Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Toll-like receptor 4 and CD14 polymorphisms in ankylosing spondylitis: Evidence of a weak association in finns

Objective To investigate the association of CD14 and Toll-like receptor (TLR4) with ankylosing spondylitis (AS). Methods A promoter variant in CD14 and 2 coding polymorphisms in TLR4 were investigated in UK and Finnish families with AS and in a UK case-control study. A metaanalysis of published TLR4 and CD14 studies was performed. Results In the Finnish study the CD74-260bp T variant showed an association (p = 0.006), and the common 2-marker TLR4 haplotype showed a weak association (global p = 0.03), with AS. No associations were seen in the UK based studies or in the metaanalyses. Conclusion CD14 and TLR4 showed an association with AS in the Finns only.

Influence of LRP5 polymorphisms on normal variation in BMD

Genetic studies based on cohorts with rare and extreme bone phenotypes have shown that the LRP5 gene is an important genetic modulator of BMD. Using family-based and case-control approaches, this study examines the role of the LRP5 gene in determining normal population variation of BMD and describes significant association and suggestive linkage between LRP5 gene polymorphisms and BMD in >900 individuals with a broad range of BMD. Introduction: Osteoporosis is a common, highly heritable condition determined by complex interactions of genetic and environmental etiologies. Genetic factors alone can account for 50-80% of the interindividual variation in BMD. Mutations in the LRP5 gene on chromosome 11q12-13 have been associated with rare syndromes characterized by extremely low or high BMD, but little is known about the contribution of this gene to the development of osteoporosis and determination of BMD in a normal population. Materials and Methods: To examine the entire spectrum of low to high BMD, 152 osteoporotic probands, their families (597 individuals), and 160 women with elevated BMD (T score > 2.5) were recruited. BMD at the lumbar spine, femoral neck, and hip were measured in each subject using DXA. Results: PAGE sequencing of the LRP5 gene revealed 10 single nucleotide polymorphisms (SNPs), 8 of which had allele frequencies of >5%, in exons 8, 9, 10, 15, and 18 and in introns 6, 7, and 21. Within families, a strong association was observed between an SNP at nucleotide C171346A in intron 21 and total hip BMD (p < 1 × 10-5 in men only, p = 0.0019 in both men and women). This association was also observed in comparisons of osteoporotic probands and unrelated elevated BMD in women (p = 0.03), along with associations with markers in exons 8 (C135242T, p = 0.007) and 9 (C141759T, p = 0.02). Haplotypes composed of two to three of the SNPs G121513A, C135242T, G138351A, and C141759T were strongly associated with BMD when comparing osteoporotic probands and high BMD cases (p < 0.003). An SNP at nucleotide C165215T in exon 18 was linked to BMD at the lumbar spine, femoral neck, and total hip (parametric LOD scores = 2.8, 2.5, and 2.2 and nonparametric LOD scores = 0.3, 1.1, and 2.2, respectively) but was not genetically associated with BMD variation. Conclusion: These results show that common LRP5 polymorphisms contribute to the determination of BMD in the general population.

The effect of transforming growth factor β1 gene polymorphisms in ankylosing spondylitis

Objectives. To determine whether genetic polymorphisms in or near the transforming growth factor β1 (TGFB1) locus were associated d with susceptibility to or severity of ankylosing spondylitis (AS). Methods. Five intragenic single-nucleotide polymorphisms (SNP) and three microsatellite markers flanking the TGFB1 locus were genotyped. Seven hundred and sixty-two individuals from 184 multiplex families were genotyped for the microsatellite markers and two of the promoter SNPs. One thousand and two individuals from 212 English and 170 Finnish families with AS were genotyped for all five intragenic SNPs. A structured questionnaire was used to assess the age of symptom onset, disease duration and disease severity scores, including the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) and BASFI (Bath Ankylosing Spondylitis Functional Index). Results. A weak association was noted between the rare TGFB1 + 1632 T allele and AS in the Finnish population (P = 0.04) and in the combined data set (P = 0.03). No association was noted between any other SNPs or SNP haplotype and AS, even among those families with positive non-parametric linkage scores. The TGFB1 +1632 polymorphism was also associated with a younger age of symptom onset (English population, allele 2 associated with age of onset greater by 4.2 yr, P = 0.05; combined data set, allele 2 associated with age of onset greater by 3.2 yr, P = 0.02). A haplotype of coding region SNPs (TGFB1 +869/ +915+1632 alleles 2/1/2) was associated with age of symptom onset in both the English parent-case trios and the combined data set (English data set, haplotype 2/1/2 associated with age of onset greater by 4.9 yr, P = 0.03; combined data set, haplotype 2/1/2 associated with greater age of onset by 4.2 yr, P = 0.006). Weak linkage with AS susceptibility was noted and the peak LOD score was 1.3 at distance 2 cM centromeric to the TGFB1 gene. No other linkage or association was found between quantitative traits and the markers. Conclusion. This study suggests that the polymorphisms within the TGFB1 gene play at most a small role in AS and that other genes encoded on chromosome 19 are involved in susceptibility to the disease.

