The association between the PTPN22 1858C>T variant and type 1 diabetes depends on HLA risk and GAD65 autoantibodies.

The single nucleotide polymorphism 1858C>T in the PTPN22 gene is associated with type 1 diabetes (T1D) in several populations. Earlier reports have suggested that the association may be modified by human leukocyte antigen (HLA), as well as by islet autoantibodies. In a large case-control study of Swedish incident T1D patients and controls, 0-34 years of age, we tested whether the odds ratio (OR) measure of association was dependent on HLA or autoantibodies against the islet autoantigens glutamic acid decarboxylase 65 kDa autoantibodies (GADA), insulin, islet antigen-2, or islet cell. The association between the carrier status of 1858C>T allele in PTPN22 (PTPN22(CT+TT)) and T1D was modified by HLA. In addition, in GADA-positive T1D, the OR was 2.83 (2.00, 3.99), whereas in GADA-negative T1D, the OR was 1.41 (0.98, 2.04) (P for comparison=0.007). The OR of association between PTPN22(CT+TT) and GADA-positive T1D declined with increasing HLA-risk category from 6.12 to 1.54 (P=0.003); no such change was detected in GADA-negative T1D (P=0.722) (P for comparison=0.001). However, the absolute difference in risk between PTPN22(CC) and PTPN22(CT+TT) subjects with high-risk HLA was five times higher than that for subjects with low-risk HLA. We hypothesize that the altered T-cell function because of the PTPN22(1858C>T) polymorphism is exclusively associated with GADA-positive T1D at diagnosis.

Polymorphisms in Toll-like receptor 4 (TLR4) are associated with protection against leprosy.

Accumulating evidence suggests that polymorphisms in Toll-like receptors (TLRs) influence the pathogenesis of mycobacterial infections, including leprosy, a disease whose manifestations depend on host immune responses. Polymorphisms in TLR2 are associated with an increased risk of reversal reaction, but not susceptibility to leprosy itself. We examined whether polymorphisms in TLR4 are associated with susceptibility to leprosy in a cohort of 441 Ethiopian leprosy patients and 197 healthy controls. We found that two single nucleotide polymorphisms (SNPs) in TLR4 (896G>A [D299G] and 1196C>T [T399I]) were associated with a protective effect against the disease. The 896GG, GA and AA genotypes were found in 91.7, 7.8 and 0.5% of leprosy cases versus 79.9, 19.1 and 1.0% of controls, respectively (odds ratio [OR] = 0.34, 95% confidence interval [CI] 0.20-0.57, P < 0.001, additive model). Similarly, the 1196CC, CT and TT genotypes were found in 98.1, 1.9 and 0% of leprosy cases versus 91.8, 7.7 and 0.5% of controls, respectively (OR = 0.16, 95% CI 0.06--.40, P < 0.001, dominant model). We found that Mycobacterium leprae stimulation of monocytes partially inhibited their subsequent response to lipopolysaccharide (LPS) stimulation. Our data suggest that TLR4 polymorphisms are associated with susceptibility to leprosy and that this effect may be mediated at the cellular level by the modulation of TLR4 signalling by M. leprae.

IA-2 autoantibodies in incident type I diabetes patients are associated with a polyadenylation signal polymorphism in GIMAP5.

