Relative predispositional effects of HLA class II DRB1-DQB1 haplotypes and genotypes on type 1 diabetes: a meta-analysis.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

Molecular aspects of a novel HLA-A*02 allele (A*0297): the first HLA class I allele mutated at codon 232.

We describe a new HLA-A*02 allele, identified in a cord blood unit and in her mother. Nucleotide sequence analysis showed the presence of a new HLA-A*02 allele identical to HLA-A*02010101 except for a non-synonymous nucleotide exchange in exon 4 modifying codon 232 from GAG (Glu) to GAC (Asp). No other human leucocyte antigen class I allele sequenced so far shows this triplet at codon 232. The amino acid exchange affects a position that is part of the membrane proximal domain of class I major histocompatibility complex (MHC), designated alpha 3, and involved in the interaction with CD8 molecule. Using molecular modelling approach, the interactions between different subunits of the native and mutated forms of MHC-I resulted in relevant changes.

Role of non-HLA genetic polymorphisms in graft-versus-host disease after haematopoietic stem cell transplantation.

Graft-versus-host disease (GvHD) is the main complication after haematopoietic stem cells transplantation (HSCT) and acute forms (aGvHD) occur in 20-40% of cases even after donor (D) and recipient (R) HLA matching, apparently because of D/R minor histocompatibility antigen (mHA) mismatches and cytokine polymorphisms. The genotype of cytokines and mHA of 77 haematological R following HSCT from HLA identical siblings were determined to detect genetic polymorphisms correlated with GvHD. We analysed TNFA (-863 C/A, -857 C/T and G/A at positions -574, -376, -308, -244, -238), IL-10 (-1082 G/A, -819 C/A, -592 C/T), IL-1B (T/C +3953), IL-1RA (VNTR), HA-1 (H/R allele) and CD-31 (C/G at codon 125, A/G at codon 563). Allele frequencies were in Hardy-Weinberg equilibrium and similar to those of 77 healthy controls. We observed positive correlations between a lower risk of clinically significant aGvHD and both the presence of -1082G -819C -592C IL-10 haplotype when both R and D are considered together and the absence of R IL-1RA allele 2. Furthermore, we observed an association between the absence of TNF-A -238 A allele and the risk of extensive chronic GvHD. mHA and cytokines genotyping would thus seem a valid source of information for the prior identification of recipients with a higher risk of aGvHD.

Identification of a new allele, HLA-DRB5*0113, through three different molecular biology techniques.

A new HLA-DRB5 allele, HLA-DRB5*0113, has been identified in an Italian patient during routine HLA typing in order to activate a bone marrow donor search. HLA typing was performed by different molecular biology techniques, and the results showed that the HLA-DRB5*0113 allele differs from HLA-DRB5*010101 allele for three nucleotide substitutions at codons 57 (GAC-->GAT; Asp) and 58 (GCT-->GAG; Ala-->Glu) of exon 2.

Identification of a new HLA-DRB1 allele in three members of an Italian family.

Abstract A new human leucocyte antigen (HLA)-DRB1 allele, HLA-DRB1*1149, has been identified in three members of an Italian family during routine sequence based typing. This new allele differs from HLA-DRB1*110101 only for a single nucleotide substitution at position 113 of exon 2 resulting in an amino acid change from Valine (GTG) to Alanine (GCG) at codon 38.

New HLA-A*11 allele, A*1112, identified by sequence-based typing.

In this report, we describe the identification of HLA-A*1112, a novel HLA-A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence-specific primer and was confirmed by high-resolution sequencing-based typing. The nucleotide sequences of HLA-A*1112 exons 2 and 3 are identical to HLA-A*11011 except for a single nucleotide substitution in codon 90 (GAC-->GCC).

Cytotoxic T lymphocyte precursor frequency as a predictor of acute graft-versus-host disease in bone marrow transplantation from HLA-identical siblings.