Interleukin 10 polymorphisms in ankylosing spondylitis

Genetic polymorphisms of the IL10 promoter region have been implicated in many autoimmune diseases, including seronegative spondyloarthropathies. We studied three SNPs (IL10-1087,-824, and -597) and two microsafellites(IL10R and IL10G) lying within the promoter region of IL10 for association with susceptibility to and clinical manifestations of ankylosing spondylitis (AS), a common form of spondyloarthritis. Four hundred and sixty-eight individuals from 182 Finnish families affected with AS were studied. No association between individual IL10 promoter region polymorphisms or marker haplotype was observed with susceptibility to AS, but weak association was noted between the IL10-597 and -824 SNPs and age of disease onset (P= 0.01 for each SNP). The IL10.G4 allele was associated with BASFI (corrected for disease duration) (P= 0.03). We conclude that IL10 promoter polymorphisms have no significant effect on susceptibility to AS, but may play a minor role in determining age of disease onset and disease severity. © 2003 Nature Publishing Group All rights reserved.

Interethnic studies of TNF polymorphisms confirm the likely presence of a second MHC susceptibility locus in ankylosing spondylitis

The objective of this study was to investigate TNF promoter region polymorphisms for association with susceptibility to ankylosing spondylitis (AS). The TNF -238 and -308 polymorphisms were genotyped in 306 English AS cases and 204 ethnically matched healthy B27-positive controls, and 96 southern German AS cases, 58 B27-positive and 251 B27-negative ethnically matched controls. Additionally, the TNF -376 polymorphism was genotyped in the southern German cases and controls. In the southern German AS patients a significant reduction in TNF -308.2 alleles was seen, compared with B27 positive controls (odds ratio 0.4, P= 0.03, 95% confidence interval 0.2-0.9), but no difference in allele frequencies was observed at TNF -238. Significant association between AS and both TNF -238 and TNF -308 was excluded in the English cases. These results confirm previous observations in the southern German population of association between TNF promoter region polymorphisms and AS, but the lack of association in the English population suggests that these polymorphisms themselves are unlikely to be directly involved. More likely, a second, non-HLA-B, MHC locus is involved in susceptibility to AS in these two populations.

Polymorphisms of the CYP2D6 gene increase susceptibility to ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly familial rheumatic disorder. The sibling recurrence risk ratio for the disease is 63 and heritability assessed in twins > 90%. Although MHC genes, including HLA-B27, contribute only 20-50% of the genetic risk for the disease, no non-MHC gene has yet been convincingly demonstrated to influence either susceptibility to the disease or its phenotypic expression. Previous linkage and association studies have suggested the presence of a susceptibility gene for AS close to, or within, the cytochrome P450 2D6 gene (CYP2D6, debrisoquine hydroxylase) located at chromosome 22q13.1. We performed a linkage study of chromosome 22 in 200 families with AS affected sibling-pairs. Association of alleles of the CYP2D6 gene was examined by both case-control and within-family means. For case-control studies, 617 unrelated individuals with AS (361 probands from sibling-pair and parent-case trio families and 256 unrelated non-familial sporadic cases) and 402 healthy ethnically matched controls were employed. For within-family association studies, 361 families including 161 parent-case trios and 200 affected sibling-pair families were employed. Homozygosity for poor metabolizer alleles was found to be associated with AS. Heterozygosity for the most frequent poor metabolizer allele (CYP2D6*4) was not associated with increased susceptibility to AS. Significant within-family association of CYP2D6*4 alleles and AS was demonstrated. Weak linkage was also demonstrated between CYP2D6 and AS. We postulate that altered metabolism of a natural toxin or antigen by the CYP2D6 gene may increase susceptibility to AS.

Study of miRNA polymorphisms and their potential association with breast cancer risk in Australian Caucasian populations

This project established a large and well characterised prospective breast cancer DNA biobank and used this biobank to conduct genetic studies in breast cancer. The thesis presented the results of these high-throughput genotyping studies in two separate Australian Caucasian case-control populations and identified association between three novel genetic variants in microRNA genes and breast cancer risk.

