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1.  A study of 82 extended HLA haplotypes in HFE-C282Y homozygous hemochromatosis subjects: relationship to the genetic control of CD8+ T-lymphocyte numbers and severity of iron overload 
BMC Medical Genetics  2006;7:16.
It has been recently demonstrated that CD8+ T-lymphocyte numbers are genetically transmitted in association with the MHC class I region. The present study was designed with the objective of narrowing the region associated with the setting of CD8+ T-lymphocyte numbers in a population of C282Y homozygous hemochromatosis subjects, in whom a high prevalence of abnormally low CD8+ T-lymphocyte counts has been described.
The study includes 43 C282Y homozygous subjects fully characterized both phenotypically and genotypically. Clinical characterization includes measurements of iron parameters at diagnosis (transferrin saturation and serum ferritin), total body iron stores and T-cell immunophenotyping determined by flow cytometry. Genetic characterization includes HLA class I alleles (A, B and C) and four additional microsatellite markers (D6S265, D6S2222, D6S105 and D6S2239) spanning 5 Megabases in the 6p21.3 region.
Eighty-two extended C282Y carrying haplotypes were defined. Single-locus analysis revealed that the HLA-A region was associated with CD8+ T-cell numbers. Multivariate analysis showed that the combinations of the most common HLA-A alleles (HLA-A*03, -A*02 and -A*01) were associated with significantly lower numbers of CD8+ T-lymphocytes (0.30 ± 0.14 × 106/ml), in comparison with subjects carrying only one copy of those alleles (0.46 ± 0.19 × 106/ml) and subjects without any copy of those alleles (0.79 ± 0.15 × 106/ml;p = 0.0001). No differences were observed in CD8+ T-cell counts among control subjects carrying the same combinations of HLA-A alleles (0.47 ± 0.14; 0.45 ± 0.21 and 0.41 ± 0.17 × 106/ml, respectively), therefore not supporting a direct effect of HLA specificity but rather an indirect association with a locus close to HLA-A. Multivariate analysis showed that the combination of the most common HLA-A alleles also have an impact on the clinical expression of HH in terms of iron stores, in males(p = 0.0009).
The present study provides evidence supporting an inextricable link between extended HLA haplotypes, CD8+ T-lymphocyte numbers and severity of iron overload in hereditary hemochromatosis(HH). It gives additional information to better define a candidate region involved in the regulation of CD8+ T-lymphocyte numbers. A new evolutionary hypothesis concerning the inheritance of the phenotype of low CD8+ T-lymphocyte numbers associated with particular ancestral HLA haplotypes carrying the C282Y mutation and its implication on the clinical heterogeneity of HH is discussed.
PMCID: PMC1413516  PMID: 16509978
2.  Effects of Highly Conserved Major Histocompatibility Complex (MHC) Extended Haplotypes on Iron and Low CD8+ T Lymphocyte Phenotypes in HFE C282Y Homozygous Hemochromatosis Patients from Three Geographically Distant Areas 
PLoS ONE  2013;8(11):e79990.
Hereditary Hemochromatosis (HH) is a recessively inherited disorder of iron overload occurring commonly in subjects homozygous for the C282Y mutation in HFE gene localized on chromosome 6p21.3 in linkage disequilibrium with the human leukocyte antigen (HLA)-A locus. Although its genetic homogeneity, the phenotypic expression is variable suggesting the presence of modifying factors. One such genetic factor, a SNP microhaplotype named A-A-T, was recently found to be associated with a more severe phenotype and also with low CD8+T-lymphocyte numbers. The present study aimed to test whether the predictive value of the A-A-T microhaplotype remained in other population settings. In this study of 304 HH patients from 3 geographically distant populations (Porto, Portugal 65; Alabama, USA 57; Nord-Trøndelag, Norway 182), the extended haplotypes involving A-A-T were studied in 608 chromosomes and the CD8+ T-lymphocyte numbers were determined in all subjects. Patients from Porto had a more severe phenotype than those from other settings. Patients with A-A-T seemed on average to have greater iron stores (p = 0.021), but significant differences were not confirmed in the 3 separate populations. Low CD8+ T-lymphocytes were associated with HLA-A*03-A-A-T in Porto and Alabama patients but not in the greater series from Nord-Trøndelag. Although A-A-T may signal a more severe iron phenotype, this study was unable to prove such an association in all population settings, precluding its use as a universal predictive marker of iron overload in HH. Interestingly, the association between A-A-T and CD8+ T-lymphocytes, which was confirmed in Porto and Alabama patients, was not observed in Nord-Trøndelag patients, showing that common HLA haplotypes like A*01–B*08 or A*03–B*07 segregating with HFE/C282Y in the three populations may carry different messages. These findings further strengthen the relevance of HH as a good disease model to search for novel candidate loci associated with the genetic transmission of CD8+ T-lymphocyte numbers.
PMCID: PMC3839968  PMID: 24282517
3.  HLA variants related to primary sclerosing cholangitis influence rejection after liver transplantation 
AIM: To investigate influence of human leukocyte antigen (HLA) and killer immunoglobuline-like receptor (KIR) genotypes on risks of acute rejection (AR) after liver transplantation (LTX).
METHODS: In this retrospective study we included 143 adult donor-recipient pairs with a minimum of 6 mo follow-up after LTX for whom DNA was available from both donor and recipients. Clinical data, all early complications including episodes and severity of AR and graft/patient survival were registered. The diagnosis of AR was based on clinical, biochemical and histological criteria. All suspected episodes of AR were biopsy confirmed. Key classical HLA loci (HLA-A, HLA-B, HLA-C and HLA-DRB1) were genotyped using Sanger sequencing. 16 KIR genes were genotyped using a novel real time PCR approach which allows for determination of the diploid copy number of each KIR gene. Immunohistochemical staining for T (CD3), B (CD20) and natural killer (NK) cells (CD56 and CD57) were performed on liver biopsies from 3 different patient groups [primary sclerosing cholangitis (PSC), primary biliary cirrhosis and non-autoimmune liver disease], 10 in each group, with similar grade of AR.
RESULTS: Fourty-four (31%) patients were transplanted on the basis of PSC, 40% of them had AR vs 24% in the non-PSC group (P = 0.04). No significant impact of donor-recipient matching for HLA and KIR genotypes was detected. In the overall recipient population an increased risk of AR was detected for HLA-B*08 (P = 0.002, OR = 2.5; 95%CI: 1.4-4.6), HLA-C*07 (P = 0.001, OR = 2.4; 95%CI: 1.4-4.0) and HLA-DRB1*03 (P = 0.03, OR = 1.9; 95%CI: 1.0-3.3) and a decreased risk for HLA-DRB1*04 (P = 0.001, OR = 0.2; 95%CI: 0.1-0.5). For HLA-B*08, HLA-C*07 and DRB1*04 the associations remained evident in a subgroup analysis of non-PSC recipients (P = 0.04, P = 0.003 and P = 0.02, respectively). In PSC recipients corresponding P values were 0.002, 0.17 and 0.01 for HLA-B*08, HLA-C*07 and DRB1*04, respectively. A dosage effect of AR prevalence according to the PSC associated HLA alleles was also notable in the total recipient population. For HLA-B*08 the frequency of AR was 56% in HLA-B*08 homozygous recipients, 39% in heterozygous recipients and 21% in recipients lacking HLA-B*08 (P = 0.02). The same was observed for the HLA-C*07 allele with AR in 57%, 27% and 18% in recipients being homozygous, heterozygous and lacking HLA-C*07 respectively (P = 0.003). Immunohistochemical analysis showed similar infiltration of T, B and NK cells in biopsies with AR in all three groups.
