Pharmacogenetic testing to individualize ACE inhibitor therapy remains controversial. We conducted a systematic review to assess the effect modification of the insertion/deletion (I/D) polymorphism of the ACE gene on any outcome in patients treated with ACE inhibitors for cardiovascular and/or renal disease.Our systematic review involved searching six electronic databases, then contacting the investigators  responsible for the creation of these databases. Two reviewers independently selected relevant randomized, placebo-controlled trials and abstracted from each study details on characteristics and quality.Eleven studies met our inclusion criteria. Despite repeated efforts to contact authors, only four of the eleven studies provided sufficient data to quantify the effect modification by genotypes. We observed a trend towards better response to ACE inhibitors in Caucasian DD carriers compared to II carriers, in terms of blood pressure, proteinuria, glomerular filtration rate, ACE activity and progression to end-stage renal failure. Pooling of the results was inappropriate, due to heterogeneity in ethnicity, clinical domains and outcomes.Lack of sufficient genetic data from the reviewed studies precluded drawing any convincing conclusions. Better reporting of genetic data are needed to confirm our preliminary observations concerning better response to ACE inhibitors among Caucasian DD carriers as compared to II carriers. Angiotensin-converting enzyme (ACE) inhibitors have become a cornerstone in the management of patients with cardiovascular disorders . A largeIn the late 1980s, researchers began investigating genetic factors to determine the origins of inter-individual variability in patients' responses to ACE inhibitor therapy. Rigat et al.  identifiThe ACE I/D polymorphism has received much attention since its discovery, and many pharmacogenetic studies have been conducted in different patient groups to assess its effect modification. Debate, however, continues regarding the extent and direction of its effect modification ,14,18. TA previous publication details our study methodology .® version Cary North Carolina from inception to 2005), EMBASE , Biosis , the Cochrane Controlled Trials Register  and the Science Citation Index. In collaboration with an information specialist, we carried out a preliminary literature search in Medline to design the final search strategy [We performed extensive literature searches in (Pre-) MEDLINE  and ordered the full text of all potential articles. The two reviewers then examined the full texts of all retrieved citations and included studies if they 1) were randomised controlled trials comparing ACE inhibitors to placebo or to a non-active treatment 2) included patients with heart failure, primary and secondary hypertension, coronary artery disease, diabetic nephropathy, primary nephropathy and patients who had undergone renal transplantation and 3) had determined the insertion/deletion polymorphism.Two reviewers  independently assessed all obtained titles and abstracts stored in Reference ManagerTwo reviewers independently abstracted the data and assessed the quality of the included studies. We corrected discordant items based on obvious reading errors and resolved through consensus discordance based on real differences in interpretation. A third reviewer (LMB) resolved any remaining discrepancies.We used a pre-designed and pilot-tested data extraction form  and reco® statistical software package .Results of the data extraction and assessment of study validity were summarized in structured tables. In comparing the intervention group (ACE inhibitor) with control groups (placebo or no active control), treatment effects for each genetic subgroup  were assessed by calculating mean differences for continuous outcomes and relative risks for dichotomous outcomes. We also calculated mean differences and relative risks between intervention and control groups for the whole study population, including all three genetic subgroups (DD/DI/II), in order to provide an overall treatment effect of the study. All statistical analyses were performed using the StataFigure Table Details about the individual trials and participant profiles are provided in Table Of the studies included in the analysis of the effect modification, three had enrolled Caucasians with non-diabetic chronic nephropathies , insulinThe studies of Perna  and Van The study of Penno et al.  was alsoIn the study of Okamura , which iBased on our systematic review, evidence quantifying the extent of effect modification related to the I/D polymorphism is sparse. We did note a trend towards better response to ACE inhibitor therapy in DD Caucasians as compared to II carriers, who seemed not to benefit.The strengths of this review include the comprehensive literature search and strict adherence to systematic review methodology. We restricted our analyses to trials with placebo controls, as studies without a placebo control group do not allow for estimation of the ACE inhibitor effect and are likely to be confounded ,36.Although we identified 11 randomised controlled trials that assessed differences in treatment effects among genotypes, only the results of four trials studying 925 patients contributed to our analyses. The authors of the remaining seven studies that included 577 patients did not provide data about genetic subgroups in the intervention and control arms -28,30. ODespite an overall lack of evidence, some of our findings merit attention. For example, the second largest study of Caucasians with chronic nephropathies  showed aFrom the patient and clinician's perspective, it is still too early to draw solid conclusions about the optimal treatment among different genotypes. We can, however, speculate that an effect modification exists and that pharmacogenetic testing of the I/D polymorphism might provide additional information about the adequate treatment for these patients. From the public health perspective, it remains unclear whether pharmacogenetic testing would be justifiable in clinical practice. Before investing additional resources to reevaluate our preliminary observations in a primary study of high methodological quality, it might be informative to assess whether screening patients for the I/D polymorphism would have potential economic value. One recent economic analysis showed, for example, that screening men for the I/D polymorphism before starting lipid-lowering therapy with statins would result in considerable cost savings . Thus, eWe conclude that evidence is still scarce as there are few pharmacogenetic studies of high methodological quality with comprehensive reporting of their results. Nevertheless we did note a trend towards better response to ACE inhibitors in Caucasian DD carriers compared to II carriers. Future efforts should focus on conducting high-quality pharmacogenetic studies, and reporting of genetic data should be improved.The author(s) declare that they have no competing interests.All authors approved the manuscript.The study was supported by unrestricted educational grants from AstraZeneca, Switzerland, and from Pfizer, Switzerland. Dr Bachmann's work (grants no. 3233B0-103182 and 3200B0-103183) was supported by the Swiss National Science Foundation.