Serum immunoglobulin G directed against porcine trypsin in the serum of cystic fibrosis children receiving porcine pancreatic enzyme supplements

Cystic fibrosis (CF) patients require pancreatic enzyme replacement therapy to correct pancreatic insufficiency. These enzymes are derived from porcine pancreas and are known to be antigenic. To determine the possible clinical consequences, a specific ELISA was developed to detect IgG antibody directed against porcine trypsin (PTAb) in the sera of CF patients. The assay was used to evaluate the occurrence of PTAb in a cross sectional study of 103 CF patients in relation to the introduction of porcine enzyme therapy, clinical status and genotype. Antibodies against porcine trypsin were detected in the sera of 63% of patients unrelated to the age of commencement or the duration of enzyme therapy. No differences were observed in the clinical status of CF patients who had developed PTAb (n = 65) and those who had no detectable PTAb (n = 38) as determined from: the current prescribed dose of porcine pancreatic enzyme capsules; Z scores for height and weight; and respiratory function tests. It is suggested that the PTAb commonly found in the sera of CF patients are of doubtful clinical significance but the prospect of PTAb contributing to immune complex disease should be examined further.

Association of microRNA 17–92 cluster host gene (MIR17HG) polymorphisms with breast cancer

Breast cancer incidence and mortality rates are increasing despite our current knowledge on the disease. Ninety-five percent of breast cancer cases correspond to sporadic forms of the disease and are believed to involve an interaction between environmental and genetic determinants. The microRNA 17–92 cluster host gene (MIR17HG) has been shown to regulate expression of genes involved in breast cancer development and progression. Study of single-nucleotide polymorphisms (SNPs) located in this cluster gene could help provide a further understanding of its role in breast cancer. Therefore, this study investigated six SNPs in the MIR17HG using two independent Australian Caucasian case–control populations (GRC-BC and GU-CCQ BB populations) to determine association to breast cancer susceptibility. Genotyping was undertaken using chip-based matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS). We found significant association between rs4824505 and breast cancer at the allelic level in both study cohorts (GRC-BC p = 0.01 and GU-CCQ BB p = 0.03). Furthermore, haplotypic analysis of results from our combined population determined a significant association between rs4824505/rs7336610 and breast cancer susceptibility (p = 5 × 10−4). Our study is the first to show that the A allele of rs4824505 and the AC haplotype of rs4824505/rs7336610 are associated with risk of breast cancer development. However, definitive validation of this finding requires larger cohorts or populations in different ethnical backgrounds. Finally, functional studies of these SNPs could provide a deeper understanding of the role that MIR17HG plays in the pathophysiology of breast cancer.

The use of a synthetic prostaglandin e1 analogue (misoprostol) as an adjunct to pancreatic enzyme replacement in cystic fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2% p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

The use of a synthetic prostaglandin-E1 analog (misoprostol) as an adjunct to pancreatic-enzyme replacement in cystic-fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2%, p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption.

Genome-wide association study identifies novel genetic variants contributing to variation in blood metabolite levels

Metabolites are small molecules involved in cellular metabolism, which can be detected in biological samples using metabolomic techniques. Here we present the results of genome-wide association and meta-analyses for variation in the blood serum levels of 129 metabolites as measured by the Biocrates metabolomic platform. In a discovery sample of 7,478 individuals of European descent, we find 4,068 genome- and metabolome-wide significant (Z-test, P<1.09 × 10−9) associations between single-nucleotide polymorphisms (SNPs) and metabolites, involving 59 independent SNPs and 85 metabolites. Five of the fifty-nine independent SNPs are new for serum metabolite levels, and were followed-up for replication in an independent sample (N=1,182). The novel SNPs are located in or near genes encoding metabolite transporter proteins or enzymes (SLC22A16, ARG1, AGPS and ACSL1) that have demonstrated biomedical or pharmaceutical importance. The further characterization of genetic influences on metabolic phenotypes is important for progress in biological and medical research.

Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 x 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

Polymorphisms in the vascular endothelial growth factor gene and the risk of familial endometriosis

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein suspected to be involved in the pathogenesis of endometriosis by establishing a new blood supply to the human exfoliated endometrium. Several transcription factor-binding sites are found in the VEGF 5'-untranslated region and variation within the region increases the transcriptional activity. Six previous studies which tested between one and three single nucleotide polymorphisms (SNPs) in samples comprising 105-215 cases and 100-219 controls have produced conflicting evidence for association between the SNPs in the VEGF region and endometriosis. To further investigate the reported association between VEGF variants and endometriosis, we tested the four VEGF polymorphisms (-2578 A/C, rs699947; -460 T/C, rs833061; +405 G/C, rs2010963 and +936 C/T, rs3025039) in a large Australian sample of 958 familial endometriosis cases and 959 controls. We also conducted a literature-based review of all relevant association studies of these VEGF SNPs in endometriosis and performed a meta-analysis. There was no evidence for association between endometriosis and the VEGF polymorphisms genotyped in our study. Combined association results from a meta-analysis did not provide any evidence for either genotypic or allelic association with endometriosis. Our detailed review and meta-analysis of the VEGF polymorphisms suggests that genotyping assay problems may underlie the previously reported associations between VEGF variants and endometriosis.