CONCLUSION: We found significant associations between the PSC-associated HLA-B*08, HLA-C*07, HLA-DRB1*03 and HLA-DRB1*04 alleles and risk of AR in liver transplant recipients.
PMCID: PMC3983454  PMID: 24744588
Liver transplantation; Primary sclerosing cholangitis; Acute rejection; Human leukocyte antigen; Killer immunoglobulin-like receptor
4.  A rapid method of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid population 
Gut  1998;42(4):566-569.
Background—HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known. 
Aims—To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies. 
Patients—A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method. 
Methods—A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population. 
Results—27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y. 
Conclusions—Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation. 

Keywords: HFE; haemochromatosis; PCR-SSP; linkage disequilibrium
PMCID: PMC1727064  PMID: 9616322
5.  Distribution of HLA-A, -B and -DRB1 Genes and Haplotypes in the Tujia Population Living in the Wufeng Region of Hubei Province, China 
PLoS ONE  2012;7(6):e38774.
The distribution of HLA alleles and haplotypes varies widely between different ethnic populations and geographic areas. Before any genetic marker can be used in a disease-associated study it is therefore essential to investigate allelic frequencies and establish a genetic database.
Methodology/Principal Findings
This is the first report of HLA typing in the Tujia group using the Luminex HLA-SSO method HLA–A, –B and -DRB1 allelic distributions were determined in 124 unrelated healthy Tujia individuals, and haplotypic frequencies and linkage disequilibrium parameters were estimated using the maximum-likelihood method. In total 10 alleles were detected at the HLA–A locus, 21 alleles at the HLA–B locus and 14 alleles at the HLA-DRB1 locus. The most frequently observed alleles in the HLA-I group were HLA–A*02 (35.48%), A*11 (28.23%), A*24 (15.73%); HLA–B*40 (25.00%), B*46 (16.13%), and B*15 (15.73%). Among HLA-DRB1 alleles, high frequencies of HLA-DRB1*09 (25.81%) were observed, followed by HLA-DRB1*15 (12.9%), and DRB1*12 (10.89%). The two-locus haplotypes at the highest frequency were A*02–B*46A (8.47%), followed by A*11–B*40 (7.66%), A*02–B*40 (8.87%), A*11–B*15 (6.45%), A*02–B*15 (6.05%), B*40–DRB1*09 (9.27%) and B*46–DRB1*09 (6.45%). The most common three-locus haplotypes found in the Tujia population were A*02–B*46–DRB1*09 (4.84%) and A*02–B*40–DRB1*09 (4.03%). Fourteen two-loci haplotypes had significant linkage disequilibrium. Construction of a neighbor-joining phylogenetic tree and principal component analysis using the allelic frequencies at HLA-A was performed to compare the Tujia group and twelve other previously reported populations. The Tujia population in the Wufeng of Hubei Province had the closest genetic relationship with the central Han population, and then to the Shui, the Miao, the southern Han and the northern Han ethnic groups.
These results will become a valuable source of data for tracing population migration, planning clinical organ transplantation, carrying out HLA-linked disease-associated studies and forensic identification.
PMCID: PMC3375274  PMID: 22719940
6.  The Inheritance of Resistance Alleles in Multiple Sclerosis 
PLoS Genetics  2007;3(9):e150.
Multiple sclerosis (MS) is a complex trait in which alleles at or near the class II loci HLA-DRB1 and HLA-DQB1 contribute significantly to genetic risk. HLA-DRB1*15 and HLA-DRB1*17-bearing haplotypes and interactions at the HLA-DRB1 locus increase risk of MS but it has taken large samples to identify resistance HLA-DRB1 alleles. In this investigation of 7,093 individuals from 1,432 MS families, we have assessed the validity, mode of inheritance, associated genotypes, and the interactions of HLA-DRB1 resistance alleles. HLA-DRB1*14-, HLA-DRB1*11-, HLA-DRB1*01-, and HLA-DRB1*10-bearing haplotypes are protective overall but they appear to operate by different mechanisms. The first type of resistance allele is characterised by HLA-DRB1*14 and HLA-DRB1*11. Each shows a multiplicative mode of inheritance indicating a broadly acting suppression of risk, but a different degree of protection. In contrast, a second type is exemplified by HLA-DRB1*10 and HLA-DRB1*01. These alleles are significantly protective when they interact specifically in trans with HLA-DRB1*15-bearing haplotypes. HLA-DRB1*01 and HLA-DRB1*10 do not interact with HLA-DRB1*17, implying that several mechanisms may be operative in major histocompatibility complex–associated MS susceptibility, perhaps analogous to the resistance alleles. There are major practical implications for risk and for the exploration of mechanisms in animal models. Restriction of antigen presentation by HLA-DRB1*15 seems an improbably simple mechanism of major histocompatibility complex–associated susceptibility.
Author Summary
Multiple sclerosis (MS) is a complex neurological disease with a strong genetic component. With the possible exception of a weak association at Chromosome 5p, the major histocompatibility complex is the only locus consistently linked to MS. Because of this the major histocompatibility complex has recently undergone renewed attention. A region at or near the gene HLA-DRB1 influences the risk of MS. HLA-DRB1 comes in over 400 different forms (or alleles). A common form in Europe, named 1501, increases risk of MS by 3-fold. In this paper, to our knowledge the largest-ever analysis of this region in MS, we examine the inheritance of newly discovered HLA-DRB1 MS resistance alleles, namely HLA-DRB1*14, HLA-DRB1*11, *10, and HLA-DRB1*01. We show that HLA-DRB1*14 and HLA-DRB1*11 are dominantly protective; e.g., HLA-DRB1*14 significantly reduces the risk associated with HLA-DRB1*15 when they are inherited together. This may explain, in part, why MS is rare in Asia; there, the HLA-DRB1*14 allele is frequent. HLA-DRB1*01 and HLA-DRB1*10 are protective only in the presence of HLA-DRB1*15. HLA-DRB1*14 and HLA-DRB1*11 haplotypes and HLA-DRB1*01 and HLA-DRB1*10 haplotypes share common ancestral origins and this may be why the alleles can be grouped in terms of their protective nature. Discovery of the mechanism of protection against MS may lead to the discovery of new treatments to make a palpable difference in the lives of those who have been affected by this devastating disease.
PMCID: PMC1971120  PMID: 17845076
7.  Allele Polymorphism and Haplotype Diversity of HLA-A, -B and -DRB1 Loci in Sequence-Based Typing for Chinese Uyghur Ethnic Group 
PLoS ONE  2010;5(11):e13458.
