This study explores whether these reductions are sustained when NVP is added to a range of commonly consumed foods or incorporated into a breakfast cereal.Reductions in postprandial glycemia have been demonstrated previously with the addition of the novel viscous polysaccharide (NVP), PolyGlycopleX2), participated in an acute, randomized controlled trial. The glycemic response to cornflakes, rice, yogurt, and a frozen dinner with and without 5 g of NVP sprinkled onto the food was determined. In addition, 3 granolas with different levels of NVP and 3 control white breads and one white bread and milk were also consumed. All meals contained 50 g of available carbohydrate. Capillary blood samples were taken fasting and at 15, 30, 45, 60, 90 and 120 min after the start of the meal. The glycemic index (GI) and the glycemic reduction index potential (GRIP) were calculated. The blood glucose concentrations at each time and the iAUC values were subjected to repeated-measures analysis of variance (ANOVA) examining for the effect of test meal. After demonstration of significant heterogeneity, differences between individual means was assessed using GLM ANOVA with Tukey test to adjust for multiple comparisons.Ten healthy subjects . The GI of cornflakes, cornflakes+NVP, rice, rice+NVP, yogurt, yogurt+NVP, turkey dinner, and turkey dinner+NVP were 83 ± 8, 58 ± 7, 82 ± 8, 45 ± 4, 44 ± 4, 38 ± 3, 55 ± 5 and 41 ± 4, respectively. The GI of the control granola, and granolas with 2.5 and 5 g of NVP were 64 ± 6, 33 ± 5, and 22 ± 3 respectively. GRIP was 6.8 ± 0.9 units per/g of NVP.Sprinkling or incorporation of NVP into a variety of different foods is highly effective in reducing postprandial glycemia and lowering the GI of a food.NCT00935350. Epidemiological evidence suggests that postprandial glucose levels have a stronger relationship with cardiovascular events than fasting glucose in individuals with diabetes ,2. RecenIncreasing viscous fiber consumption however is hampered by both palatability issues and variability in effectiveness. The hig® (PGX®) . This complex consists of three viscous non starch polysaccharides which are processed using proprietary technology (EnviroSimplex®) which delays the onset of viscosity and thus increases palatability of the fiber when added to food. Previously, we reported the glucose lowering effect of this fiber in a dose responsive manner when mixed with a glucose drink and sprinkled onto a standard white bread demonstrating that the processing did not interfere with the effectiveness of the fiber [Recently, a commercial novel viscous polysaccharide complex has been developed which is marketed in both the USA and Canada under the trade name PolyGlycopleXhe fiber . From th2 participated in an acute randomized, controlled study. Subjects were recruited from the Glycemic Index Laboratories clinic volunteer roster. Entry criteria included BMI < 35 kg/m2 and fasting blood glucose < 5.5 mmol/L, subjects taking medications or dietary supplements were excluded. The study was approved by the Western Institutional Review Board  and conducted in compliance with HIPAA guidelines. Informed written consent was obtained from all subjects prior to the start of the study. Subjects received a financial reward for their participation.Ten healthy subjects  and is manufactured from konjac-xanthan gum and sodium alginate using a proprietary process (EnviroSimplex®) to create a novel polysaccharide complex which exceeds the viscosity of the constituent ingredients. PGX® is 87.4% dietary fiber, of which 81.8% is soluble. A typical recommended serving ranges from 2.5 to 5 grams depending on the application.The NVP used in this study is marketed under the trade name PGX® (Kellogg Canada) with 125 mL milk, Jasmine long grain rice, turkey dinner , strawberry yogurt (Astro Yogurt) with or without 5 g of NVP sprinkled onto it. In addition three different granolas were also consumed into which either 0 g, 2.5 g or 5 g of NVP had been incorporated. To control for the additional carbohydrate from the milk taken with the cornflakes, an additional control white bread was consumed with 125 ml of 2% butterfat milk. Finally, all subjects also consumed the control white bread on three separate occasions to allow calculation of the glycemic index of the meals . Comparison of the meals with and without NVP, showed that addition of NVP to the meals significantly reduced incremental postprandial glucose levels at 30, 45, and 60 min after consumption of the rice meal and at 45 min after both the cornflakes and turkey dinner meals , and the granolas with 2.5 g and 5 g of NVP were 64 ± 6, 33 ± 5, and 22 ± 3 respectively. Percent reductions in GI of the cornflakes, rice, turkey dinner and yogurt meals with NVP were 26%, 45%, 24% and 12% respectively when compared to the meals without NVP. Percent reductions for the 2.5 g NVP blueberry granola and 5 g NVP blueberry granola were 46% and 64% respectively when compared to the control granola. The average GRIP of all meals was 6.8 ± 0.9 GI units per g of NVP.®, is effective in lowering postprandial glycemia when either sprinkled or incorporated into commonly eaten foods and confirms earlier predictions that the GI of a food would be reduced.This study demonstrates that this particular viscous fiber preparation, PGXEnrichment of the diet using soluble fiber is particularly attractive as it both increases the dietary fiber levels of the diet as is recommended by health agencies such as the American and Canadian Diabetes Associations and the American Dietetic Association -17 and iTo allow a direct comparison of the effectiveness of different substances in affecting the GI of a food, we proposed the term "GRIP"  concept (CMJ). Previous studies estimated the GRIP of NVP to be 5 when added to liquids and 7 when added to a solid food ,25. ThisLimitations of this study include the relatively small number of meals and subjects tested and the acute nature of this study. Further studies need to be undertaken to confirm consistent effects across a wider range of foods, and whether the acute reductions seen translate in flattening of the fluctuations seen in glycemia during the day.In conclusion, use of a soluble dietary fiber supplement may be an effective means of increasing the dietary fiber intake as recommended for individuals and simultaneously reduce the GI of the diet. However, reduced palatability of the high fiber foods and the large quantities needed to confer a health benefit are often barriers to increasing consumption. This particular NVP preparation has an advantage over most fibers in that it is a standardized product with a consistently high viscosity which allows a reduction in the amounts required. It has a powerful GRIP value with respect to glucose lowering effect of high GI foods, which is either equivalent or higher than other fiber preparations. The use of this fiber may potentially have positive health implications and further research is therefore warranted to confirm the long term health benefits of this novel viscous polysaccharide.ALJ received financial remuneration for the preparation of the manuscript and is a consultant to the parent company of the sponsor.VK was an employee of InovoBiologic Inc. during the planning and execution of these studies.MRL is a consultant to the parent company of the sponsor.TMSW - no competing interestsALJ, VK, and MRL participated in the design of the study. ALJ and TW performed the statistical analysis. ALJ coordinated the research and drafted the manuscript. All authors read and approved the final manuscript.


To review systematically the neurodevelopmental characteristics of individuals with sex chromosome trisomies (SCTs).A bibliographic search identified English-language articles on SCTs. The focus was on studies unbiased by clinical referral, with power of at least 0.69 to detect an effect size of 1.0.We identified 35 articles on five neonatally identified samples that had adequate power for our review. An additional 11 studies were included where cases had been identified for reasons other than neurodevelopmental concerns. Individuals with an additional X chromosome had mean IQs that were within broadly normal limits but lower than the respective comparison groups, with verbal IQ most affected. Cognitive outcomes were poorest for females with XXX. Males with XYY had normal-range IQs, but all three SCT groups  had marked difficulties in speech and language, motor skills, and educational achievement. Nevertheless, most adults with SCTs lived independently. Less evidence was available for brain structure and for attention, social, and psychiatric outcomes. Within each group there was much variation.Individuals with SCTs are at risk of cognitive and behavioural difficulties. However, the evidence base is slender, and further research is needed to ascertain the nature, severity, and causes of these difficulties in unselected samples. Errors in the process of maternal or paternal meiosis can lead to chromosome trisomies. Among the most common trisomies compatible with live birth are those involving the X and Y chromosomes. Early studies established that the frequency of each of the sex chromosome trisomies (SCTs), XXY (Klinefelter syndrome), XXX, and XYY, was about one in 1000 same-sex individuals.4Although SCTs are not rare, there is a scarcity of rigorous evidence on prognosis and cognitive outcomes. One reason is that SCTs are not associated with gross physical or cognitive impairments and so may go unidentified. A corollary of this is that children who are identified may be atypical, with the SCT discovered only after clinical referral for cognitive or behavioural difficulties.There are three reasons why it is important to obtain an unbiased picture of the cognitive and behavioural outcomes of children with SCTs. First, SCTs can be identified on prenatal screening for other conditions, and parents might be offered termination of pregnancy on this basis. Those counselling them need accurate information about the range of outcomes that might be expected, in order to offer objective advice to parents.7Our aim in this review is to synthesize what is known about neurocognitive and behavioural outcomes in individuals with the XXY, XXX, or XYY karyotype, and to highlight areas where there are gaps in existing knowledge. For each karyotype, the research literature will be evaluated for information in five domains of development: general intelligence; scholastic strengths and weaknesses; attention and executive control; motor skills, speech and language; and social communication, interaction, and adaptation. We also review what is known about the risk of psychopathology and neurological correlates associated with each karyotype.We conducted a systematic review of the past five decades of published data on cognitive and behavioural outcomes of individuals with an extra sex chromosome. The data were obtained primarily through published articles, although authors were contacted for clarification where appropriate. The online Web of Knowledge database was used to identify English-language articles, using the search terms  OR Klinefelter) AND (cognit* OR language OR behav* OR brain OR neuro*). The search was confined to articles in databases for genetics, neuroscience, behavioural science, and psychiatry. Further articles were traced through PubMed and from reference lists in identified papers.Because letter combinations such as XXX are used with various meanings in other disciplines, the search criteria generated many irrelevant papers, but these were readily identified, leaving 702 papers with a focus on chromosome abnormalities. As shown in n=64), that is, studies of individuals recruited through institutions for cognitive or psychiatric disorders, as well as those who were identified via patient support groups or through postnatal chromosome testing. Our rationale was that it was not usually possible to tell why a chromosome test had been conducted, or why an individual had joined a support group. Thus, we erred on the side of caution and excluded those who may have come to attention because of cognitive or behavioural difficulties. However, we did not exclude males with XXY who were identified as adults because of infertility or endocrinological concerns, because there is no reason to suppose that they were cognitively atypical. Studies of a sample of adults identified via population chromosome screening of tall males were also included.As we aimed to characterize the phenotype in as unbiased a way as possible, we next excluded articles with major ascertainment bias  power of at least 0.69 to detect an effect size (Cohen’s d) of at least 1.0 on a one-tailed test, computed using