The measurement of precursor frequencies of donor anti-recipient cytotoxic T lymphocytes (CTL-p) has been shown to predict the incidence and the severity of acute graft-versus-host disease (aGVHD) in unrelated donor bone marrow transplantation (BMT). In HLA-identical sibling BMT, where aGVHD is most likely caused by minor histocompatibility antigen mismatches, this assay did not appear to be sensitive enough to provide similar predictive information. In this study, the CTL-p frequencies and the incidence and severity of aGVHD in 51 onco-hematological patients transplanted from HLA-identical siblings were compared. Sibling donors were selected on the basis of HLA identity using serological typing for HLA-A, B, C antigens, whereas HLA-DRB was tested by molecular analysis. Sibling identity was also confirmed by DNA heteroduplex analyses. Fifteen out of 21 (71%) patients with high precursor frequency (>1:100 x 10(3)) and 12 out of 30 (40%) with low precursor frequency (<1:100 x 10(3)) experienced clinically significant (II-IV) aGVHD. A significant correlation (P = 0.04) between CTL-p frequency and severe aGVHD was demonstrated. Moreover there was a positive trend for a high frequency response according to an increasing grade of aGVHD, which was statistically significant (P = 0.04). In our experience the CTL-p assay is a helpful predictive test for aGVHD in HLA-identical sibling BMT, indicating high risk patients possibly requiring additional prophylaxis.

Analysis of the turin umbilical cord blood bank registry.

BACKGROUND: The polymorphic nature of the HLA system reduces a patient's probability of finding an HLA-compatible unrelated bone marrow (BM) donor, even though more than 6 million individuals are enrolled in international registries. Recently, umbilical cord blood (UCB) has been successfully employed as a source of HPCs. The use of such cells reduces the risk of GVHD and allows transplants with one or two HLA mismatches. UCB represents an expensive resource: therefore, it is necessary to carefully manage the UCB unit inventory. STUDY DESIGN AND METHODS: The current study analyzed the genetic heterogeneity of HLA-A, -B, and -DR gene frequencies between pools of UCB and unrelated-donor BM in the Piedmont (an administrative region of Italy). An Italian hematology patient's probability of finding complete or partial matches as a function of donor pool size was determined by considering subsamples randomly selected from the local unrelated BM donors. RESULTS: The HLA gene frequencies in UCB and unrelated-donor BM pools were not significantly different. The search simulation, based on actual HLA phenotypes, showed that the percentage of Italian patients matched with an HPC unit increases remarkably if 1 or 2 mismatches are accepted, reaching a proportion of 90 percent with an inventory of only about 500 units, while the increment is not so remarkable if the number of UCB units is greater. CONCLUSION: To optimize economic resources and to be internationally competitive, UCB banks should aim to increase the genetic heterogeneity of their units rather than increasing the UCB inventory, acquire efficient quality control systems, and acquire and preserve UCB units with a greater number of nucleated cells.

Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection.

BACKGROUND: A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. MATERIALS AND METHODS: To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. RESULTS: No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). CONCLUSIONS: Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.

Polymorphisms in angiotensin-converting enzyme gene and severity of renal disease in Henoch-Schoenlein patients. Italian Group of Renal Immunopathology.

BACKGROUND: The influence of angiotensin converting enzyme (ACE) gene polymorphism on the progression of primary IgA nephropathy (pIgAN) is still debated. Even though the allele frequency was reported to be similar to controls, in some studies D/D patients had a faster decline of renal function and need of dialysis. Since Henoch-Schoenlein purpura (HSP) nephritis is considered a systemic vasculitis with renal lesions indistinguishable from pIgAN, we investigated the effect of the ACE polymorphism on presentation and progression of HSP IgAN. METHODS: We examined the insertion (I) and deletion (D) polymorphism in intron 16 of ACE gene by PCR amplification of genomic DNA of 82 patients (37 children), with biopsy-proven IgAN associated with HSP enrolled in a collaborative study. RESULTS: No significant association with clinical presentation at onset or with final outcome was found (functional impairment at outcome in 31.8%, D/D, 27.4%, I/D and 44% I/I, heavy proteinuria in 36.3% D/D, 21.6% I/D, and 11.1% I/I). Patients homozygous for the D allele had a greater number of extrarenal relapses (P=0.0028). No association was found between the ACE genotype and the presence of hypertension at onset and at the end of the follow-up. No difference was found between adults and children. CONCLUSIONS: In this cohort of HSP IgAN, no ACE I/D polymorphisms were found to be associated with progressive deterioration of renal function. Different genes possibly involved in vasculitis might more strictly modulate expression and evolution of HSP IgAN.

Are HLA class II and immunoglobulin constant region genes involved in the pathogenesis of mixed cryoglobulinemia type II after hepatitis C virus infection?

