The Path to Conserved Extended Haplotypes: Megabase-Length Haplotypes at High Population Frequency.

This minireview describes the history of the conceptual development of conserved extended haplotypes (CEHs): megabase-length haplotypes that exist at high (≥0.5%) population frequency. My career began in internal medicine, shifted to pediatrics, and clinical practice changed to research. My research interest was initially in hematology: on plasma proteins, their metabolism, synthesis, and function. This narrowed to a focus on proteins of the human complement system, their role in immunity and their genetics, beginning with polymorphism and deficiency of C3. My group identified genetic polymorphisms and/or inherited deficiencies of C2, C4, C6, and C8. After defining glycine-rich beta glycoprotein as factor B (Bf) in the properdin system, we found that the genes for Bf (CFB), C2, C4A, and C4B were inherited as a single haplotypic unit which we named the "complotype." Complotypes are located within the major histocompatibility complex (MHC) between HLA-B and HLA-DRB1 and are designated (in arbitrary order) by their CFB, C2, C4A, and C4B types. Pedigree analysis revealed long stretches (several megabases) of apparently fixed DNA within the MHC that we referred to as "extended haplotypes" (later as "CEHs"). About 10 to 12 common CEHs constitute at least 25 - 30% of MHC haplotypes among European Caucasian populations. These CEHs contain virtually all the most common markers of MHC-associated diseases. In the case of type 1 diabetes, we have proposed a purely genetic and epigenetic model (with a small number of Mendelian recessive disease genes) that explains all the puzzling features of the disease, including its rising incidence.

A New Pedigree-Based SNP Haplotype Method for Genomic Polymorphism and Genetic Studies.

Single nucleotide polymorphisms (SNPs) are usually the most frequent genomic variants. Directly pedigree-phased multi-SNP haplotypes provide a more accurate view of polymorphic population genomic structure than individual SNPs. The former are, therefore, more useful in genetic correlation with subject phenotype. We describe a new pedigree-based methodology for generating non-ambiguous SNP haplotypes for genetic study. SNP data for haplotype analysis were extracted from a larger Type 1 Diabetes Genetics Consortium SNP dataset based on minor allele frequency variation and redundancy, coverage rate (the frequency of phased haplotypes in which each SNP is defined) and genomic location. Redundant SNPs were eliminated, overall haplotype polymorphism was optimized and the number of undefined haplotypes was minimized. These edited SNP haplotypes from a region containing HLA-DRB1 (DR) and HLA-DQB1 (DQ) both correlated well with HLA-typed DR,DQ haplotypes and differentiated HLA-DR,DQ fragments shared by three pairs of previously identified megabase-length conserved extended haplotypes. In a pedigree-based genetic association assay for type 1 diabetes, edited SNP haplotypes and HLA-typed HLA-DR,DQ haplotypes from the same families generated essentially identical qualitative and quantitative results. Therefore, this edited SNP haplotype method is useful for both genomic polymorphic architecture and genetic association evaluation using SNP markers with diverse minor allele frequencies.

A stochastic epigenetic Mendelian oligogenic disease model for type 1 diabetes.

The incidence of type 1 diabetes (T1D) and some other complex diseases is increasing. The cause has been attributed to an undefined changing environment. We examine the role of the environment (or any changing non-genetic mechanism) in causing the rising incidence, and find much evidence against it: 1) Dizygotic twin T1D concordance is the same as siblings of patients in general; 2) If the environment is responsible for both the discordance among identical twins of patients with T1D and its rising incidence, the twin concordance rate should be rising, but it is not; 3) Migrants from high-to low-incidence countries continue to have high-incidence children; 4) TID incidence among the offspring of two T1D parents is identical to the monozygotic twin rate. On the other hand, genetic association studies of T1D have revealed strong susceptibility in the major histocompatibility complex and many optional additive genes of small effect throughout the human genome increasing T1D risk. We have, from an analysis of previously published family studies, developed a stochastic epigenetic Mendelian oligogenic (SEMO) model consistent with published observations. The model posits a few required recessive causal genes with incomplete penetrance explaining virtually all of the puzzling features of T1D, including its rising incidence and the specific low T1D incidence rates among first-degree relatives of patients. Since historic selection against any causal gene could prevent T1D, we postulate that the rising incidence is because of increasing population mixing of parents from some previously isolated populations that had selected against different causal genes.

