Dominant sequences of human major histocompatibility complex conserved extended haplotypes from HLA-DQA2 to DAXX.

We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight "common" European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population's haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots.

The role of TNF-α in mice with type 1- and 2- diabetes.

BACKGROUND: Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. METHODOLOGY AND PRINCIPAL FINDINGS: To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-α) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-α interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-α treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-α treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-α treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. CONCLUSIONS: In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-α dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-α mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling.

A protein multiplex microarray substrate with high sensitivity and specificity.

The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three-dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen-specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specific signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laboratory environment.

Frequent occurrence of conserved extended haplotypes (CEHs) in two Caucasian populations.

Conserved extended haplotypes (CEHs) are large (>or=1Mb) regions of identical DNA of the major histocompatibility complex (MHC) region of chromosome 6p in unrelated individuals. They are recognized by family studies and constitute nearly half of MHC haplotypes among European Caucasians. We studied 49 Hungarian Caucasian families in comparison with the previous findings in 2675 normal American Caucasian chromosomes from families in the Boston area. Besides HLA-A, -B and HLA-DRB1/-DQB1 alleles, copy number polymorphism of C4A and C4B genes and several SNPs encoded in the central (class III) MHC region were determined. By comparing 188 Caucasian haplotypes in Hungary to 2675 normal Caucasian chromosomes in Boston, we found that 11 of 12 of the most common CEHs (with a frequency of at least 1%) among the Boston chromosomes also occurred in Hungary. Moreover, there was a significant correlation (R=0.789; p=0.0023) in the frequency order of these haplotypes between the two Caucasian populations. Of 10 haplotypes found in >or=2 copies among the Hungarian chromosomes, all but one occurred in one to 14 copies among the Boston haplotypes. These findings indicate that CEHs are commonly shared by distinct European Caucasian populations; however, lower frequency CEHs may differ.

The MHC type 1 diabetes susceptibility gene is centromeric to HLA-DQB1.

HLA-DQB1 is widely considered to be the major histocompatibility complex (MHC) susceptibility gene for type 1 diabetes (T1D). However, since inheritance of the gene in T1D is recessive, the presence of the protective HLA-DQB1 0602 allele with normal nucleotide sequence in some patients raises the question of whether HLA-DQB1 is not the susceptibility locus itself but merely a good marker. HLA-DQB1 0602 is part of a conserved extended haplotype (CEH) [HLA-B7, SC31, DR2] (B7, DR2) with fixed DNA over more than 1Mb of genomic DNA that normally carries a protective allele at the true susceptibility locus. We postulated that, in patients with HLA-DQB1 0602, the protective allele at the susceptibility locus has been replaced by a susceptibility allele through an ancient crossover at meiosis centromeric to HLA-DQB1. We analyzed single nucleotide polymorphisms (SNPs) distinguishing the HLA-DQA2 (the first expressed gene centromeric to HLA-DQB1) allele on the normal HLA-B7, DR2 CEH from those on susceptibility CEHs in T1D patients and controls with HLA-DQB1 0602. All but 1 of 20 healthy control HLA-DQB1 0602 haplotypes had identical (consensus) first intron HLA-DQA2 5-SNP haplotypes. Fifteen of 19 patients with HLA-DQB1 0602 were homozygous for 1 or more HLA-DQA2 SNPs differing from consensus HLA-DQA2 SNPs, providing evidence of crossover involving the HLA-DQA2 locus. The remaining 4 patients were heterozygous at all positions and therefore uninformative. The loss of dominant protection usually associated with HLA-DQB1 0602 haplotypes is consistent with a locus centromeric to HLA-DQB1 being a major determinant of MHC-associated susceptibility, and perhaps the true T1D susceptibility locus.

A DNA oligonucleotide microarray for detecting human astrovirus serotypes.

Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of low degeneracy capable of detecting all eight serotypes of human astroviruses. RT-PCR products are then hybridized against a microarray consisting of short oligonucleotide probes 17-18 nucleotides in length. Cy3-labeled ssDNA targets are generated using a Cy3-labeled primer in the RT-PCR. The non-labeled strand is enzymatically digested, and the labeled target is rescued by column purification. This method of generating labeled target uses equimolar concentrations of the amplifying primers and does not compromise assay sensitivity for initial detection of the virus. Hybridization can be performed without the need for additional amplification. Although the amplicon spans a relatively conserved region of the astrovirus genome, the use of short probes enables type distinction despite such limited diversity. Probes differing by as little as a single nucleotide can be used to distinguish isolates. The microarray developed was capable of distinguishing representatives of the eight known serotypes of human astroviruses.

Genetic fixity in the human major histocompatibility complex and block size diversity in the class I region including HLA-E.

BACKGROUND: The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association. RESULTS: Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments. CONCLUSION: We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.

The haplotype structure of the human major histocompatibility complex.

There is great interest in the use of single-nucleotide polymorphisms (SNPs) and linkage disequilibrium (LD) analysis to localize human disease genes. The results suggest that the human genome, including the major histocompatibility complex (MHC), consists largely of 5- to 200-kb blocks of sequence fixity between which random recombination occurs. Direct determination of MHC haplotypes from family studies also demonstrates similar-sized blocks, but otherwise gives a very different picture, with a third to a half of Caucasian haplotypes fixed from HLA-B to HLA-DR/DQ (at least 1 Mb) as conserved extended haplotypes (CEHs), some of which encompass more than 3 Mb. These fixed haplotypes differ in frequency both in different Caucasian subpopulations and in Caucasian patients with HLA-associated diseases, complicating disease susceptibility gene localization. The inherent inability of LD analysis to "see" DNA fixity beyond three markers contributes to the failure of SNP/LD analysis to define in detail or even detect CEHs in the MHC and probably elsewhere in the genome. More importantly, the use of statistical analysis, rather than direct haplotype determination and counting, fails to reveal the details of haplotype structure essential for gene localization. Given the oversimplified picture of the MHC (and probably the rest of the genome) provided only by SNP/LD-defined blocks, it is questionable whether this approach will be of great help in disease susceptibility gene localization or identification.

Linkage disequilibrium between HLA-DPB1 alleles and retinoid X receptor beta haplotypes.

The human retinoid X receptor beta (RXRB) gene maps to the major histocompatibility complex (MHC) region, between KE4 and COL11A2, approximately 130-kb centromeric to HLA-DPB1. We have recently reported a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to detect the G to T single nucleotide polymorphism (SNP) located seven nucleotides after the tenth exon of the RXRB gene, or 3'end+7 position according to existing nomenclature. We also reported strong linkage disequilibrium between the HLA-DPB1*0401 and RXRB+7*T alleles. In the present study, we describe two PCR-RFLP methods to detect additional SNPs in the RXRB gene, T to A, at exon10+378 and A to T at 3'end+140. This new methodology permitted the unambiguous assignment of three distinct SNPs at RXRB exon10+378, 3'end+7 and 3'end+140 to form an "RXRB haplotype." The data generated from this study were used to determine linkage disequilibrium between several MHC markers and the RXRB alleles and haplotypes. Family studies revealed significant linkage disequilibrium between the RXRB alleles and a number of HLA-DPB1 alleles.