Ischaemic stroke in persons of European descent has a genetic basis, but whether the stroke-susceptibility alleles, the strength of any association, and the extent of their attributable risks are the same in persons of non-European descent remains unanswered. Whether ethnicity itself has a relevant or substantial contribution on those effect estimates is controversial. Comparative analyses between the ethnic groups may allow general conclusions to be drawn about polygenic disorders.angiotensin I converting enzyme (ACE) insertion/deletion (I/D) polymorphism with a mean OR of 1.90 (95% CI 1.23–2.93) in the Chinese and 1.74 (95% CI 0.88–3.42) in the Japanese; the summary OR for the C677T variant of 5,10-methylenetetrahydrofolate reductase (MTHFR) was 1.18 (95% CI 0.90–1.56) in Chinese and 1.34 (95% CI 0.87–2.06) in Koreans; and the pooled OR for the apolipoprotein E (APOE) gene was 2.18 (95% CI 1.52–3.13) in Chinese and 1.51 (95% CI 0.93–2.45) in Japanese. Comparing the commonly investigated stroke genes among the Asian groups against studies in persons of European descent, we found an absence of any substantial qualitative or quantitative interaction for ORs by ethnicity. However, the number of individuals recruited per study in the studies of persons of non-European descent was significantly smaller compared to studies of persons of European descent, despite a similar number of studies conducted per gene.We performed a literature-based systematic review of genetic association studies in stroke in persons of non-European descent. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined for each gene–disease association using fixed and random effect models. We further performed a comparative genetic analysis across the different ethnic groups  to determine if genetic risks varied by ethnicity. Following a review of 500 manuscripts, eight candidate gene variants were analysed among 32,431 individuals , comprising mainly Chinese, Japanese, and Korean individuals. Of the eight candidate genes studied, three were associated with ischaemic stroke: the These data suggest that genetic associations studied to date for ischaemic stroke among persons of non-European descent are similar to those for persons of European descent. Claims of differences in genetic effects among different ethnic populations for complex disorders such as stroke may be overstated. However, due to the limited number of gene variants evaluated, the relatively smaller number of individuals included in the meta-analyses of persons of non-European descent in stroke, and the possibility of publication bias, the existence of allele variants with differential effects by ethnicity cannot be excluded. This meta-analysis found that genetic associations so far studied for ischemic stroke among non-Europeans are similar to those found for persons of European descent. A stroke occurs when the blood supply to part of the brain is interrupted, either because a blood vessel supplying the brain becomes blocked or because one ruptures. Strokes are a substantial cause of death and disability worldwide, with most of the burden affecting people living in developed countries. Most strokes fall into a category termed ischemic stroke. This type is caused by blockages in the blood vessels supplying the brain, which can happen when there is a buildup of fatty deposits or clots within the blood vessels. Many of the risk factors for this particular type of stroke are affected by an individual's behavior, including for example smoking, high blood pressure, diabetes, inactivity, and so on. In addition, variations in an individual's genetic makeup might affect his or her chance of having a stroke. Previous research studies have shown that variants in many different genes are likely to be involved in determining the overall risk of having a stroke, each variant contributing in a small way to the risk.The group performing this study had previously carried out a systematic review of existing research, looking specifically at the genetics of ischemic stroke among people of European origin . However, it was not obvious whether the genetic risk factors for stroke they found would be the same for people from a different ethnic background. Therefore the research group wanted to find out what the genetic risk factors were for stroke among people of non-European origin and to compare these findings with those of their previous systematic review. This research might help to find out whether the genetic risk factors for stroke were different in people from different parts of the world.ACE I/D ; a variant in MTHFR ; and a variant in the gene APOE, which codes for a protein that plays a role in breaking down fats. The researchers then compared their findings from this study with the findings of a previous systematic review they had carried out among people of European origin. Overall, each gene studied seemed to have a similar effect in the different populations, with the exception of APOE, which seemed to be associated with stroke in the Asian studies but not in the studies from people of non-European origin. The researchers also found that generally the Asian studies suggested a slightly greater effect of each gene variant than the studies in people of non-European origin did.As a starting point, these researchers wanted to find all the different studies that had already been carried out examining the effect of genetic risk factors on stroke among people of non-European origin. To do this, searches were carried out of electronic databases using a particular set of terms. All resulting studies that involved genetic research in people of non-European origin and in which strokes were confirmed by brain scanning were then evaluated in more detail. The findings of different studies were combined if at least three studies were available for the same genetic variant. Eventually 60 studies were found that looked at the association between eight specific gene variants and stroke. The only data that could be included in a combined analysis came from Chinese, Japanese, and Korean populations. Three of the eight gene variants were associated with an increased risk of stroke. Those three gene variants were APOE, similar gene variants play a role in determining stroke risk in people of European origin and Asian populations. Although generally the studies examined here suggested a slightly greater effect of these gene variants in Asian populations, this is not necessarily a real finding. This greater effect may just be due to small-study bias. Small-study bias describes the observation that small research studies are more likely to produce a false positive result than are large research studies. Therefore, future studies that examine the genetic basis of stroke should recruit much larger numbers of participants from populations made up of people of non-European origin than has previously been the case.These findings suggest that, with the possible exception of http://dx.doi.org/10.1371/journal.pmed.0040131.Please access these Web sites via the online version of this summary at stroke from NHS Direct Health Encyclopedia entry on National Institute of Neurological Disorders and Stroke Stroke Information page from the The Stroke Association, a UK charity funding this studyWorld Health Organization on the distribution and burden of stroke worldwideInformation from the world atlas of heart disease and strokeThe WHO has a  The incidence and standardised mortality ratios for stroke are known to vary between different ethnic groups , which mHowever, whether stroke susceptibility alleles, their genetic effect sizes, or attributable risks are qualitatively (similarity of gene variants) or quantitatively (size of effects of such variants) homogenous across groups of differing ancestry  remains We performed a systematic review of genetic association studies of sporadic ischaemic stroke in persons of non-European descent and compared the findings to a prior overview we conducted among persons of European descent , which ahttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed; EMBASE, http://www.embase.com; Google Scholar, http://scholar.google.com; and Yahoo, http://www.yahoo.com) were searched up to and including January 2005 for all genetic-association studies evaluating any candidate gene in stroke in persons of non-European descent. The medical subject headings, terms, and text words used for the search were: cerebrovascular disease, stroke, brain infarction, and cerebrovascular disorder, in combination with polymorphism, genotype, gene, allele, or mutation. The search results were limited to humans and all languages were included. The references of all the computer-identified publications were searched for any additional studies, and the related articles option (a feature available on Medline) was used to search for any further possible related articles. Another search to identify all previous genetic meta-analyses in stroke was also performed.Electronic databases  confirmation of an ischaemic stroke diagnosis, and were analysed as a dichotomous trait. Studies were excluded if: (i) patients were under 18 years of age; (ii) they evaluated quantitative or intermediate phenotypes exclusively; or (iii) genotype frequency was not reported, in which case authors were contacted for this information where possible. For duplicate publications the smaller dataset was discarded.http://www.cc-ims.net/RevMan) and Comprehensive Meta Analysis version 2.2.023 .Data were analysed using software for preparing and maintaining Cochrane reviews  and 5,10-methylenetetrahydrofolate reductase (MTHFR), followed by apolipoprotein E (APOE) and SERPINE1 (plasminogen activator inhibitor-1 [PAI-1]) (The Chinese population was by far the most extensively studied across the various candidate genes . The sec[PAI-1]) .ACE (ACE I/D) was the most investigated polymorphism in all three ethnic groups . The overall OR for the nine studies in the Chinese population was 1.90 (95% CI 1.23–2.93)  among studies within this ethnic grouping. For the six Japanese studies .–23.ACE , and although the results were dominated by the relatively large study of Li et al.  –46 and t52–3.13) . No hete studies  was 1.51n groups , studiesapoliproprotein A (APOA) gene on ischaemic stroke with a pooled OR of 1.28 , the total number of cases and controls  was substantially higher than for all other gene variants. It was mainly due to the large case-control study by Sun et al. [Although only three studies –52 in ChSERPINE1 (PAI-1) [tumour necrosis factor-alpha (TNF) [angiotensin II type 1 receptor (AT1R) [angiotensinogen (AGT) [TNF and SERPINE1 (PAI-1), with the latter result consistent with previous results in persons of European descent 10. However, the number of studies for each genetic polymorphism was considerable smaller than that for other gene variants  –55, tumoha (TNF) –58, angir (AT1R) ,59–61, aen (AGT) ,49,61,62variants . In addiTNF gene ,57., the total number of cases studied were significantly greater in the samples of persons of European descent compared with the combined ethnic group  had also been extensively studied in persons of European descent, which enabled us to compare their effects against our previous meta-analysis in persons of European descent . Interestingly, the genetic effect observed in the Chinese population was even higher than that observed in other Asian ethnic groups, such as the Japanese population, although some overlap of their CIs remains . In contrast, for the MTHR C677T variant, the effect estimate within the groups of non-European descent (Chinese and Koreans), as well as when compared with that in persons of European descent, was homogenous  for the different candidate genes. Equally likely is that, since association studies conducted in persons of non-European descent are smaller compared to persons of European descent, ethnicity may act as a proxy for the presence of small-study bias, whether it represents publication bias, poorer study quality, or greater random error. Study quality itself is difficult to measure, but heterogeneity forms one aspect of this measurement. Whether the positive gene–disease ORs observed here will reduce toward a null effect in very large studies conducted in different ethnic groups remains to be seen. A plausible scenario is that the genetic effect is indeed genuine but lower than it currently appears to be, such as the case of the MTHFR C677T gene–coronary heart disease association in which the authors, in contrast to our findings, also observed a larger genetic effect in Chinese samples [The larger genetic effect observed in these candidate gene–stroke meta-analyses for Chinese