BACKGROUND/AIMS: Hepatitis C virus infection is known to play an important role in the pathogenesis of essential mixed cryoglobulinemia type II. Progression of hepatitis C virus infection to mixed cryoglobulinemia may be influenced by host immune response. To analyze the immunogenetic background of mixed cryoglobulinemia, we studied HLA-DR, DQ loci and the switch regions of immunoglobulin heavy chain gamma1 and gamma4 constant genes. METHODS: HLA typing was performed in 84 hepatitis C virus-infected patients (46 with cryoglobulins and 38 without), and 109 healthy controls, through analysis of restriction fragment length polymorphisms, supplemented with other techniques. Immunoglobulin heavy chain gamma1 and gamma4 polymorphisms, detected by restriction fragment length polymorphisms, were studied in 41 patients with mixed cryoglobulinemia and 51 controls. RESULTS: The gene frequency of DRB1*11 was significantly higher in patients with mixed cryoglobulinemia than in controls (0.36 and 0.20, respectively; p= 0.0035). However, DRB1*11 was also increased in the subgroup of patients without mixed cryoglobulinemia who did not develop severe liver disease, while it was decreased in those with severe liver damage (0.50 and 0.13; p=0.0035). The frequency of 5.4 kb allele of the immunoglobulin heavy chain gamma1 switch region was higher in patients with mixed cryoglobulinemia than in controls (0.47 and 0.22; pc=0.002), while the frequency of 5.5 kb allele was lower (0.51 and 0.78; pc= 0.001). CONCLUSIONS: Susceptibility to develop cryoglobulins after hepatitis C virus infection was not associated with HLA-DR or DQ. HLA-DRB1*11-positive individuals were protected from serious chronic liver disease after hepatitis C virus infection. Immunoglobulin heavy chain constant gamma1 switch region restriction fragment length polymorphisms were associated with mixed cryoglobulinemia.

Immunogenetics of Henoch-Schoenlein disease.

In order to investigate the genetic basis of susceptibility to Henoch-Schoenlein purpura (HS), blood samples of 152 patients, 105 of whom had renal disease, were collected in a two-step study. The evaluation of DRB, DQB and DQA polymorphism was done by analysis of the restriction polymorphisms produced by TaqI enzyme. DRB1*07 was less frequent in patients than in the control group (gene frequency 0.09 and 0.18, respectively; P = 0.0023), whereas 64% of the patients were positive for DRB1*01 and/or DRB1*11 compared with 48% of the control group (P = 0.0069). Polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) typing of DRB1*01- and DRB1*11-positive individuals did not show any deviation of frequencies of DRB1*01 subtypes between patients and controls, whereas among DRB1*11 subtypes DRB1*1104 was significantly increased in the patients (Pc = 0.033). The comparison between patients with renal disease and those without renal disease showed no significant differences in the frequency of the single DRB, DQB and DQA alleles. The study of restriction polymorphisms in the switch region of the constant genes alpha 1, alpha 2 and mu of the heavy chains of immunoglobulins, using the enzyme Sacl and a specific probe, did not show any difference between 44 patients and 54 controls. This study demonstrates that susceptibility to HS also has a genetic origin: on one hand, the presence of DRB1*01 or DRB1*11 makes disease onset easier; on the other hand, DRB1*07 could induce some resistance to the disease. It is suggested that, as well as for other diseases caused by an impaired immune response, single amino acids in a key position in the HLA-DRB molecule make it more or less easy to recognize some antigenic peptide, towards which an immune response leading to disease is triggered.

Human leukocyte antigen class II polymorphisms and genetic susceptibility of IDDM in Egyptian children.

OBJECTIVE: To analyze the association between human leukocyte antigen (HLA) and insulin-dependent diabetes mellitus (IDDM) in the Egyptian population for the first time and, thus, to determine the frequency of risk-associated alleles identified by a genomic HLA class II typing. Egyptians are genetically classified as North Africans and considered to be between Caucasoids and Africans (closer to Caucasoids). RESEARCH DESIGN AND METHODS: HLA class II typing was performed for 50 IDDM patients and 50 healthy control subjects by a restriction fragment-length polymorphism (RFLP) technique. The analysis of position 57 of the DQB1 molecules was conducted by polymerase chain reaction and specific sequence oligonucleotide hybridization. RESULTS: The frequency of DRB1*0301-DRB3*0201-DQA1*0501-DQB1*0201 haplotype was 43.9% in the IDDM patients and 7.1% in the control subjects (P < 0.00001), reflecting the increased prevalence of DQA1*0501 susceptibility allele coding for arginine (Arg) in position 52 and DQB1*0201 susceptibility allele non-coding aspartic acid (Asp) at position 57. Alleles DQB1*0601 and 0603, both carrying Asp at position 57 of the beta-chain, and DQA1*0103, encoding a non-Arg 52 alpha-chain, were significantly decreased among the IDDM patients. The presence of four susceptibility residues (two DQA1 Arg 52+ and two DQB1 Asp 57-) conferred the highest relative risk at 20.2. On the other hand, homozygous genotypes for DQA1 non-Arg 52 and DQB1 Asp 57 were found only in the control group. CONCLUSIONS: IDDM susceptibility and resistance in the Egyptian population is strongly associated with the expressed DQ alpha- and beta-heterodimers in a dose-effective manner, as already defined in many different ethnic groups.