In a large case-control study of Swedish incident type I diabetes patients and controls, 0-34 years of age, we tested the hypothesis that the GIMAP5 gene, a key genetic factor for lymphopenia in spontaneous BioBreeding rat diabetes, is associated with type I diabetes; with islet autoantibodies in incident type I diabetes patients or with age at clinical onset in incident type I diabetes patients. Initial scans of allelic association were followed by more detailed logistic regression modeling that adjusted for known type I diabetes risk factors and potential confounding variables. The single nucleotide polymorphism (SNP) rs6598, located in a polyadenylation signal of GIMAP5, was associated with the presence of significant levels of IA-2 autoantibodies in the type I diabetes patients. Patients with the minor allele A of rs6598 had an increased prevalence of IA-2 autoantibody levels compared to patients without the minor allele (OR=2.2; Bonferroni-corrected P=0.003), after adjusting for age at clinical onset (P=8.0 x 10(-13)) and the numbers of HLA-DQ A1*0501-B1*0201 haplotypes (P=2.4 x 10(-5)) and DQ A1*0301-B1*0302 haplotypes (P=0.002). GIMAP5 polymorphism was not associated with type I diabetes or with GAD65 or insulin autoantibodies, ICA, or age at clinical onset in patients. These data suggest that the GIMAP5 gene is associated with islet autoimmunity in type I diabetes and add to recent findings implicating the same SNP in another autoimmune disease.

Glutamate cysteine ligase catalytic subunit promoter polymorphisms and associations with type 1 diabetes age-at-onset and GAD65 autoantibody levels.

The purpose of this study was to test the hypothesis that glutamate cysteine ligase catalytic subunit (GCLC) promoter polymorphisms are susceptibility factors for type 1 diabetes (T1D), T1D age-at-onset and T1D autoantibodies. T1D patients and control subjects from the Swedish Childhood Diabetes Registry and the Swedish Diabetes Incidence Study registry were genotyped for two GCLC promoter polymorphisms; the GCLC -129 C to T single nucleotide polymorphism (GCLC -129 SNP) and the GCLC GAG trinucleotide repeat polymorphism (GCLC TNR). Glutamate decarboxylase antibody (GAD65Ab) positive T1D patients with the GCLC -129 SNP C/T genotype have increased GAD65Ab levels (p-value, <0.05) compared to the GCLC -129 SNP C/C genotype. T1D patients with an age-at-onset of 14-35 years who possess the GCLC -129 SNP T/T genotype have a higher GAD65Ab index than T1D patients with the GCLC -129 SNP C/C genotype (p-value <0.05). In addition, T1D patients with an age-at-onset of 14-35 years possess the GCLC TNR 7/8 genotype at a lower frequency than the control subjects (OR, 0.33, 95% CI, 0.13-0.82). The GCLC -129 SNP and GCLC TNR appear to be in linkage disequilibrium (p-value<0.0001). These results suggest that GCLC promoter polymorphisms may influence GAD65Ab levels and may influence the age at which T1D is diagnosed.

[Neonatal conjunctivitis in a hospital at Gran Buenos Aires. Last 5 years up-date]. / Conjuntivitis neonatal en un hospital del Gran Buenos Aires. Situación de los últimos 5 años.

Neonatal conjunctivitis is one of the most important consultation reasons in the first days of life. Causes may be chemical or infectious. Neonatal conjunctivitis study for common microorganisms and Chlamydia trachomatis is being performed at Posadas Hospital since 1995, in a progressive form. The objective of this study was to know the epidemiological situation in the last five years (1999-2003), and to describe the variations detected between two periods, 1995-1998, and 1999-2003. It was observed no change about C. trachomatis prevalence (8%), during all the time in the population studied. With regard to common microorganisms it was showed a decrease in the second period (36.6%) with respect to the first (52.4%). Likely, the causes may be due to changes about sample collection conditions, or to epidemiological variations. The most frequent microorganisms found were: Staphylococcus aureus 69 (27.6%), Streptococcus pneumoniae 68 (27.2%), and Haemophilus influenzae 64 (25.6%). We consider important to evaluate the kind of ocular secretions at the first consultation, which can lead to a symptomatic treatment that will be corroborated or not, according to microbiological results.

