Migraine association and linkage studies of an endothelial nitric oxide synthase (NOS3) gene polymorphism

Migraine shows strong familial aggregation. However, the number of genes involved in the disorder is unknown and not identified. Nitric oxide is involved in the central processing of pain stimuli and plays an important role in the regulation of basal or stimulated vasodilation. Nitric oxide synthase, which controls the synthesis of nitric oxide, could possibly be a cause, or candidate gene, in migraine etiology. In this study, we detected a polymorphism for endothelial nitric oxide synthase by polymerase chain reaction and tested this for association and linkage to migraine. Results from the study did not show an association of the nitric oxide synthase microsatellite when tested in 91 affected and 85 unaffected individuals. Using the FASTLINK program for parametric linkage analysis, the polymorphism did not show significant linkage to migraine when tested in four migraine pedigrees composed of 116 individuals, 52 affected. Total LOD scores excluded linkage up to 8.5 cM between the nitric oxide synthase polymorphism and migraine. Results using the nonparametric affected pedigree member form of analysis also did not support a role for this gene in migraine etiology.

Glutathione transferase Ti and M1 genotype polymorphism in the normal population of Shanghai

Glutathione transferases are known to be important enzymes in the metabolism of xenobiotics. In humans genetic polymorphisms have been reported for the hGSTM1 and hGSTT1 genes leading to individual differences in susceptibility towards toxic effects, such as cancer. This study describes the distribution of the two polymorphisms of hGSTT1 and hGSTM1 in the normal Chinese population of Shanghai. Out of 219 healthy individuals having been genotyped for GSTTI and GSTMI, 108 (49%) were identified to be homozygously deficient for the GSTT1 gene and 107 (49%) for the GSTM1 gene.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Genetic susceptibility to environmental toxicants : the interface between human and experimental studies in the development of new toxicological concepts

The growing knowledge of the genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual susceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. The results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification, and experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these possibilities may be combined to lead to new strategies of human-oriented toxicological research. This overview will present some outstanding examples for such strategies taken from the practically very important field of occupational toxicology. The main focus is placed on the effects of enzyme polymorphisms of the xenobiotic metabolism in association with the induction of bladder cancer and renal cell cancer after exposure to occupational chemicals. Also, smoking and induction of head and neck squamous cell cancer are considered.

The F158V polymorphism in FcγRIIIA shows disparate associations with rheumatoid arthritis in two genetically distinct populations

Objectives: To investigate the association of the FcγRIIIA gene with rheumatoid orthritis (RA) in two genetically distinct groups: a white group from the United Kingdom and a northern Indian group. Methods: The distributions of the two alleles of the FcγRIIIA F158V polymorphism were determined in 398 white patients from the United Kingdom and 63 Indian patients with RA and compared with those from 289 United Kingdom and 93 Indian healthy controls, respectively. Results: Among the Indian patients, the frequency of the rare 158V allele and the proportion of 158VV homozygotes were reduced (relative risk (RR)=0.3, 95% confidence interval (95% CI) 0.1 to 1.1, p<0.06), reaching statistical significance for carrying the 158VV phenotype relative to 158FV or FF (RR=0.2, 95% CI 0.05-0.9, p<0.02). Conversely, no significant deviation in allelic frequencies was noted between the patients and controls from the United Kingdom. Conclusions: The 158VV phenotype showed a weak protective effect against developing RA in the Indian group. However, this sample was small (resulting in a low power for statistical analysis) and no independent confirmation was found in the larger white United Kingdom group. Thus the FcγRIIIA locus is unlikely to be of major importance in causing RA.

Genetic testing for haemochromatosis in patients with chondrocalcinosis

Hereditary haemochromatosis (HH) is the most common lethal monogenic human disease, affecting roughly 1 in 300 white northern Europeans. Homozygosity for the C282Y polymorphism within the HFE gene causes more than 80% of cases, with compound heterozygosity of the C282Y and H63D polymorphism also increasing susceptibility to disease. The aim of this study was to determine the frequency of the C282Y and H63D polymorphisms in the disease, and to assess the risk of HH in heterozygotes for the C282Y polymorphism. 128 patients were recruited because of either radiographic chondrocalcinosis (at least bicompartmental knee disease or joints other than the knee involved) or CPPD pseudogout. Genotyping of the HFE C282Y and H63D mutations was performed using PCR/SSP and genotypes for the C282Y polymorphism confirmed by PCR/RFLP. Historical white European control data were used for comparison. Two previously undiagnosed C282Y homozygotes (1.6%), and 16 C282Y heterozygotes (12.5%), including four (3.1%) C282Y/ H63D compound heterozygotes were identified. This represents a significant overrepresentation of C282Y homozygotes (relative risk 3.4, p-0.037), but the number of heterozygotes was not significantly increased. At a cost per test of £1 for each subject, screening all patients with chondrocalcinosis using the above ascertainment criteria costs only £64 for each case of haemochromatosis identified, clearly a highly cost effective test given the early mortality associated with untreated haemochromatosis. Routine screening for haemochromatosis in patients with appreciable chondrocatcinosis is recommended.

