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.

Linkage analysis of the C4A/C4B copy number variation and polymorphisms of the adjacent steroid 21-hydroxylase gene in a healthy population.

Genes encoding the steroid 21-hydroxylase (CYP21A2) and the complement component C4 proteins (C4A and C4B) are located in the MHC region in a strongly linked structure named RCCX module. Previous studies found that carriers of C4B gene deficiency (C4B*Q0) have higher risk for cardiovascular diseases. A potential explanation is that lacking the C4B gene may result in altered function of the neighboring CYP21A2 gene. Therefore we sequenced the CYP21A2 gene in 96 healthy individuals to identify polymorphisms and to characterize their linkage pattern. Fifty-three variations were detected including a new one which alters the TATA-box of the gene. Only three known mutations (V281L, Q318X and R479L) associated with congenital adrenal hyperplasia, were found in 7, 2 and 1 subjects, respectively. Linkage analysis revealed that some variations exhibit strong correlation with the C4 copy number polymorphism and constituents of the MHC III region. Rare alleles of three polymorphisms were identified as components of the 8.1 ancestral haplotype. Haplotyping and family study confirmed that the variant alleles of two intronic SNPs were constituents of haplotype blocks lacking the C4B gene. These results suggest that variations of CYP21A2 gene can be involved in disease associations of the 8.1 haplotype and the C4B*Q0 genotype.

HLA-association of serum levels of natural antibodies.

Natural antibodies of IgM or IgG types are present in sera of most healthy individuals and are important participants of the immune response. Little is known, however, about the genetic regulation of their plasma levels in humans. We determined the concentrations of three IgM type natural autoantibodies (NAAbs) reactive to certain conserved self-antigens (citrate synthase (A-CIT), chondroitin sulphate C (A-COS) and 60 kDa heat shock proteins (A-HSP) in the sera of 78 healthy individuals and in their 86 children. In case of all the 164 individuals alleles of several polymorphisms were determined in class II (HLA-DQ, -DR), class III (AGER-429T>C, HSP70-2 1267A>G, TNF-308G>A, CFB S/F, copy number of the C4A and C4B genes), and class I (HLA-A, -B) regions of the major histocompatibility complex (MHC). Since the samples originated from a family study, extended MHC haplotypes were also determined for each study participant. Our results show that children of parents with low NAAb concentration have significantly lower serum concentrations of all the three NAAbs, as compared to offsprings of parents without reduced serum concentration. This indicates that the serum levels of these NAAbs were partly regulated by factors which are inherited from the parents to offsprings. In further studies performed only in genetically independent parents, we found significant differences in the serum levels of the IgM type A-CIT and A-COS antibodies (Abs) between carriers and non-carriers of the HLA-DR2 (15 and 16) antigens. In both cases the Ab concentrations were higher in the HLA-DR15 carriers (p=0.002 and p=0.008, respectively) and lower in DR16 carriers (p=0.029 and p=0.049, respectively) than in the non-carriers. Even more significant differences were found when the levels of two Abs were evaluated together. Frequency of the DR15 carriers was significantly lower among subjects with one or two low (in the lowest quartile) titers of A-CIT/A-COS Abs (p=0.014), A-CIT/A-HSP Abs (p=0.016) and A-COS/A-HSP Abs (p=0.013) as compared to those with normal Ab titers for both antigens. By contrast, frequency of the DR16 carriers was significantly higher among subjects with one or two low A-CIT/A-COS Abs (p=0.001), A-CIT/A-HSP Abs (p=0.002) and A-COS/A-HSP Abs (p=0.021) as compared to those with normal Ab titers for both antigens. Similar differences were found for both IgM type antibodies when carriers and non-carriers of the HLA-DR15-DQ6 and HLA-DR16-DQ5 haplotypes were considered. These novel observations indicate that not only adaptive immune response but also natural autoantibody pattern, as a part of innate immune response, is influenced by the MHC allele composition.