Pedigree-Defined Haplotypes and Their Applications to Genetic Studies.

A haplotype is a string of nucleotides or alleles at nearby loci on one chromosome, usually inherited as a unit. Within the major histocompatibility complex (MHC) region on human chromosome 6p, independent population studies of multiple families have identified conserved extended haplotypes (CEHs) that segregate as long stretches (≥1 megabase) of essentially identical DNA sequence at relatively high (≥0.5 %) population frequency ("genetic fixity"). CEHs were first identified through segregation analysis in the early 1980s. In European Caucasian populations, the most frequent 30 CEHs account for at least one-third of all MHC haplotypes. These CEHs provide all of the known individual MHC susceptibility and protective genetic markers within those populations for several complex genetic diseases. Haplotypes are rigorously determined directly by sequencing single chromosomes or by Mendelian segregation analysis using families with informative genotypes. Four parental haplotypes are assigned unambiguously using genotypes from the two parents and from two of their haploidentical (to each other) children. However, the most common current technique to phase haplotypes is probabilistic statistical imputation, using unrelated subjects. Such probabilistic techniques have failed to detect CEHs and are thus of questionable value in identifying long-range haplotype structure and, consequently, genetic structure-function relationships. Finally, with haplotypes rigorously defined, association studies can determine frequencies of alleles among unrelated patient haplotypes vs. those among only unaffected family members (i.e., control alleles/haplotypes). Such studies reduce, as much as possible, the confounding effects of population stratification common to all genetic studies.

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.

Interaction between immunoglobulin allotypes and NK receptor genes in diabetes post-hepatitis C virus infection.

Genetic interactions between natural killer (NK) cells immunoglobulin-like receptor (KIR) genes and immunoglobulin allotypes have been previously reported in type 2 diabetes mellitus (DM) patients. Puerto Rican Americans with a history of intravenous drug use who developed DM following HCV infection (n=32) were compared to individuals infected with HCV without diabetes (n=121) and to DM non-infected individuals (n=95). Subjects were genotyped for KIRs and immunoglobulin allotypes. We found interactions of immunoglobulin allotypes KM3/KM3 with NK inhibitory receptors 2DL3/2DL3, 2DL1 in the absence of 2DS4 associated with susceptibility to DM in HCV infected individuals. These data suggest the possibility that a subset of patients with HCV could have an immune-mediated component contributing to the development of DM.

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.

Protective KIR-HLA interactions for HCV infection in intravenous drug users.

Intravenous drug use has become the principal route of hepatitis C virus (HCV) transmission due to the sharing of infected needles. In this study, we analyzed the distribution of HLA-KIR genotypes among 160 Puerto Rican intravenous drug users (IDUs) with HCV infection and 92 HCV-negative Puerto Rican IDUs. We found a significant association between the presence of different combinations of KIR inhibitory receptor genes (KIR2DL2 and/or KIR2DL3, pC=0.01, OR=0.07; KIR2DL2 and/or KIR2DL3+KIR2DS4, pC=0.01, OR=0.39) and HLA-C1 homozygous genotypes (HLA-C1+KIR2DS4, pC=0.02, OR=0.43; HLA-C1+KIR2DL2+KIR2DS4, pC=0.02, OR=0.40) together with the activating receptor KIR2DS4 (HLA-C1+KIR2DS4+KIR2DL3 and/or KIR2DL2, pC=0.004, OR=0.38) with protection from HCV infection. Our findings in HCV-infected and non-infected IDUs suggest an important role for KIRs (KIR2DL2 and KIR2DL3) with group HLA-C1 molecules, in the presence of activating KIR2DS4, in protection from HCV infection. These results support the hypothesis that activator signaling, mediated by KIR2DS4, plays a determinant role in the regulation of NK cell antiviral-activity.

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.

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.

A genetic explanation for the rising incidence of type 1 diabetes, a polygenic disease.