G*Power 3;Overall, 46 articles met our criteria .51.Direct studies of educational skills confirmed an average deficit in reading accuracy and reading comprehension for males with XXY relative to unaffected males, with a large effect size , but only a trend for impairment in fine motor skills (d=0.3). Motor deficits were also seen in adults, with Boone et al.A study of the Toronto newborn sample used population norms to identify impairment on the revised Yale Developmental Inventory in toddlers. A deficit in gross motor skills was found in 11 of 25 males with XXY, and a deficit in fine motor skills was found in seven of the 25 males..Although it has been claimed that there is a high rate of non-right-handedness in males with XXY karyotype,48d=0.7–0.8) and verbal memory (d=1.1–1.2). Differences in receptive language were mostly non-significant, but effect sizes were around 0.6, suggesting that they might be reliable in a larger sample.Significant delays in the earliest stages of language development, assessed by developmental schedules, were reported in the Edinburgh,n=24) did not differ at age 6 years on the Vineland Social Maturity Scale.Children from the Edinburgh cohort did not differ from comparison groups on the Behaviour Screening Questionnaire at age 3 years,Taken together, these studies suggest that males with XXY do not usually have major problems with social interaction and adaptation, although they may be timid and unassertive.Götz et al.23Overall brain volumetry. It has been suggested that the lower IQ in males with XXY may be related to reduced brain growth, given that head circumference during childhood is smaller than that of unaffected individuals, despite greater height (d≈1).d=1.6). In a further study of 18 adults being treated for hypogonadism, the investigators did not find any difference in brain volume compared with unaffected males who were closely similar in IQ.53r=−0.51, p=0.003), as well as with overall verbal processing speed.It has been suggested that increases in ventricle volume are associated with the XXY karyotype,Left-hemisphere structure and function. There has been a long-standing interest in the notion that language deficits in individuals with SCTs may be linked to atypical cerebral lateralization, starting with a study by Netley and RovetAmygdala. A brain basis for problems with socio-emotional development in XXY has been suggested in terms of a significant reduction in amygdala volume compared with comparison groups.d=1.7) is all the more striking when compared with the results from hippocampal volume, which gave identical mean values in the XXY and comparison groups. However, the authors’ interpretation, linking this finding to risk of psychopathology, seems highly speculative, given that the participants came from the Denver cohort, who showed very little evidence of psychopathology in adulthood (see above).Cerebellum.Little is known about the neurological origins of motor difficulties in Klinefelter syndrome. On magnetic resonance imaging, Itti et al.Brain function. There is very little direct evidence relating to differences in brain function in individuals with SCTs. In the only electrophysiological study meeting our sampling criteria, the authors reported reduced amplitude of the P300 event-related potential component in a sample of 32 individuals with XXY compared with males with untreated idiopathic hypogonadotrophic hypogonadism and healthy males, and they construed this difference as a measure of overall central nervous system function.54d=1.24) was significantly greater than that in performance IQ (d=1.05).Despite the small evidence base from only four cohorts of unselected females with an third X chromosome, data on IQ are consistent, indicating a significant reduction in IQ of approximately 20 points see . Both ved>1).21All studies of literacy skills have shown significant reading impairments, with most females needing some extra assistance with reading , visual association (0.8), visual closure (0.7), and auditory closure (1.9). The presence of such pervasive difficulties with language skills suggests that the difficulties are not simply a secondary consequence of motor or articulatory limitations. However, this result must be interpreted with some caution because the comparison was with test norms rather than a matched comparison group.Difficulties with speech and language have been noted in females with an extra X chromosome, with 40 to 91% needing speech therapy at some point.In middle childhood and adolescence, females with XXX appeared to develop good relationships, and were described as well liked by friends.23d=2.1 in adolescence, d=1.6 in mid-20s) and eight of 11 adolescents had DSM-III-RBender et al.One other study with some pertinent information is the brain-imaging study of the Edinburgh sample, where the Structured Interview for Schizotypy was administered to 11 females with the XXX karyotype and 13 unaffected females.Magnetic resonance imaging of brain volume has been conducted in 10 XXX females from the Denver cohort34The similarity between individuals with XXY and XXX has been suggested to indicate that ‘the presence of a supernumerary X chromosome has a demonstrable effect on brain development’Regional brain volumes and asymmetries did not differ significantly between females with the XXX karyotype and unaffected females after adjustment for whole-brain volume.25As shown in Netleyd=0.9). The two groups did not differ in parental socio-economic status. This fits with the IQ data in suggesting that XYY males underachieve relative to their social background.Witkin et al.RatcliffeThe only other information about educational progress in an unselected sample comes from Linden and Bender’s surveyThree of the 14 XYY males in Linden and Bender’s sample had a diagnosis of attention deficit disorder, and three more were described as distractible.Data on motor abilities were available for only four XYY males from the Denver newborn study; three of these males scored below the 10th centile on a composite score based on a battery of motor tests. In the Edinburgh newborn cohort, the XYY group scored 1.29 SD below the comparison group on a test of fine motor coordination, and 1.42 SD below the comparison group on balance.33Delayed motor development or lack of motor coordination was also remarked on for five of 14 cases studied by Linden and BenderIt is worth considering whether some of the cognitive and communicative difficulties affecting males with XYY may be related to motor impairments, but this has not been possible to establish, because of lack of data on different domains in the same individuals.There is a dearth of information on speech and language development in males with an extra Y chromosome. In an uncontrolled study, Geerts et al.Ratcliffe et al. found little evidence of behaviour difficulties in nine 3-year-olds with XYY, on the Behaviour Screening Questionnaire28Interview data from adults in the Copenhagen cohort49One of the first studies of XYY was conducted at a Scottish special hospital for individuals with mental health problems and behaviour dangerous to either themselves or to the public.The first unbiased sample, recruited by Witkin et al.This issue was further assessed by a detailed investigation of the Edinburgh newborn cohort in adulthood,A single study of 10 XYY males met inclusion criteria for this review.34Our review highlights that, although outcomes are variable, individuals with an extra sex chromosome are at risk of a range of neurodevelopmental difficulties. Deficits in IQ have been reported in those with an extra X chromosome, especially in females with XXX. Rates of speech and language impairments are elevated across all three groups, and difficulties with reading and educational achievement are often seen. Motor skills are impaired in all groups, and there is some suggestion of difficulties in attention and executive control, although this has barely been investigated. Although outcomes are variable, difficulties with social interaction, communication, and adaptation seem most apparent in females with XXX, who also showed an elevated risk of psychiatric disturbances such as anxiety and depression. Across all domains, most authors describe considerable variability within their samples, with some individuals performing in the normal or above-average range. The handful of studies on adult outcomes indicates that most individuals with a SCT are able to live independently and form normal adult relationships.Although all three trisomies are characterized by an increased risk of educational problems, the specific profiles differ. Males with XYY have average IQs, consistent with the one study showing normal brain volume on magnetic resonance imaging. In contrast, both XXY and XXX groups tend to have below-average verbal IQ and small cerebral volume. However, whereas both verbal and performance IQ tend to be low in females with XXX, there is more selective impairment of verbal IQ in males with XXY. Neurological studies showed that ventricle volume was increased in males with XXY but not in females with XXX, whereas amygdala volume was significantly reduced only in males with XXY, once brain size had been taken into consideration.Interpretation of such findings is complicated by uncertainty about robustness of results from small samples, and it is clear from our review that there are many contradictory findings in the literature, even in domains that have been assessed repeatedly, suggesting substantial heterogeneity within each of these groups.Our review has revealed a number of important limitations that characterize the existing literature. A serious issue is sample size, which constrains statistical power. To have sufficient studies to review, we needed to relax the power criterion for inclusion to 0.69 to detect an effect size of 1, whereas power is ordinarily set at a minimum of 0.8.Small study samples of children with SCTs are understandable when one considers that to obtain an unbiased sample one has to screen around 1000 children to identify a single case with XYY or XXX. To compound the difficulties of studying such children, neonatal screening raises major ethical concerns, and studies adopting this approach are very unlikely to be conducted in future.More information is available on XXY because this karyotype affects endocrinological development and fertility in late adolescence and adulthood, and so hitherto unidentified cases come to attention.A recommendation for future research is to ensure that there is a large comparison sample. It was frustrating to find that small groups of cases of SCT were often compared with equally small comparison groups, or sometimes no comparison group at all, when recruitment should not pose undue difficulty. It is not adequate to rely on test norms or clinical impression to identify neurodevelopmental deficits; a sizeable comparison group from a similar socio-economic background should always be included in a study. As illustrated in the studies reviewed here, comparison groups may be recruited from the same community, from siblings, or, as in one case, from children with a familial balanced autosomal rearrangement that did not affect the phenotype.In general, research in this area has progressed from early somewhat anecdotal observations to studies placing greater reliance on standardized assessments, which allow one to quantify impairment in relation to a normative group, and to estimate error of measurement. However, this does not remove the need for information of a more descriptive and categorical kind, such as whether the child requires special help at school or will be able to live independently in adulthood, which may be of more importance to a parent than a test score. Here too, it is important to have a comparison group to interpret findings. Because IQ is often depressed in SCTs, IQ-matched comparison groups can be informative in establishing whether any problems are compatible with generally lower intellectual functioning or are a more specific correlate of the SCT. This approach was adopted in the Copenhagen sampleWork on the neurological bases of cognitive deficits in SCTs is still in its infancy, and there is a substantial imbalance in the published literature, with an almost exclusive focus on individuals with XXY, in contrast with very limited information on XXX and XYY. There is a paucity of studies of brain function rather than structure alone, which makes links to cognitive outcomes and difficulties rather speculative, and no researchers to date have investigated brain connectivity or neurotransmitter function. Furthermore, in the case of Klinefelter syndrome, studies of adults are complex to interpret, as