Fas promoter polymorphisms: genetic predisposition to sarcoidosis in African-Americans.

Apoptosis may perpetuate some forms of inflammation. Of the apoptotic pathway proteins, Fas is particularly overexpressed in sarcoidosis. We hypothesized that Fas promoter single nucleotide polymorphisms (SNPs) contribute to the development and severity of sarcoidosis. Associations of known Fas promoter SNPs (-670, -690 and -1377) and deduced haplotypes with sarcoidosis and sarcoidosis severity were evaluated using matched case-control (n = 656 pairs) and case-comparison (n = 656) studies, respectively, using conditional logistic regression. Hardy-Weinberg equilibrium was confirmed for all three polymorphisms in African-Americans (AA), and for the -670 and -1377 in whites. Genotype and allele frequencies were significantly different between whites and AA. Race-stratified analysis revealed that a common haplotype, -1377G/-690T/-670G, was associated with sarcoidosis [odds ratio (OR) = 1.78, P = 0.05] only in AA. The haplotype -1377G/-690C/-670A was negatively associated with sarcoidosis (OR = 0.39, P = 0.03) only in AA. In conclusion, the consistency of these findings suggests that Fas promoter genetic variants may be related to sarcoidosis disease risk in AA.

Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF.

BACKGROUND: While idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease, the aetiology of IPF is poorly understood. Familial cases of pulmonary fibrosis suggest a genetic basis for some forms of the disease. Recent reports have linked genetic mutations in surfactant protein C (SFTPC) with familial forms of pulmonary fibrosis, including one large family in which a number of family members were diagnosed with usual interstitial pneumonitis (UIP), the pathological correlate to IPF. Because of this finding in familial cases of pulmonary fibrosis, we searched for SFTPC mutations in a cohort of sporadic cases of UIP and non-specific interstitial pneumonitis (NSIP). METHODS: The gene for SFTPC was sequenced in 89 patients diagnosed with UIP, 46 patients with NSIP, and 104 normal controls. RESULTS: Ten single nucleotide polymorphisms in the SFTPC sequence were found in IPF patients and not in controls. Only one of these created an exonic change resulting in a change in amino acid sequence. In this case, a T to C substitution resulted in a change in amino acid 73 of the precursor protein from isoleucine to threonine. Of the remaining polymorphisms, one was in the 5' UTR, two were exonic without predicted amino acid sequence changes, and six were intronic. One intronic mutation suggested a potential enhancement of a splicing site. CONCLUSIONS: Mutations in SFTPC are identified infrequently in this patient population. These findings indicate that SFTPC mutations do not contribute to the pathogenesis of IPF in the majority of sporadic cases.

High beryllium-stimulated TNF-alpha is associated with the -308 TNF-alpha promoter polymorphism and with clinical severity in chronic beryllium disease.

Beryllium (Be)-antigen stimulates tumor necrosis factor-alpha (TNF-alpha) from bronchoalveolar lavage (BAL) cells in chronic beryllium disease (CBD). This study tested the hypothesis that high concentrations of Be-stimulated TNF-alpha are related to polymorphisms in the TNF-alpha promoter and clinical markers of disease severity in CBD. Demographic and clinical information was obtained from patients with CBD (n = 20). TNF-alpha concentrations were measured in BAL cell culture supernatant by ELISA. A priori, we categorized CBD subjects as either high or low TNF-alpha producers using a cutoff of 1,500 pg/ml. The TNF-alpha promoter sequence, +64 to -1045, was determined by direct sequencing. Human leukocyte-associated antigen (HLA)-DPB1 and -DRB1 genotyping was determined by polymerase chain reaction (PCR). High Be-stimulated TNF-alpha was associated with TNF2 alleles, Hispanic ethnicity, presence of HLA-DPB1 Glu69, and absence of HLA-DR4. Be-stimulated TNF-alpha concentrations correlated with markers of disease severity, including chest radiograph, beryllium lymphocyte proliferation, and spirometry. We found no novel TNF-alpha promoter polymorphisms. These data suggest that the TNF2 A allele at -308 in the TNF-alpha promoter region is a functional polymorphism, associated with a high level of Be-antigen-stimulated TNF-alpha and that these high TNF-alpha levels indicate disease severity in CBD.