Conjuntivitis neonatal en un hospital del Gran Buenos Aires. Situación de los últimos 5 años / [Neonatal conjunctivitis in a hospital at Gran Buenos Aires. Last 5 years up-date]

Neonatal conjunctivitis is one of the most important consultation reasons in the first days of life. Causes may be chemical or infectious. Neonatal conjunctivitis study for common microorganisms and Chlamydia trachomatis is being performed at Posadas Hospital since 1995, in a progressive form. The objective of this study was to know the epidemiological situation in the last five years (1999-2003), and to describe the variations detected between two periods, 1995-1998, and 1999-2003. It was observed no change about C. trachomatis prevalence (8

), during all the time in the population studied. With regard to common microorganisms it was showed a decrease in the second period (36.6

) with respect to the first (52.4

). Likely, the causes may be due to changes about sample collection conditions, or to epidemiological variations. The most frequent microorganisms found were: Staphylococcus aureus 69 (27.6

), and Haemophilus influenzae 64 (25.6

). We consider important to evaluate the kind of ocular secretions at the first consultation, which can lead to a symptomatic treatment that will be corroborated or not, according to microbiological results.

High GAD65 autoantibody levels in nondiabetic adults are associated with HLA but not with CTLA-4 or INS VNTR.

OBJECTIVES: To explore the relationship between genetic background and antibody levels in a nondiabetic population. We evaluated if high levels of autoantibodies against the 65 kDa isoform of glutamic acid decarboxylase (GAD65Ab), were associated with high-risk genes, i.e. HLA, CTLA-4 and INS VNTR genes. DESIGN AND SUBJECTS: Seventy-five (M/F 39/36) subjects exceeding the 95th percentile of GAD65 autoantibody index and 75 age and sex matched subjects below the 95th percentile, randomly selected amongst participants in the Västerbotten Intervention Programme. METHODS: The GAD65 Ab were measured in a radioligand-binding assay. HLA class II typing was performed by an oligoblot hybridization method. CTLA-4 repeat length was analysed and divided into short forms and long forms. Class I and class III alleles of INS VNTR were detected. Differences in distribution were tested by Pearson chi-square with Yates correction. Odds ratios (OR) were used to compare groups calculated with Cochran's and Mantel-Haenszel statistics. RESULTS: The DQB1*0201-DQA1*0501-DRB1*03 haplotype was increased in subjects with high GAD65Ab levels (P = 0.04). This increase seemed to be explained by a difference in haplotype frequencies amongst men (P = 0.01). Calculating OR showed a significant association between the DQB1*0201-DQA1*0501-DRB1*03 haplotype and elevated levels of GAD65Ab in all subjects (OR 2.2, 95% CI 1.02-4.9) as well as in men (OR 4.6, 95% CI 1.3-15.9). There was no association between high levels of GAD65Ab and either INS VNTR or CTLA-4 polymorphisms. CONCLUSION: Our study suggests that adult males with the DQB1*0201-DQA1*0501-DRB1*03 haplotype tend to develop high GAD65Ab titres. As none of these subjects have developed diabetes these data suggest that HLA may be important in GAD65Ab formation but that additional factors are required for the progression to overt type 1 diabetes.

Oestrogen-induced bone marrow aplasia in a dog with a Sertoli cell tumour.

A 10-year-old male Drahthaar dog with unilateral cryptorchidism was examined because of feminisation and myelotoxicity. Radiography and ultrasonography revealed an abdominal mass which was surgically removed. The mass was identified as a Sertoli cell tumour on histological examination. Findings on bone marrow examination were compatible with aplasia due to the oestrogens secreted by the tumoral cells. Treatment with fluids, antibiotics, whole blood transfusions, corticosteroids and lithium carbonate was unsuccessful. Survival time was 22 days after surgery.

Incidence of Chlamydia trachomatis and other potential pathogens in neonatal conjunctivitis.