Identification of unrelated HLA-identical bone marrow donors: RFLP, oligotyping and PCR fingerprinting for HLA class II compared to MLC responses.

22 patients awaiting bone marrow transplantation (BMT) and 65 unrelated healthy HLA-A, B and DR serologically identical donors were studied. 21 patients were non reactive on mixed lymphocyte culture (MLC) towards at least one BMT donor, resulting in 32 pairs MLC-negative. 33 other HLA-matched donors gave proliferative responses on MLC. The phenotype DR3/DR7 was significantly higher in patients (P) and donors (D) studied (p < 0.00001). P and D pairs were DNA typed by RFLP analysis for DRB, DQA and DQB genes, and DNA matched by PCR Fingerprinting (PCRF) for DRB, DQA, DQB and DPB. Six patients and 18 donors were also oligotyped for the subtypes of DR1, DR3, DR4 and DR5. Patients and donors were divided according to identity on RFLP, PCRF and responsiveness on MLC, represented by RRI. In the group of MLC non responder pairs, 11% had differences on DRB PCRF compared to 57% in the group of MLC responders (p = 0.0002). The mean RRI value of PCRF DRB incompatible pairs was significantly higher compared to RRI of compatible pairs (p = 0.012). With respect to PCRF for DPB, 75% of MLC-ve pairs were different. Also the mean RRI value was significantly lower in DPB identical pairs compared to non identical ones (p = 0.048). The compatibility between P/D pairs assessed by oligotyping was in accordance with DRB PCRF. PCRF for DQB, but not for DQA, corresponded to MLC responses. Our findings confirm that PCRF offers a precise and fast alternative in DNA matching for DRB. We also suggest that PCRF for DQB and DPB, together with DRB, could eventually substitute MLC.

Immunoglobulin and HLA-DP genes contribute to the susceptibility to juvenile dermatitis herpetiformis.

HLA-DQ genes and gluten diet are the main factors involved in the pathogenesis of Dermatitis Herpetiformis (DH), as well as Coeliac Disease (CD). However other genetic factors are probably relevant, since about 10% of the patients with DH and CD lack the DQA1*0501/B1*0201 heterodimer while the majority of individuals presenting this genotype and also being exposed to gluten diets did not suffer from these diseases. To evaluate the role of other genes, 36 Northern Italian children with DH were analysed for DNA polymorphisms at HLA-DP and immunoglobulin (Ig) heavy chain loci. DPA1*0201 and DPB1*1301 frequencies were higher in patients than in controls (Pc = 0.0357 and Pc = 0.0273). With respect to immunoglobulin heavy chain restriction fragment length polymorphisms (RFLP), the 4.6 kb SacI RFLP at the switch alpha 2 gene was more frequent in patients (0.13) than in controls (0.019; Pc = 0.036). Moreover, rare alleles or duplications in the switch regions occurred more frequently in the patients than in the controls. These results support the hypothesis of a multifactorial inheritance of DH, the HLA and Ig constant heavy chain genes being some of the loci contributing to the susceptibility. In accordance with previous CD studies, these data also confirm that DP subregion is probably involved in the pathogenesis of DH.

HLA class II gene frequencies in Italy.

The frequency of HLA alleles at HLA-DR and DQ loci, and that of the related HLA-D specificities, were estimated in the Italian population. 109 healthy unrelated subjects, born in several Italian regions and living in the district of Torino, were studied. DNA typing was achieved by the restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta, DQ alpha and beta genes, hybridizing specific probes with TaqI digested DNAs. The present study allowed to define in more detail the HLA class II polymorphisms in the Italian population.