The CR1 C5507G polymorphism is not involved in susceptibility to idiopathic pulmonary fibrosis in two European populations.

Idiopathic pulmonary fibrosis (IPF), a severe lung disease with unknown aetiology, is thought to have an important genetic component. Single nucleotide polymorphism, C5507G, of the complement receptor 1 (CR1) gene, which affects the number of CR1 molecules on erythrocytes, has been associated with susceptibility to IPF in a single European population. To replicate this finding, 53 Czech IPF patients with 203 Czech healthy control subjects and 70 English IPF patients with 149 English controls were investigated. In both populations, there were no significant differences in distribution of CR1 C5507G variants between IPF patients and their appropriate control groups. In conclusion, the association of the CR1 C5507G polymorphism with susceptibility to IPF was not reproducible in Czech and English populations.

A polymorphism in the promoter region of the CD86 (B7.2) gene is associated with systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral autoimmunity. Strong T-cell-dependent autoantibody and HLA associations are found in SSc subsets. The co-stimulatory molecule, CD86, expressed by antigen-presenting cells, plays a crucial role in priming naïve lymphocytes. We hypothesized that SSc, or one of the disease subsets, could be associated with single-nucleotide polymorphisms of the CD86 gene. Using sequence specific primer-polymerase chain reaction (SSP-PCR) methodology, we assessed four CD86 polymorphisms in 221 patients with SSc and 227 healthy control subjects from the UK. Haplotypes were constructed by inference and confirmed using PHASE algorithm. We found a strong association between SSc and a specific haplotype (haplotype 5), which was more prevalent in patients than in controls (29% vs 15%, OR = 2.3, chi(2) = 12, P = 0.0005). This association could be attributed to the novel -3479 promoter polymorphism; a significant difference was observed in the distribution of the CD86 -3479 G allele in patients with SSc compared to controls (43.7% vs. 32.4%, OR = 1.7, chi(2) = 12.1, P = 0.0005). TRANSFAC analyses suggest that the CD86-3479T allele contains putative GATA and TBP sites, whereas G allele does not. We assessed the relative DNA protein-binding activity of the -3479 polymorphism in vitro using electromobility gel shift assays (EMSA), which showed that the -3479G allele has less binding affinity compared to the T allele for nuclear proteins. These findings highlight the importance of co-stimulatory pathways in SSc pathogenesis.

Single-nucleotide polymorphisms in the SPARC gene are not associated with susceptibility to scleroderma.

OBJECTIVE: SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that modulates cell-cell and cell-extracellular matrix interactions. SPARC expression is restricted mainly to sites of tissue remodelling and wound repair, and is prominent in fibrotic disorders. Single-nucleotide polymorphisms (SNPs) in the SPARC gene are reportedly linked to scleroderma in four ethnic groups: Choctaw Indians, Caucasians, African Americans and Mexican Americans. We set out to reproduce and to positionally clone these disease associations in a set of UK Caucasian scleroderma patients and ethnically matched controls. METHODS: One hundred and twenty-one scleroderma subjects and 200 controls were genotyped by polymerase chain reaction with sequence-specific primers differing only in the 3' nucleotide corresponding to each allele of the biallelic SNPs. Scleroderma patients were analysed against controls and on the basis of their fibrosing alveolitis status as judged by high-resolution computed tomography evaluation and the extent of cutaneous involvement. RESULTS: Eight biallelic SNPs were genotyped: three from the last untranslated exon, which had been described previously, and an additional five novel SNPs: two in the promoter region, one in exon three and two in the 3' untranslated region. Six major haplotypes were constructed across all eight SNP positions. No significant differences in genotype, allele or haplotype frequency were observed between scleroderma and controls or within scleroderma subgroups. CONCLUSIONS: SNPs in the SPARC gene are not associated with susceptibility to scleroderma. This research adds to the genetic knowledge of the SPARC gene by identifying five novel SNPs spanning the whole gene and inserting these within the context of clearly defined haplotypes.

TNF and TNFR polymorphisms in severe sepsis and septic shock: a prospective multicentre study.

Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced in sepsis. Studies examining the association of individual TNF single nucleotide polymorphisms with sepsis have produced conflicting results. This study investigated whether common polymorphisms of the TNF locus and the two receptor genes, TNFRSF1A and TNFRSF1B, influence circulating levels of encoded proteins, and whether individual polymorphisms or extended haplotypes of these genes are associated with susceptibility, severity of illness or outcome in adult patients with severe sepsis or septic shock. A total of 213 Caucasian patients were recruited from eight intensive care units (ICU) in the UK and Australia. Plasma levels of TNF (P = 0.02), sTNFRSF1A (P = 0.005) and sTNFRSF1B (P = 0.01) were significantly higher in those who died on ICU compared to those who survived. There was a positive correlation between increasing soluble receptor levels and organ dysfunction (increasing SOFA score) (sTNFRSF1A R = 0.51, P < 0.001; sTNFRSF1B R = 0.53, P < 0.001), and in particular with the degree of renal dysfunction. In this study, there were no significant associations between the selected candidate TNF or TNF receptor polymorphisms, or their haplotypes, and susceptibility to sepsis, illness severity or outcome. The influence of polymorphisms of the TNF locus on susceptibility to, and outcome from sepsis remains uncertain.

A single round PCR method for genotyping human surfactant protein (SP)-A1, SP-A2 and SP-D gene alleles.

The genes coding for the human surfactant proteins (SP)-A and SP-D are located on chromosome 10q22-q23.1. SP-D is the product of a single gene whereas SP-A is the product of two highly homologous genes SP-A1 and SP-A2. Several single nucleotide polymorphisms (SNP) are present in the SP-A1, SP-A2 and SP-D genes. Because of this high degree of sequence homology between the SP-A1 and SP-A2 genes, current genetic analysis studies employ a nested PCR/radioactive hybridization or restriction fragment length polymorphism approach to initially isolate the genes and subsequently to detect the SNP in these isolates. In this manuscript, we report the primers and conditions of a sequence specific primer-PCR methodology that enables the identification of SP-A1, SP-A2 and SP-D gene allelic variants directly on genomic DNA material.