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Summary Several studies have demonstrated an association of cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) (IDDM 12) alanine 17 with type 1 diabetes, but we wished to study the parental effect of CTLA-4 49 A/G dimorphism in diabetic families. The CTLA-4 exon 1 polymorphism (49 A/G), HLA-DRB1 and insulin gene (INS) variable number tandem repeats (VNTR) were analysed in 134 type 1 diabetic patients vs. 273 control subjects. The segregation analysis for transmission was carried out in 70 informative diabetic families using the transmission distortion test (TDT). All genotyping was performed by PCR-RFLP. CTLA-4 49 G allele frequency was not increased in diabetic patients compared to controls (41 vs. 38%, not significant). The distribution of GG, AG and AA CTLA-4 genotypes was similar in the two groups (13, 57 and 30% vs. 11, 54 and 35%, respectively) and was independent of HLA-DRB1 or INS VNTR polymorphism. The CTLA-4 49 G allele showed weak distorted transmission to the diabetic offspring, whereas random transmission was observed in unaffected offspring. This distortion is attributable to a maternal effect (71% compared to the 50% expected ratio; tdt = 4.8; P

Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs increase in diabetes and modulate cellular functions through binding to a specific cell surface receptor (RAGE). The RAGE gene maps to chromosome 6p in the HLA class III area and is telomeric to the class II region at 250 kb from DRA. A recent report described the characterization of a major RAGE gene variant as a biallelic single base polymorphism (G/A 557) in the exon 3 sequence leading to a change of a glycine to a serine at position 82. Using DGGE and PCR-RFLP, we have investigated the distribution of this dimorphism in conjunction with HLA class II genes in large populations of type 1 diabetic patients and healthy subjects. Although no association of this RAGE gene polymorphism with disease susceptibility was found, we report a strong linkage disequilibrium between the variant carrying the serine amino acid at position 82 and two HLA-DR2 and HLA-DR4 specificities. In particular, we describe two major extensive HLA class II haplotypes associated with this serine variant and identified as DRB1*0401-DQA1*0301-DQB1*0301 in the diabetic group and DRB1*1501-DQA1*0102-DQB1*0602 in control individuals. These data were partially confirmed by family transmission analysis.

The HLA class II typing of 167 unrelated Gabonese individuals from the Banzabi ethnic group was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The most frequent alleles at each locus were DRB1*1501-3 (0.31), DQA1*0102 (0.50), DQB1*0602 (0.42) and DPB1*0402 (0.29). The estimation of the haplotype frequencies as well as the observation of the segregation of several haplotypes using additional HLA typing of relatives, revealed that the three-locus haplotype DRB1*1501-3-DQA1*0102-DQB1*0602 was found at the highest frequency (0.31) among these individuals. This haplotype is not typically African and has already been described in Caucasians, but its presence at high frequency is exclusive to populations originating from Central Africa, and can thus be designated as a particular genetic marker of these populations.

Gestational diabetes mellitus (GDM) and impaired glucose tolerance during pregnancy (IGT) are associated with an increased risk of perinatal morbidity and then further development of diabetes among 30-50% of affected women. This is a real public health problem that deserves investigation of phenotypic and genotypic predisposing markers. However, the involvement of genetic background in GDM and IGT remains unclear. In particular, association with HLA class II polymorphism has been poorly studied and has produced conflicting results. In attempt to clarify these discrepancies, we investigated HLA class II polymorphism in 95 GDM and 95 IGT women from the north of France using DNA amplification followed by restriction enzyme digestion (PCR-RFLP). Ninety-five pregnant women with normal glucose tolerance (NGT) were chosen as a control reference group. The distribution of HLA class II polymorphism was not found to be significantly different between GDM, IGT and NGT samples. In particular, we did not find any significant variation of DRB1*03 and DRB1*04 allele frequencies between these three groups. These data provide further evidence that insulin-dependent diabetes mellitus (IDDM) HLA class II susceptibility alleles cannot serve as genetic markers for susceptibility to glucose intolerance during pregnancy. However, GDM and IGT were not equivalent to the NGT control group and presented particular HLA patterns. In particular, we observed an increase of the DRB1*0701-DQA1*0201-DQB1*02 haplotype in GDM women (P = 0.02; Pc not significant) and an increase of DRB1*0101-DQA1*0101-DQB1*0501 and DRB1*1302-DQA1*0102-DQB1*0604 haplotypes in the IGT group (P = 0.02 and 8 x 10(-3), respectively; Pc not significant). In contrast, we found a decrease in the DRB1*1101 allele in IGT samples (P = 0.03; Pc not significant) and a decrease of DRB1*1103-*1104 alleles in the GDM group (P = 9 x 10(-3); Pc not significant). Although these findings are only descriptive, it points out the genetic heterogeneity of glucose intolerance during pregnancy.