Previous studies indicate that the frequency distributions of HLA alleles and haplotypes vary from one ethnic group to another or between the members of the same ethnic group living in different geographic areas. It is necessary and meaningful to study the high-resolution allelic and haplotypic distributions of HLA loci in different groups.
Methodology/Principal Findings
High-resolution HLA typing for the Uyghur ethnic minority group using polymerase chain reaction-sequence-based-typing method was first reported. HLA-A, -B and -DRB1 allelic distributions were determined in 104 unrelated healthy Uyghur individuals and haplotypic frequencies and linkage disequilibrium parameters for HLA loci were estimated using the maximum-likelihood method. A total of 35 HLA-A, 51 HLA-B and 33 HLA-DRB1 alleles were identified at the four-digit level in the population. High frequency alleles were HLA-A*1101 (13.46%), A*0201 (12.50%), A*0301 (10.10%); HLA-B*5101(8.17%), B*3501(6.73%), B*5001 (6.25%); HLA-DRB1*0701 (16.35%), DRB1*1501 (8.65%) and DRB1*0301 (7.69%). The two-locus haplotypes at the highest frequency were HLA-A*3001-B*1302 (2.88%), A*2402-B*5101 (2.86%); HLA-B*5001-DRB1*0701 (4.14%) and B*0702-DRB1*1501 (3.37%). The three-locus haplotype at the highest frequency was HLA-A*3001-B*1302-DRB1*0701(2.40%). Significantly high linkage disequilibrium was observed in six two-locus haplotypes, with their corresponding relative linkage disequilibrium parameters equal to 1. Neighbor-joining phylogenetic tree between the Uyghur group and other previously reported populations was constructed on the basis of standard genetic distances among the populations calculated using the four-digit sequence-level allelic frequencies at HLA-A, HLA-B and HLA-DRB1 loci. The phylogenetic analyses reveal that the Uyghur group belongs to the northwestern Chinese populations and is most closely related to the Xibe group, and then to Kirgiz, Hui, Mongolian and Northern Han.
The present findings could be useful to elucidate the genetic background of the population and to provide valuable data for HLA matching in clinical bone marrow transplantation, HLA-linked disease-association studies, population genetics, human identification and paternity tests in forensic sciences.
PMCID: PMC2973946  PMID: 21079793
8.  Human leukocyte antigen alleles, genotypes and haplotypes frequencies in renal transplant donors and recipients from West Central India 
Indian Journal of Human Genetics  2013;19(2):219-232.
Human leukocyte antigen (HLA) is comprised of a highly polymorphic set of genes which determines the histocompatibility of organ transplantation. The present study was undertaken to identify HLA class I and class II allele, genotype and haplotype frequencies in renal transplant recipients and donors from West Central India.
HLA typing was carried out using Polymerase Chain Reaction-Sequence Specific Primer in 552 live related and unrelated renal transplant recipients and donors.
The most frequent HLA class I and class II alleles and their frequencies in recipients were HLA-AFNx0101 (0.1685) and AFNx0102 (0.1649), HLA-BFNx0135 (0.1322), and HLA-DR beta 1 (DRB 1)FNx0115 (0.2192), whereas in donors, these were HLA-AFNx0102 (0.1848) and AFNx0101 (0.1667), HLA-BFNx0135 (0.1359), and HLA-DRB1FNx0115 (0.2409). The two-locus haplotype statistical analysis revealed HLA-AFNx0102-B61 as the most common haplotype with the frequency of 0.0487 and 0.0510 in recipients and donors, respectively. Further, among the three locus haplotypes HLA-AFNx0133-BFNx0144-DRB1FNx0107 and HLA-AFNx0102-BFNx0161-DRB1FNx0115 were the most common haplotypes with frequencies 0.0362 and 0.0326, respectively in recipients and 0.0236 and 0.0323, respectively in donors. Genotype frequency revealed a high prevalence of genotype HLA-AFNx0102/AFNx0124 in recipients (0.058) compared to donors (0.0109) whereas low prevalence of HLA-AFNx0101/AFNx0102 in recipients (0.0435) than in donors (0.0797). The phylogenetic and principal component analysis of HLA allele and haplotype frequency distribution revealed genetic similarities of various ethnic groups. Further, case control analysis provides preliminary evidence of association of HLA-A genotype (P < 0.05) with renal failure.
This study will be helpful in suitable donor search besides providing valuable information for population genetics and HLA disease association analysis.
PMCID: PMC3758731  PMID: 24019626
Allele; genotype; haplotype; human leukocyte antigens frequencies; polymorphism; renal transplant
9.  Allelic associations and homozygosity at loci from HLA-B to D6S299 in genetic haemochromatosis. 
Journal of Medical Genetics  1995;32(6):446-452.
Haemochromatosis (GH) is an autosomal recessive disorder in which increased iron absorption causes iron overload. The gene (HFE) is closely linked to HLA-A on chromosome 6 (6p21.3) but has not yet been identified. We have examined eight polymorphic loci, HLA-B (most centromeric), I82, D6S265, HLA-A, D6S128, HLA-F, D6S105, and D6S299 (most telomeric) in 37 unrelated patients and 60 control subjects. There are also significant positive associations between GH and alleles at all loci except D6S299. Analysis of 48 GH chromosomes in which haplotypes could be established showed that the most common haplotype was I82-2:D6S265-1:HLA-A3:D6S128-2:HLA-F1:D6S105-8. This was present in 28 of 48 chromosomes. In 14 the haplotype included HLA-B7 but only in seven did this extend beyond the telomere to D6S299-2 (the most common allele on GH chromosomes at this locus). In 36 out of 48 chromosomes the two locus haplotype, F1:D6S105-8 was present. Since haemochromatosis appears to originate from a founder mutation we have examined linkage disequilibrium between these various loci and GH using calculations of pexcess. The maximum value (0.72, 95% CI 0.55-0.85) is given by D6S105-8 but is not significantly different from values for HLA-A3 and HLA-F1 (0.50, 95% CI 0.34-0.61 and 0.49, 0.25-0.66 respectively). However, both HLA-A and D6S105 give a value for pexcess which is significantly higher than that for the most centromeric marker, HLA-B (0.17, 95% CI 0.02-0.30). We have counted the number of patients who are homozygous for the common allele at each locus. At D6S105, 22 patients are homozygous for allele 8, with 18 homozygous for HLA-F1 and 10 homozygous for A3. The pattern of cumulative homozygosity suggests a gene location closer to D6S105 than HLA-A. We have also analysed our data for divergence from the apparent founder haplotype (A3:F1:105-8) and have calculated the theoretical frequencies of crossovers between loci. These data suggest a location telomeric to D6S105. A more precise localisation of the gene may be possible with the identification of new markers around D6S105.
PMCID: PMC1050484  PMID: 7666396
10.  IFN-γ production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1*0401 and HLA-DQ8 
Arthritis Research  1999;2(1):75-84.
IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.
Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5].
It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses.
To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum.