OBJECTIVE: Ocular infection in neonatology is a permanent and important health problem. To improve primary attention, prevention, and control, the study of the potential bacterial etiology of all consecutive cases of conjunctivitis was incorporated as a regular procedure in primary care from July 1995 to December 1998. MATERIALS AND METHODS: Prof. A. Posadas Hospital (Great Buenos Aires) has an average of 4294 births per year. This report analyzes the results obtained in 332 infants (age range, 0-30 d) with conjunctivitis. Clinical conjunctivitis was diagnosed in inpatients and outpatients by the same specialized staff. Isolation and characterization of bacteria were done by conventional microbiologic methods, including specific search for Neisseria gonorrhoeae and Chlamydia trachomatis. Chlamydia trachomatis was studied by antigen immunodetection and polymerase chain reaction, and genotyped by restriction fragment length polymorphism. RESULTS: Conjunctivitis had an incidence (cases per 1000 live births) of 39.6 in 1995, 25.3 in 1996, 15.4 in 1997, and 15.2 in 1998. Microbial growth was detected in 167 (50.3%) of 332 cases. Ocular C. trachomatis infection was detected in 26 cases (7.83%). Five of seven isolates in tissue cultures belonged to type E and two to type G. Bacteria from respiratory ecology were the main isolates: Haemophilus influenzae (16.9%), Streptococcus pneumoniae (12.3%), and Staphylococcus aureus (8.7%). Haemophilus influenzae isolates were not serotyped and 17.2% of them were b-lactamase producers. In 15 cases both H. influenzae and S. pneumoniae were isolated together. Of S. pneumoniae, 4.9% were oxacillin resistant. CONCLUSIONS: There has been a decline in the total number of cases of neonatal conjunctivitis, but the disease is still an important health problem. Chlamydia trachomatis also shows a decreasing profile with an incidence of (cases per 1000 live births) 4.39 in 1995, 1.85 in 1996, 1.01 in 1997, and 0.78 in 1998, and a tendency to show more incidence in spring-summer and significant accumulation of cases in babies between 7 and 9 days of age. Haemophilus influenzae alone (12.3%) or associated with S. pneumoniae (4.5%) appears as a prevalent potential bacterial pathogen. A significant accumulation of H. influenzae and S. pneumoniae cases occurs in winter. In 47.6% of cases, there was no bacterial growth. No significant seasonal differences in percentage of negative cultures or among the three-day age groups were detected. Neisseria gonorrhoeae was not found associated with ophthalmia neonatorum in this series.

Clinical characteristics of prostate cancer in an analysis of linkage to four putative susceptibility loci.

PURPOSE: Hereditary prostate cancer is an etiologically heterogeneous disease with six susceptibility loci mapped to date. We aimed to describe a collection of high-risk prostate cancer families and assess linkage to multiple markers at four loci: HPC1 (1q24-25), PCaP (1q42.2-43), HPCX (Xq27-28), and CAPB (1p36). EXPERIMENTAL DESIGN: Medical record data on 505 affected men in 149 multiply-affected prostate cancer families were reviewed, and correlations of clinical traits within each family were calculated. Logarithm of odds (LOD) score and nonparametric (NPL) linkage analyses were performed; white families were stratified by age of diagnosis, grade and stage of disease, and evidence of linkage to the other loci to increase genetic homogeneity. RESULTS: Age at diagnosis was the most correlated clinical trait within families. A maximum NPL score of 2.61 (P = 0.007) appeared to confirm HPC1 linkage for families that had a prevalence of high-grade or advanced-stage prostate cancer and which were not likely to be linked to PCaP, HPCX, or CAPB. Because the NPL scores improved when families more likely to be linked to the other loci were excluded, HPC1 may act independently of the other loci. The relationship of HPC1 and aggressive disease was strongest in families with median age at diagnosis > or =65 years (NPL, 3.48; P = 0.0008). CONCLUSIONS: The current results suggest that HPC1 linkage may be most common among families with more severe prostate cancer. Stratification by clinical characteristics may be a useful tool in prostate cancer linkage analyses and may increase our understanding of hereditary prostate cancer.

Germline mutations in the p73 gene do not predispose to familial prostate-brain cancer.