studies, despite the smaller sample sizes, is in agreement with a recent systematic review of 13 gene–disease associations from a wide variety of complex disorders, in which gene effects in samples from Chinese people tended to be larger than those observed in non-Chinese Asian people, and more accentuated with studies conducted in persons of European descent. An interesting difference with that analysis is the case of the  samples .ACE I/D in persons of European descent versus Chinese individuals), the impact of similar genetic effects may be different in other ethnic populations.We are aware of one previous study that has attempted to address the question of ethnic differences in genetics of a wide variety of complex traits . These ihttp://www.hugenet.org.uk) will prove to play an essential role for an adequate study of gene effects in persons of non-European descent [Despite our attempt to evaluate the gene effects in all ethnic groups, we were limited by the fact that the vast majority of data in persons of non-European descent came from Chinese, Japanese, and Korean populations. The nature and extent of the genetic contribution to stroke and other multifactorial disorders in groups of differing ancestry should be considered an important endeavour for risk evaluation and primary prevention measure across all populations. First, evaluation of genetic effects across multiple ethnic backgrounds with substantial difference in cultural behaviour may prove to be the best scenario for the study of gene–environment interactions. Second, the presence of concordance of gene effects on disease risk, as well as on its effect on the direct intermediate phenotype, are strong tools to reliably identify putative functional variants. Third, since the uses of a genetic approach by using gene variants as tools of common intermediate phenotype is expected to provide great advantage in aetiological research, adequately powered genetic studies may help to unravel aetiological factors for the increasing public health disorders in persons of non-European descent such as stroke, which is rapidly becoming a major cause of mortality in Asia with over 1.6 million deaths in China alone in 2002 . Certain descent .As with all meta-analyses based on aggregate data, our results are dependent on published data. Although, as described above, efforts were conducted to limit small-study bias, (the most likely cause of which is publication bias), significant results of the test for small-study bias indicate that this bias has to be carefully considered..In addition, even ethnicity can be a difficult trait to define and sometimes is inconsistently reported in the literature. That the included studies have tended to come from their native countries, should minimize such misclassification. In addition, self-reported ethnic grouping, which is likely to take place in native countries, is known to be very reliable for categorization purposes . FinallyWe conclude that genetic associations so far studied for ischaemic stroke among persons of non-European descent are similar to those found for persons of European descent. However, all the published data on persons of non-European descent are largely derived from Chinese, Japanese, and Korean populations. To date, the evidence does not support ethnicity playing a major part in the genetic aetiology of common ischaemic stroke. However, people from a more diverse range of ethnic groups need to be recruited into genetic studies in much greater numbers than is currently the case.


Several narrative reviews have been conducted on the literature examining environmental correlates of physical activity (PA). To date these reviews have been unable to provide definitive summaries of observed associations. This study utilizes meta-analytical techniques to calculate summaries of associations between selected environmental characteristics and PA.Published studies were identified from electronic databases and searches of personal files. Studies were examined to determine the environmental constructs most frequently studied. Included studies (N = 16) examined at least one identified construct and determined associations between perceived environmental constructs and PA using logistic regression. Data were analyzed separately for crude and adjusted ORs using general-variance based fixed effect models.No significant associations emerged between environmental characteristics and PA using crude OR. The perceived presence of PA facilities , sidewalks , shops and services  and perceiving traffic not to be a problem  were positively associated with activity using adjusted ORs. Variance in PA accounted for by significant associations ranged from 4% (heavy traffic not a problem) to 7% (presence of shops and services).Results of the meta-analysis support the relevance of perceived environmental characteristics for understanding population PA. These results should encourage the use of comprehensive ecological models that incorporate variables beyond basic demographic information. The burden of disease attributable to physical inactivity is estimated at $377 million in Australia , $2.1 biFindings of existing primary studies and narrative reviews studying the associations between the perceived environment and PA are ambiguous. For example, pathways in close proximity to the home have been found to have a positive association with PA in some instances  while shMeta-analyses permit the strength and direction of associations between dependent and independent variables to be detailed ,21. In cphysical activity, exercise, walking, environment, built environment, and perceived environment. Second, a manual search was performed, for the same time period, on the following Journal titles: Medicine and Science in Sports and Exercise, Preventive Medicine, American Journal of Public Health, American Journal of Health Promotion, and American Journal of Preventive Medicine. These titles were included for manual searches as the majority of studies identified from electronic searches were published in these titles. Third, to identify previously unidentified studies, reference lists from narrative reviews on the topic 2              Variance ORu is the upper confidence interval of the ith OR, and ORi is the OR of the ith study. Confidence intervals and the crude OR from each case were used to estimate the overall OR for each environment variable using a general variance-based fixed effect model [Where ORct model .s = e∑[(wi × ln ORi)]/∑wi              ORs is the summary OR for the specified variable, wi is the weight of the ith study, which is the inverse of ith study's variance and ln ORi is the natural log of the OR for the ith study [Where ORth study . HomogenQ = ∑[wi × (ln ORmh - ln ORi)2]              i is the weight of the ith study, and ORmh is the natural log of the summary OR for the dependent variable. The Q statistic provides a test of whether the group of effect sizes represents a common population effect. A significant Q statistic indicates the effect does not represent the population of studies and that a search for moderator variables is warranted. Variance in PA explained by each variable (R2) was calculated using the procedure proposed by Nagelkerke [Where Wgelkerke .Table Using crude ORs, no variables demonstrated a significant association with PA Table .PA facilities was the only variable to exhibit significant heterogeneity among effect sizes . Unfortunately, the limited number of effect sizes within PA facilities precludes any search for moderators to resolve this heterogeneity.Age, income and education level were the most commonly adjusted for variables in the original studies. When individually adjusted ORs were combined, four significant associations emerged. People who reported the presence of PA facilities were more likely to engage in PA than those not reporting proximal infrastructure . Similarly, people reporting the presence of sidewalks, compared to those reporting the absence of sidewalks, were more likely to be physically active . Those people reporting the presence of shops and services within the neighborhood were more likely to engage in activity compared to people not reporting their presence . People reporting that heavy traffic was not a problem were more likely to engage in PA compared to those reporting heavy traffic was a problem . Examination of Q statistics revealed that PA facilities Q(7) = 22.58, p < 0.05, and the presence of shops and services Q(6) = 25.22, p < 0.05, both displayed significant heterogeneity of variance. Once again, the limited number of effect sizes within these two variables prevents any search for moderators to resolve this heterogeneity.2 = 0.04), PA facilities and sidewalks explained 5% and 6% of physical activity variance respectively, whilst shops and services explained the greatest amount, accounting for 7%.Individual constructs displaying significant associations with PA were further analysed using Nagelkerke's R to determine the amount of variance accounted for in PA. The presence of heavy traffic explained the least amount of variance  . The curSome characteristics identified in this study, while likely difficult to alter, may be more readily manipulated than existing land densities and land-use mixes, which have received much attention within transportation literature . CompariThe current research had several limitations that should be considered when examining the results. First, the sample was limited to studies that were published or accepted for publication, included adult populations, and used logistic regression analysis to determine associations between the perceived environment and activity. The limit to adult populations was imposed because very few studies examined features of the built or perceived environment in relation to the PA of children and youth. Thus, more research should be conducted with children and youth.Second, two variables  displayed significant heterogeneity of variance suggesting that those groups of effect sizes do not represent a common population and that potential sources of heterogeneity should be sought. Unfortunately, the limited number of studies included in the analysis prohibited any search for moderators in the sample and thus the results for these two variables should be interpreted with caution. Likely sources of heterogeneity are the measurement instruments used to assess PA and the types of PA that were assessed in the original studies . The possibility of PA measures and outcome measures as sources of heterogeneity should be examined in future analyses. Third, while several self-report measures with acceptable reliability ,69 are aThis quantitative review provides an objective summary of the association between a number of perceived environment variables and PA. The results should assist researchers in designing future research in the area. The small proportions of PA variance explained by the constructs reviewed suggest a need for the application of more comprehensive ecological models that include demographic, psychosocial, environmental, and biological variables.The author(s) declare that they have no competing interests.MD collated the literature, conducted the synthesis and meta-analysis and drafted the overall manuscript. JS assisted in the meta-analytic procedures and editing of the manuscript. KM conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Objective: Multiple studies suggest that bacterial vaginosis (BV) causes preterm labor; yet its routine treatmentremains controversial. In order to help to elucidate this controversy, we performed a thorough review of studieswith levels of evidence ranging from I to II–II.Methods:  We searched for all of the studies from the years 1994 to 2001 via Medline’s database, includingMD Consult and Ovid Mednet.Results:  Several trials discovered a decrease in the incidence of preterm labor when BV was treated, but mostof those trials were performed on women with a history of preterm labor. However, the majority of trialsreviewed advise against treatment of a general low-risk obstetric population, as there was no significant decreasein preterm labor.Conclusions: Therefore, based on the above studies and the current guidelines of the Centers for DiseaseControl and Prevention (CDC), treating pregnant women in high-risk populations who are diagnosed with BVprovides the clinician with an opportunity to possibly prevent preterm labor in this population. In nulliparouswomen without a history of preterm birth, treatment is recommended if other risk factors are present (e.g.gonorrhea or chlamydia). However, in the general low-risk populations, routine screening is not indicated.