there is evidence to suggest structural brain effects of testosterone treatment in males with XXY.There is also a need for more longitudinal data. The Denver, Edinburgh, and Toronto studies have all followed the same cases over time, but, probably because of small sample sizes and sample attrition, investigators have seldom considered how far early variables predict later outcomes in individuals. It would be interesting, for example, to consider whether a tendency to withdraw from novelty at the age of 3 years in males with XXYBender et al. have emphasized that future studies should be focused not only on the average outcomes of children with SCTs but also on individual variation and the causes thereof. In their sample they were able to show that children with SCTs seemed particularly sensitive to adverse environmental factors, whereas a supportive home background appeared to act as a protective factor.The information collected so far on individuals with SCTs indicates that this is a vulnerable group at increased risk of educational failure and neurodevelopmental disorder. However, there is considerable variation in outcomes within each of the three groups reviewed here. Furthermore, the majority of children with SCTs will be able to live independently and form close relationships as adults. Nevertheless, these conclusions are based on a remarkably small evidence base. The limited data from unselected studies suggest varied outcomes, but considerable work needs to be done to chart and understand this variability, and to explain the risks and protective factors that may be associated with outcomes at the cognitive and neurological level.Because of difficulties in recruiting participants, recent studies have tended to be focused on cases identified in childhood or adulthood via self-help groups or genetics clinics, and most of these have been restricted to Klinefelter syndrome. Although these studies may provide useful insights into neuropsychological profiles and brain correlates in individuals with an extra X chromosome, they can give a misleading picture of the typical outcome, as they will inevitably exclude individuals who are not giving cause for concern.Neonatal screening is no longer feasible, but there is potential for studying larger groups of children identified on prenatal screening. Although such a sample is not totally unbiased – it is likely to contain a disproportionate number of older parents and will inevitably include only those parents who decided to continue the pregnancy – it could provide valuable information on typical outcomes for children with SCTs, without the bias that comes from selecting only those cases diagnosed because of childhood problems. A dilemma for such a study is that the child may not have been told about the trisomy. Nevertheless, one could use standardized methods of parental reporting, which have provided valuable information in some of the studies reviewed here and typically agree well with direct testing.In addition to informing our understanding of brain–behaviour relationships in people with an extra sex chromosome, we urgently need more information about the impact of SCTs to guide clinicians as they advise parents receiving a prenatal diagnosis about possible future outcomes, and to set the scene for targeted intervention early in life, where appropriate.


Background. Genetic polymorphisms of drug metabolizing enzymes involved in the detoxification pathways of carcinogenic substances may influence cancer risk. Methods. Case-control study that investigates the relationship between CYP1A1 Ile/Val, exon 4 mEH, and GSTM1 null genetic polymorphism and the risk of oral and pharyngeal cancer examining the interaction between these genes, tobacco, and alcohol. 92 incident cases and 130 consecutive hospital-based controls have been included. Results. No significant associations were found for any of the genotypes assessed. The estimated risk was slightly elevated in subjects with the wild type of the mEH gene and the null GSTM1 genotype. For exon 4 mEH heterozygous polymorphism, the risk was slightly lower for heavy smokers than for light smokers. The inverse association was observed for the GSTM1 null genotype. Conclusions. The results suggest that exon 4 mEH and GSTM1 null polymorphisms might influence oral and pharyngeal cancer. Oral and pharyngealcancers represent an important problem worldwide. The incidence and prevalencerates for these tumors are double in men than in women. Cancers of the oralcavity rank as the eighth most common cancer among men, being responsible for3% of the cancers diagnosed in this gender . MortaliTobacco and alcoholare the main risk factors for oral and pharyngeal cancers. In USA and Europe,they are responsible for 75–80% of thesetumors , 3. EpidThe reason why someindividuals develop cancer and others do not can be attributed, at least partly,to variations in genetic polymorphisms responsible for metabolizingcarcinogenic substances found in tobacco and alcohol. Although many genes havebeen associated with metabolism of these compounds, some with the highestrational of those beinginvolved are CYP1A1, mEH, and GSTM1.The CYP1A1 genebelongs to the CYP1 subfamily and encodes for the enzyme aryl hydrocarbonhydrolase, which is involved in the activation of many polycyclic aromatichydrocarbons and aromatic amines  and is pThe microsomal formof epoxide hydrolase is primarily associated with the metabolism of exogenousxenobiotic compounds. Its interest in oral and pharyngeal cancers comes fromthe fact that it has been detected in alltissues including the aerodigestive tract and catalyzes the hydrolosis of arene, alkene, and aliphatic epoxides frompolycyclic aromatic hydrocarbons and aromatic amines . EnzymatThe glutathione Stransferase (GST) comprises a family of phase II detoxifying enzymes thatcatalyze a great number of detoxification reactions that take place between thecytosolic glutathione and compounds containing an electrofilic centre . TheGSTIn the present case-controlstudy, we aimed to examine the relationship between the CYP1A1 Ile/Val, exon 4 mEH(139 Arg → His), and GSTM1 nullgenetic polymorphism and the risk of oral and pharyngeal cancers, investigatingalso the association with smoking, drinking, and the gene-gene interactions.The presenthospital-based case-control study was conducted at the Santiago de CompostelaUniversity Hospital Complex  between January 1996 and January 2000. Data was collected on a totalof 92 incident Caucasian male cases with histopathologically confirmeddiagnosis of primary oral or pharyngeal cancers. The study was restricted tonewly diagnosed patients over 20 years of age without a prior history ofcancer. For study purposes, tumors of the lip were excluded.A total of 130 consecutivecontrols were included from patients attending the Hospital ComplexPreoperative Unit for nonsmoking- and nonalcohol-related trivial surgery. Theinclusion criterion for controls was absence of prior history of cancer. Subjectsunder 20 years of age were excluded. The types of surgical procedures controls were scheduled to undergo mainlycomprised inguinal hernias, cataracts, and orthopedic surgery. Informed consentwas obtained from all study subjects prior to the interview and the extractionof total blood. Only 1 case and 6 controls refused to participate in the study. The study protocol was approved by the Galician Ethical Research Committee.All study subjectswere interviewed by a person purpose-trained to administer a structuredquestionnaire addressing various aspects of lifestyle, with special emphasis onsmoking habit, alcohol consumption, occupation, and other activities related to the development oforal and pharyngeal cancers. For cases, the interview was done as soon aspossible after cancer was detected and always within 15 days from diagnosis. The questionnaire used was previously applied to a group of individualsfulfilling the characteristics of the subjects going to be included and allquestions with difficult interpretation were duly amended.μg/mL. The genotypingassays were performed at the Molecular Medicine Unit of the Santiago deCompostela University Hospital Complex.After cases andcontrols were identified, whole blood samples of 6 mL were collected from eachsubject in heparin-containing tubes. The samples were stored at 4°C and centrifuged at 2800 rpmfor at least 10 minutes within the next 24 hours. The three independentfractions were isolated and stored at −84°C until analysis. DNA was extracted fromthe buffy coat of middle layer containing monocytes. The cells were washed withTE (10x) and centrifuged at 3000 rpm during 10 minutes several times until theywere cleaned. The pellet was then treated with lysis buffer and proteinase K in1% SDS previous to the extraction of DNA with phenol-chloroform and ethanolprecipitation similarly as previously described . The DNAμL containing 20 mM Tris-HCl (PH 8.4), 50 mM KCl, 3 mM MgCl2, 0.25 μMof each GSTM1 primer, 0.25 μM of each β-globin primer , 1 U Taq DNApolymerase (Promega), 2 μMeach dNTPs, and 600–900 ng of DNA. The GSTM1 primers were 5′-GAACTCCCTGAAAAGCTAAGC and 5′-GTTGGGCTCAAATATACGGTGG. A negative control was included in allbatches. The PCR conditions were 94° for 3 minutes, followed by 35 cyclesof 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 45 seconds, and afinal extension of 72°C for 7 minutes.Genotyping for GSTM1was carried out in the whole sample using a modified PCR method describedpreviously . Reactio139 arg), a 357-bp fragment containing thepolymorphic site was amplified pyrene-7,8-diol epoxide, the activated form ofbenzo[a]pyrene  but the Our study suggeststhat the effect of the GSTM1 null polymorphism is more noticeable among lightdrinkers, although this association was nonsignificant. This relationship wasmaintained when we took out the nondrinkers from the analysis (data not shown). This differential effect could be explained by the fact that the GSTM1isoenzyme, together with the alcohol dehydrogenase, is involved in theoxidation of ethanol to acetaldehyde . Even thEven though theCYP1A1 mutation has been shown to increase microsomal activity for convertingprocarcinogens, including PAH aromatic amines, the results of various reportson smoking-related cancers are inconsistent , 66–70. This paper hasseveral limitations. The main one is the small sample size included. This is especiallyimportant when analyzing polymorphisms that have a very low frequency in thepopulation, such as CYP1A1 Ile/Val, but also limits the power to identifygene-environmental and gene-gene interactions in any polymorphismsinvestigated. Nevertheless, it has to be taken into account that for cancerswith a low incidence such as oral and pharyngeal ones, 92 cases can be arelatively good number. Another limitation could be the fact that our study washospital-based and this could result in selection bias. Even though it has beensuggested that studies with hospital controls can provide lower risk estimates,since diseases of controls could be associated with the polymorphisms understudy, previous meta- and pooled analysis that assessed these polymorphisms onhead and neck cancers found no differences for hospital-based studies inrelation to population-based studies , 66. TheThe present studyhas also some advantages. One of them is that the participation of cases andcontrols was very high. All the cases belonged to the same catchment area,which has a unique reference hospital, and were collected consecutively, makingthem representative of the cases in that area. The controls also belonged tothe same area and did not have any symptom or disease related to alcohol or tobaccoconsumption. Another advantage is the fact that the genes analysed here were ofphase I and phase II, which adds value to the results obtained. When studyingsusceptibility genes in many occasions, both types of genes are not studied andindividuals can have susceptibility in type I compensated with the activity of the other type suchas it was shown in Park et al. investigation .As a conclusion, itseems that even though none of the three genotypes analysed have a significantassociation with the risk of oral and pharyngeal cancer in this population, therisks of tobacco and alcohol consumption might be modified by GSTM1 null andexon 4 mEH polymorphisms. The small number of nonsmoking and nondrinking subjectsin our population limited our analysis, so we propose that further studies are carriedout to clarify this question. Even though we found no significant interactionbetween the GSTM1null genotype and the exon 4 mEH genotype, the GSTM1 absencedid show a slight rise in risk so this should also be investigated taking intoaccount other polymorphisms including the CYP1A1.