Autoimmune-associated HLA-B8-DR3 haplotypes in Asian Indians are unique in C4 complement gene copy numbers and HSP-2 1267A/G.

The classical AH8.1 (HLA-A1-B8-DR3-DQ2) is the most common Caucasian haplotype, associated with several autoimmune diseases, immunologic hyperreactivity and rapid progression to the acquired immunodeficiency syndrome. However, in Asian Indians, there are multiple unique B8-DR3 haplotypes that are associated with autoimmunity and differ significantly from the common Caucasian AH8.1. The Indian HLA-A1-B8-DR3 is therefore referred to as an AH8.1 variant. The aims of this study were to compare C4A and C4B copy numbers and to identify alleles in HSP70-2 and LTA in these haplotypes. The Indian B8-DR3 haplotypes differ from the Caucasian AH8.1 at C4A and HSP70-2 loci. The Indian B8-DR3 haplotypes have 1 copy each at C4A and C4B, while the Caucasian AH8.1 has 1 copy at C4B but no C4A gene. Moreover, the Indian and Caucasian B8-DR3 haplotypes had HSP70-2 1267 *A, and *G alleles, respectively. By contrast, the LTA 252 *G allele occurred both in the Indian and Caucasian haplotypes. The Indian haplotypes also contained Bf*F and TNF-308*G that were different from the Caucasian equivalents Bf*S and TNF-308*A. These differences and previous studies support the hypothesis that B8-DR3-DQ2 haplotypes in Asian Indian population might have originated independently of Caucasian AH8.1 selectively through recombination and mutations. Because autoimmune disease associations are shared among these otherwise diverse haplotypes, these data strongly suggest that some shared component(s) of all these associated haplotypes may be playing a key role in such associations.

3'UTR C2370A allele of the IL-23 receptor gene is associated with relapsing-remitting multiple sclerosis.

The interleukin (IL) -23/IL-17 cytokine axis has been suggested to play an important role in the development of several autoimmune diseases including multiple sclerosis. Here, we compared the prevalence of C2370A single nucleotide polymorphism (SNP) in the 3' untranslated region (3'UTR) of the IL-23 receptor (IL23R) between 223 patients with relapsing-remitting multiple sclerosis (RRMS) and 200 healthy controls. The A2370A genotype was significantly over-represented among patients with RRMS (10.8%) and RRMS exhibiting oligoclonal bands in the cerebrospinal fluid (12.9%) when compared to healthy subjects (5.50%). Multiple regression analysis revealed that presence of AA genotype provides a two-fold risk for the development of multiple sclerosis (OR=2.072, 95% CI: 0.988-4.347, p<0.05). These data indicate that IL23R represents a novel shared susceptibility gene as its association with inflammatory bowel disease (IBD) has recently been verified.

Frequency of carriers of 8.1 ancestral haplotype and its fragments in two Caucasian populations.

Within the human MHC region larger stretches of conserved DNA, called conserved ancestral haplotypes exist. However, many MHC haplotypes contain only fragments of an ancestral haplotype. Little is known, however, on relative distribution of the ancestral haplotypes to their fragments. Therefore we determined the frequency of carriers of the whole ancestral haplotype 8.1 (AH8.1) and its fragments in 127 healthy Hungarian people, 101 healthy Ohioian females, and in nine Hungarian families. The HLA-DQ2, HLA-DR3(17), RAGE -429C allele, the mono-S-C4B genotype, the HSP70-2 1267G allele and the TNF -308A (TNF2) allele were used as markers of the AH8.1. Frequency of carriers of the whole AH8.1 and its fragments was similar in the both populations. 18% of the subjects carried the whole AH8.1 in at least one chromosome, while 17-20%, 36-39%, and 24-29%, respectively carried two or three constituents of the haplotype, only one constituent or none of them. Similar results were obtained in the family study. In addition, marked differences were found in the relationship of the constituents' alleles to the whole AH8.1. In both populations, 29%, 50-59%, 52-56% and 76-96%, respectively of the carriers of HSP70-2 1267G, RAGE-429C, TNF2, and mono-S carriers carried the whole 8.1 haplotype. These findings may have important implications for studies of the disease associations with different MHC ancestral haplotypes.

