have been discovered in the last 5 years. Defining the polymorphism of these new genes may elucidate their function and their relevance to diseases with MHC associations. Polymerase chain reaction and single stranded conformation polymorphism (PCR SSCP) analyses were used to detect sequence polymorphisms of PERB1 demonstrated by comparing the available genomic sequence of four haplotypes. This study showed that PCR SSCP of PERB1 is reproducible. In addition, PERB1 alleles segregate within families together with MHC haplotypes. Typing results from the Forth Asia and Oceania Histocompatibility Workshop (4AOHW) cell panel indicate that the identified polymorphisms of PERB1 are "haplotypic', i.e., unrelated individuals carrying the same MHC ancestral haplotypes carry the same PERB1 SSCP pattern. Interestingly, PERB1 SSCP patterns allow the distinction of ancestral haplotypes which share HLA-B serological specificities, such as HLA-B44 and therefore this analysis can be used to further define MHC haplotypes and thus to improve our understanding of the evolution of this complex.

We have used the novel strategy of overlapping yeast artificial chromosome (YAC) clones to localize a series of new transcripts within a 170 kb region of the human major histocompatibility complex (MHC), containing genes likely to be of importance for disease susceptibility. Using cloned genomic probes we have further localized these transcripts to a region 15 kilobases (kb) centromeric of HLA-B. In the liver there are at least four transcripts ranging in size between 7.5 kb and 3.4 kb, while in the lung two transcripts of 5.8 kb and 4.2 kb are detected. The possible implications of these transcripts for autoimmune disease are discussed, given that they are located in a region previously shown to be of importance for susceptibility to insulin-dependent diabetes mellitus and myasthenia gravis. Furthermore, we conclude that YACs as large as 360 kb are able to be used as probes to identify new transcripts and that the MHC region between HLA-B and BAT1 is the site of a large multiply spliced gene, provisionally designated PERB6.

We have used genomic analysis to characterize a region of the central major histocompatibility complex (MHC) spanning approximately 300 kilobases (kb) between TNF and HLA-B. This region has been suggested to carry genetic factors relevant to the development of autoimmune diseases such as myasthenia gravis (MG) and insulin dependent diabetes mellitus (IDDM). Genomic sequence was analyzed for coding potential, using two neural network programs, GRAIL and GeneParser. A genomic probe, JAB, containing putative coding sequences (PERB11) located 60 kb centromeric of HLA-B, was used for northern analysis of human tissues. Multiple transcripts were detected. Southern analysis of genomic DNA and overlapping YAC clones, covering the region from BAT1 to HLA-F, indicated that there are at least five copies of PERB11, four of which are located within this region of the MHC. The partial cDNA sequence of PERB11 was obtained from poly-A RNA derived from skeletal muscle. The putative amino acid sequence of PERB11 shares approximately 30% identity to MHC class I molecules from various species, including reptiles, chickens, and frogs, as well as to other MHC class I-like molecules, such as the IgG FcR of the mouse and rat and the human Zn-alpha 2-glycoprotein. From direct comparison of amino acid sequences, it is concluded that PERB11 is a distinct molecule more closely related to nonmammalian than known mammalian MHC class I molecules. Genomic sequence analysis of PERB11 from five MHC ancestral haplotypes (AH) indicated that the gene is polymorphic at both DNA and protein level. The results suggest that we have identified a novel polymorphic gene family with multiple copies within the MHC.

As ancestral haplotypes of the major histocompatibility complex (MHC) appear to define identical MHC haplotypes in unrelated individuals, unrelated individuals sharing the same ancestral haplotype should also share the same NK-defined allospecificities that have recently been shown to map to the human MHC. To test this prediction, multiple cell lines from unrelated individuals sharing the same ancestral haplotypes were tested for the NK-defined allospecificities. It was found that cells sharing the same ancestral haplotypes do have the same NK-defined specificities. Furthermore, the NK-defined phenotype of cells that possess two different ancestral haplotypes can be predicted from the NK-defined phenotypes of unrelated cells that are homozygous for the ancestral haplotypes concerned. Although the group 1 and 2 NK-defined allospecificities can be explained to some extent by HLA-C alleles, evidence is presented that additional genes may modify the phenotype conferred by HLA-C.

Chimpanzees (Pan Troglodytes) and humans are closely related and belong to the same subfamily, Homininae. The approximately 1.8% genetic difference that exists between humans and the chimpanzees must be responsible for observed differences between these two species. It has been shown that chimpanzees can be infected with HIV, but AIDS has not been reported. Furthermore, the prevalence of autoimmune diseases may be low in this species. For instance, type II diabetes occurs, but type I (autoimmune) diabetes (IDDM), to our knowledge, has not been reported. In humans, susceptibility genes for MG and IDDM have been localized to the region between TNF and HLA-B. This region may also influence the rate of progression to death after HIV infection. We have identified differences in this region between humans and the chimpanzees. As shown by PFGE, the TNF to Patr-B region in the chimpanzees is approximately 130-160 kb shorter than the equivalent in humans. Southern and sequence analyses indicate that the deletions in chimpanzees (insertions in humans) include one copy of CL (approximately 10 kb) and the X sequences (< 30 kb). Obviously, other deletions/insertions (approximately 120 kb) need to be identified. Since CL has been shown to be transcribed, the results imply the lack of the gene or, at least, a different gene copy number in the chimpanzees, and we propose that such differences may be relevant to the observed functional differences. We demonstrate here a strategy to identify critical genes responsible for disease development.