P<0.01), under the control of the influence of BCP double mutations. Most of the pre-S deletions occurred in, or involved, the 5′ half of the pre-S2 region and the difference between HCC  and controls  was significant for this region (P=0.015). There was no significant difference in pre-S deletions between the BCP mutant group and BCP wild-type group (P>0.05), nor was the prevalence of pre-S deletions significantly different between genotypes B and C (P>0.1). These results suggest that pre-S deletions constitute an independent risk factor for HCC and their emergence and effect are independent of BCP mutations. The 5′ terminus of pre-S2 is the favoured site for the deletion mutations, especially in HCC cases. Further prospective studies are required to confirm the role of these mutations in the development of HCC.A matched nested case–control study of 33 paired cases and controls was conducted, based on a study cohort in Long An county, Guangxi, China, to determine whether infection with hepatitis B virus (HBV) with pre-S deletions is independently associated with the development of hepatocellular carcinoma (HCC), without the confounding effects of basal core promoter (BCP) double mutations. The prevalence of pre-S deletions was significantly higher in HCC  than the controls  ( HoweverThe emergence of persistently infected individuals of HBV with deletions in the pre-S region has been recognized for many years  of Long An county and the CPDC of Guangxi Province. From 1 March 2004, the study teams travelled to 128 villages in each of the 12 townships of Long An county to visit agricultural workers aged 30–55 to collect a 3 ml sample of blood by venepuncture for screening for HBsAg. All samples were tested for HBsAg and positive samples were tested in China for HBV DNA by using nested PCR. We also detected and excluded those samples positive for anti-hepatitis C virus (HCV) to eliminate the confounding effect of HCV infection on the incidence of HCC. We started to follow up the study subjects from 1 July 2004. Each study subject completed a one-page questionnaire at the first visit and provided a serum sample every six months for the assessment of virological parameters and alpha fetoprotein (AFP) concentrations and was monitored for HCC by ultrasonography (US). All cases of HCC diagnosed were confirmed at the Medical University of Guangxi, the Cancer Institute of Guangxi or the Hospital of Guangxi (Nanning) using criteria set by the Chinese Anti-Cancer Association.After 36 months follow-up, 61 individuals were diagnosed with HCC and sufficient volumes of serum remained from 33  for this study. A control was selected from the cohort for each case, matched for age , sex and the status of BCP sequence (wild type or double mutation) . Alanine aminotransferase (ALT) levels were determined using a Reitman kit (Sichuan Mike Scientific Technology Company).DNA was extracted from 85 μl serum by Pronase digestion followed by phenol/chloroform extraction. For nested PCR, first round PCR was carried out in a 50 μl reaction using primers LSOB1  and MDN5R , or P2 (Products from the second round were confirmed by agarose gel electrophoresis and then purified using the GenElute PCR Clean-up kit (Sigma) according to the manufacturer's instructions. Cycle sequencing was carried out directly on both strands using 2 μl purified amplicon DNA and primer LSBI1 or ADELN  and a BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's instructions. The nucleotide sequences derived in this study have been submitted to GenBank/EMBL/DDBJ under accession numbers FM211353–FM211418.http://www.vgb.ucl.ac.uk/starn.shtml (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi).HBV genotyping for both cases and controls were determined using the sequences above and the STAR program [χ2 tests, McNemar's test and Fisher's exact test. All P-values were two-tailed and P<0.05 was considered to be significant. Univariate and multivariate conditional logistic regression analyses were performed using the Statistical Package of Statistical Analysis System (SAS version 9.0). Variables with P<0.05 on univariate analysis were analysed by stepwise multivariate analysis for independent risk factors associated with HCC development. All P-values were two-tailed and P<0.1 was considered to be significant.The statistical comparisons were performed using Pearson's P<0.01). There was no significant difference in the prevalence of deletions between males and females (P>0.1). On univariate analysis, pre-S deletion was independently associated with the development of HCC but HBeAg, anti-HBe, ALT concentrations and genotypes were not. On multivariate analysis, pre-S deletion remained independently associated with the development of HCC  (Table 3Pre-S deletions were found in HBV DNA from 15 of 33 (45.5 %) HCC cases tested (Table 2P=0.015).The locations of the deletions are shown in Fig. 1P>0.1). The ATG initiation codon was mutated to ATA in six study subjects and to ATC, ACG, GGG and GTA (one study subject each) in the remainder. In addition, six cases with a mixture of ATG and ATA (one subject), AGG (two subjects) or GTG (three subjects) were not included in the analysis.Expression of the middle surface protein also may be abrogated by point mutations in the pre-S2 initiation codon. That codon was changed in 18.2 % (6/33) of HCC cases but only 12.1 % (4/33) of controls, although the difference is not statistically significant  and 74.2 % (49/66) of the subjects, respectively. The remainder  are infected with a recombinant of genotype C and HBV sequences of unknown genotype (U/C recombinant); details of this recombinant, described originally in Vietnam (which neighbours Guangxi), have been presented previously (4–105 copies ml−1 was suggested to be the cut-off (3–105 genomes ml−1 (1653T) and elsewhere in the basal core promoter (T1753V) were found not to be associated with the development of HCC in our study cohort  is small, so we cannot carry out stratification analysis of a synergistic effect with BCP double mutations in the development of HCC. HBV viral loads have been reported to be associated with the development of HCC and 10mes ml−1 . In the As noted above, the polymerase ORF is maintained in all isolates regardless of the size and number of deletions. All of the deleted viruses seem to be able to synthesize truncated versions of the large surface protein, with the exception of the isolate from HCC case BO105 which has two extensive deletions and seems unable to make either the large or middle surface protein, although the major surface ORF is intact. It is not clear that such a virus is viable. The sequence from HCC case DH230 is noteworthy; although it is a U/C recombinant  and probPre-S2 initiation codon mutations may abrogate the expression of M, resulting in pre-S2-defective variants (It has been reported that the development of pre-S deletion mutations is associated with HBV genotype and their prevalence is higher in genotype C than genotype B in Taiwan, Japan and mainland China  but theiIn summary, the results of this matched nested case–control study showed that the pre-S deletion mutations, particularly deletions involving the pre-S2 region, are associated with the development of HCC. The pre-S deletion mutations act independently of BCP mutations. The development of pre-S deletions and BCP mutations is independent. There is no significant difference in pre-S deletions between genotypes B and C but the pre-S deletions are more common in the U/C recombinant. However, this is a case–control study and further prospective studies are needed to confirm the role of these mutations in the development of HCC.