Genetic studies in osteoporosis: The end of the beginning

Osteoporosis and disorders of bone fragility are highly heritable, but despite much effort the identities of few of the genes involved has been established. Recent developments in genetics such as genome-wide association studies are revolutionizing research in this field, and it is likely that further contributions will be made through application of next-generation sequencing technologies, analysis of copy number variation polymorphisms, and high-throughput mouse mutagenesis programs. This article outlines what we know about osteoporosis genetics to date and the probable future directions of research in this field.

Genetic studies of osteoporosis: A rethink required

It has been 10 years since the seminal paper by Morrison and colleagues reporting the association of alleles of the vitamin D receptor and bone density [1], a paper which arguably kick-started the study of osteoporosis genetics. Since that report there have been literally thousands of osteoporosis genetic studies published, and large numbers of genes have been reported to be associated with the condition [2]. Although some of these reported associations are undoubtedly true, this snow-storm of papers and abstracts has clouded the field to such a great extent that it is very difficult to be certain of the veracity of most genetic associations reported hereto. The field needs to take stock and reconsider the best way forward, taking into account the biology of skeletal development and technological and statistical advances in human genetics, before more effort and money is wasted on continuing a process in which the primary achievement could be said to be a massive paper mountain. I propose in this review that the primary reasons for the paucity of success in osteoporosis genetics has been: •the absence of a major gene effect on bone mineral density (BMD), the most commonly studied bone phenotype; •failure to consider issues such as genetic heterogeneity, gene–environment interaction, and gene–gene interaction; •small sample sizes and over-optimistic data interpretation; and •incomplete assessment of the genetic variation in candidate genes studied.

Genetic aspects of susceptibility, severity, and clinical expression in ankylosing spondylitis

While twin studies have previously demonstrated high heritability of susceptibility to ankylosing spondylitis (AS), it is only recently that the involvement of genetic factors in determining the severity of the disease has been demonstrated. The genes involved in determining the rate of ankylosis in AS are likely to be different from those involved in the underlying immunologic events, and represent important potential targets for treatment of AS. This article will describe the progress that has been made in the genetic epidemiology of AS, and in identifying the genes involved.

Genetic associations and functional characterization of M1 aminopeptidases and immune-mediated diseases

Endosplasmic reticulum aminopeptidase 1 (ERAP1), endoplasmic reticulum aminopeptidase 2 (ERAP2) and puromycin-sensitive aminopeptidase (NPEPPS) are key zinc metallopeptidases that belong to the oxytocinase subfamily of M1 aminopeptidase family. NPEPPS catalyzes the processing of proteosome-derived peptide repertoire followed by trimming of antigenic peptides by ERAP1 and ERAP2 for presentation on major histocompatibility complex (MHC) Class I molecules. A series of genome-wide association studies have demonstrated associations of these aminopeptidases with a range of immune-mediated diseases such as ankylosing spondylitis, psoriasis, Behçet's disease, inflammatory bowel disease and type I diabetes, and significantly, genetic interaction between some aminopeptidases and HLA Class I loci with which these diseases are strongly associated. In this review, we highlight the current state of understanding of the genetic associations of this class of genes, their functional role in disease, and potential as therapeutic targets.

Effect of an estrogen receptor-α intron 4 polymorphism on fat mass in 11-year-old children

Context: in the ESR1 gene encoding estrogen receptor (ER)-α may be associated with fat mass in adults. Objectives: The objective of the study was to establish whether ESR1 polymorphisms influence fat mass in childhood. Design: This was a cross-sectional analysis after genotyping of rs9340799, rs2234693, and rs7757956 ESR1 polymorphisms. Setting: The Avon Longitudinal Study of Parents and Children (ALSPAC) was a population-based prospective study. Participants: Participants included 3097 11-yr-old children with results for ESR1 genotyping, puberty measures, and dual-energy x-ray absorptiometry results. Outcomes: Relationships between ESR1 polymorphisms and indices of body composition were measured. Results: The rs7757956 polymorphism was associated with fat mass (P = 0.002). Total body fat mass (adjusted for height) was reduced by 6% in children with TA/AA genotypes, and risk of being overweight (≥85th centile of fat mass) was decreased by 20%. This genetic effect appeared to interact with puberty in girls (P = 0.05 for interaction): in those with the TT genotype, total body fat mass (adjusted for height) was 18% higher in Tanner stages 3-5 vs. stages 1-2; the equivalent difference was 7% in those with TA/AA genotypes. Furthermore, the risk of being overweight was 36% lower in girls with TA/AA genotypes in Tanner stages 3-5, but no reduction was seen in those in stages 1-2. Neither rs9340799 nor rs2234693 polymorphisms were associated with body composition measures. Conclusions: Fat mass in 11-yr-old children was related to the rs7757956 ESR1 polymorphism. This association was strongest in girls in more advanced puberty, in whom the risk of being overweight was reduced by 36% in those with the TA/AA genotype.

Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton

Osteoporosis is a common, increasingly prevalent and potentially debilitating condition of men and women. Genetic factors are major determinants of bone mass and the risk of fracture, but few genes have been definitively demonstrated to be involved. The identification of these factors will provide novel insights into the processes of bone formation and loss and thus the pathogenesis of osteoporosis, enabling the rational development of novel therapies. In this article, we present the extensive genetic and functional data indicating that the LRP5 gene and the Wnt signalling pathway are key players in bone formation and the risk of osteoporosis, and that LRP5 signalling is essential for normal morphology, developmental processes and bone health.

Genetic and genomic studies of PADI4 in rheumatoid arthritis

Objectives. Strong genetic association of rheumatoid arthritis (RA) with PADI4 (peptidyl arginine deiminase) has previously been described in Japanese, although this was not confirmed in a subsequent study in the UK. We therefore undertook a further study of genetic association between PADI4 and RA in UK Caucasians and also studied expression of PADI4 in the peripheral blood of patients with RA. Methods. Seven single-nucleotide polymorphisms (SNP) were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism in 111 RA cases and controls. A marker significantly associated with RA (PADI4_100, rs#2240339) in this first data set (P = 0.03) was then tested for association in a larger group of 439 RA patients and 428 controls. PADI4 transcription was also assessed by real-time quantitative PCR using RNA extracted from peripheral blood mononuclear cells from 13 RA patients and 11 healthy controls. Results. A single SNP was weakly associated with RA (P = 0.03) in the initial case-control study, a single SNP (PADI4_100) and a two marker haplotype of that SNP and the neighbouring SNP (PADI4_04) were significantly associated with RA (P = 0.02 and P = 0.03 respectively). PADI4_100 was not associated with RA in a second sample set. PADI4 expression was four times greater in cases than controls (P = 0.004), but expression levels did not correlate with the levels of markers of inflammation. Conclusion. PADI4 is significantly overexpressed in the blood of RA patients but genetic variation within PADI4 is not a major risk factor for RA in Caucasians.

Polymorphism in codon 17 of the CTLA-4 gene (+49 A/G) is not associated with susceptibility to rheumatoid arthritis in British Caucasians

The role of the CTLA-4 antigen in the development of autoimmune diseases is well documented, with several autoimmune disorders showing association or linkage with the CTLA-4 locus. Its role in the aetiology of rheumatoid arthritis (RA) however, remains unclear, as the functional studies of the B7-CTLA-4 pathway in mouse models of RA and genetic studies in humans have given contrasting results. We have studied the single nucleotide polymorphism at position +49 (A/G) of the CTLA-4 gene, in a cohort of 421 RA cases and 452 healthy controls from the UK. Despite the high statistical power to detect even a weak susceptibility effect, no significant association was found. We also analysed the distribution of the allele and genotype frequencies with respect to the presence of the shared epitope (a known RA susceptibility factor) and found no statistically significant differences. We conclude that, although the importance of the B7-CTLA-4 interaction in the development of RA can not be excluded, the CTLA-4 gene is unlikely to be a predisposing factor to this disease.

UHT milk contains multiple forms of αS1-casein that undergo degradative changes during storage

Milk proteins are susceptible to chemical changes during processing and storage. We used proteomic tools to analyse bovine αS1-casein in UHT milk. 2-D gels of freshly processed milk αS1-casein was presented as five or more spots due to genetic polymorphism and variable phosphorylation. MS analysis after phosphopeptide enrichment allowed discrimination between phosphorylation states and genetic variants. We identified a new alternatively-spliced isoform with a deletion of exon 17, producing a new C-terminal sequence, K164SQVNSEGLHSYGL177, with a novel phosphorylation site at S174. Storage of UHT milk at elevated temperatures produced additional, more acidic αS1-casein spots on the gels and decreased the resolution of minor forms. MS analysis indicated that non-enzymatic deamidation and loss of the N-terminal dipeptide were the major contributors to the changing spot pattern. These results highlight the important role of storage temperature in the stability of milk proteins and the utility of proteomic techniques for analysis of proteins in food.