Objectives Fetal macrosomia is a common complication of maternal diabetes mellitus and is associated with substantial morbidity, but the precise cellular and molecular mechanisms that induce fetal macrosomia are not well understood. The imprinted genes IGF-II and H19 are crucial for placental development and fetal growth. The term placentas from diabetic pregnancies express more insulin-like growth factor II (IGF-II) than those from normal pregnancies. Deregulation of their imprinting status is observed in the macrosomia-associated syndrome, the Beckwith–Wiedemann syndrome. The aim of this study was to determine whether loss of imprinting hence biallelic expression was also a hallmark of macrosomia in diabetic pregnancies. Design and methods IGF-II and H19 maternal and paternal expressions were studied in placentas from two groups of type 1 diabetic mothers: one with macrosomic babies and the other with babies of normal weight. Maternal or paternal allele specific expressions were defined by using DNA polymorphic markers of the IGF-II and H19 genes. RFLP analysis was performed on PCR products from genomic DNA of the father, the mother and the child, and on RT-PCR products from placental mRNA. Results RFLP analysis showed that the IGF-II gene remains paternally expressed and the H19 gene remains maternally expressed in all placentas examined, independently of the birth weight status. Conclusions These results suggest that, in contrast with Beckwith–Wiedemann syndrome-associated macrosomia, loss of imprinting for IGF-II or H19 is not a common feature of diabetic pregnancies associated with macrosomia.

Summary Objectives We investigated the association of the RAGE (Receptor for Advanced Glycation End products) exon3 gene polymorphisms with stages of nephropathy in type 1 diabetes. Methods: The RAGE exon 3 genotype was assessed by Denaturing Gradient Gel Electrophoresis (DGGE) procedure in 487 type 1 diabetic patients with proliferative retinopathy subdivided into four groups according to their level of renal involvement and in 351 control subjects (GENEDIAB study). Results We reported here three main low frequency dimorphisms, previously submitted to data banks, Gly82Ser, Val89 CTC/CTG, and Arg77Cys. The genotype distribution of these polymorphisms was not statistically different in type 1 diabetic patients compared to healthy controls (p = 0.37). Among the three described polymorphisms, only the RAGE Gly82Ser genotype frequency was significantly increased in the group with advanced nephropathy (11%) defined by a chronic renal failure compared to the three others groups: no nephropathy, 5%; incipient (microalbuminuria) 5%; established (macroalbuminuria), 2%) (P = 0.04). The 82 Ser allele was identified as an independent risk marker for the stage of advanced nephropathy: adjusted odds ratio 3.17(95% CI 1,32-7,85, p = 0.008). Conclusion These data suggest that the 82 Ser allele of the RAGE gene is a risk allele for developing advanced nephropathy. This suggests that some RAGE gene polymorphisms may be associated with progression to diabetic advanced nephropathy in Caucasian type 1 diabetic patients.

Several studies have demonstrated an association of cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) (IDDM 12) alanine 17 with type 1 diabetes, but we wished to study the parental effect of CTLA-4 49 A/G dimorphism in diabetic families. The CTLA-4 exon 1 polymorphism (49 A/G), HLA-DRB1 and insulin gene (INS) variable number tandem repeats (VNTR) were analysed in 134 type 1 diabetic patients vs. 273 control subjects. The segregation analysis for transmission was carried out in 70 informative diabetic families using the transmission distortion test (TDT). All genotyping was performed by PCR-RFLP. CTLA-4 49 G allele frequency was not increased in diabetic patients compared to controls (41 vs. 38%, not significant). The distribution of GG, AG and AA CTLA-4 genotypes was similar in the two groups (13, 57 and 30% vs. 11, 54 and 35%, respectively) and was independent of HLA-DRB1 or INS VNTR polymorphism. The CTLA-4 49 G allele showed weak distorted transmission to the diabetic offspring, whereas random transmission was observed in unaffected offspring. This distortion is attributable to a maternal effect (71% compared to the 50% expected ratio; tdt = 4.8; P0.03). The combined transmission of maternal CTLA-4 G with HLA-DRB1*03 (90%; tdt = 6.4; P0.01) and VNTR class I (80%; tdt = 5.4; P0.02) enhanced the susceptibility effect of each marker separately. We noted a slight CTLA-4 49 G and HLA-DRB1*04 distortion of transmission shared in paternal and maternal diabetic meiosis. In non-diabetic offspring, the CTLA-4 49 A allele confers a protective effect in the presence of maternal HLA-DRB1*03 and paternal HLA-DRB1*04 alleles. Despite the absence of a positive association of the CTLA-4 49 G allele with type 1 diabetes, our segregation analysis supports the hypothesis of a modulation by CTLA-4 49 G/A dimorphism of the susceptibility conferred by maternal HLA-DRB1*03 inheritance. This potential parental effect needs to be confirmed in a larger data set.