We had earlier hypothesized, if parents originated from previously isolated populations that had selected against different critical susceptibility genes for a polygenic disease, their offspring could have a greater risk of that disease than either parent. We therefore studied parents of patients with type 1 diabetes (T1D). We found that parents who transmitted HLA-DR3 to HLA-DR3/DR4 patients had different HLA-A allele frequencies on the non-transmitted HLA haplotype than HLA-DR4-transmitters. HLA-DR3-positive parents also had different insulin (INS) gene allele frequencies than HLA-DR4-positive parents. Parent pairs of patients had greater self-reported ethnicity disparity than parent pairs in control families. Although there was an excess of HLA-DR3/DR4 heterozygotes among type 1 diabetes patients, there were significantly fewer HLA-DR3/DR4 heterozygous parents of patients than expected. These findings are consistent with HLA-DR and INS VNTR alleles marking both disease susceptibility and separate Caucasian parental subpopulations. Our hypothesis thus explains some seemingly disconnected puzzling phenomena, including (1) the rising world-wide incidence of T1D, (2) the excess of HLA-DR3/DR4 heterozygotes among patients, (3) the changing frequency of HLA-DR3/DR4 heterozygotes and of susceptibility alleles in general in patients over the past several decades, and (4) the association of INS alleles with specific HLA-DR alleles in patients with T1D.

Incomplete penetrance of susceptibility genes for MHC-determined immunoglobulin deficiencies in monozygotic twins discordant for type 1 diabetes.

Incomplete intrinsic penetrance is the failure of some genetically susceptible individuals (e.g., monozygotic twins of those who have a trait) to exhibit that trait. For the first time, we examine penetrance of susceptibility genes for multiple MHC gene-determined traits in the same subjects. Serum levels of IgA, IgD, IgG3, but not IgG4, in 50 pairs of monozygotic twins discordant for type 1 diabetes (T1D) correlated more closely in the twins than in random paired controls. The frequencies of subjects deficient in IgA (6%), IgD (33%) and IgG4 (12%), but not in IgG3, were higher in the twins than in controls. We postulate that this was because the MHC haplotypes (and possible non-MHC genes) that predispose to T1D also carry susceptibility genes for certain immunoglobulin deficiencies. Immunoglobulin deficiencies were not associated with T1D. Pairwise concordance for the deficiencies in the twins was 50% for IgA, 57% for IgD and 50% for IgG4. There were no significant associations among the specific immunoglobulin deficiencies except that all IgA-deficient subjects had IgD deficiency. Thus, intrinsic penetrance is a random process independently affecting different MHC susceptibility genes. Because multiple different external triggers would be required to explain the results, differential environmental determinants appear unlikely.

Increased apoptosis of CD20+ IgA + B cells is the basis for IgA deficiency: the molecular mechanism for correction in vitro by IL-10 and CD40L.

IgA deficiency is the most common primary immunodeficiency in humans. Comparative analysis of gene expression in PBMC from IgA-deficient (IgAd) and normal donors using functional multiplex panels showed overexpression of the Caspase-1 (CASP-1) gene. Cells from all the IgAd donors (n=7) expressed 4-10-fold caspase-1 mRNA over normal controls (n=5). CD19(+) B cells from all IgAd donors produced IgA in cultures following IL-10 and CD40L with Staphylococcus aureus (Cowan) (SAC) or tetanus toxoid (TT) treatments. In CD19(+) B cells from IgAd donors, reconstitution of IgA secretion was associated with protection of the CD20(+) B cell population that underwent apoptosis in the absence of IL-10, CD40L, and TT (triple treatment). Caspase-1 gene expression was decreased in the reconstituted cells. Furthermore, treatment with a caspase-1 inhibitor also independently protected against B cell apoptosis in vitro. An apoptosis-specific cDNA array showed differential expression of 4 out of 96 genes and a shift towards survival-related gene expression from the apoptotic to the protected B cells after triple treatment. There was an increase in the expression of the IAP-2 (inhibitor of apoptosis) gene in the reconstituted cells. Upregulation of the IAP-2 gene protects B cells from deletion and allows for IgA secretion in this system. The inability to detect secreted IgA in IgAd patients could result from the loss of IgA-committed B cells that express high levels of caspase-1.

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.

Mendelian inheritance of polygenic diseases: a hypothetical basis for increasing incidence.