Human leukocyte antigen-DRB1 position 11 residues are a common protective marker for sarcoidosis.

Genetic factors, in particular human leukocyte antigens (HLAs) are important determinants of susceptibility to sarcoidosis, a chronic granulomatous disease of undetermined etiology. To clarify the role of HLA in sarcoidosis we determined HLA-DR and -DQ alleles in case-control samples from three European populations (United Kingdom, Czech, and Polish) and compared these results with those published for three additional populations (Italian, Japanese, and Scandinavian) to determine whether the HLA-DR and/or -DQ alleles act as ethnic-dependent, or ethnic-independent modifiers of disease risk. Although variations were apparent in the alleles associated with susceptibility, reductions in the frequency of alleles associated with protection were remarkably consistent in the six populations. Previously detected associations between single-nucleotide polymorphisms at the TAP2 locus and sarcoidosis were shown to be due to linkage disequilibrium with the HLA-DR locus. The protective HLA-DR alleles, which encode the DR1 and DR4 antigens, were found to share characteristic small hydrophobic residues at position 11, which were replaced by small hydrophilic residues in the remaining, nonprotective, HLA-DR alleles. This residue position is within a pocket of the HLA-DR complex antigen binding groove (designated P6), where it is the only variable amino acid and therefore determines the peptide binding preferences of this pocket. A highly significant reduction in the frequency of individuals carrying HLA-DR alleles with a hydrophobic residue at position 11 was observed in the sarcoidosis cases in the three populations we examined. This suggests this HLA-DR residue is an important protective marker in sarcoidosis.

Familial sarcoidosis is linked to the major histocompatibility complex region.

Sarcoidosis is a systemic granulomatous disorder associated with high CD4+ cell activity, but no pathogen is detectable. Clustering in families occurs, and the existence of a genetic predisposition to sarcoidosis is widely accepted. The major histocompatibility complex (MHC) is believed to contribute to this susceptibility. Many studies testing this hypothesis have produced conflicting results. We have genotyped 122 affected siblings from 55 families for seven DNA polymorphisms that flank and cover the MHC region on chromosome 6, and for HLA-DPB1, a candidate gene for granulomatous disorders. Multipoint nonparametric linkage (NPL) analysis showed linkage (NPL score > 2.5; p < 0.006) for the entire MHC region with a maximum NPL score of 3.2 (p = 0.0008) at marker locus D6S1666 in the Class III gene cluster. There was a significant excess of marker haplotype sharing among affected siblings. However, the frequency of HLA-DPB1 alleles on 104 shared chromosomes did not differ from that of a control group of founders from the family panel. Transmission disequilibrium was found for allele DPB1*0201, but only nine families contributed to this result. We conclude that genes of the MHC are involved in the genetic predisposition to sarcoidosis, but HLA-DPB1 alone does not sufficiently explain this fact.

Epidemiology of familial sarcoidosis in the UK.

BACKGROUND: The last comprehensive epidemiological studies on familial sarcoidosis in the UK were more than 25 years ago, reporting another affected family member in 1.7% of index cases. A significant proportion of like-sex over unlike-sex pairs, an excess of mother-child over father-child associations, and a preponderance of monozygous over dizygous twins was noted. Another study reported ethnic heterogeneity in familial disease. This study was undertaken to identify the risk ratio (lambda(S)) for siblings of familial sarcoidosis in the UK, to determine if the previous epidemiological findings have persisted, and to reassess whether ethnic heterogeneity prevails in familial disease. METHOD: Questionnaires were sent to 406 index patients. RESULTS: Two hundred and sixty eight replies (66%) were received. Twenty four of the original 406 index patients (5.91%) were found to have at least one other relative (first, second or third degree) with biopsy proven sarcoidosis. A lambda(S) value of 36-73 was calculated indicating significant familial clustering of the disease. Ethnically the families comprised 62.5% Caucasian, 29.2% Afro-Caribbean, and 8.3% Asian. Mean age at diagnosis was 39.8 years for women and 40.9 years for men with a male to female ratio of 1:1.7. This differed for the Asian families in which all the affected members were male. Three sets of female twins (two monozygous and one dizygous) were included. There was an equal distribution of like-sex (primarily female) and unlike-sex families as well as mother-child and father-child pairs. Pulmonary involvement was predominant irrespective of ethnicity, as was the need for corticosteroid treatment. CONCLUSIONS: These results support the theory that a shared determinant (either genetic or environmental) is operating in familial sarcoidosis and suggest that this determinant is similar for all ethnic groups.