Cellular responses to synthetic peptides from the Liver Stage Antigen-1 (LSA-1) from Plasmodium falciparum were determined in 229 Gabonese children. HLA class I and II typing (by PCR-SSP and -RFLP, respectively) revealed that HLA-A*19, -B*17 (and -B*70), -DRB1*05, -DQA1*0102, -DQB1*0602 and -DPB1*0402 were the most frequent types or alleles at each locus. The DQB1*0201 and DQB1*0301 alleles were present at a higher frequency among IL-6 and IFN-gamma responders to the LSA-Rep and LSA-CTL peptides, respectively, and a higher proportion of these responders carried A*19 or B*53. The DRB1*06 type was positively related to the IL-10 production in response to the LSA-CTL peptide, and responders presented mainly A*2. The specificity A*10 was negatively associated with the cellular response to the LSA-J peptide. These results suggest a degree of genetic regulation of specific immune responses by HLA-A, operating at the pre-erythrocytic stage of development of P. falciparum in this Central African population.

Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs increase in diabetes and modulate cellular functions through binding to a specific cell surface receptor (RAGE). The RAGE gene maps to chromosome 6p in the HLA class III area and is telomeric to the class II region at 250 kb from DRA. A recent report described the characterization of a major RAGE gene variant as a biallelic single base polymorphism (G/A 557) in the exon 3 sequence leading to a change of a glycine to a serine at position 82. Using DGGE and PCR-RFLP, we have investigated the distribution of this dimorphism in conjunction with HLA class II genes in large populations of type 1 diabetic patients and healthy subjects. Although no association of this RAGE gene polymorphism with disease susceptibility was found, we report a strong linkage disequilibrium between the variant carrying the serine amino acid at position 82 and two HLA-DR2 and HLA-DR4 specificities. In particular, we describe two major extensive HLA class II haplotypes associated with this serine variant and identified as DRB1*0401-DQA1*0301-DQB1*0301 in the diabetic group and DRB1*1501-DQA1*0102-DQB1*0602 in control individuals. These data were partially confirmed by family transmission analysis.

The HLA class II typing of 167 unrelated Gabonese individuals from the Banzabi ethnic group was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The most frequent alleles at each locus were DRB1*1501-3 (0.31), DQA1*0102 (0.50), DQB1*0602 (0.42) and DPB1*0402 (0.29). The estimation of the haplotype frequencies as well as the observation of the segregation of several haplotypes using additional HLA typing of relatives, revealed that the three-locus haplotype DRB1*1501-3-DQA1*0102-DQB1*0602 was found at the highest frequency (0.31) among these individuals. This haplotype is not typically African and has already been described in Caucasians, but its presence at high frequency is exclusive to populations originating from Central Africa, and can thus be designated as a particular genetic marker of these populations.

Insulin-dependent diabetes mellitus is a polygenic disease with an environmental component. Technological advances and large collection families allowed genetic factors understanding. On clinical practice, two questions could be raised. First, will the genetic markers be of interest in disease prediction either in families studies or in the general population? Second, will the genetic approach explain the physiopathological process of the disease? Initially, the gene candidate approach led to the identification of two important loci. Linkage with the HLA locus showed the importance of the autoimmune part. Linkage of insulin-dependent diabetes mellitus with insulin locus gave a mechanistic answer for disease susceptibility. These two loci can be used as prediction markers, but only in family studies. Since 1993, a whole genome approach was performed and led to the identification of other susceptibility loci. These initial results are in progress and should have important implications for public health strategies.