The incidence of common polygenic diseases, such as type 1 diabetes, bronchial asthma, and gluten-sensitive enteropathy, is increasing. Although this is usually attributed to environmental factors, it is possible that this rising incidence also has a genetic basis. The hypothesis is put forth that, in the past, these diseases, with their increased morbidity and mortality, were selected against. In contrast to monogenic diseases, the incidence of polygenic diseases can be reduced by selection against susceptibility alleles of any of the genetic loci necessary for disease to occur. In different isolated populations, different disease susceptibility loci may have been selected against. Parents who derive from different isolated populations in which there are inversely different susceptibility allele frequencies because of selection or genetic drift, would be expected to have offspring with an increased risk for that polygenic disease. It is shown mathematically that the incidence of a hypothetical polygenic disease increases under these circumstances. The increased risk in these offspring results from a kind of genetic complementation in which they have inherited a more complete set of susceptibility alleles at all susceptibility loci than is carried by either of their parents. Hallmarks of this hypothesized phenomenon would be increased heterozygosity for specific population markers (whether susceptibility alleles or not) among the disease-affected offspring and a paucity of such heterozygotes among their parents. The parents and patients would also be expected to give more evidence of ethnic or subethnic disparity than that observed in controls.

The genetics of HLA-associated disease.

Type 1 diabetes mellitus (T1D) remains the most intensively studied, and thus the best paradigm, of MHC-associated diseases. Accumulating evidence suggests that MHC susceptibility for T1D is recessive, with susceptibility alleles more common than protective alleles. Updated allele-level and nucleotide sequence analysis of MHC class II T1D susceptibility markers of conserved extended haplotypes underscore the uncertainty surrounding the actual T1D MHC susceptibility locus. Recent studies have established that disease concordance in dizygotic twins is the same as that in siblings generally, for both T1D and the MHC-associated autoimmune disease gluten-sensitive enteropathy, leaving little room for a differential environmental trigger. Epigenetic mechanisms are probably involved in many MHC-associated phenomena, including autoimmunity, and appear to be the best explanation for incomplete penetrance.

Immunoglobulin deficiencies and susceptibility to infection among homozygotes and heterozygotes for C2 deficiency.

About 25% of C2-deficient homozygotes have increased susceptibility to severe bacterial infections. C2-deficient homozygotes had significantly lower serum levels of IgG2, IgG4, IgD, and Factor B, significantly higher levels of IgA and IgG3 and levels of IgG1 and IgM similar to controls. Type 1 (28 bp deletion in C2 exon 6 on the [HLA-B18, S042, DR2] haplotype or its fragments) and type II (non-type I) C2-deficient patients with increased susceptibility to bacterial infection had significantly lower mean levels of IgG4 (p < 0.04) and IgA (p < 0.01) than those without infections (who had a higher than normal mean IgA level) but similar mean levels of other immunoglobulins and Factor B. Of 13 C2-deficient homozygotes with infections, 85% had IgG4 deficiency, compared with 64% of 25 without infections. IgD deficiency was equally extraordinarily common among infection-prone (50%) and noninfection-prone (70%) homozygous type I C2-deficient patients. IgD deficiency was also common (35%) among 31 type I C2-deficient heterozygotes (with normal or type II haplotypes), but was not found in 5 type II C2-deficient heterozygotes or 1 homozygote. Thus, C2 deficiency itself is associated with many abnormalities in serum immunoglobulin levels, some of which, such as in IgG4 and IgA, may contribute to increased susceptibility to infection. In contrast, IgD deficiency appears not to contribute to increased infections and appears to be a dominant trait determined by a gene or genes on the extended major histocompatibility complex (MHC) haplotype [HLA-B 18, S042, DR2] (but probably not on type II C2-deficient haplotypes) similar to those previously identified on [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3].

CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation.

We have shown previously that primary dendritic cells and monocytes express equal levels of CD14 but are distinguishable by the presence of CD2 on dendritic cells. CD2 is known to mediate the activation of T and natural killer (NK) cells through its interaction with CD58. CD2 epitopes recognized by anti-T111, -T112, and -T113 monoclonal antibodies (mAbs) are present on dendritic cells. Here we show that CD2 engagement significantly increases class II, costimulatory (CD40, CD80, CD86), adhesion (CD54, CD58), and CCR7 molecule expression on primary dendritic cells. Conversely, minimal or no change in the expression of the above antigens occurs on monocyte-derived dendritic cells, because these molecules are already maximally expressed. However, both kinds of dendritic cells release interleukin-1beta (IL-1beta) and IL-12 after CD2 engagement. Lastly, interference with dendritic cell CD2-T-cell CD58 engagement decreases naive CD4+CD45RA+ T-cell proliferation. Collectively, our results suggest another role of the CD2-CD58 pathway that allows nonimmune and immune cells to interact directly with dendritic cells and initiate innate and adaptive immune responses.