Mannose-binding lectin promoter and structural gene variants in sarcoidosis.

BACKGROUND: Sarcoidosis is a chronic granulomatous disease of unknown aetiology. Studies have suggested that the causative agent may be an infectious micro-organism. The mannose binding lectin (MBL) is involved in innate immunity to a wide range of micro-organisms. Mutations in the promoter region and exon 1 of the MBL gene occur with high frequency and are associated with reduced serum levels of MBL and increased susceptibility to microbial diseases. This study investigated whether MBL variants predispose to sarcoidosis by increasing their susceptibility to micro-organisms. METHODS: MBL gene promoter and exon 1 variants were detected by sequence specific primer polymerase chain reaction (SSP-PCR) in 167 UK Caucasian sarcoidosis patients and 164 control subjects. Severity of pulmonary disease outcome among patients was assessed by radiography after a minimum of 4 years from disease onset and classified as mild, moderate, and severe disease categories, accordingly. RESULTS: MBL variant frequencies were similar in patients and controls studied. Among sarcoidosis patients, the frequencies of variants were similar regardless of severity of disease outcome. The average patient ages at time of diagnosis were similar for all MBL genotypes. CONCLUSIONS: MBL gene variants do not appear to influence susceptibility to sarcoidosis, age of disease onset, or severity of disease.

Analysis of MHC encoded antigen-processing genes TAP1 and TAP2 polymorphisms in sarcoidosis.

Sarcoidosis is a chronic granulomatous disease of unknown etiology. Several studies have suggested involvement of human leukocyte antigen (HLA) genes in sarcoidosis susceptibility. HLA associations described have not been consistent, possibly because of additional susceptibility genes adjacent to or within the major histocompatibility complex (MHC) such as genes for the transporter associated with antigen processing (TAP). The aim of this study was to analyze TAP gene polymorphisms in patients with sarcoidosis using the amplificatory refraction mutation system (ARMS) PCR. To determine whether any association between TAP gene variation and sarcoidosis was ethnic-independent we examined two European populations: 117 unrelated UK Caucasoid patients with sarcoidosis and 290 healthy UK control subjects, and 87 unrelated Polish Slavonic patients with sarcoidosis and 158 healthy Polish control subjects. We detected significant differences in TAP2 between the UK control and patient groups, and in TAP2 between the Polish control and patient groups. Comparing the UK and Polish control groups, we observed a difference in TAP1. Examination of HLA-DPB1 in our UK population showed no associations with disease or between variants at the TAP gene loci and HLA-DPB1 variants. These results suggest associations at the TAP loci occur independently of HLA-DPB1 associations, that TAP associations seen may be involved in determining sarcoidosis susceptibility, and that such susceptibilities differ between UK and Polish populations. This first study of TAP genes in UK and Polish sarcoid populations has demonstrated the importance of using multiple defined ethnic populations in defining the role genetic factors play in sarcoidosis susceptibility and the importance of candidate gene studies.

T-cell receptor gene usage in patients with fibrosing alveolitis and control subjects.