The role of the Fcγ receptor IIa (FcγRIIa), a receptor for C-reactive protein (CRP), the classical acute phase protein, in atherosclerosis is not yet clear. We sought to investigate the association of FcγRIIa genotype with risk of coronary heart disease (CHD) in two large population-based samples.FcγRIIa-R/H131 polymorphisms were determined in a population of 527 patients with a history of myocardial infarction and 527 age and gender matched controls drawn from a population-based MONICA- Augsburg survey. In the LURIC population, 2227 patients with angiographically proven CHD, defined as having at least one stenosis ≥ 50%, were compared with 1032 individuals with stenosis <50%.In both populations genotype frequencies of the FcγRIIa gene did not show a significant departure from the Hardy-Weinberg equilibrium. FcγRIIa R(-131) → H genotype was not independently associated with lower risk of CHD after multivariable adjustments, neither in the MONICA population (odds ratio (OR) 1.08; 95% confidence interval (CI) 0.81 to 1.44), nor in LURIC .Our results do not confirm an independent relationship between FcγRIIa genotypes and risk of CHD in these populations. BC019931] is expressed on various cells involved in atherogenesis like monocytes, macrophages, platelets and neutrophils, and though it has been detected immunocytochemically in human atherosclerotic lesions, its potential active role in atherosclerosis is still a matter of controversy [Fcγ receptors (FcγR) bind the Fc portion of immunoglobulin (Ig) and thereby link antigen recognition by antibodies with cell-based effector mechanisms -4. Althotroversy ,6. Calvetroversy ,8.A specific genetic polymorphism affecting its function has been described in the FcγRIIa, causing an amino acid exchange from arginine (R) to histidine (H) at position 131. H131 is the only FcγR that recognizes IgG2 efficiently. The codominantly expressed R131 allele has affinity for IgG3 and IgG1 subtypes and exhibits a strongly diminished binding to IgG2 .Increasing evidence suggests that immune processes play a substantial role in atherosclerosis . It has On the other hand, Gavasso et al. assessed the relationship between polymorphisms in three different FcγR genes and coronary artery disease (CAD). In 882 patients undergoing coronary angiography, no association was found between the FcγRIIa genotypes and CAD .FcγRIIa has been identified as the major receptor for human C-reactive protein (CRP) . Pan et CRP, the classical acute phase protein, is a strong cardiovascular risk marker, and responds rapidly to various proinflammatory stimuli -18. FurtDevaraj et al. showed that CRP binds to FcγR, mainly FcγRIIa on human aortic endothelial cells (HAEC) to mediate its biological activity, and incubation of HAEC with monoclonal antibodies to FcγRIIa before addition of CRP markedly reversed the proatherogenic effects of CRP on prostacyclin synthase and IL-8. Similar results were observed with intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, cGMP and PAI-1 . CRP stiRecently, research in this field has focused on the two allelic variants of FcγRIIa. It has been shown that CRP binding is allele-specific. CRP binds with high avidity to monocytes and neutrophils from R131 homozygotes, but shows strongly decreased binding to cells from FcγRIIa H131 homozygotes and intermediate binding to FcγRIIa heterozygotes, implying that the HH131 genotype may thus be less susceptible to coronary heart disease (CHD) .Since there is no direct evidence that the polymorphism may be associated with a lower risk of CHD in vivo we sought to investigate the potential association between the allelic status of FcγRIIa and risk of CHD in two large independent populations.The MONICA (MONItoring of trends and determinants in CArdiovascular disease) Augsburg study was part of the multinational World Health Organisation MONICA project. From 1984/85 to 1994/95, three population-based MONICA surveys had been conducted, and a regional myocardial infarction registry had been established. Altogether 13,427 subjects of caucasian background  were prospectively followed within the frame of the KORA (Cooperative Research in the Region of Augsburg). The design of the project has been described in detail elsewhere [The presence of CHD was defined as a validated diagnosis of non- fatal and fatal MI according to MONICA diagnostic criteria . DiabeteA total of 3,297 patients with suspect CHD participating in the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study were included in these analyses. The design of the study has been described in detail elsewhere . AnalyseThus, 2,227 patients with significant CHD were compared with 1,032 individuals with stenoses <50% (control group). DM was diagnosed according to the criteria of the American Diabetes Association (ADA) if plasma glucose was >1.25 g/L in the fasting state or 2.00 g/L two hours after an oral glucose load, which was performed in all subjects not previously diagnosed as having DM, or if individuals received oral hypoglycemic drugs or insulin. Hypertension was defined as having a systolic and/or diastolic blood pressure > 140 and/or 90 mm Hg or having a history of hypertension.All subjects underwent a standardized interview conducted by trained personnel. The information obtained included current health status -especially physician diagnosed hypertension and DM, medical history, and current medication. Furthermore, sociodemographic characteristics and lifestyle habits were recorded.Both studies have been approved by the appropriate ethics committees and have therefore been performed in accordance with the Declaration of Helsinki.2+ . CRP measurements were done by a high-sensitivity latex enhanced nephelometric assay on a BN II analyser .A non-fasting venous blood sample was obtained from all study participants while sitting. Total cholesterol was determined with an enzymatic method . LDLc was measured after precipitation with dextran sulphate  and HDLc after precipitation with phosphatungstate acid and Mg2+ . CRP concentrations were determined using the same high-sensitivity latex-enhanced nephelometric assay on a BN II analyser . All laboratory analyses in both studies were done in a blinded fashion.Venous blood was drawn from all participants early in the morning before cardiac catheterization. Total cholesterol was determined enzymatically. HDLc was determined after precipitation with phosphatungstate acid and MgGenomic DNA has been isolated from white blood cells by standard methods . FcγRIIaBstUI (New England BioLabs) and analysed by agarose gel electrophoresis. The G allele (H131) yielded fragments of 120 and 23 bp; the A allele (R131) was not digested (143 bp).Genomic DNA was prepared from EDTA anticoagulated blood. Receptor polymorphism was determined from polymerase chain reaction and restriction typing. The primers for amplification were 5'-CCTTGGACAGTGATGGTCAC-3' and 5'-TGGAAAATCCCAGAAATTCTCGC-3'. Amplification products were digested with In both populations demographic, clinical and biochemical characteristics were compared in a descriptive way. Differences in genotype distribution among cases were compared with values predicted by the Hardy-Weinberg equilibrium. The odds ratio (OR) and 95% confidence intervals (CI) for the presence of CHD were assessed by multivariable logistic regression analysis, adjusted for traditional cardiovascular risk factors, given the HH131 genotype compared to RR131 and/or RH131 genotypes. The following established risk factors were used in the fully adjusted models: in MONICA, age, sex, years of education, alcohol consumption, smoking status, BMI, history of hypertension, history of diabetes, HDLc; in LURIC, age, sex, smoking status, BMI, history of hypertension, history of diabetes, and HDLc. Finally, we calculated a power assessment: A two group Chi-square test based on a two-sided α of 0.050 and a power of 80%was used to estimate which statistically significant excess risk (Odds ratio) between cases and control of each study could be detected at minimum based on the proportions of the genotype HH131 versus the other two genotypes combined.Table Table Table Table In the LURIC population, the OR for CHD associated with the HH genotype in the LURIC population was 0.99  after adjustment for age and gender and 1.01  after further adjustment for other traditional cardiovascular risk factors. Again, if the RR and RH genotypes were taken as reference, the OR for the association of CHD with the HH genotype was 0.96  after partial adjustment for age and gender, and did not change  after multivariable adjustment.We expected carriers of the HH131 genotype to have a lower risk of CHD because of the strongly decreased binding of CRP to cells expressing the specific FcγIIa receptor. However, results from these two large independent case-control studies do not suggest that the HH131 genotype of FcγRIIa may be associated with a lower risk of CHD.Recently published experimental data are in support of the negative findings in our populations. It was shown that CRP binding to monocytes from R131 homozygotes and RH131 heterozygotes was dose-dependent and saturable at approximately 100 mg/L of CRP. The binding avidities of CRP for monocytes from R131 homozygous and RH131 heterozygous donors were 24 and 75 mg/L, respectively . Since ain vitro studies demonstrated reduced phagocytosis of IgG2-opsonized particles in cells from individuals homozygous for FcγRIIa-R131 [S. pneumoniae and H. influenza, and maybe in this way provides partial protection from encapsulated infectious pathogens in individuals bearing the R131 allele of FcγRIIa, contributing to a reduction of atherosclerotic risk [S. pneumoniae, in the presence of CRP, primarily activates the classical complement pathway and complement is required for the protection by CRP. However, CRP protection does not require FcγR, indicating that CRP is not primarily protective by direct opsonization but by complement and the subsequent opsonophagocytosis [H. influenza. Using a CRP transgenic mouse model, CRP has also been shown to be protective against gram-negative bacterium [As expected, crystal structure analyses showed that the amino-acid position 131 is critical for the interaction with the IgG-Fc-tail . In certIIa-R131 . Howevertic risk . The opsocytosis . CRP andacterium -39. ThisIn summary, one can hypothesize that there is a critical counterbalance between complement activation by CRP and subsequent protection of bacteria-induced inflammation and cardiovascular disease and the proatherogenic role of CRP in both, the case of strong binding and the case of weak binding of CRP.Recently three studies suggested the possibility that FcγRI could be the high-affinity receptor for CRP -42. UsinOur study has several strengths that need to be mentioned. We carefully selected cases and controls and, in addition, we extensively recorded conventional risk factors in order to assess their potential for confounding and carried out adjustments in multivariate models. The large sample sizes and the independency of the two populations also contribute to the strength of this study. In the MONICA population, all analyses were done in patients with a history of prior MI and compared to population-based controls. In the LURIC population, consecutive cases with angiographically proven stenosis of a major coronary artery of at least 50% were included. Since the FcγRIIa could not be found in mice, such well designed epidemiological studies are of considerable interest in investigating the receptor.Our study also has some limitations. We used a case-control design in both populations, therefore the temporal relationship of the plasma parameters and disease is difficult to establish. However the temporal relationship of genotype and CHD is certain. Selection and survival bias may represent a further problem. The appropriateness of the control group of the LURIC study deserves discussion. Early lesions undetectable by angiography could not be ruled out in controls, nor was coronary