Forty-five patients who met the 1987 American College of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera.
Heparinized blood was density gradient separated and PBMCs were cultured at 1 × 106/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 μ g/ml), killed influenza virus (Vaxigrip, Pasteur Mérieux, Lyon, France; diluted 1 : 1000) or purified protein derivative (PPD; 10 μ g/ml). CII was heat-denatured in 56°C for 30 min.
Cell supernatants were collected after 7days and IFN-γ contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study.
PBMCs from both RA patients and healthy control individuals responded with inteferon-γ production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for healthy control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-γ response to both native and denatured CII than did healthy control individuals (P = 0.02 and 0.04, respectively).
Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-γ responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II.
The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6,7,8]. That no difference between the RA group and the control group was recorded in CII-induced IFN-γ production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-γ production and the PPD-induced and killed influenza virus-induced IFN-γ production, and compared these fractions. A highly significant difference between the RA and healthy control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-γ values as the denominator (Fig. 2b).
When comparing the compensated IFN-γ response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8).
No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach.
We identified an association between the IFN-γ response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404).
CII, a major autoantigen candidate in RA, can elicit an IFN-γ response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.
PMCID: PMC17806  PMID: 11219392
collagen type II; human leucocyte antigen-DR; IFN-γ; rheumatoid arthritis; T cell
11.  Diversity of Extended HLA-DRB1 Haplotypes in the Finnish Population 
PLoS ONE  2013;8(11):e79690.
The Major Histocompatibility Complex (MHC, 6p21) codes for traditional HLA and other host response related genes. The polymorphic HLA-DRB1 gene in MHC Class II has been associated with several complex diseases. In this study we focus on MHC haplotype structures in the Finnish population. We explore the variability of extended HLA-DRB1 haplotypes in relation to the other traditional HLA genes and a selected group of MHC class III genes. A total of 150 healthy Finnish individuals were included in the study. Subjects were genotyped for HLA alleles (HLA-A, -B, -DRB1, -DQB1, and -DPB1). The polymorphism of TNF, LTA, C4, BTNL2 and HLA-DRA genes was studied with 74 SNPs (single nucleotide polymorphism). The C4A and C4B gene copy numbers and a 2-bp silencing insertion at exon 29 in C4A gene were analysed with quantitative genomic realtime-PCR. The allele frequencies for each locus were calculated and haplotypes were constructed using both the traditional HLA alleles and SNP blocks. The most frequent Finnish A∼B∼DR -haplotype, uncommon in elsewhere in Europe, was A*03∼B*35∼DRB1*01∶01. The second most common haplotype was a common European ancestral haplotype AH 8.1 (A*01∼B*08∼DRB1*03∶01). Extended haplotypes containing HLA-B, TNF block, C4 and HLA-DPB1 strongly increased the number of HLA-DRB1 haplotypes showing variability in the extended HLA-DRB1 haplotype structures. On the contrary, BTNL2 block and HLA-DQB1 were more conserved showing linkage with the HLA-DRB1 alleles. We show that the use of HLA-DRB1 haplotypes rather than single HLA-DRB1 alleles is advantageous when studying the polymorphisms and LD patters of the MHC region. For disease association studies the HLA-DRB1 haplotypes with various MHC markers allows us to cluster haplotypes with functionally important gene variants such as C4 deficiency and cytokines TNF and LTA, and provides hypotheses for further assessment. Our study corroborates the importance of studying population-specific MHC haplotypes.
PMCID: PMC3836878  PMID: 24278156
12.  HLA-G UTR Haplotype Conservation in the Malian Population: Association with Soluble HLA-G 
PLoS ONE  2013;8(12):e82517.
The HLA-G molecule plays an important role in immunomodulation. In a previous study carried out on a southern French population our team showed that HLA-G haplotypes, defined by SNPs in the coding region and specific SNPs located in 5′URR and 3′UTR regulatory regions, are associated with differential soluble HLA-G expression (sHLA-G). Furthermore, the structure of these HLA-G haplotypes appears to be conserved in geographically distant populations.
The aim of our study is to confirm these expectations in a sub-Saharan African population and to explore additional factors, such as HLA-A alleles, that might influence sHLA-G expression.
DNA and plasma samples were collected from 229 Malians; HLA-G and HLA-A genotyping were respectively performed by the Snap Shot® method and by Luminex™ technology. sHLA-G dosage was performed using an ELISA kit. HLA-G and HLA-A allelic and haplotypic frequencies were estimated using an EM algorithm from the Gene[Rate] program. Associations between genetic and non genetic parameters with sHLA-G were performed using a non-parametric test with GRAPH PAD Prism 5.
Our results reveal a good conservation of the HLA-G UTR haplotype structure in populations with different origins and demographic histories. These UTR haplotypes appear to be involved in different sHLA-G expression patterns. Specifically, the UTR-2 haplotype was associated with low sHLA-G levels, displaying a dominant negative effect. Furthermore, an allelic effect of both HLA-G and HLA-A, as well as non genetic parameters, such as age and gender possibly linked to osteogenesis and sexual hormones, also seem to be involved in the modulation of sHLA-G.
These data suggest that further investigation in larger cohorts and in populations from various ethnical backgrounds is necessary not only to detect new functional polymorphism in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion and this might therefore have an impact on transplantation practice.
PMCID: PMC3871591  PMID: 24376542
13.  Polymorphisms of FCRL3 in a Chinese population with Vogt-Koyanagi-Harada (VKH) syndrome 
Molecular Vision  2009;15:955-961.
The polymorphisms of the Fc receptor-like 3 gene (FCRL3), a novel immunoregulatory gene, have been shown to be associated with certain autoimmune diseases. This study was designed to examine whether the polymorphisms of FCRL3 are associated with susceptibility to Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese population.
A case-control study was performed in 230 Chinese VKH patients and 301 healthy controls. Four single nucleotide polymorphisms (SNPs; −169C/T, −110A/G, +358C/G, and +1381A/G) in FCRL3 were detected using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). human leukocyte antigen -DR4 (HLA-DR4) and HLA-DRw53 genotyping was performed using PCR techniques.
The results showed that the frequency of haplotype CACG was significantly lower in patients when compared with that in controls (p=0.0018, corrected p [pc]=0.0288). A significantly higher frequency was found for haplotype CGGG in HLA-DR4 negative patients than in HLA-DR4 negative controls (p=9.94×10−8, Pc=1.59×10−6). There were no significant differences in the allele and genotype frequencies of the four investigated SNPs between VKH patients and controls. HLA-DR4 and HLA-DRw53 were significantly associated with VKH syndrome (p=3.21×10−16 and p=7.08×10−5, respectively). Stratification analysis according to HLA-DR4 and HLA-DRw53 did not show any association of FCRL3 polymorphisms with VKH syndrome.
Our study confirms the previous association of HLA-DR4 and HLA-DRw53 with VKH syndrome but fails to demonstrate an association between FCRL3 polymorphisms and VKH syndrome. Haplotype CACG might be a protective haplotype for VKH syndrome, and haplotype CGGG may be a risk haplotype in HLA-DR4 negative individuals.
