Further evidence of a primary, causal association of the PTPN22 620W variant with type 1 diabetes.

OBJECTIVE: The minor allele of the nonsynonymous single nucleotide polymorphism (SNP) +1858C>T within the PTPN22 gene is positively associated with type 1 diabetes and other autoimmune diseases. Genetic and functional data underline its causal effect, but some studies suggest that this polymorphism does not entirely explain disease association of the PTPN22 region. The aim of this study was to evaluate type 1 diabetes association within this gene in the Sardinian population. RESEARCH DESIGN AND METHODS: We resequenced the exons and potentially relevant portions of PTPN22 and detected 24 polymorphisms (23 SNPs and 1 deletion insertion polymorphism [DIP]), 8 of which were novel. A representative set of 14 SNPs and the DIP were sequentially genotyped and assessed for disease association in 794 families, 490 sporadic patients, and 721 matched control subjects. RESULTS: The +1858C>T variant, albeit rare in the general Sardinian population (allele frequency 0.014), was positively associated with type 1 diabetes (P(one tail) = 3.7 x 10(-3)). In contrast, the background haplotype in which this mutation occurred was common (haplotype frequency 0.117) and neutrally associated with disease. We did not confirm disease associations reported in other populations for non +1858C>T variants (rs2488457, rs1310182, and rs3811021), although they were present in appreciable frequencies in Sardinia. Additional weak disease associations with rare variants were detected in the Sardinian families but not confirmed in independent case-control sample sets and are most likely spurious. CONCLUSIONS: We provide further evidence that the +1858C>T polymorphism is primarily associated with type 1 diabetes and exclude major contributions from other purportedly relevant variants within this gene.

Somatic deletion of the normal beta-globin gene leading to thalassaemia intermedia in heterozygous beta-thalassaemic patients.

Two beta-thalassaemia patients, whose constitutive genotype was beta(39C)/beta(39C-->T), had the clinical phenotype beta-thalassaemia intermedia. Analysis of leucocyte DNA showed the presence of the mutated beta(39C-->T)-gene exclusively, while the normal beta(39C)-gene was also present in reticulocyte RNA. Deletional analysis of chromosome 11p15.5 on leucocyte DNA showed large deletions including the beta-globin gene. Two populations of erythroid progenitors, one heterozygous and the other hemizygous for the beta(39C-->T) mutation, were demonstrated in one case. This confirms that, in heterozygous individuals, beta-thalassaemia intermedia may be caused by inactivation of the beta-locus in trans as a result of chromosome 11p15.5 deletions in a subpopulation of haematopoietic cells.

Beta-thalassemia microelectronic chip: a fast and accurate method for mutation detection.

BACKGROUND: beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area. METHODS: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. RESULTS: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. CONCLUSIONS: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.