BACKGROUND: Analysis of high-risk prostate cancer (PC) families with at least one confirmed case of primary brain cancer (BC) has identified a region of genetic linkage on chromosome 1p36 termed CAPB. The p36 region of chromosome one has been reported to have frequent loss of heterozygosity (LOH) in brain and central nervous system (CNS) tumors and epidemiological studies have shown an increased relative risk of BC and tumors of the CNS in PC families. In 1997 a reported tumor suppressor with high homology to p53, termed p73, was mapped to the p36 region of chromosome one. Here, we examine the p73 gene as a potential candidate for CAPB. METHODS: Ninety-four members from the 12 prostate-brain cancer families in which linkage was originally found were examined. The complete coding region and intron-exon boundaries of the p73 gene were analyzed for germline mutations by Single Stranded Conformational Polymorphism analysis (SSCP) and direct DNA sequencing. RESULTS: Silent nucleotide substitutions only were detected within the coding regions of the gene in affected individuals. Nucleotide changes were detected in introns 1, 6, 8, 9, and 10, but all were located >or=16 base pairs from the splice site, and are thus unlikely to be deleterious mutations. CONCLUSIONS: Germline mutations in the p73 gene are unlikely to be critical for inherited susceptibility to PC in this specified subset of families.

Genetic linkage analysis of prostate cancer families to Xq27-28.

OBJECTIVES: A recent linkage analysis of 360 families at high risk for prostate cancer identified the q27-28 region on chromosome X as the potential location of a gene involved in prostate cancer susceptibility. Here we report on linkage analysis at this putative HPCX locus in an independent set of 186 prostate cancer families participating in the Prostate Cancer Genetic Research Study (PROGRESS). METHODS: DNA samples from these families were genotyped at 8 polymorphic markers spanning 14.3 cM of the HPCX region. RESULTS: Two-point parametric analysis of the total data set resulted in positive lod scores at only two markers, DXS984 and DXS1193, with scores of 0.628 at a recombination fraction (theta) of 0.36 and 0.012 at theta = 0.48, respectively. The stratification of pedigrees according to the assumed mode of transmission increased the evidence of linkage at DXS984 in 81 families with no evidence of male-to-male transmission (lod = 1.062 at theta = 0.28). CONCLUSIONS: Although this analysis did not show statistically significant evidence for the linkage of prostate cancer susceptibility to Xq27-28, the results are consistent with a small percentage of families being linked to this region. The analysis further highlights difficulties in replicating linkage results in an etiologically heterogeneous, complexly inherited disease.

A genomic scan of families with prostate cancer identifies multiple regions of interest.

A 10-cM genomewide scan of 94 families with hereditary prostate cancer, including 432 affected men, was used to identify regions of putative prostate cancer-susceptibility loci. There was an average of 3.6 affected, genotyped men per family, and an overall mean age at diagnosis of 65.4 years. A total of 50 families were classified as early onset (mean age at diagnosis <66 years), and 44 families were classified as later onset (mean age at diagnosis > or =66 years). When the entire data set is considered, regions of interest (LOD score > or =1.5) were identified on chromosomes 10, 12, and 14, with a dominant model of inheritance. Under a recessive model LOD scores > or =1.5 were found on chromosomes 1, 8, 10, and 16. Stratification by age at diagnosis highlighted a putative susceptibility locus on chromosome 11, among the later-onset families, with a LOD score of 3. 02 (recombination fraction 0) at marker ATA34E08. Overall, this genomic scan suggests that there are multiple prostate cancer loci responsible for the hereditary form of this common and complex disease and that stratification by a variety of factors will be required for identification of all relevant genes.

A susceptibility region for myasthenia gravis extending into the HLA-class I sector telomeric to HLA-C.