TP53 is an attractive candidate for modifying age of onset (AO) in Huntington disease (HD): The amino-terminus of the mutated huntingtin (htt) exon 1 translation product has functional properties which may affect critically the TP53 pathway in HD neurons. The pathogenic domain of mutant htt interacts with nuclear transcription factors, and it potentially modulates TP53-induced transcriptional events. A single nucleotide polymorphism (SNP) resulting in the R72P exchange in TP53 protein might modulate the variation in AO. In addition, also the R196K replacement in human caspase activated DNase (hCAD) may theoretically affect the AO.We have genotyped the polymorphisms R72P and R196K in a well established cohort of 167 unrelated HD patients.The expanded CAG repeat explained 30.8% of the variance in AO. Adding the genotypes of the SNPs investigated did not affect the variance of the AO variance explained.In this replication study, no association was found explaining a significant amount of the variability in AO of HD thus contradicting a recent report. TP53 is the most important tumor suppressor gene. A variety of intracellular stress signals activate TP53 to induce either transient cell cycle arrest with stimulation of repair activities, senescence or apoptosis [Arg /Pro) in the TP53 gene differ with respect to the ability to suppress cellular transformation [hCAD), since DNase is responsible for DNA degradation during apoptosis [TP53 and hCAD variations modulate the AO of HD in our established cohort.Huntington disease (HD) is an autosomal dominantly transmitted, neurodegenerative disorder characterized by motor abnormalities, cognitive dysfunction and psychiatric symptoms . HD is cpoptosis . It has ormation  and induormation . Therefoormation . In the poptosis . VariatiTP53 gene was demonstrated after PCR amplification using the following primers: 5'-GAGGACCTGGTCCTCTGACT-3' and 5'-GTAGGTTTTCTGGGAAGGGA-3'. PCR was carried out in a final volume of 10 μl with 50 ng of DNA, 200 μM dNTP and 1 U Taq Polymerase. Thermal cycling was performed with an initial denaturing step for 5 minutes at 94°C.; 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were digested with the restriction enzyme Bsh1236I I at 37°C overnight and visualised on 2% agarose gels stained with ethidium bromide. This digestion yielded altogether 3 bands of different sizes: a 254 bp fragment (restriction site absent) corresponding to the C allele as well as a set of 160 and 94 bp fragments corresponding to the G allele (restriction site present). The polymorphism R196K in the hCAD gene was demonstrated using the following primers: 5'-CCTCTGACCACAGGACTGG-3' and 5'-TCTGTCGAAGTACGTGCCAT-3' under the same conditions mentioned above. PCR products were digested with the restriction enzyme Alu I at 37°C overnight. Digested DNA was electrophoresed on 3% agarose gels. The restriction fragments of the G allele were 129, 99 and 13 bp in length. The A allele harbors an additional restriction site so that the 99 bp fragment is digested into 73 and 26 bp. The variability in AO attributable to the CAG repeat number was calculated by linear regression. For the regression analysis, we used the AO as the dependent variable, the respective genotypes as independent variables.Our study population consists of 167 unrelated patients with the clinical diagnosis of HD , recruitThe CAG repeat number was considered as numerical variables, the other putative modifying genotypes were considered as nominal variables. SPSS Ver.11.0 for Windows (SPSS Inc.) was used for all statistical analyses.TP53 gene to be a significant risk factor for HD. In order to retest also this assumption, we performed a case-control study as well. As expected, there was no difference between the case and control groups. All frequencies observed were in Hardy-Weinberg equilibrium and correspond to those reported for the general Caucasian population [As detailed in an earlier study concerning our clinically thoroughly characterised HD cohort, the expanded CAG repeat explained 30.8% of the variance in AO . Adding pulation .TP53 and R196K polymorphism in hCAD genes with the AO of HD. Since we employed a larger cohort of exceptionally well-characterized HD patients the initial association may have been due to chance or to bias as introduced by population stratification. It is, however, possible that a true association exists in Indians. The effect size may also be quite small, requiring an even larger sample size than ours for Caucasians.Our study failed to replicate the association between the genotypes at the R72P polymorphism in The author(s) declare that they have no competing interests.LA initiated the study, carried out the molecular genetic studies and drafted the manuscript. PHK participated in the data analysis. JA and CS had ascertained the clinical status of the patients, and JTE participated in the study design, the coordination and finalized the analyses as well as the paper.The pre-publication history for this paper can be accessed here:


ALAD G177C polymorphism in the gene coding for ALAD is associated with risk of intracranial tumors of the brain and nervous system. We use data from a case–control study with 782 incident brain tumor cases and 799 controls frequency matched on hospital, age, sex, race/ethnicity, and residential proximity to the hospital. Blood samples were drawn and DNA subsequently sent for genotyping for 73% of subjects. ALAD genotype was determined for 94% of these samples . Having one or more copy of the ALAD2 allele was associated with increased risk for meningioma , with the association appearing stronger in males  than in females . No increased risk associated with the ALAD2 variant was observed for glioma or acoustic neuroma. These findings suggest that the ALAD2 allele may increase genetic susceptibility to meningioma.The enzyme δ -aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme synthesis, can be inhibited by several chemicals, including lead, a potential risk factor for brain tumors, particularly meningioma. In this study we examined whether the  ALAD gene codes for the enzyme δ -aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme synthesis involving the condensation of two molecules of aminolevulinic acid (ALA) to form porphobilinogen. The most commonly studied polymorphism in the gene, ALAD G177C (dbSNP ID: rs1800435) contains a G-to-C transversion at position 177 of the coding region, resulting in the substitution of asparagine for lysine. ALAD G177C has two codominant alleles: ALAD1 and ALAD2  in Caucasian populations, 3–11% in Asian populations, and 3% in African-American populations . Analyses were conducted using data from a hospital-based case–control study of brain tumors conducted by the National Cancer Institute (NCI) between 1994 and 1998.Given that the Subjects for the brain tumor study were enrolled from 1994 through 1998 from three hospitals specializing in brain tumor treatment, located in Phoenix, Arizona; Boston, Massachusetts; and Pittsburgh, Pennsylvania. The study protocol was approved by the institutional review board of each participating institution. Study methods have been described in detail elsewhere .Eligible cases for the parent study were ≥ 18 years of age with a first intracranial glioma, meningioma, or acoustic neuroma diagnosed during or within the 8 weeks preceding hospitalization. Ninety-two percent of eligible brain tumor patients agreed to participate in the study. All diagnosed cases of glioma and meningioma were confirmed by microscopy, as were 96% of acoustic neuroma cases. A total of 489 subjects with glioma, 197 subjects with meningioma, and 96 subjects with acoustic neuroma were enrolled.Controls were patients admitted to the same hospitals as cases for a variety of non-neoplastic conditions, with the most common being injuries (25%), circulatory system disorders (22%), musculoskeletal disorders (22%), and digestive disorders (12%). More than 90% of patients were interviewed within 1 year of symptom onset. Control subjects were frequency matched in a 1:1 ratio to all brain tumor cases based on hospital, age, sex, race/ethnicity, and proximity of residence to the hospital; 799 controls  were enrolled.Informed written consent was obtained from all cases and controls. Blood samples were collected and sent for genotyping for 73% of all subjects: 382 subjects with glioma (78%), 158 subjects with meningioma (80%), 71 subjects with acoustic neuroma (74%), and 540 control subjects (68%). The main obstacle to obtaining