The HLA 8.1 ancestral haplotype is strongly linked to the C allele of -429T>C promoter polymorphism of receptor of the advanced glycation endproduct (RAGE) gene. Haplotype-independent association of the -429C allele with high hemoglobinA1C levels in diabetic patients.

Previously we reported on strong linkage disequilibrium (LD) between the mono-S-C4B-RCCX module (mono-S) and the TNF2 allele (both known constituents of the 8.1 ancestral haplotype (8.1 AH)) in two Caucasian populations. The gene for the receptor of advanced glycation endproducts (RAGE) is encoded between the RCCX module and the HLA class II genes in the central MHC region. In order to assess the relationship between the promoter polymorphisms of the RAGE gene and the 8.1 AH, we performed a family study in eight informative families affected with type 1 diabetes mellitus; haplotypes of a RAGE promoter SNP (-429T>C) with the HLA-DQ2, -DR-3(17) and TNF2 alleles, as well as the mono-S genotype were determined. A similar analysis was performed in 82 unrelated patients with type 1 diabetes mellitus, and in unrelated healthy individuals of three different Caucasian populations (Hungarians, Ohioian females, Icelandics). In the diabetic patients clinical correlations were also investigated. Out of the 32 paternal and maternal chromosome 6 from the eight families, 15 different MHC haplotypes were found. Haplotypes containing at least three of the known constituents of the 8.1 AH (HLA-DQ2, -DR17, mono-S, TNF2) were always linked to the RAGE -429C allele. The RAGE -429C allele exhibited highly significant (p<0.0001) LD coefficients to known constituents of the 8.1 AH both in healthy persons and patients with type 1 diabetes. In the group of patients with diabetes we found significantly (p=0.013) higher maximal hemoglobinA1C concentration in the carriers of the RAGE -429C allele, this trait, however was not linked to the 8.1 AH. Our present findings indicate that the RAGE -429C allele can be considered as a candidate member of the 8.1 AH. The results also reveal a spectrum of recombinant MHC haplotypes in addition to the conserved ancestral haplotypes.

The 8.1 ancestral MHC haplotype is associated with delayed onset of colonization in cystic fibrosis.

Major cause of death in patients with cystic fibrosis (CF) is colonization with Staphylococcus aureus and Pseudomonas aeruginosa. The wide phenotypic variation in CF patients suggests that genes other than the cystic fibrosis transmembrane conductance regulator (CFTR) gene modify the disease. The 8.1 ancestral haplotype (8.1AH) in main histocompatibility complex is associated with alterations of the immune response. To study the influence of carriage of 8.1AH on frequency and onset of colonization in CF patients, DNA samples of 72 CF patients (39 homozygous and 33 heterozygous for DeltaF508) were genotyped for member alleles of the 8.1AH: HLA-DQB1*0201, HLA-DRB1*0301, receptor for advanced glycation end products (AGER) -429C, HSP70-2 -1267G (HSP70-2G) and tumor necrosis factor-alpha (TNF-alpha) -308A (TNF2). Colonization was verified by regular clinical and bacteriological screening. Frequency of colonization was significantly (P = 0.012) lower in the 8.1AH carriers; age, gender and DeltaF508 genotype-adjusted odds ratio to be colonized of the carriers versus non-carriers was 0.112 (0.024-0.520). According to survival analysis, patients with 8.1AH had significantly (P < 0.0001) longer colonization-free period compared with non-carriers. Our novel observations demonstrate that the 8.1AH is associated with delayed onset of colonization in CF, presumably by influencing defense mechanisms against infections.

In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines.

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.

Similar genetic features and different islet cell autoantibody pattern of latent autoimmune diabetes in adults (LADA) compared with adult-onset type 1 diabetes with rapid progression.