PMCID: PMC2683028  PMID: 19452015
14.  High Resolution Human Leukocyte Antigen Class I Allele Frequencies and HIV-1 Infection Associations in Chinese Han and Uyghur Cohorts 
PLoS ONE  2012;7(12):e50656.
Host immunogenetic factors such as HLA class I polymorphism are important to HIV-1 infection risk and AIDS progression. Previous studies using high-resolution HLA class I profile data of Chinese populations appeared insufficient to provide information for HIV-1 vaccine development and clinical trial design. Here we reported HLA class I association with HIV-1 susceptibility in a Chinese Han and a Chinese Uyghur cohort.
Methodology/Principal Findings
Our cohort included 327 Han and 161 Uyghur ethnic individuals. Each cohort included HIV-1 seropositive and HIV-1 seronegative subjects. Four-digit HLA class I typing was performed by sequencing-based typing and high-resolution PCR-sequence specific primer. We compared the HLA class I allele and inferred haplotype frequencies between HIV-1 seropositive and seronegative groups. A neighbor-joining tree between our cohorts and other populations was constructed based on allele frequencies of HLA-A and HLA-B loci. We identified 58 HLA-A, 75 HLA-B, and 32 HLA-Cw distinct alleles from our cohort and no novel alleles. The frequency of HLA-B*5201 and A*0301 was significantly higher in the Han HIV-1 negative group. The frequency of HLA-B*5101 was significantly higher in the Uyghur HIV-1 negative group. We observed statistically significant increases in expectation-maximization (EM) algorithm predicted haplotype frequencies of HLA-A*0201-B*5101 in the Uyghur HIV-1 negative group, and of Cw*0304-B*4001 in the Han HIV-1 negative group. The B62s supertype frequency was found to be significantly higher in the Han HIV-1 negative group than in the Han HIV-1 positive group.
At the four-digit level, several HLA class I alleles and haplotypes were associated with lower HIV-1 susceptibility. Homogeneity of HLA class I and Bw4/Bw6 heterozygosity were not associated with HIV-1 susceptibility in our cohort. These observations contribute to the Chinese HLA database and could prove useful in the development of HIV-1 vaccine candidates.
PMCID: PMC3520934  PMID: 23251376
15.  Human Leukocyte Antigens and Cellular Immune Responses to Anthrax Vaccine Adsorbed 
Infection and Immunity  2013;81(7):2584-2591.
Interindividual variations in vaccine-induced immune responses are in part due to host genetic polymorphisms in the human leukocyte antigen (HLA) and other gene families. This study examined associations between HLA genotypes, haplotypes, and homozygosity and protective antigen (PA)-specific cellular immune responses in healthy subjects following immunization with Anthrax Vaccine Adsorbed (AVA). While limited associations were observed between individual HLA alleles or haplotypes and variable lymphocyte proliferative (LP) responses to AVA, analyses of homozygosity supported the hypothesis of a “heterozygote advantage.” Individuals who were homozygous for any HLA locus demonstrated significantly lower PA-specific LP than subjects who were heterozygous at all eight loci (median stimulation indices [SI], 1.84 versus 2.95, P = 0.009). Similarly, we found that class I (HLA-A) and class II (HLA-DQA1 and HLA-DQB1) homozygosity was significantly associated with an overall decrease in LP compared with heterozygosity at those three loci. Specifically, individuals who were homozygous at these loci had significantly lower PA-specific LP than subjects heterozygous for HLA-A (median SI, 1.48 versus 2.13, P = 0.005), HLA-DQA1 (median SI, 1.75 versus 2.11, P = 0.007), and HLA-DQB1 (median SI, 1.48 versus 2.13, P = 0.002) loci, respectively. Finally, homozygosity at an increasing number (≥4) of HLA loci was significantly correlated with a reduction in LP response (P < 0.001) in a dose-dependent manner. Additional studies are needed to reproduce these findings and determine whether HLA-heterozygous individuals generate stronger cellular immune response to other virulence factors (Bacillus anthracis LF and EF) than HLA-homozygous subjects.
PMCID: PMC3697592  PMID: 23649091
16.  Complementarity of Binding Motifs is a General Property of HLA-A and HLA-B Molecules and Does Not Seem to Effect HLA Haplotype Composition 
Different human leukocyte antigen (HLA) haplotypes (i.e., the specific combinations of HLA-A, -B, -DR alleles inherited together from one parent) are observed in different frequencies in human populations. Some haplotypes, like HLA-A1-B8, are very frequent, reaching up to 10% in the Caucasian population, while others are very rare. Numerous studies have identified associations between HLA haplotypes and diseases, and differences in haplotype frequencies can in part be explained by these associations: the stronger the association with a severe (autoimmune) disease, the lower the expected HLA haplotype frequency. The peptide repertoires of the HLA molecules composing a haplotype can also influence the frequency of a haplotype. For example, it would seem advantageous to have HLA molecules with non-overlapping binding specificities within a haplotype, as individuals expressing such an haplotype would present a diverse set of peptides from viruses and pathogenic bacteria on the cell surface. To test this hypothesis, we collect the proteome data from a set of common viruses, and estimate the total ligand repertoire of HLA class I haplotypes (HLA-A-B) using in silico predictions. We compare the size of these repertoires to the HLA haplotype frequencies reported in the National Marrow Donor Program (NMDP). We find that in most HLA-A and HLA-B pairs have fairly distinct binding motifs, and that the observed haplotypes do not contain HLA-A and -B molecules with more distinct binding motifs than random HLA-A and HLA-B pairs. In addition, the population frequency of a haplotype is not correlated to the distinctness of its HLA-A and HLA-B peptide binding motifs. These results suggest that there is a not a strong selection pressure on the haplotype level favoring haplotypes having HLA molecules with distinct binding motifs, which would result the largest possible presented peptide repertoires in the context of infectious diseases.
PMCID: PMC3827838  PMID: 24294213
haplotypes; HLA antigens; selection; genetic; peptide binding; bioinformatics; computational biology
17.  High-Density SNP Mapping of the HLA Region Identifies Multiple Independent Susceptibility Loci Associated with Selective IgA Deficiency 
PLoS Genetics  2012;8(1):e1002476.
Selective IgA deficiency (IgAD; serum IgA<0.07 g/l) is the most common form of human primary immune deficiency, affecting approximately 1∶600 individuals in populations of Northern European ancestry. The polygenic nature of IgAD is underscored by the recent identification of several new risk genes in a genome-wide association study. Among the characterized susceptibility loci, the association with specific HLA haplotypes represents the major genetic risk factor for IgAD. Despite the robust association, the nature and location of the causal variants in the HLA region remains unknown. To better characterize the association signal in this region, we performed a high-density SNP mapping of the HLA locus and imputed the genotypes of common HLA-B, -DRB1, and -DQB1 alleles in a combined sample of 772 IgAD patients and 1,976 matched controls from 3 independent European populations. We confirmed the complex nature of the association with the HLA locus, which is the result of multiple effects spanning the entire HLA region. The primary association signal mapped to the HLA-DQB1*02 allele in the HLA Class II region (combined P = 7.69×10−57; OR = 2.80) resulting from the combined independent effects of the HLA-B*0801-DRB1*0301-DQB1*02 and -DRB1*0701-DQB1*02 haplotypes, while additional secondary signals were associated with the DRB1*0102 (combined P = 5.86×10−17; OR = 4.28) and the DRB1*1501 (combined P = 2.24×10−35; OR = 0.13) alleles. Despite the strong population-specific frequencies of HLA alleles, we found a remarkable conservation of these effects regardless of the ethnic background, which supports the use of large multi-ethnic populations to characterize shared genetic association signals in the HLA region. We also provide evidence for the location of association signals within the specific extended haplotypes, which will guide future sequencing studies aimed at characterizing the precise functional variants contributing to disease pathogenesis.