blood samples was subject refusal, with nonparticipation in the blood draw being higher for control subjects than for case subjects.Shortly after hospitalization, a trained research nurse administered a structured in-person interview for each subject. Information on known or possible risk factors for brain tumors  was collected for all subjects.ALAD genotyping was conducted by the NCI’s Core Genotyping Facility using a medium-throughput TaqMan assay .DNA was extracted from the peripheral white blood cells (buffy coat or granulocytes) of blood samples using a phenol-chloroform method described by ALAD1 allele, contained the FAM dye reporter. Probe 2 (TGTGAACCGGCTGG) was specific to the ALAD2 allele and contained the VIC dye reporter. The primers used were primers F (TGCCTTCCTTCAACCCCTCTA) and R (CAAGGGCCTCAGCATCTCTT). Step 1 of the assay-specific thermocycling process involved 2 min of UNG (uracil-DNA glycosylase) activation using AmpErase UNG (Applied Biosystems) at 50°C. This was followed by 10 min of enzyme activation at 95°C (step 2), 0.30 sec of template denaturation at 92°C if using 3′MGB quencher, or at 95°C if using 3′TAMRA quencher (step 3), and 1 min of assay-specific annealing at 60°C (step 4). Steps 3 and 4 were repeated 49 times, after which the reaction was held at 4°C. The plate was then read on the ABI 7900HT sequence detection system (Applied Biosystems), and the results of the allelic discrimination were graphed as a scatter plot of allele 1 reaction versus allele 2 reaction , with each well of the 384-well plate represented as a spot on the graph. Four distinct clusters on the allelic plot represented the NTCs and three possible genotypes, ALAD1–1, ALAD2–2, and ALAD1–2, respectively. Calls were determined manually by a technician using SDS software.Reactions for the assay were done in a 384 (96 × 4)-well plate format. Lyophilized sample DNA (10 ng) was used for a 5 μL TaqMan reaction. Four Coriell DNA controls  for each genotype as well as no-template controls (NTCs) were put on the plate along with the samples; 2.5 μL of the 2X Universal Master Mix (Applied Biosystems), 200 nM of each primer, and 900 nM of each probe was used in the reaction. Probe 1 (TGTGAAGCGGCTGG), specific to the n = 34; QC-B, n = 20; QC-C, n = 15) and 76 duplicates from study subjects. These specimens were submitted for genotyping in a masked fashion and were collected and processed in a manner identical to that for study samples. Ninety-eight percent agreement was achieved between the three non-study replicates. The concordance rate for study duplicates (study samples compared with masked relabels) was 87% for glioma, 100% for meningioma, 85% for acoustic neuroma, and 89% for controls.Quality control specimens for the study included multiple samples from three individuals who were not study subjects . Missing values  were generally equally likely to be from case or control samples.Using a conservative call strategy that labeled a call as missing if the genotype was unclear, ALAD2 allele, adjusting for study matching factors. We entered adjustment variables as indicator variables in the following categories: age in years ; race/ethnicity ; sex ; hospital ; and residential proximity to the hospital in miles . Because the small number of ALAD2–2 homozygotes precluded accurate estimation of risk for ALAD1–2 heterozygotes and ALAD2–2 homozygotes separately, these categories were combined for analysis. ALAD1–1 homozygotes were the reference group. In order to test for the influence of control group composition on the results, the models were run excluding each major category of control discharge diagnosis, one at a time.We assessed statistically significant departure from Hardy-Weinberg equilibrium for controls using the chi-square test. Unconditional logistic regression was used to estimate odds ratios (ORs) and calculate 95% confidence intervals (CIs) for the effect of the variant The distribution of demographic characteristics for genotyped subjects  was compStudy participants in the youngest and oldest age brackets were less likely to have given a blood sample, as were control subjects who lived closest to the hospital. Brain tumor cases that were male, of “other” race/ethnicity, or from the Phoenix study site were also less likely to have given a blood sample (results not shown).p = 0.3). No significant departure from Hardy-Weinberg equilibrium was detected for controls (We found that the ALAD2 allele of the G177C polymorphism was associated with increased risk of meningioma, especially in males. Confirmation of our findings will require replication in other studies with a larger number of meningioma cases. If risk of meningioma is truly increased in individuals with the ALAD2 allele, the question arises as to whether the effect depends upon exogenous chemical exposures that act on the heme synthesis pathway or is independent of such exposures. A direct effect of the ALAD2 polymorphism might be indicated if the ALAD2 allele has lower enzyme activity than the ALAD1 allele, given that the precursor ALA is thought to be neurotoxic and genotoxic . HoweverThe observation that the relationship between ALAD2 and risk of meningioma was stronger in men than in women could be due to biologic differences or differential exposure to a chemical agent modified by ALAD genotype. Given the small number of male meningioma cases with the variant allele, we also cannot rule out the possibility that the observed effect modification is due to chance.The specific question of ALAD genotype and brain tumor risk has not been addressed previously in the literature. In a previously published analysis of this same data set, we found elevated risk of meningioma in individuals who had worked in military occupations or as autobody painters, designers and decorators, industrial production supervisors, teachers, or managers . Aside fIn a study with hospital controls, study results can be biased if the exposure under study is associated with conditions enrolled in the control series . To asseThe relatively low concordance rate for study duplicates is another potential concern. However, the concordance for duplicates from meningioma cases was 100%, so our observed association for meningioma was probably not affected by genotyping concerns. Moreover, if we assume that 10% nondifferential misclassification did occur, this would have biased our findings toward the null, making our observed association a conservative estimate.We chose to study the ALAD polymorphism based on a priori biologic and functional considerations and not by screening a large number of associations. Nonetheless, the possibility that our findings are due to chance cannot be ruled out. These findings should be viewed as hypothesis generating and need to be confirmed by replication in other studies.ALAD genotype and risk of meningioma, it will be important to conduct a detailed exposure assessment and evaluate the joint effect of exposure and ALAD genotype in this, or another, study population.Although our study had limited power for evaluating risk with respect to subtypes of tumor, it remains one of the largest case–control studies of brain tumors to date. Aside from a small percentage of brain tumors that can be explained by familial syndromes or exposure to ionizing radiation, very little is known about the etiology of brain tumors . In orde