BACKGROUND: Fibrosing alveolitis is characterized by inflammation, fibrosis and increased numbers of activated CD4+ T-cells in the lower respiratory tract. The aims of this study were to compare the T-cell antigen receptor repertoire in the lungs of subjects with fibrosing alveolitis systemic sclerosis (FASSc) with cryptogenic fibrosing alveolitis (CFA) and normal control subjects, to determine whether FASSc is driven by a specific T-cell trigger and is determined by a T-cell driven immune response, and to assess the clonality of CD4+ and CD8+ TcR usage in subjects with FASSc. MATERIALS AND METHODS: We used reverse transcription polymerase chain reaction with specific V alpha- and V beta-chain primers to identify the TcR gene usage in biopsy material, bronchoalveolar lavage fluid or peripheral blood from our subjects. RESULTS: We found individual-specific restriction of V alpha- and V beta-chain usage in lung biopsies from patients and control subjects. To establish whether this was due to expression bias in the CD4+ or CD8+ T-cells and was restricted to the lung, the alpha beta-T-cell receptor chain usage was assessed in T-cell subsets separated from the lungs of patients with fibrosing alveolitis and was compared with that of the peripheral blood. There was no consistent difference in the expression of any variable family chain among the population studied, although there was a significant difference between lung and peripheral blood lymphocyte V beta-families in CD8+ T-cells (P = 0.0007). CONCLUSION: We conclude that there is individual TcR V alpha- and V beta-expression bias in subjects with fibrosing alveolitis.

Polymorphic analysis of the high-affinity tumor necrosis factor receptor 2.

The tumor necrosis factor receptor 2 (TNF-RII, CD120b, TNF-R p75/80) gene has recently been characterised. It is located on chromosome 1p362 and consists of 10 exons and 9 introns A number of biallelic polymorphisms have been found in exons 4, 6, 9 and 10 based on differences between published sequences. In this study we have used polymerase chain reaction methodology in association with sequence-specific primers (PCR-SSP) incorporating mismatches at the 3' end to identify these polymorphisms. We were able to confirm the presence of a single biallelic polymorphism in exon 6 corresponding to a (T/G) at nucleotide 676 of TNF-RII mRNA (gb:M32315) which results in an amino acid change and three biallelic polymorphisms in exon 10 (in the3'UTR) corresponding to (A/G) at nucleotide 1663, (T/G) at nucleotide 1668 and a (C/T) at nucleotide 1690 of gb:M32315, whereas no polymorphisms were observed in exons 4 and 9. Here we report that in 192 unrelated UK Caucasian individuals the allele frequencies determined by direct counting were: 676-T (0.77), 1663-G (0.51), 1668-T (0.95), and 1690-T (0.64) and the calculated gene frequencies were; 676-T (0.52), 676-G (0.12); 1663-G (0.30), 1663-A (0.28); 1668-T (0.77), 1668-G (0.025); and 1690-T (0.40), 1690-C (0.20). Furthermore, the presence of an A allele at nucleotide position 1663 was found to be strongly associated with the presence of a C allele at nucleotide position 1690 and a G allele at nucleotide position 1668 whereas the presence of a G allele at position 1663 was associated with the absence of a C allele at nucleotide position 1690.

HLA-DPB polymorphisms: Glu 69 association with sarcoidosis.

Sarcoidosis is a chronic granulomatous disorder, which is characterized by the accumulation of activated CD4+ T lymphocytes (T cells) at disease sites. There is up-regulation of cell surface expression of MHC molecules in sarcoidosis, and it has been suggested that specific MHC class II alleles are associated with the disease. A study of chronic beryllium disease (CBD), a granulomatous disorder which is pathologically similar to sarcoidosis, has identified an association between this disease and the presence of a glutamine residue at position 69 (Glu 69+) of the B1 chain of the HLA-DPB molecule. A further study also suggested the importance of Glu at position 55 of the same chain. The aims of the present study were to attempt to define MHC class II alleles associated with sarcoidosis by comparison of their frequency in two groups of subjects and to compare the frequency of HLA-DPB1 Glu 69+/- and Glu 55+/-alleles in the same subjects. Forty-one subjects with sarcoidosis and 76 normal subjects were studied. The polymorphic regions of the class II MHC were identified by PCR in association with sequence-specific oligonucleotide probes. There were no significant differences in the phenotype frequencies of MHC class II or Glu 55+ alleles between the two groups of subjects. However, there was a significant increase (P = 0.02) in the frequency of HLA-DPB1* Glu 69+ alleles compared with the control population. We therefore suggest that the presence of a Glu residue at position 69 on the DPB1 chain may play an important role in antigen presentation and recognition in chronic granulomatous diseases.