Chronic diseases are polymorph and influenced by many environmental and genetic factors. The HLA system is implicated in the modulation the onset and the evolution of chronic diseases. These observations are important to be considered for the prediction, prognosis and treatment adaptation. Evaluation tests are mainly statistical and are based on epidemiological studies. Thus, results must be considered with caution. Two aspects are to be considered:Associations between HLA alleles and chronic diseases are well known but concern very few diseases like narcolepsy or rheumatoid arthritis. Insulin dependent diabetes mellitus is particular because the prediction is limited to familial studies.This point is less documented in clinical applications but is of interest particularly in inflammatory bowel diseases or systemic affections. This observation can be considered as a compartmental response which is a kind of adaptation to stress.

Some studies have suggested that distribution of HLA DRB1 alleles in polymyalgia rheumatica (PMR) resembles that found in giant cell arteritis (GCA). However these data are controversial. OBJECTIVE--To evaluate in French native patients whether PMR immunogenetically resembles GCA in determining HLA DRB1 alleles. PATIENTS AND METHODS--Fourty-five patients were included in the study. Twenty-one patients with PMR alone (Bird's criteria) and 24 with GCA (ACR criteria). In 11 patients, GCA was associated with PMR. HLA DRB1 genotype was determined by PCR-RFLP analysis. Statistical analysis was performed by the chi 2 test and determination of the odds ratio (OR). Two hundred and thirty-three unselected normal healthy subjects served as controls. RESULTS--A significant increased prevalence of HLA DR1 was observed in patient with PMR alone and an absence of DR7 (0% vs 10.3%, p = 0.02, OR = 0.1). An increased incidence of DR4 and particularly *0401 allele was only found in patients with GCA (OR = 2.4). No patient with isolated PMR had DR7 genotype compared with 25% in GCA (p0.001, OR = 0.03). A comparative study between isolated PMR versus GCA showed a significant increased in DR1 and DR3 alleles in isolated PMR and a significant increased prevalence of DR4 and DRB1 *0701 allele in GCA. CONCLUSION--The present study emphasizes the absence of similarity in HLA DRB1 allele distribution between PMR and GCA. The association of DR7 in patient with GCA seems characteristic in French native patients.

The aim of the present study was to determine HLA DRB1* genotype by PCR-RFLP analysis in patients with rheumatoid arthritis (N = 122) associated with extra-articular manifestations (N = 24). A significant increased of DRB1* 0405 was found in RA patients with extra-cellular involvement (odd-ratio = 8.33, confidence interval = 1.44 - 56.7). In patients with RA, HLA DRB1*0405 allele might constitute a susceptibility marker of extra-articular involvement such as vasulitis or cardio-pulmonary manifestations.

The authors have conducted a comparative retrospective study between polymyalgia rheumatic (N = 26) and rheumatoid arthritis in the elderly (N = 44), including HLA DRB1 genotype determination by PCR-RFLP analysis. No clinical nor biological differences were significant between the 2 groups of patients. However 70% of RA patients had one ore more susceptibility alleles (shared epitope hypothesis) and 50% in polymyalgia rheumatica.

To investigate the relationships between predominant HLA class II alleles and immune responses to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), 50 individuals from the highlands of Madagascar were followed-up from 1988 to 1991. The T cell reactivity and antibody responses to synthetic peptides (EENV) 4 , (EENVEHDA) 4 , and (DDEHVEEPTVA) 3 , representing major T and B epitopes of Pf155/RESA antigen, were assessed with an average of five determinations per individual over the four-year follow-up period. The T cell reactivity was investigated by lymphocyte proliferation and assays for interferon-γ and interleukin-2 release. Anti-peptide antibodies were measured using the Falcon assay screening test-enzyme-linked immunosorbent assay. The cumulative prevalence rates of cellular (range for the three peptides = 64-68%) and antibody responders (range = 70-74%) were similar for each peptide. The HLA class II typing was performed using polymerase chain reaction- restriction fragment length polymorphisms. The prevalent alleles or groups of alleles (frequency > 20%) were similar in responders and nonresponders, both for cellular and antibody responses to each peptide. These were HLA-DR 5 group and HLA-DQAI *0601, *0101-0102-0104, HLA-DQB1 *0301, and HLA-DPB1 *0101-2601 alleles. Allelic distribution was similar in individuals presenting with (74%) or without (26%) a malaria attack during a 20-week follow-up conducted when malaria was hyperendemic (P > 0.05, by Fisher's exact test). Despite repeated immunologic measures that better identify the responders, no relationship was found between HLA class II alleles and the cellular or antibody responses to Pf155/RESA epitopes. If immune responses to Pf155/RESA epitopes or susceptibility to malaria attacks are genetically regulated, our data suggest the HLA class II region is not involved