Author Summary
The human leukocyte antigen (HLA) locus is robustly associated with many immune-mediated conditions. However, identification of the genetic variants contributing to the disease pathophysiology has been greatly hampered by the extensive chromosomal conservation within this genomic region. To better understand the association of the HLA locus in selective IgA deficiency (IgAD), we used an extensive genotyping database from a recent genome-wide association study (GWAS) to generate a high-density SNP map of this region in a combined sample of >2,700 individuals from 3 independent European populations. In addition, we took advantage of recent methodological advances to impute the more common HLA-B, -DRB1, and -DQB1 alleles in all subjects. We confirmed the strong disease-association of the HLA locus and identified several different signals located in specific conserved HLA haplotypes contributing independent risk or protection for IgAD. Further analysis of the chromosomal sequences associated with the associated HLA alleles allowed us to refine the mapping of the susceptibility variants. These findings represent the most comprehensive high-density SNP mapping of the HLA locus in IgAD to date and provide important new information as to the location of the genetic variants contributing to this common immune deficiency.
PMCID: PMC3266887  PMID: 22291608
18.  Extended major histocompatibility complex haplotypes in type I diabetes mellitus. 
Journal of Clinical Investigation  1984;74(2):449-454.
We have studied major histocompatibility complex markers in Caucasian patients with type I diabetes mellitus and their families. The frequencies of extended haplotypes that were composed of specific HLA-B, HLA-DR, BF, C2, C4A, and C4B allelic combinations, which occurred more commonly than expected, were compared on random diabetic and normal chromosomes in the study families. We demonstrated that all of the previously recognized increases in HLA-B8, B18, B15, DR3, and perhaps DR4 could be ascribed to the increase among diabetic haplotypes of a few extended haplotypes: [HLA B8, DR3, SC01, GLO2]; [HLA-B18, DR3, F1C30]; [HLA-B15, DR4, SC33]; and [HLA-BW38, DR4, SC21]. In fact, HLA-DR3 on nonextended haplotypes was "protective", with a relative risk considerably less than 1.0. There was a paucity or absence among diabetic patients of several extended haplotypes of normal chromosomes, notably [HLA-B7, DR2, SC31] and [HLA-BW44, DR4, SC30]. The extended haplotype [HLA-BW38, DR4, SC21] is found only in Ashkenazi Jewish patients, which suggests that extended haplotypes mark specific mutations that arise in defined ethnic groups. The data show that no known MHC allele, including HLA-DR3 and possibly HLA-DR4, is per se a marker for or itself a susceptibility gene for type I diabetes. Rather, extended haplotypes, with relatively fixed alleles, are either carriers or noncarriers of susceptibility genes for this disease. Thus, the increased frequency (association) or the decreased frequency (protection) of individual MHC alleles is largely explainable by these extended haplotypes.
PMCID: PMC370496  PMID: 6746903
19.  Type 1 Diabetes in the Spanish Population: additional factors to Class II HLA-DR3 and -DR4 
BMC Genomics  2005;6:56.
The Major Histocompatibility Complex is the main genetic contributor to susceptibility to type 1 diabetes (T1D); genome-wide scans have consistently mapped increased predisposition to this region. The highest disease risk has been associated with HLA-DR3 and HLA-DR4. In particular, the DR3-positive ancestral haplotype 18.2 was reported as highly diabetogenic. We aimed to corroborate whether this haplotype increases the susceptibility conferred by the DQ2-DR3 alleles in a Mediterranean population. We also searched for additional susceptibility factors to the classic DQ2-DR3 and DQ8-DR4.
Genetic MHC markers were analysed in a case-control study with 302 T1D patients and 529 ethnically matched controls. DR3-TNFa1b5 carrier rate was significantly higher in DR3-positive heterozygous T1D patients than in DR3-positive heterozygous controls (p = 0.0019; odds ratio OR [95% confidence interval CI] = 2.26 [1.3–3.93]). This data was confirmed analysing the allelic frequency, which includes the information corresponding to the DR3-homozygous individuals (p = 0.001; OR = 2.09) and by using the Arlequin software to check the DR3-positive haplotypes (p = 0.004;OR = 1.93). The present results provide strong evidence of a second susceptibility region in the ancestral haplotype 18.2 in the Spanish population.
Moreover, we searched for T1D susceptibility factors in addition to the MHC classical ones, within the DR2-DQ6/DR3-DQ2/DR4-DQ8 negative population. Several genetic markers in both MHC class II (DQA1*0101-DQB1*0501 [p = 0.007;OR = 2.81], DQA1*0201-DQB1*0202 [p = 0.03; OR = 2.35]) and III (TNFa2b1 [p = 0.01 OR = 2.74], BAT-2*2 [p = 0.004; OR = 3.19]) were found. These different alleles associated with T1D were not independent and we observed linkage disequilibrium among them leading us to describe two new risk haplotypes (DQA1*0101-DQB1*0501-TNFa2b1 and DQA1*0201-DQB1*0202- BAT-2*2). Finally, we studied a T1D susceptibility/protection marker located in extended class I, D6S2223; however, no association was observed in our population.
Our results suggest that other associated MHC haplotypes might present susceptibility factors in loci different from HLA-class II and that the class II molecules are not necessarily the universal etiologic factor in every MHC haplotype.
PMCID: PMC1097726  PMID: 15842729
20.  Epistatic Interaction of ERAP1 and HLA-B in Behçet Disease: A Replication Study in the Spanish Population 
PLoS ONE  2014;9(7):e102100.
Behçet's disease (BD) is a multifactorial disorder associated with the HLA region. Recently, the ERAP1 gene has been proposed as a susceptibility locus with a recessive model and with epistatic interaction with HLA-B51. ERAP1 trims peptides in the endoplasmic reticulum to optimize their length for MHC-I binding. Polymorphisms in this gene have been related with the susceptibility to other immune-mediated diseases associated to HLA class I. Our aim was, the replication in the Spanish population of the association described in the Turkish population between ERAP1 (rs17482078) and BD. Additionally, in order to improve the understanding of this association we analyzed four additional SNPs (rs27044, rs10050860, rs30187 and rs2287987) associated with other diseases related to HLA class I and the haplotype blocks in this gene region. According to our results, frequencies of the homozygous genotypes for the minor alleles of all the SNPs were increased among patients and the OR values were higher in the subgroup of patients with the HLA-B risk factors, although differences were not statistically significant. Moreover, the presence of the same mutation in both chromosomes increased the OR values from 4.51 to 10.72 in individuals carrying the HLA-B risk factors. Therefore, although they were not statistically significant, our data were consistent with an association between ERAP1 and BD as well as with an epistatic interaction between ERAP1 and HLA-B in the Spanish population.