Individuals belonging to the same family share a number of genetic as well as environmental circumstances that may condition a common SBP level. Among the genetic factors, the angiotensin converting enzyme (ACE) gene I/D polymorphism appears as a possible candidate as it might influence both SBP and the pharmacological effect of ACE inhibitors. We aimed to combine genetic epidemiology with public health ideas concerning life-course and multilevel epidemiology in order to understand the role of familial factors regarding individual SBP.We applied multilevel regression analysis on 1926 individuals nested within 438 families from South Sweden. Modelling familial SBP variance as a function of age and use of ACE inhibitors we calculates a variance partition coefficient and the proportional change in familial SBP variance attributable to differences in ACE gene I/D polymorphismOur results suggest the existence of genetic or environmental circumstances that produce a considerable familial clustering of SBP, especially among individuals using ACE-inhibitors. However, ACE gene I/D polymorphism seems to play a minor role in this context. In addition, familial factors – genetic, environmental or their interaction – shape SBP among non-users of ACE inhibitors but their effect is expressed later in the life-course.Strategies directed to prevent hypertension should be launched in younger rather than in older ages and both prevention of hypertension and its treatment with ACE inhibitors should be focused on families rather than on individuals. Individuals belonging to the same family share a number of genetic as well as environmental factors like common physical environment, socioeconomic position, dietary habits, familial attitudes and health believes, etc. Such circumstances may condition a common level of systolic blood pressure (SBP) that may express itself as a clustering of SBP level within families.It is known that SBP is a central risk factor for cardiovascular diseases . SBP levOne of the main mechanisms for blood pressure homeostasis is the renin angiotensin aldosterone system (RAAS) and a common effective group of antihypertensive drugs are angiotensin converting enzyme (ACE) inhibitors. By blocking the ACE these drugs reduce the levels of angiotensin II and thereby decrease vasoconstriction and renal sodium reabsorption which, in turn, leads to lower blood pressure . Among tTherefore, we expected to find a familial clustering for both ACE I/D polymorphism and for SBP level. If this were true, it might also be possible that familial differences in ACE I/D polymorphism could explain some of the familial differences in SBP and thus clustering of SBP. This possible phenomenon might differ by age and use of ACE inhibitors.Considering all those factors  simultaneously is a methodological challenge. In the present study we aim to apply a methodological approach based on multilevel regression analysis ,11 that In this study we pooled three population based family materials from South Sweden consisting of at least two siblings and, when available, their relatives and spouses. The information was collected during 1996–2000 in all the three samples. Two of them  included siblings with established hypertension, whereas one  included siblings with a positive family history of established hypertension but presence of established hypertension was not a necessary inclusion criterion. In Malmö-A, probands were identified in the Malmö Diet and Cancer  and MalmThe local ethics committee at Gothenburg and Lund University approved the study protocols and all participants gave their informed consent.® sphygmomanometer for adjustment of cuff size to arm circumference  and the relative [((VF before - VF after)/VF before) × 100] change in variance.Using the Vp-values. A p-value greater than 0.05 was considered non-significant (NS).The precision of the estimates was appraised by their SE. Parameters were tested in an approximate manner using a normal test  and Wald statistic with a chi-square distribution , and calIn addition to familial clustering  of SBP we also calculated the familial clustering of homozygosis II, homozygosis DD, and heterozygosis ID, as well as the familial clustering of ACE inhibitors use. Since these variables are binary we applied multilevel logistic regression analysis  and calcThe software MLwiN, Version 2  was usedThe characteristics of the population are presented in Table Table Table Figure The results of the complementary logistic regression analyses showed a strong familial genetic clustering for the different forms of the ACE gene I/D polymorphism, specially for homozygous forms DD (VPC = 43%) and II (VPC = 37%) and in a lower degree for the heterozygous form ID (VPC = 15%). Use if ACE inhibitors was also clustered within families (VPC = 9%).The multilevel regression analysis allowed us to study the distribution of blood pressure in families along individual age and use of ACE inhibitors and simultaneously investigate the role of ACE gene I/D polymorphism in this context.We observed that SBP level clustered within families to an appreciable degree, and that this clustering increased considerably with age in people that did not use ACE-inhibitors. This fact suggests the existence of specific familial genetic or environmental factors influencing individual SBP, and that the effect of these familial factors express later in the life course. In fact, familial differences in SBP were large in the elderly but very small in youth people.It is possible that both familial genetic and environmental circumstances  affecting SBP act only after latency period. It is also probable that genetic susceptibility affects SBP level only when the effects of familial environmental factors become fully developed through the individual's life course. As a hypothetical example, learned familial behaviours may lead to obesity in adulthood, which in turn increases the risk of hypertension later in life in people with certain genetic predisposition. It is possible that these kinds of mechanisms could explain our results. However, the database used in this investigation was not primarily design to answer those question and information on socioeconomic and behavioural factors was not available. Moreover our study was cross-sectional and we cannot exclude – even if we dot believe it probable – the existence of a cohort effect confounding the age related familial differences in SBP.In the present study we cannot differentiate between genetic effects (apart from the minor effect of the ACE I/D polymorphism) and effects of familial environment on SBP. However, it is reasonable to believe that, to a large extent, genetic and environmental factors determining SBP interact with each other throughout life. Thus, genetic susceptibility to high SBP is likely to be modified by reducing environmental risk factor load. Therefore, our results suggest that acting on familial environmental factors early in the life course, rather than in late adulthood, might be a more effective strategy to prevent high blood pressure in the population.  Future rThe degree of familial clustering of SBP level was much higher in users of ACE-inhibitors than in non-users Figure  and thisThe role of the ACE I/D polymorphism in blood pressure regulation has been extensively studied. Although there is no doubt that the polymorphism is associated with plasma ACE activity, there is less evidence on the role of ACE I/D polymorphism in blood pressure regulation. ,27 BloodACE-inhibition was not a first-line antihypertensive therapy in Sweden at the time of patient-recruitment in the present study, indicating that these patients had responded less well on conventional treatment (thiazides and beta-blockers) or that they had concomitant diseases such as diabetes with microalbuminuria or heart failure. One possible explanation for the greater familial clustering of SBP in users than in non-users of ACE inhibitors could thus be that patients treated with ACE-inhibitors had more atherosclerosis and endothelial dysfunction than non-users of ACE inhibitors. In analogy with the effect of age on familial SBP clustering in non-users, it can be speculated that genetic and familial environmental factors affecting SBP have a greater impact when acting on the background of a relatively more pathological or "biologically aged" vascular system.Most patients treated with ACE inhibitors were also on treatment with other antihypertensive agents, mostly beta-receptor blockers  and calcium channel blockers  introducing some uncertainty as to how much of the on-treatment blood pressure level was influenced by overall drug effects as opposed to ACE-inhibitor specific effects. However, adjustment for use of those medications in model III had only a minor effect on the slope variance, which was reduced from 131 to 128 (not show in tables).We advocate a study design that may be useful for studying the influence of genetic and environmental familial factors in public health. This approach may facilitate the investigation of common processes that with multifaceted expression and development across time in different contexts. Multilevel regression analysis  emerges Our study suggest that familial circumstances  condition individual SBP level and seems to modify the effect of ACE inhibitors, a fact that deserves more investigation in order to improve the effectiveness of pharmacological treatment of hypertension. Familial circumstances also seem to play a relevant role for understanding SBP level in non-users of ACE inhibitors but those effects express later in the life course. Those facts suggests that strategies directed to prevent hypertension should be launched in younger rather than in older ages and both prevention of hypertension and its treatment with ACE inhibitors should be focused on families rather than on individuals.The author(s) declare that they have no competing interests.JM had the original idea of this article, performed the design, the analyses and the discussion of the results and write the manuscript. OM and KB contributed to the design, the discussion of the results and the writing of the manuscript. UL and LR contributed to the discussion of the results and the writing of the manuscript. OM, KB, UL and LR supplied the databases. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The relationship of apoA1 -75 bp subjects for genotype and phenotype studies.The M1+ and M1- alleles frequencies were 0.808 and 0.192 for M1+ and M1-, respectively, comparable to the frequency of apoA1 (M1+ and M1-) amongst a healthy Omani population, 0.788 and 0.212, respectively.-) of the apoA1 gene were in Hardy-Weinberg equilibrium.The frequencies of the hetero- and homozygous subjects for the MspI polymorphism at -75 , irrespective of gender and the diabetic status.The mean Lp(a) concentration was significantly higher(P = 0.02) in subjects carrying M1-) polymorphism is relatively common and is positively associated with Lp(a) and therefore, may confer a potential risk for cardiovascular disease (CVD).ApolipoproteinA1-75 G/A (M1 An inverse relationship has been reported between the apoA1 and HDL and CVD in the general population . The prooA1 gene ,6. NumeroA1 gene .- allele was 22% [-) allele polymorphism on lipoprotein phenotypes and Lp(a) among a cohort of high-risk of Omani diabetic and non-diabetic population.Previously, our group has reported the alleles frequency of apoA1 gene among a healthy Omani population. The frequency M1 was 22% . This stLp(a), TG, and HDLc were positively skewed, therefore, natural logarithms of the data were used in all parametric significance testing.-) of the apoA1 gene were in Hardy-Weinberg equilibrium.The frequencies of the hetero- and homozygous subjects for the MspI polymorphism at -75 (M1-) gene polymorphism on lipoprotein phenotype. The sample size of subjects carrying the homozygous minus alleles was small and therefore the hetero and homozygous minus alleles were combined together in one group. Neither the demographics nor most of the lipoprotein parameters other than Lp(a) have reached statistical significance. The mean Lp(a) concentration was significantly higher (P = 0.02) in subjects carrying the hetero or homozygous M1-+/-- gene compared to M1++ gene with an odd ratio of 2.3 .The M1+ and M1- alleles frequencies were 0.808 and 0.192 for M1+ and M1-, respectively. Table -+/-- gene) compared to the M1++ gene, irrespective to gender  concentrations compared to men. Despite the confounding influence of gender on various lipids and lipoproteins, Lp(a) was significantly higher in subjects carrying the hetero or homozygous minus allele  concentration was significantly higher in subjects carrying the hetero or homozygous M1y, Table .-) is relatively common, occurring in around 20% of the adult Omani diabetic and non-diabetic population. Quite comparable to what our group have reported previously on healthy Omani adult population.The G-to-A transition in the promoter region of apoA1 gene -75 (M1-) in the promoter region of APOA1 gene was associated with a significantly higher Lp(a) concentration in both diabetic and non-diabetic subjects irrespective of gender. We underwent a Medline search; we were only able to find one study where an association between the apoA1 alleles polymorphism and Lp(a) was reported. This study was on newborn babies from an Asian origin  and on samples collected from cord plasma. They undertook this study free of the confounding effect of environmental factors to determine the influence of apoA1 gene -75 (M1-) polymorphism on apoA1 and HDL concentrations. To their surprise no significant association was revealed between the apoA1 polymorphism and the apoA1 and HDL, however, a significant association was found between the above polymorphic sites and Lp(a) in male Asian Indians, and the apoA1 gene could account for 14% of the variation in Lp(a) concentration [The present study has revealed that the G-to-A transition . Partly due to the fact that diabetes and metabolic syndrome are highly prevalent in Oman, according to a national survey, 20–30% of the adult Omani population are either T2DM or have metabolic syndrome partly due to the increase in prevalence of obesity, sedentary life and consanguinity rate. T2DM is associated with dyslipidaemia that is characterized by raised triglyceride, and low HDL cholesterol and therefore, it is tempting to speculate such confounding factors could mask the influence of apoA1 genotyping on lipoprotein phenotype -11. Neveot girls ; in healot girls ; in Finnot girls  and in Fot girls . In contra et al , Lopez-Mra et al , Mata etra et al , Carmenara et al , Akita era et al . Moreoveof apoA1 . Pukkinesubejcts . In contOA1 gene .- allele of the APOA1 gene. Future studies are required on larger samples to confirm our findings and perhaps look into some of the molecular mechanism that could explain the interaction of apoA1 polymorphism and Lp(a).Numerous studies have confirmed the pro- and anti-atherogenic properties, respectively, of Lp(a) and apoA1 -26. Howe-) in the promoter region of APOA1 gene was significantly associated with higher Lp(a) concentration in irrespective of gender and diabetic status.The G-to-A transition (M1c (>7%) were fulfilled by all patients with type 2 diabetes. The proportions of T2DM subjects on insulin, oral hypoglycaemic agents or dietary control were 3%, 57% and 40%, respectively. History of smoking, alcohol, exercise and prophylactic drugs  was collected. Patients were labeled CVD+ if they had a previous myocardial infarction more than three months prior to entry to the study or having stable angina pectoris and/or they had a positive thallium stress test or coronary angiogram. Exclusion criteria included myocardial infarction within three months prior to entry to the study, uncontrolled thyroid disease (hypo or hyperthyroidism), macro-proteinuria (positive urine protein dip-stick x2), severe hepatic impairment or renal impairment (serum creatinine level >114 (μmol/L), and those on lipid modifying agents.The institutional Ethics Committee approved the study and all subjects gave their informed consent prior to participating in this study. Ninety non-diabetic subjects (27 women and 63 men) and one hundred and forty nine diabetic subjects (53 women and 96 men) were recruited from cardiac/lipid and diabetic clinics, respectively. All patients had cardiovascular, respiratory, and central nervous systems examination. Blood pressure was measured to the nearest even digit using a sphygmomanometer with the subject in the sitting position after a 5–10 minutes rest and considered hypertensive if the blood pressure was equal or greater than 140/90 mmHg on two repeated occasions or by 24 BP monitoring. Those on anti-hypertensive therapy were also assumed to be hypertensive. Diagnosis of T2DM was based on clinical characteristics; prior history of use of oral hypoglycaemic agents, the presence of obesity, no history of ketosis, or strong family history of diabetes. World Health Organization criteria for diagnosis of T2DM either by an abnormal oral glucose tolerance test (OGTT), two abnormal fasting blood glucose (>7.0 mmol/L) or high HbA1An overnight fasting blood samples were taken for measurement of total cholesterol (TChol), triglycerides (TG), HDL-cholesterol (HDLc), Lp(a) and apoA1 and apolipoprotein B (ApoB). Cholesterol and triglycerides were measured using timed endpoint enzymatic methods on the Synchron CX system (Beckman).ApoA1, apoB and Lp(a) were measured using rate nephelometric immunochemistry assay by IMMAGE system (Beckman). Both the ApoB and ApoA-1 methods used have been standardized according to the International Federation of Clinical Chemistry. Reference material for ApoB and ApoA-1 are lot SP3-07 and lot SP1-01, respectively, which were approved by WHO . Low denM Mgd2, 200 μM dNTPs, 10 pmol of each primer , AND 1 u Taq DNA polymerase (GibcoBRL). PCR conditions consisted of a 5-min initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 0.5 min, and extension at 72°C for 0.5 min, with a final extension at 72°C for 5 min. Fifteen microliters of the 434-bp PCR product containing here MspI restriction sites at -75, +37, and +83-bp of