microvascular dysfunction. Nevertheless, coronary angiography remains the gold standard for the diagnosis of clinically relevant CAD, and it is well known that the future incidence of cardiac events is low in subjects with normal coronary angiograms. Although the present study did not have the power to detect a very weak association between FcγRIIa genotypes (i.e. HH131 vs. others) and CHD, it had a power of 80% to detect an OR of 1.46 (α = 0.05) or larger in the MONICA study and an OR of 1.26 or larger in the LURIC study.Our results do not suggest an independent and clinically relevant relationship of the FcγRIIa gene polymorphism with risk of CAD. Consequently, it seems unlikely that, in the population studied, FcγRIIa R(-131) → H genotype represents a promising tool for identifying those at increased risk for CHD.AAI: ankle-arm index; ADA: American Diabetes Association; AHA: American Heart Association; BMI: body mass index; CAD: coronary artery disease; CHD: coronary heart disease; CI: confidence interval; CRP: C-reactive protein; DM: diabetes mellitus; EC: endothelial cell; ERK: extracellular signal-regulated kinase; HAEC: human aortic endothelial cell; HDLc: high-density lipoprotein cholesterol; ICAM: intercellular adhesion molecule; Ig: immunoglobulin; IL: interleukin; KORA: Cooperative Research in the Region of Augsburg; LDLc: low-density lipoprotein cholesterol; LURIC: LUdwigshafen RIsk and Cardiovascular Health; MI: myocardial infarction; MMP: matrix metalloproteinase; MONICA: MOnitoring trends and determinants in CArdiovascular disease; OR: odds ratio; PAI: plasminogen activator inhibitor; TNF: tumour necrosis factor; VCAM: vascular cell adhesion molecule.The authors declare that they have no competing interests.MK drafted the manuscript. TI, CV, WM and MHH carried out molecular genetic studies, participated in the study design and coordination. DR and WM analysed the data. BB, CM, BW and NK contributed to sample collection and critically revised the manuscript. WK conceived the study and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


CFH), complement component 3 (C3) and age-related maculopathy susceptibility 2 (ARMS2) have repeatedly been shown to confer significant risks for AMD; however, their role in disease progression and thus their potential relevance for interventional therapeutic approaches remains unknown.Age-related macular degeneration (AMD) is a prevalent cause of blindness in Western societies. Variants in the genes encoding complement factor H  due to AMD. A quantitative phenotype of disease progression was computed based on longitudinal observations by fundus autofluorescence imaging. In a subset of 99 cases with pure bilateral GA, variants in CFH (Y402H), C3 (R102G), and ARMS2 (A69S) are associated with disease  when compared to 612 unrelated healthy control individuals. In cases, median progression rate of GA over a mean follow-up period of 3.0 years was 1.61 mm2/year with high concordance between fellow eyes. No association between the progression rate and any of the genetic risk variants at the three loci was observed (P>0.13).Here, we analyzed association between variants in CFH, C3, and ARMS2 confer significant risks for GA due to AMD. In contrast, our data indicate no association of these variants with disease progression which may have important implications for future treatment strategies. Other, as yet unknown susceptibilities may influence disease progression.This study confirms that variants at  Age-related macular degeneration (AMD [MIM 603075]) is the leading cause of blindness in Western countries C3 [MIM 120700]) gene ARMS2 [MIM 611313]) HTRA1 [MIM 602194]) AMD is a complex condition with both genetic and environmental factors contributing to disease CFH-Y402H and ARMS2-A69S and progression from early or intermediate stages of AMD to the advanced forms Going beyond cross-sectional association between AMD and genetic variants, Seddon et al. reported a significant association between the polymorphisms CFH, C3, and ARMS2 genes.A quantitative measure of geographic atrophy progression represents the detection and quantification of atrophic areas on the fundus at different time points in order to calculate atrophy enlargement rates over time. The gold standard to define such areas has been fundus photography; however, the reproducibility for defining and measuring GA by fundus photography has been reported by several groups to be only moderate, particularly for smaller lesions Table S1). Mean follow-up was 3.0 years . Mean number of measurements of GA area was 3.3 per eye  and 3.5 per patient . Mean age of patients was 71.8±7.4 years  and 76.2±5.3 years  for the controls . Overall, 61% of the AMD patients and 62% of the controls were female (p = 0.837). Considering the most general measurement of smoking history as a binary “ever or never” variable, AMD patients were significantly more likely to smoke than controls .Among the 619 participants of the FAM study cohort, 136 exhibited GA with no sign of CNV throughout subsequent follow-up examinations. Of these, 99 patients exhibited pure GA in both eyes throughout the study period and were included in the further analysis  in ARMS2 and R102G (rs2230199) in the C3 gene were strongly associated with GA due to AMD , which is very similar to our previous report 2 . As a measure of concordance of the GA progression rate between the right and left eye, the concordance correlation coefficient In a case-control association analysis , polymorphisms Y402H (rs1061170) in e to AMD Table 1.CFH (Y402H), ARMS2 (A69S), and C3 (R102G), patients were grouped for genotypes of these three major risk genes , 0.275 mm2/year  and 0.292 mm2/year , respectively. There was 95% power to detect an effect of less than 0.4 mm2/year for any of the three genetic risk factors. A detailed power calculation is given in Table S2.To visualize the distribution of GA growth rate for individual genotypes of sk genes Figure 2CFH, C3, and ARMS2. The odds ratios for heterozygotes and homozygotes for any of the three genes are highly consistent with previous reports C3 as a significant risk variant for AMD where similar results were found for CNV and GA HTRA1 as a risk gene for CNV due to AMD, implicating that polymorphisms in the ARMS2-HTRA1 region are responsible for this specific phenotype of late AMD. This is a surprising notion since others, including our own study, did not find any difference in the association data between GA and CNV due to AMD ARMS2-HTRA1 variants and suggests that the association between the CFH and ARMS2 polymorphisms is independent of disease evolution towards the atrophic or the neovascular form of AMD.Here, we demonstrate a significant association between our study group with late AMD manifestations of pure GA and known variants at CFH, C3 and ARMS2 contribute to GA progression once GA has developed. It is of note that our analysis had good power to detect an additive allele effect of less than 0.3 mm2 of GA progression per year. A GA progression rate of 0.3 mm2 or less represents a relatively small effect with less than 1/5 of the mean effect in our study population. Moreover, it is clearly below the threshold of recent phase II studies with therapeutic intervention in GA  which aim to slow GA progression by at least 0.6 mm2/year.A significant original finding of this study is that none of the AMD-associated polymorphisms in Although the number of patients  may appear limited, the significance of this study mainly originates from its dependency on the major outcome variable which is a biologically based continuous variable. To put the size of our patient study group into perspective, the major population-based studies such as the Beaver Dam Eye Study as well as the major clinical trial sponsored by the National Eye Institute  all include similarly limited numbers of longitudinally phenotyped patients with pure bilateral GA. Specifically, AREDS has 70 such patients  The strength of the current data is based on the phenotypic assessment of GA by cSLO-based FA imaging, semi-automated image analysis and complex statistics condensing longitudinal data into one continuous phenotypic variable. Although its accuracy may improve with the number of measurements over time, we have previously shown that GA progression is linear, which makes two consecutive measurements sufficient to estimate GA growth rate, although the number of longitudinal observations improves the accuracy of the estimated GA progression rate. ARMS2-HTRA1 region makes it difficult to distinguish between ARMS2 HTRA1ARMS2 (*372_815del443ins54) that is in almost perfect LD with the A69S variant and directly affects the stability of the transcript by removing the polyadenylation signal and inserts a 54-bp element known to mediate rapid mRNA turnover. Strong linkage disequilibrium (LD) across the CFH is associated with AMD susceptibility. Our data confirm this finding specifically for GA. Recently, we have detected systemic complement activation in AMD and found that markers of chronic complement activation were associated with the CFH risk haplotype (including the Y402H polymorphism). CFH, C3, and ARMS2 are not associated with progression of GA and since our previous analysis did not detect a significant contribution of other characteristics (including smoking and BMI) Experimental CFH, C3, and ARMS2 confer risk for susceptibility of GA but appear unrelated to its progression, addressing the complement cascade or ARMS2-, and possibly HTRA1-associated pathways per se may not be promising strategies for alleviating disease progression once GA has developed. Instead, defining the exogenic and possibly the genetic factors responsible for GA progression could be crucial in helping those patients already suffering from advanced stages of the disease.Our findings may have strong implications for future efforts to design therapeutic interventions for AMD, although replication in an independent cohort and empirical treatment data are needed for further substantiation. Since variants within The study followed the tenets of the Declaration of Helsinki and was approved by the local Ethics Review Board at the University of Bonn. Informed written consent was obtained from each patient after explanation of the nature and possible consequences of the study.Fundus Autofluorescence in Age-Related Macular Degeneration; registration www.clinicaltrials.gov: NCT00393692). The study procedures have been previously reported. Patients with GA secondary to AMD were included from the longitudinal natural history arm of the multicenter FAM–Study , the optical and technical principles of which have been described previously. 2/year, 95% CI  previously 2/year, 95% CI .Only patient eyes with at least two follow-ups in a 6 month interval and with sufficient image quality to accurately determine the size of atrophy were included. To further improve on our previous analytical strategy Genomic DNA was extracted from peripheral blood leukocytes according to established protocols. Genotyping was done by TaqMan SNP Genotyping or by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry method . TaqMan Pre-Designed SNP Genotyping Assays  were performed according to the manufacturer's instructions and were analyzed with a 7900HT Fast Real-Time PCR System (Applied Biosystems). All SNPs showed high genotyping quality with an average call rate of 98.5%.http://www.r-project.org/), and the R-library “non-linear mixed effects” (NLME) was applied for hierarchical regression modelling. A linear mixed effects model was used to quantify GA growth. A detailed description of the longitudinal modelling process is provided elsewhere. Data management and statistical analysis were conducted using SAS Version 9.1.3 . The statistical analyses were carried out using software package R, Version 2.7.0 Click here for additional data file.Table S2Power values for alpha = 0.05 and additive allele effects of growth of geographic atrophy (n = 99)(0.04 MB DOC)Click here for additional data file.