We have analyzed a series of HLA region markers in 207 UK Caucasoids with early-onset myasthenia gravis (EOMG, onset before age 40), where there is a strong female bias. The well known associations with HLA-DR3 and -B8 have now proved to be significantly stronger in the 165 females than in the 42 males. In patients (of either sex) lacking -DR3, there was also a significant increase in HLA-DR2. Although the muscle weakness in EOMG is clearly mediated by autoantibodies, the associations are consistently stronger with HLA-B8 (in class I) than with HLADR3 (in class II), as confirmed here. We therefore typed 87-137 cases for polymorphisms at four loci in the intervening class III region, and also at three in the adjacent stretch of class I. At each locus, one allele tended to co-occur with HLA-B8 and showed strong and highly significant associations in the patients. There appeared to be a region of maximal susceptibility extending from HSP70 (in class III) past HLA-B and HLA-C at least 600 kb telomerically into the class I region, which is now being mapped in detail. Any candidate genes here that act shortly after puberty may allow more precise localization of susceptibility.

Mapping HLA for single nucleotide polymorphisms.

Knowledge of DNA sequence variation may help us understand how genetic variability gives rise to functional variability and, in so doing, revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1,000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human leukocyte antigen (HLA) complex. Interestingly, the precise molecular defects in the HLA genes are unknown and the notion that non-HLA genes, within the same chromosomal region, are involved remains a formal possibility. We have determined the nucleotide sequence of a contiguous 2.2 Mbp segment of chromosome six that includes all of the HLA class I region, and have identified over 10,000 SNPs therein. Because of the derivative knowledge of gene and SNP content and position, the scientific community is now uniquely poised to identify disease-contributory SNPs that lie within the MHC.

The human major histocompatibility complex: 42,221 bp of genomic sequence, high-density sequence-tagged site map, evolution, and polymorphism for HLA class I.

We report the isolation and characterization of newly identified yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones spanning the HLA class I region between HLA-C and HLA-E and of YACs extending telomeric of HLA-F. When included with previously characterized HLA class I YACs, a contiguous stretch of over 2.4 Mb pairs including the entire class I region has been isolated as a series of overlapping YAC and BAC clones. Evidence that the cloned DNA faithfully represents the source genomic DNA was obtained by extensive characterization of the YACs and by independent isolation of two or more overlapping YACs or BACs spanning the entire region. As a result of this work, over 80 unique sequence probes were identified, the majority of which were sequenced to yield 42,221 bp of new major histocompatibility complex (MHC)-derived sequence. Some of these data were reduced to sequenced tagged site primer sets, facilitating the isolation of all or nearly all of HLA class I from a variety of genomic libraries. The sequence data were analyzed for protein coding capacity and homology to existing expressed tagged sites and tested for conservation of sequences in other mammalian genomes. These results indicated that large portions of the HLA class I region are conserved among mammals. Measurements of polymorphism within non-HLA class I loci generated additional data pointing toward information of potential relevance to MHC-associated diseases. The combined data and clones presented here set the stage for the determination of the complete nucleotide sequence of HLA class I.

The complete genomic sequence of 424,015 bp at the centromeric end of the HLA class I region: gene content and polymorphism.

We report here the genomic sequence of the centromeric portion of HLA class I, extending 424,015 bp from tumor necrosis factor alpha to a newly identified gene approximately 20 kb telomeric of Otf-3. As a source of DNA, we used cosmids centromeric of HLA-B that had been mapped previously with conventional restriction digestion and fingerprinting and previously characterized yeast artificial chromosomes subcloned into cosmids and mapped with multiple complete digest methodologies. The data presented provide a description of the gene content of centromeric HLA class I including new data on intron, promoter and flanking sequences of previously described genes, and a description of putative new genes that remain to be characterized beyond the structural information uncovered. A complete accounting of the repeat structure including abundant di-, tri-, and tetranucleotide microsatellite loci yielded access to precisely localized mapping tools for the major histocompatibility complex. Comparative analysis of a highly polymorphic region between HLA-B and -C was carried out by sequencing over 40 kb of overlapping sequence from two haplotypes. The levels of variation observed were much higher than those seen in other regions of the genome and indeed were higher than those observed between allelic HLA class I loci.