The MHC is a region of some 4 megabases that has been studied intensively owing to the large number of diseases that are associated with susceptibility genes within this region of the genome. The total number of genes located within the MHC is now approximately 100, but more can be predicted. Recently identified genes within the MHC include PERB6, a large gene producing multiple transcripts located between HLA-B and TNF, and PERB1, a member of the protein tyrosine kinase-gene family. PERB6 was identified by YAC probing of tissue blots, while PERB1 was identified by genomic sequencing.

To characterize the genomic structure and gene content between HLA-B and TNF we studied three overlapping yeast artificial chromosome (YAC) clones derived from the Boleth cell line carrying the 62.1 MHC ancestral haplotype (AH). In particular, we have evaluated the stability of haplospecific sequence motifs (haplospecific geometric elements) in YACs. By PCR and gene scanning we show that the two larger YAC clones (318G11 and 8F2) exhibit features characteristic of the 62.1 AH carried by the parental cell line as well as those of a separate cell line (BSM) which also carries 62.1. It is concluded that a highly polymorphic duplicated region is represented faithfully in spite of the fact that it contains sequences that could be interpreted as simple repeats liable to mutations. In contrast, microsatellites AFM031 and AFM158, which are also located in the MHC, showed greater instability and the structure in the YACs was not consistent with genomic structure. Likewise, different examples of the same ancestral haplotype show differences at these microsatellite loci. We conclude that polymorphic repeat sequences may exhibit a range of stabilities in vivo and that this may be reflected in their stability in YACs. Northern blotting using the largest YAC clone revealed several novel transcripts which await localization.

It remains unclear which MHC loci are involved in susceptibility to autoimmune diseases and immune deficiencies. We have chosen to evaluate whether different alleles of tumour necrosis factor-alpha (TNF-alpha) are important, as TNF has been implicated in the etiology of many immunological disorders. We have shown previously that a restriction fragment length polymorphism in the TNF region correlates with MHC ancestral haplotypes. We therefore examined the effect of ancestral haplotype on the activity of TNF-alpha in culture supernatants of lymphoblastoid cell lines. The results demonstrate that TNF-alpha activity in supernatants of 8.1 (A1, B8, DR3) cell lines was higher than that present in the supernatants from cells homozygous for eight different MHC ancestral haplotypes, and indicate that polymorphisms in TNF-alpha, or in other MHC genes that regulate TNF, may be responsible for the 8.1 phenotype.

We describe here a number of Caucasoid MHC haplotypes that extend from HLA-B to DR and that have been conserved en bloc. These haplotypes and recombinants between any two of them account for 73% of unselected haplotypes in our Caucasoid population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. Irrespective of the mechanisms involved in preservation of ancestral haplotypes, it is clear that these haplotypes carry several MHC genes, other than HLA, which may be relevant to antigen presentation, autoimmune responses, and transplantation rejection. In light of the existence of ancestral haplotypes, it is critical to evaluate MHC associations with disease and transplantation outcome in terms of associations with ancestral haplotypes rather than individual alleles.

The major histocompatibility complex (MHC) contains multiple and diverse genes which may be relevant to the induction and regulation of autoimmune responses in insulin dependent diabetes mellitus (IDDM). In addition to HLA class I and II, the possible candidates include TNF, C4, and several other poorly defined polymorphic genes in the central MHC region. This study describes two approaches which take advantage of the fact that the relevant genes are carried by highly conserved ancestral haplotypes such as 8.1 (HLA-B8, TNFS, C4AQ0, C4B1, DR3, DQ2). First, three "diabetogenic" haplotypes (two Caucasoid and one Mongoloid) have been compared and it has been shown that all three share a rare allele of BAT3 as well as sharing DR3, DQ2. In 43 sequential patients with IDDM the cross product ratio for BAT3S was 4.8 (p less than 0.01) and 6.9 for HLA-B8 plus BAT3S (p less than 0.001). Second, partial or recombinant ancestral haplotypes with either HLA class I (HLA-B8) or II (HLA-DR3, DQ2) alleles were identified. Third, using haplotypic polymorphisms such as the one in BAT3, we have shown that all the patients carrying recombinants of the 8.1 ancestral haplotype share the central region adjacent to HLA-B. These findings suggest that both HLA and non-HLA genes are involved in conferring susceptibility to IDDM, and that the region between HLA-B and BAT3 contains some of the relevant genes. By contrast, similar approaches suggest that protective genes map to the HLA class II region.

The TNFB genes from two major histocompatibility complex (MHC) ancestral haplotypes have been compared. The genes carried by the ancestral haplotypes 8.1 (A1,B8,BfS,C4AQ0, C4B1,DR3) and 57.1 (A1, B57. BfS, C4A6, C4B1, DR7) were cloned and sequenced to determine the degree of polymorphism. In this report we show that the respective TNF genes are allelic and have unique nucleotide sequences. The data demonstrate the presence of three nucleotide differences between the TNFB alleles of 8.1 and 57.1. Two of the differences occur in untranslated regions of the gene but the third nucleotide change results in an amino acid difference in the mature TNFB protein. These polymorphisms may have implications with respect to differential regulation in disease- and nondisease-associated haplotypes.