It has been suggested that dopamine dysfunction may play a role in bipolar disorder (BD). An indirect approach to examine this issue was developed, focusing on associations between dopamine proxy measures observed in BD .3459 eligible bipolar patients were enrolled in an observational study. Incidence rates of tardive movement syndromes  were examined. A priori hypothesized associations between incident TDD and other dopamine proxies  were tested over a 2 year follow-up period.The incidence rate of tardive syndromes was 4.1 %. Incident TDD was independently associated not only with use of antipsychotics, but also with more severe bipolar symptoms, other extrapyramidal symptoms and prolactin-related adverse effects of medication.Apart from the well-known association with antipsychotics, development of TDD was associated with various other dopamine proxy measures, indirectly supporting the notion of generalised dopamine dysregulation in BD. Extrapyramidal symptoms (EPS) in general and tardive dyskinesia (TD) in particular have been extensively studied in schizophrenia. Even though a number of studies suggest that bipolar patients experience higher rates of EPS  and TD compared to patients with a diagnosis of schizophrenia ,2, reseaBerk and colleagues, reviewing converging data on the role of dopamine (DA) in bipolar disorder, recently postulated the Dopamine Dysregulation Syndrome , based oWhile of major interest, hypotheses about alterations in DA function in severe mental illness are difficult to examine directly. It has been suggested, therefore, that alterations in DA function in severe mental illness  may be explored using clinical variables serving as proximity measures (i.e. indirect measures) for DA neurotransmission in different DA tracts. These may include, for example, presence of (i) prolactin-related adverse effects as a proxy for alterations in the tuberoinfundibular tract , ii) TD The Dopamine Dysregulation Syndrome, as applied to BD by Berk and colleagues , is currEMBLEM  was a 2-year prospective, observational study on the outcome of pharmacological treatment of mania across 14 European countries. A total of 3459 eligible in- and outpatients were enrolled at the discretion of the treating psychiatrist. Patients were eligible for participation if they were at least 18 years old and they initiated/changed oral medication for treatment of acute mania in bipolar disorder  within the standard course of care. During the acute treatment phase, assessments took place at baseline and 1, 2, 3, 6 and 12 weeks after baseline. The maintenance phase consisted of assessments at 6, 12, 18, and 24 months after baseline. ERB approval and patient informed consent were obtained according to local legal requirements. The study design has been described in detail in previous reports ,11.Investigators were requested to assess presence and severity of parkinsonism, akathisia, dystonia and TD that they judged to be associated with medications used to treat bipolar disorder. This assessment was based on the investigator's clinical experience and judgment and rated as follows: 0 = not present; 1 = present, but does not significantly interfere with patient's functioning; 2 = present, and significantly interferes with patient's functioning. Guided by previous analyses using these measures , movemenThe scales used to measure extrapyramidal symptoms were simple and did not include instructions on or specific anchors for differential diagnosis. Therefore, in order to avoid diagnostic misclassification, only persistent dystonia and TD were used in the current analyses. Using the persistence measure ensured differentiation from the acute syndromes, as, for example, acute dystonia would be unlikely to persist over two subsequent visits whereas tardive dystonia would. Persistence was defined as the presence of the individual syndromes over at least 2 consecutive visits. Thus, the variable "persistent dystonia" was rated as follows: 0 = no or acute/incidental dystonia; 1 = persistent dystonia. Similarly, "persistent TD" was rated as 0 = no or incidental TD; 1 = persistent TD. Persistent TD and persistent dystonia were analyzed together as a single group . The rational for combining TD and dystonia comes from (i) their strong association ,13, (ii)Incidence rates of TDD were determined by allocating each patient person-time for the TDD outcome according to the interval from baseline to the visit in which a patient was diagnosed with TDD. If no such diagnosis was made, the interval covered baseline to the final visit of each patient. Nine time bands were constructed . The incidence of TDD was calculated by dividing the total number of incident cases of TDD by the total person-years. The same procedures were followed for calculating separate incidence rates for tardive dystonia and TD. All analyses were conducted in the risk set of patients free of TDD at baseline.Cox proportional hazard regression was used to assess survival time without TDD associated with various time-varying clinical variables. The following clinical measures were used as proxies for DA dysregulation:(i) Psychotic and manic symptoms may be associated with high DA transmission in the mesolimbic pathway ,7. Depre(ii) Both amenorrhea and sexual dysfunction are associated with elevated prolactin levels induced by low DA transmission ,20 origi(iii) Extrapyramidal symptoms, including TD, have been hypothesized to reflect low DA transmission in the nigrostriatal DA tract in the brain . ResearcTherefore, presence of EPS as a proxy measure for dysfunctional DA transmission in the nigrostriatal tract, was tested for association with incident TDD.(iv) Use of antipsychotics (APs) is known to affect dopamine transmission  and in aThe four clusters of proxy measures for DA dysfunction  were individually included as independent variables in the Cox models in order to determine associations with incident TDD. Finally, all variables were entered simultaneously in the model in order to determine which associations persisted independently of other factors. Effect sizes were expressed as Hazard Ratio's (HR) and 95% confidence intervals. The two-sided significance level was 5%.Analyses were performed with and without confounders . For each analysis, all patients with non-missing values on the dependent and independent variables were included, as well as on all confounding variables in case of adjusted analyses. Confounders were based on a review of the literature within patient populations diagnosed with bipolar disorder, schizophrenia or psychotic disorders in general. The following confounders were introduced in the Cox regression models: social economic status , country, compliance , age per decade ,23, age change in CGI-BP Overall illness score relative to the previous visit was included as confounder.It is common practice to increase the dosages of antipsychotics or lithium in response to increased symptom severity. It is widely accepted that antipsychotic use and lithium in itself are associated with an increased risk for developing movement disorders and other adverse effects . Thus, aAs many confounding variables were included in the models, traditional control for confounding by inclusion of covariates in the model may not be sufficient, as the degree of 'control' afforded by such models depends on the overlap in characteristics between the two outcome groups. The use of the propensity score has been suggested as a means to obtain more complete control in these circumstances . The proNot all countries participated in the maintenance phase of the study (12 weeks onwards), resulting in a decrease in sample size after the 12 weeks . Apart from the decrease in overall sample size, the samples for the individual analyses varied somewhat on the basis of the availability of complete data for variables included in the separate models. All analyses were performed using the computer package STATA, version 10.0 .as well as at visit 2 (one week post-baseline). First occurrence of any incident tardive syndrome could therefore occur at visit 3 (two weeks post-baseline), while for the purpose of the current analyses incidence could first occur at visit 4, due to the requirement of persistence of symptoms for at least 2 consecutive visits. Consequently, misclassification of the acute form of dystonia, which usually has an onset within 5 days of new antipsychotic treatment [Additionally, sensitivity analyses were conducted using a stricter criterion for incidence in order to exclude any possibility of bias due to carry-over from influences occasioned by factors acting during the period before baseline. To this end, a stricter risk set was defined as the sample of patients free from dystonia or TD at baseline reatment , could bA total of 3459 subjects participated in the EMBLEM study and fulfilled the CGI-BP eligibility criteria (CGI-BP mania ≥ 3). 355 Patients were excluded from the analyses, either due to missing baseline ratings of dystonia and/or TD, or due to presence of dystonia and/or TD at baseline. Of the remaining 3104 patients, 43.5% were male and the mean age was 44.5 years (sd 13.4). The mean age of onset was 29.9 years (sd 11.1). Table The TDD incidence was calculated by dividing the total number of incident cases of TDD by the total person-years. Over two years of treatment, the sample contributed 3163 patient years, while there were 129 new cases of TDD, yielding an incidence rate of 4.1% . The separate incidence rates for TD and Tardive Dystonia were 1.0%  and 3.3%  respectively. Overall, incidence rates decreased with time, as illustrated by Table In order to examine the validity of combining the tardive dystonia and TD variables into a combined outcome, associations between these tardive movement disorders were calculated using Cox analyses. The probability of incident tardive dystonia in the presence of TD was high , while the probability of incident TD in the presence of tardive dystonia was also high .Table per CGI point = 1.13, 95% CI: 1.01, 1.28; P = .041), sexual dysfunction  and presence of EPS  were associated with incident TDD independent of other factors.In order to test the degree of independence of the different associations between TDD and the various DA-proxies, a model with all predictors included together was examined. This revealed that the CGI-BP Hallucinations/delusions  and TDD was slightly reduced and no longer significant. Results are included in Tables One in twenty-five participants experienced new onset tardive dystonia and/or TD over a period of 2 years. Although carry-over effects from previous medications cannot be excluded because (i) the visits used to define persistence were close to each other in time and (ii) there was a linear decrease in incidence of TDD over the entire study period, the results from the sensitivity analyses suggest that carry-over effects did not play an important role. Indeed, even if carry-over effects existed, there is no reason to assume that these should per se affect the reported associations with third variables. Reported incidence rates of tardive syndromes in bipolar populations vary widely in the literature, probably due to variation in use of medication , tardive syndrome definitions, characteristics of patient populations, study designs and mood state dependent fluctuations [To our knowledge, rates of tardive dystonia have not been reported in a similar population. One of the few reports available on tardive dystonia originates from a sample of chronic psychiatric patients from Curacao, mostly suffering from schizophrenia. The reported incidences for tardive dystonia and TD were 0.7%, and 10.2%, respectively , whereasA strong association was demonstrated between TDD incidence and various clinical factors, beyond antipsychotic medication, that were regarded as proxy measures for DA dysfunction in different tracts: high symptom severity scores, presence of sexual dysfunction, amenorrhea and EPS. Thus, (proxy) dysfunction in the nigrostriatal tract was found to be associated with (proxy) dysfunctions in the mesolimbic and the tuberoinfundibular DA tracts, indirectly suggesting generalised dopaminergic dysfunction.The association between EPS and TDD was anticipated based on similar findings in schizophrenia , and theExplaining the associations between more severe mania and psychotic symptoms on the one hand, and incident TDD on the other, is more complicated, as these symptom clusters seemingly represent high rather than low states of DA transmission, the latter being associated with TDD. To the best of our knowledge, an association between psychosis severity and TDD has not been reported before within a bipolar population. One might argue that the increase in psychotic symptoms and the emergence of TDD may be occasioned simultaneously by withdrawal of APs . AlternaThe association between more severe mania and TDD incidence may be considered surprising, as the limited reports available suggest a decrease of TD severity during manic episodes . It may This hypothesis could also explain the finding of decreasing TDD incidence over time, as better BD symptom