PMCID: PMC4096596  PMID: 25019531
21.  Comparing HLA Shared Epitopes in French Caucasian Patients with Scleroderma 
PLoS ONE  2012;7(5):e36870.
Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc), none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, 67FLEDR71, shared by HLA-DRB susceptibility alleles, or 71TRAELDT77, shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient’s clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ2 = 28.4, p<10−6) and with anti-topoisomerase antibody (ATA) production (χ2 = 43.9, p<10−9) whereas TRAELDT association is weaker in both subgroups (χ2 = 7.2, p = 0.027 and χ2 = 14.6, p = 0.0007 respectively). Moreover, FLEDR motif- association among patients with diffuse SSc remains significant only in ATA subgroup. The risk to develop ATA positive SSc is higher with double dose FLEDR than single dose with respectively, adjusted standardised residuals of 5.1 and 2.6. The increase in FLEDR motif is mostly due to the higher frequency of HLA-DRB1*11 and DRB1*15 haplotypes. Furthermore, FLEDR is always carried by the most abundantly expressed ß chain: ß1 in HLA DRB1*11 haplotypes and ß5 in HLA-DRB1*15 haplotypes.
In French Caucasian patients with SSc, FLEDR is the main presenting motif influencing ATA production in dcSSc. These results open a new field of potential therapeutic applications to interact with the FLEDR peptide binding groove and prevent ATA production, a hallmark of severity in SSc.
PMCID: PMC3352938  PMID: 22615829
22.  The immune response to hepatitis B vaccine in humans: inheritance patterns in families 
We have recently shown that the human antibody response to the hepatitis B virus surface antigen (HBsAg) vaccine is major histocompatibility complex (MHC) associated. In studies of nonresponders to the vaccine, we found an increased incidence of individuals homozygous for human histocompatibility leukocyte antigen (HLA) proteins associated with the extended (conserved) haplotype [HLA- B8,SC01,DR3]. In later prospective vaccination trials, we showed that none of five individuals homozygous for this haplotype developed more than 1,300 radioimmunoassay (RIA) units of antibody (mean, 467 RIA units), while all heterozygotes made at least 2,500 RIA units (mean antibody level, 15,608 units). Our results suggested that [HLA- B8,SC01,DR3] lacks an immune response gene for HBsAg, and that response is inherited in a dominant fashion. To provide further evidence for this hypothesis, we have now analyzed the results of HBsAg immunization in families. 43 members of 10 families were immunized with the hepatitis B vaccine, including seven families where at least one member bore the haplotype [HLA-B8,SC01,DR3], and three families where one member had already received, but failed to respond to, the vaccine. In two of these three families, the presence of [HLA-B8,SC01,DR3] was subsequently found. Of nine MHC-identical sibling pairs in the study, both members of eight pairs had similar antibody responses (five nonresponder and three responder pairs). In all families with such sibling pairs, including the discordant pair, rank-ordering members by antibody level demonstrated that no relative's value came between the sibling pair values. Furthermore, of nine [HLA-B8,SC01,DR3]-haplotype- homozygous individuals, six were nonresponders, and two others had only low-normal responses. [HLA-B8,SC01,DR3]-heterozygous family members always had higher levels of antibody than their homozygous relatives. Linkage analysis of nonresponse to HLA haplotypes revealed a maximum likelihood LOD (logarithm of the odds) score of 6.3 at a recombination fraction of 0.1. The MHC association with lack of antibody response to HBsAg was not seen with tetanus immunization, where 1 of 20 HBsAg responders and 1 of 21 poor or nonresponders had tetanus titers of less than 1:512; both tetanus nonresponders were [HLA-B8,SC01,DR3] heterozygotes. Our results indicate that: (a) response to the HBsAg vaccine is MHC linked, and inherited in a dominant fashion; (b) an abnormal or missing immune response (Ir) gene for HBsAg is a characteristic of most examples of the extended haplotype [HLA- B8,SC01,DR3]; and (c) other haplotypes also have abnormal or missing Ir genes for HBsAg.
PMCID: PMC2119114  PMID: 1531063
23.  Identification of HLA-DRPheβ47 as the susceptibility marker of hypersensitivity to beryllium in individuals lacking the berylliosis-associated supratypic marker HLA-DPGluβ69 
Respiratory Research  2005;6(1):94.
Susceptibility to beryllium (Be)-hypersensitivity (BH) has been associated with HLA-DP alleles carrying a glutamate at position 69 of the HLA-DP β-chain (HLA-DPGlu69) and with several HLA-DP, -DQ and -DR alleles and polymorphisms. However, no genetic associations have been found between BH affected subjects not carrying the HLA-DPGlu69 susceptibility marker.
In this report, we re-evaluated an already described patient populations after 7 years of follow-up including new 29 identified BH subjects. An overall population 36 berylliosis patients and 38 Be-sensitization without lung granulomas and 86 Be-exposed controls was analysed to assess the role of the individual HLA-class II polymorphisms associated with BH-susceptibility in HLA-DPGlu69 negative subjects by univariate and multivariate analysis.
As previously observed in this population the HLA-DPGlu69 markers was present in higher frequency in berylliosis patients (31 out of 36, 86%) than in Be-sensitized (21 out of 38, 55%, p = 0.008 vs berylliosis) and 41 out of 86 (48%, p < 0.0001 vs berylliosis, p = 0.55 vs Be-sensitized) Be-exposed controls.
However, 22 subjects presenting BH did not carry the HLA-DPGlu69 marker. We thus evaluated the contribution of all the HLA-DR, -DP and -DQ polymorphisms in determining BH susceptibility in this subgroup of HLA-Glu69 subjects. In HLA-DPGlu69-negatives a significant association with BH was found for the HLA-DQLeu26, for the HLA-DRB1 locus residues Ser13, Tyr26, His32, Asn37, Phe47 and Arg74 and for the HLA-DRB3 locus clusterized residues Arg11, Tyr26, Asp28, Leu38, Ser60 and Arg74. HLA-DRPhe47 (OR 2.956, p < 0.05) resulting independently associated with BH. Further, Be-stimulated T-cell proliferation in the HLA-DPGlu69-negative subjects (all carrying HLA-DRPhe47) was inhibited by the anti-HLA-DR antibody (range 70–92% inhibition) significantly more than by the anti-HLA-DP antibody (range: 6–29%; p < 0.02 compared to anti-HLA-DR) while it was not affected by the anti-HLA-DQ antibody.
We conclude that HLA-DPGlu69 is the primary marker of Be-hypersensitivity and HLA-DRPhe47 is associated with BH in Glu69-negative subjects, likely playing a role in Be-presentation and sensitization.