OBJECTIVE: To compare the clinical parameters, C-peptide levels, pattern of islet cell-specific autoantibodies, and prevalence of predisposing genotypes in subjects with latent autoimmune diabetes in adults (LADA) and those with adult-onset type 1 diabetes with rapid progression. RESEARCH DESIGN AND METHODS: We evaluated the clinical parameters, C-peptide levels, and islet cell-specific autoantibodies in 54 LADA, 57 adult-onset type 1 diabetic, and 190 type 2 diabetic patients. Islet cell autoantibodies were also compared between subgroups of newly diagnosed patients with LADA and those with newly diagnosed adult-onset and childhood-onset type 1 diabetes. The genetic study was performed in subjects with LADA and those with adult-onset type 1 diabetes in comparison with a control population. RESULTS: There were no differences in the clinical parameters between LADA and adult-onset type 1 diabetes. Patients with LADA had lower BMI (P < 0.0001), waist-to-hip ratio (0.0029), total cholesterol (P = 0.001), and triglycerides (P = 0.001); higher HDL cholesterol levels (P < 0.0001); and lower prevalence of hypertension (P = 0.0028) compared with patients with type 2 diabetes. C-peptide levels were similar at onset (P = 0.403) but decreased less rapidly in LADA than in adult-onset type 1 diabetes (P = 0.0253). Single-autoantibody positivity was more often seen in LADA than in type 1 diabetes (P = 0.0001). The prevalence of predisposing HLA-DQB1*0302, -DR4, -DR3, and -DR3/DR4 genotypes and the DR4-DQB1*0302 haplotype were increased in both LADA and adult-onset type 1 diabetic subjects compared with the control population. There were no differences in the frequencies of these risk alleles and haplotypes between the two patient groups. CONCLUSIONS: Subjects with LADA had clinical characteristics similar to those with adult-onset type 1 diabetes with rapid progression. C-peptide levels did not differ at onset but decreased less rapidly in LADA. Patients with LADA rather had single islet cell-specific autoantibody positivity. The prevalence of HLA-DQB1*0302, -DR4, -DR3, and -DR3/DR4 risk alleles and the DR4-DQB1*0302 high-risk haplotype did not differ in the two forms of autoimmune diabetes.

Differences in the genetic background of latent autoimmune diabetes in adults (LADA) and type 1 diabetes mellitus.

OBJECTIVES: According to the recent classification of diabetes mellitus the Latent Autoimmune Diabetes in Adults (LADA) belongs to the group of type 1 autoimmune diabetes, as a slowly progressive form. Our aim was to determine (i) the prevalence of HLA-DRB1 and DQB1 genotypes, and (ii) to determine the tumor necrosis factor (TNF) alpha promoter polymorphism at position -308 (the G-->A substitution, designated the TNF2 allele) in patients with type 1 diabetes and with LADA compared with the healthy population. METHODS: The major histocompatibility complex (MHC) II genotypes and the TNF alpha promoter polymorphism were determined by PCR method. We examined 69 type 1 diabetic and 42 LADA patients. As control samples of 336 cadaver kidney donors and 138 volunteers were used. RESULTS: Both type 1 diabetes mellitus and LADA were positively associated with the DRB1*04-DQB1*0302 (DR4/DQ8) haplotype (P=0.00001, and P=0.0005, respectively), and negatively associated with the DRB1*11-DQB1*0301 (DR11/DQ7) haplotype (P=0.00006, and P=0.007, respectively) compared with control population. There were differences between the two disease entities in the frequency of the DRB1*03-DQB1*02 (DR3/DQ2) haplotype (P=0.00008 vs. P=0.177) compared with control group. The presence of the TNF2 allele was significantly lower in LADA than type I diabetes (P=0.022) or control group (P=0.017). CONCLUSION: Our findings indicate that there are marked differences in the genetic background of type 1 diabetes and LADA. The low presence of TNF2 allele (known to be associated with high amount of TNF alpha production) in LADA could be one of the factors responsible for the relatively slow progression.