control might represent a dampening of supersensitivity, which might be extended to multiple tracts. Another explanation for this finding could be that patients who are highly sensitive to TDD development would develop the syndrome early in the study, whereas less sensitive patients either develop the syndrome at a later stage or not at all.Even though the apparent contrast in the direction of proxy DA transmission status in the mesolimbic (up regulated) and nigrostriatal (down regulated) pathways that were associated with TDD is difficult to explain, it is compatible with the notion of broad dysfunction in the DA system. Alternatively, rather than an absolute interpretation of "high" or "low" states, DA instability or alterations in regulatory influences among the different DA tracts may instead represent the core characteristic driving the observed associations. The limited conclusion that can be drawn at this time is that more severe BD related symptoms are associated with an increased probability of tardive syndromes, the reason for which remains speculative.The findings of the current study are subject to a number of limitations. First, we depended on proxy measures of DA transmission. The literature indicates that the investigated symptoms and adverse effects are, to a certain extent, related to DA dysfunction in certain areas. However, various external factors may influence these symptoms and effects without (necessarily) altering DA transmission. For instance, use of concomitant medication or antagonism of other receptors by APs may affect sexual dysfunction ,20.Second, if a pan-dopaminergic dysregulation exists within BD, it remains unknown what may cause such a state. For example, a reduction in sensitivity of postsynaptic DA receptors, altered pre-synaptic activity, a reduction in absolute or relative DA concentrations or another mechanism yet to be revealed may be involved. Obviously, epidemiological research is not the appropriate methodology to investigate these underlying mechanisms; more neuroimaging and pre-clinical data are needed to shed light on the nature and extent of DA dysfunction within the framework of the hypothesized dopamine dysregulation syndrome proposed by Berk and colleagues  as well Third, as cogently discussed by Berk and colleagues, DA transmission is certainly not the sole underlying factor for neural dysfunction in BD; other neurotransmitters are likely to play a role. Even though the current model is far from able to explain all pathology associated with BD, the current literature is not incompatible with a major role of DA. Therefore, the model used may be regarded as an interesting scientific starting point for epidemiological research on the extent and nature of any involvement of DA in the bipolar spectrum.Fourth and final, the direct effects of lithium or antipsychotics dose adjustments on tardive movement disorders are complicated. Tardive dyskinesia has been reported to abate after dose increases , usuallyApart from the well-known association with antipsychotics, development of TDD was associated with various other dopamine proxy measures, indirectly supporting the notion of generalized dopamine dysregulation in BD.Inge van Rossum is employed by Lilly and has no further interests.Diederik Tenback has received honoraria related to time and expertise devoted to Lilly and BMS advisory boards.Jim van Os has received honoraria related to time and expertise devoted to EMBLEM study design and data analysis from Eli Lilly. He is/has also been an unrestricted research grant holder with, or received financial compensation as an independent symposium speaker from Eli Lilly, BMS, Lundbeck, Organon, Janssen-Cilag, GSK, AstraZeneca, Pfizer and Servier.IvR has been involved in the conception of the hypotheses, the statistical analyses, interpretation of the data and has drafted the manuscript. DT has been involved in the conception of the hypotheses, interpretation of the data and has reviewed the manuscript. JvO has been involved in the conception of the hypotheses, the statistical analyses, interpretation of the data and has reviewed the manuscript. All authors have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:


In the time period January 1998–December 2000, a case–control study on squamous cell cancer of the oesophagus was conducted in Montevideo, Uruguay. The main objective of the study was to estimate the odds ratios (ORs) associated with main food groups. For this purpose, 166 patients afflicted with squamous cell oesophageal cancer and 664 hospitalised controls were frequency matched on age and sex. Both series of patients were administered with a structured questionnaire. Aside from queries related with tobacco smoking, alcohol drinking and maté drinking, patients were interviewed with a food-frequency questionnaire (FFQ) on 64 items, representative of the usual Uruguayan diet. Red meat, salted meat and boiled meat displayed strong direct associations . On the other hand, fish and total white meat showed moderate protective effect . Total fruit intake displayed a strong inverse association , whereas total vegetable consumption presented a weak inverse association . These results suggest that vegetables, mainly cooked vegetables, are rich in thermolabile protective substances. On the other hand, boiled (stewed) meat, which is ingested at high temperature could be, like maté, a risk factor for squamous cell cancer of the oesophagus. Nutritional deficiencies have been suspected as risk factors for squamous cell oesophageal cancer for more than 20 years in Eastern countries, like Iran and China . On the This study, like other hospital based case–control studies, is limited by a number of drawbacks. Recall bias is an important limitation, mainly for cases, and could result in misclassification bias. For this reason, we decided to interview patients about their consumption habits 2 years before the date of the interview. Interviewer bias was avoided by careful training of the workers who personally conducted the interviews. Finally, the lack of validation study of the FFQ is a limitation. On the other hand, the high response rates, both for cases and controls are a strength of this research. Therefore, we decided to examine the role of foods in the aetiology of squamous cell carcinoma of the oesophagus.In the time period January 1998–December 2001, a case–control study on squamous cell carcinoma of the oesophagus was carried out in the four major hospitals in Montevideo, Uruguay. In this period, 175 newly diagnosed and histologically verified cases with squamous cell cancer of the oesophagus were considered eligible for the study. Nine patients (5.1%) were too ill and it was decided to exclude this subset of patients. The final number of cases was 166 (response rate 94.8%).In the same time period and in the same hospitals, 692 patients with conditions not related with tobacco smoking, alcohol drinking and without recent changes in their diets were considered eligible for the study. From this initial number of patients, 28 refused the interview, leading a final total of 664 controls (response rate 95.9%). These controls were frequency matched to cases on age (in decennia) and sex. Controls were afflicted by the following diseases: eye disorders , abdominal hernia , acute appendicitis , diseases of the skin , injuries , varicose veins , urinary stones , hydatid cyst  blood disorders , fractures  and osteoarticular disorders .Both cases and controls were administered with a questionnaire shortly after admission. The interviewers were two trained social workers, mostly unawere of the dietary risk factors for oesophageal cancer. The questionnaire included the following sections: (1) a sociodemographic section, (2) a complete tobacco smoking section, (3) a complete alcohol drinking section, (4) a maté section, including queries on daily amount, years of ingestion and temperature, (5) a complete occupation history, including job titles, industry and years of employment, (6) a family history of cancer in first-degree relatives, (7) self-reported height and weight 5 years before the date of the inteview, (8) menstrual and reproductive events and (9) a FFQ on 64 food items, considered representative of the usual Uruguayan diet. This FFQ was not validated but was tested for reproducibility with reasonably good results. In brief, foods and beverages were recorded as servings per year. Foods and food groups are shown in Food groups were categorised in quartiles, following the distribution of the control series. In order to compensate overeating or undereating, foods were energy adjusted by the residuals method of P=0.005), whereas total calory intake was slightly higher among cases as compared with controls (P=0.56). Cases consumed a higher amount of cigarettes than controls (P<0.001), and ingested more alcohol (P=0.02). Maté ingestion was higher among cases compared with controls (P=0.01) and maté drinked very hot was associated with a strong elevation in risk .The distribution of cases and controls by sociodemographic variables and selected risk factors is shown in P-value for trend=0.002). White meat, fish, cheese, raw vegetables, citrus fruits, other fruits, total fruits, total vegetables and fruits and pulses were inversely associated with risk of squamous cell carcinoma of the oesophagus .Odds ratios of squamous cell cancer of the oesophagus for significant foods are shown in Total processed meat see , fried mP-value <0.001). Among nondietary factors, tobacco smoking remained significantly associated with risk of squamous cell carcinoma of the oesophagus . Also, maté temperature was significantly and positively associated with the malignancy . Finally, alcohol drinking displayed a significant increased risk  , followed by stewed meat  and fresh vegetables . Tobacco smoking, alcohol drinking and maté temperature remained as significant factors but with slightly reduced risks.Several studies on oesophageal cancer reported a final model including nondietary and dietary factors (In summary, this study on squamous cell carcinoma of the oesophagus replicates previous studies on the protective effects of raw vegetables, citrus fruits and noncitrus fruits. Also, salted meat, a rich source of nitrosamines, was a strong risk factor. Finally, boiled meat, a component of stews that are ingested very hot is, possibly, a risk factor for this malignancy. The effect of this type of meat is much higher among smokers of black tobacco and hand-rolled cigarettes, compared with smokers of blond tobacco and commercial cigarettes.


We thank Cetta et al. for the interesting comments regarding our article . In theiWe agree that these are important issues. We have addressed the problem of inherited predisposition for DNA damage from a different angle, namely using the frequency of chromosomal aberration (CA) as a response indicator for occupational and environmental exposure to genotoxic agents. An increase in CA level in exposed individuals compared with controls has been documented since the 1990s . The conThe key issue—whether the association with cancer risk is attributable to exposure to carcinogenic agents or reflects inherited susceptibility and accumulated damages—was addressed with a nested case–control study on incident and deceased cancer cases in the Nordic and Italian cohorts . The maiIn all these studies, cancer has been studied as one entity. This summarization was mostly due to statistical needs, although the very early occurrence of chromosome damage in the carcinogenic pathway of most solid cancers provided a valuable rationale . A furthGSTM1 [gluta-thione S-transferase M1] and GSTT1 [gluta-thione S-transferase theta 1]polymorphisms [as all individual polymorphisms] . . . are not expected to have a dramatic influence on baseline CA [chromosomal aberration] or overall cancer risk” is not a conclusion of the study, but describes the conclusions of the extensive literature supporting this evidence . Our main concern was identify individuals more susceptible to damage from known genotoxic exposure. Because only GSTM1 and GSTT1 polymorphisms have been extensively evaluated in human surveillance studies, we tested only these genotypes. Within the consortium of studies included in this project (The beginning of Cetta et al.’s letter is misleading. The statement from our article  that “GSevidence . We agre project , furtherThe issue raised by Cetta et al. of decreased attention to pathogenetic mechanisms in favor of larger studies, with a consequent lack of biological plausibility, is only partially correct. Actually, in association studies that link a genetic polymorphism to the effect of exposure or to the risk of cancer, the lack of specificity is the main reason for failure. Another reason for their failure is small study size, which generates meaningless and often contrasting results. The conflict noted by Cetta et al. is apparent because, as demonstrated by the success of genome-wide association studies, the need of reaching a proper statistical power is as important as studying a genetic polymorphism in a specific pathway.