PMCID: PMC1198259  PMID: 16098233
24.  Effect of HLA-B and HLA-DR genes on susceptibility to and severity of spondyloarthropathies in Mexican patients 
Annals of the Rheumatic Diseases  2002;61(8):714-717.
Objective: To investigate the role of HLA-B and HLA-DR genes as contributors to genetic susceptibility and clinical expression of the spondyloarthropathies (SpA) in the Mexican population.
Methods: The study included 172 patients with SpA (undifferentiated SpA 83, ankylosing spondylitis (AS) 64, and reactive arthritis 25) and 99 healthy controls. The HLA-B and HLA-DR alleles were detected by the polymerase chain reaction with sequence-specific primers technique. Patient assessment included demographic data, diagnostic categories, and disease patterns. Statistical methods included the Mantel-Haenzel χ2 test, Fisher's exact test, and Woolf method for odds ratio (OR). Differences of continuous variables between HLA allele groups were calculated by Student's t test.
Results: Increased frequencies of HLA-B27 (pCh10-3, OR=28.7), HLA-DR1 (pC=0.045, OR=2.77), and HLA-B15 (p=0.034, pC=NS, OR=2.04) alleles in the whole group were found. HLA-B27 strength of association (OR) was 41.4 in AS; 20.9 in undifferentiated SpA; 27.2 in reactive arthritis. HLA-DR1 and HLA-B15 were increased in undifferentiated SpA (pC=0.045, OR=2.98 and p=0.004, pC=NS, OR=2.75). By analysing 58 HLA-B27 negative patients it was found that HLA-B15 and HLA-DR1 associations with SpA were independent of HLA-B27; increased frequencies of HLA-B15 were found in the whole SpA group and in patients with undifferentiated SpA (pC=0.03, OR=3.09 and pCh0.01, OR=3.77) and of HLA-DR1 in the latter (p=0.04, pC=NS, OR=3.15). HLA-B27 positive patients were younger than HLA-B27 negative patients at onset (p=0.03), but HLA-DR1 positive patients were older than HLA-DR1 negative patients (p=0.03). Bath indices for disease activity and functioning were higher in HLA-B27 positive patients (p=0.006 and p=0.004 v HLA-B27 negative patients). In contrast, neither HLA-DR1 nor HLA-B15 influenced these indices.
Conclusion: Apart from HLA-B27, there is a significant association of HLA-DR1 and HLA-B15 with SpA in Mexicans which is independent of B27. HLA-B27 is associated with younger age at onset and increased disease severity and HLA-DR1 with older age at onset. The strength of HLA-B15, HLA-B27, and HLA-DR1 associations varied in different forms of SpA.
PMCID: PMC1754177  PMID: 12117677
25.  MHC Haplotype Matching for Unrelated Hematopoietic Cell Transplantation 
PLoS Medicine  2007;4(1):e8.
Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined.
Methods and Findings
We isolated DNA strands extending across 2 million base pairs of the MHC to determine the physical linkage of HLA-A, -B, and -DRB1 alleles in 246 HCT recipients and their HLA-A, -B, -C, -DRB1, -DQB1 allele-matched unrelated donors. MHC haplotype mismatching was associated with a statistically significantly increased risk of severe acute GVHD (odds ratio 4.51; 95% confidence interval [CI], 2.34–8.70, p < 0.0001) and with lower risk of disease recurrence (hazard ratio 0.45; 95% CI, 0.22–0.92, p = 0.03).
The MHC harbors genes that encode unidentified transplantation antigens. The three-locus HLA-A, -B, -DRB1 haplotype serves as a proxy for GVHD risk among HLA-identical transplant recipients. The phasing method provides an approach for mapping novel MHC-linked transplantation determinants and a means to decrease GVHD-related morbidity after HCT from unrelated donors.
A novel method of MHC haplotype matching provides a means to decrease graft-versus-host disease-related morbidity after transplantation from unrelated donors.
Editors' Summary
Graft rejection and graft-versus-host disease (GVHD) are feared complications of hematopoietic cell transplantation (HCT). GVHD can affect all parts of the body, and, if severe (grade III to IV out of a scale of IV), can lead to the death of the transplant recipient. GVHD or rejection of the graft occurs when there are differences in specific proteins involved in the immune response (known as HLA antigens) between donor and recipient that stimulate the immune reaction. GVHD and graft rejection occur most often in people who receive transplants from unrelated donors because, although when donors are matched to recipients matching is done for the most important HLA antigens known to be involved, it has not technically been possible to match for all possible antigens. However, the human genome is organized into segments or blocks of closely linked genetic variants that are inherited as “haplotypes” on the same DNA strand of a chromosome. Most of the genes that code for HLA antigens are physically located together in one part of the human genome, known as the MHC region. Currently three HLA markers from this region (HLA-A, -B, -DRB1) are matched when matching donors and recipients. If it were possible to better map the structure of this region, it would be possible to better match recipients and donors (especially unrelated donors) for the unidentified transplantation antigens and reduce the chance of recipients getting GVHD or rejecting their grafts.
Why Was This Study Done?
Current strategies to define MHC haplotype blocks look at, on average, a length of only 18,000 base pairs and hence cannot define extended MHC haplotypes. Previously, this group of researchers developed a method of defining the HLA-A, B, DR haplotypes in recipients and their HLA-matched unrelated transplant donors using high-quality DNA containing 2 million base pairs across the MHC region. They wanted see if using this technique might provide a way to better assess the risk recipients have of developing GVHD or of having recurrent disease.
What Did the Researchers Do and Find?
They studied 246 HCT recipients and their donors who had been matched for HLA-A, -B, -C, -DRB1, -DQB1 by current techniques. The recipients were having HCT for a variety of hematological cancers: acute lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, or myelodysplastic syndrome. They found that, using the new technique, 22% of the donor–recipient pairs were haplotype-mismatched. Taking various other factors into account, including age, and patient and donor gender, MHC haplotype mismatching was associated with an approximately four times greater risk of severe acute GVHD but with a lower risk of disease recurrence. The lower risk of recurrence is believed to be because transplanted cells do not only replace abnormal cancerous cells but also react against them and therefore decrease the chance of the cancer recurring; mismatched cells are known to be more stimulated to react against the cancerous cells.
What Do These Findings Mean?
The results here suggest that this new haplotype matching method can provide a way to assess the risk of GVHD after HCT from unrelated donors, and in future could be considered as a technique to match donors and recipients.
Additional Information.
Please access these Web sites via the online version of this summary at
• Medline Plus has a page of information on stem cell transplantation, including HCT
• The Anthony Nolan Trust holds one of the largest databases of unrelated donors in the world
• The National Cancer Institute has a page of questions and answers on HCT
• The Center for International Blood & Marrow Transplant Research describes outcomes research in transplantation
• The National Marrow Donor Program describes how HLA-typed unrelated donors are identified
• The World Marrow Donor Association is involved in facilitating stem cell donation across international boundaries
PMCID: PMC1796628  PMID: 17378697

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