the APOA1 gene  was digested with 3 U of MspI (Gibco BRL) overnight. Accordingly, the digested PCR products gave 67-, 113-, 45-, and 209-bp fragments for the wild type and a combination of these fragments for the rare alleles. These fragments were analyzed on 8% polyacrylamide gel stained with ethidium bromide. The genotypes were determined by comparing the relative mobilitys of DNA fragments with reference to size molecular weight markers. The genotypes and alleles were assigned the names adopted by Wand, Liu et al (1996) [Polymerase chain reaction (PCR) was performed with a modification of the Wang, Badenhop et al (1996) method. Approximately 100 ng of genomic DNA was amplified in a 25-μl reaction mixture containing 1.5 mData was analyzed with the Statistical Package for the Social Sciences Software (SPSS version 10).Descriptive analysis including the estimation of mean values and standard error of the mean (SEM) for continuous variables were calculated. Skewed parameters were logarithmically transformed and a parametric test was used.The significance of differences for the means of plasma Tchol, LDLc, HDLc, Triglycerides, apoB, apoA1, and Lp(a) between genders and various groups were determined by the t test or ANOVA as appropriate.Chi square tests were used for establishing the relationship between the lipid parameters and the apoA1 alleles. The non-parametric Mann-Whitney test was used when the underlying assumptions were not met.ANOVA was performed to determine the effects of the apoA1 gene polymorphism on the various lipid traits in the different groups after adjustment for significant covariates. In order to verify whether age and sex were effective modifiers in the estimation of odds ratios, separate binary logistic regression models were fitted for each of the lipid profiles after adjusting for age and sex as covariates. To study the relationship between apoA1 polymorphism and IHD, two separate binary logistic regression models were considered. Once the probability model for IHD due to only apoA1 polymorphism was fitted and then the model was refitted including all the six lipid profiles, for examining whether they were potentially effect modifying variables or not. A p-value (two-tailed) of less than 0.05 was considered as statistically significant.We declare that there have been no competing or financial interests.AAB, is the major author of the above study, he had a substantial contribution to the conception, design, analysis as well as interpretation of the data. Jeannette Usher's main role was the statistical analysis of the data. EM and LR have contributed to the drafting, revising as well as critically scrutinizing the manuscript. SA and MK have contributed to the recruitment and analysis of chemistries. All authors read and approved the final manuscript.


EHP 115:116–121; Wolff et al.][. The discovery of detectable urine levels of a range of hormonally active substances in the children may shed light on how various biomarkers relate to pubertal development.Urinary biomarkers are useful measures of environmental agents in the body. However, little is known about levels of such biomarkers in children and how they may vary by race, age, body mass index, and sex. A three-city pilot study reported this month used urinary biomarkers to better characterize a number of exposures in young girls The study authors measured parent compounds and metabolites of phytoestrogens, phthalates, and phenols in the urine of girls aged 6 to 9. They tested for 25 biomarkers from 22 agents including triclosan , enterolactone (a micronutrient from seeds and grains), and monoethyl phthalate . The chemical classes studied were chosen because of their suspected hormonal activity and because they have been widely detected in the general population.The 90 girls in the pilot study were recruited in New York, Cincinnati, and San Francisco, and included members of four racial/ethnic groups. The pilot study sampled a relatively small population to determine whether urinary biomarkers of the chemicals of concern would be detectable and variable enough for meaningful comparisons of their concentrations in relation to outcomes of female growth and development.Most of the markers analyzed were found in more than 94% of the participants. Nine of them—including metabolites of isoflavones found in foods containing soy and of di(2-ethylhexyl) phthalate, a softening agent used in plastics—were found in all of the girls.The team established that urinary metabolites of phenols, phthalates, and phytoestrogens in children are detectable and variable enough to make meaningful comparisons. And though the number of subjects was small, preliminary results point to variations in concentrations of some metabolites between girls of different races, as well as variation according to body mass index, and seasonal variation during the year—all factors that will be useful in dissecting the roles of the studied chemical classes in breast cancer and other diseases.


O-methyltransferase (COMT) gene.The role of coffee intake as a risk factor for coronary heart disease (CHD) has been debated for decades. We examined whether the relationship between coffee intake and incidence of CHD events is dependent on the metabolism of circulating catecholamines, as determined by functional polymorphism of the catechol-In a cohort of 773 men who were 42 to 60 years old and free of symptomatic CHD at baseline in 1984–89, 78 participants experienced an acute coronary event during an average follow-up of 13 years. In logistic regression adjusting for age, smoking, family history of CHD, vitamin C deficiency, blood pressure, plasma cholesterol concentration, and diabetes, the odds ratio  comparing heavy coffee drinkers with the low activity COMT genotype with those with the high activity or heterozygotic genotypes was 3.2 (1.2–8.4). Urinary adrenaline excretion increased with increasing coffee intake, being over two-fold in heavy drinkers compared with nondrinkers (p = 0.008 for trend).Heavy coffee consumption increases the incidence of acute coronary events in men with low but not high COMT activity. Further studies are required to determine to which extent circulating catecholamines mediate the relationship between coffee intake and CHD. Despite decades of research, controversy persists regarding the effects of coffee consumption on cardiovascular health We have recently reported a U-shaped dose–response relationship between intake of caffeine-containing coffee and incidence of acute coronary events O-methyltransferase (COMT) gene, resulting in several-fold differences in the metabolism of circulating catecholamines, modifies the effect of heavy consumption of caffeine-containing coffee on the risk of acute coronary events in a cohort of middle-aged eastern Finnish men initially free from symptomatic CHD.To test this hypothesis, we examined whether the functional polymorphism of the catechol-The Kuopio Ischaemic Heart Disease Risk Factor Study is a population-based prospective follow-up study of 2682 men aged 42, 48, 54, or 60 years at the baseline examination carried out in 1984–89 LL genotype), homozygous for the high activity allele , and heterozygous .COMT polymorphism is generated by the presence of guanine or adenine at nucleotide 475 encoding a valine or methionine at codon 158 and is recognized by the heat-shock protein 92 II restriction endonuclease. The polymorphism results in three different genotypes: homozygous for the low activity allele  was treated with two units of heat-shock protein 92 II for 16 hours at 37°C and then analyzed by 2% agarose gel electrophoresis. The diagnostic bands were 179  and 139 (methionine).Consumption of foods and beverages was assessed with an instructed and interview-checked 4-day food recording by household measures, including cups of coffee and tea The examination protocol and measurements have been described previously Urinary adrenaline and noradrenaline were purified with commercial phenylboronic acid columns and analyzed under isocratic conditions by reverse-phase liquid chromatography  until the end of 1992 is previously described LH and LL genotypes were combined into a single category and coffee non-drinkers were combined with light drinkers. Logistic regression was used to assess the association between the composite coffee intake–COMT variable and acute coronary events; heavy coffee drinkers with either the HH or LH genotype were used as the reference category. Continuous covariates without a meaningful zero value were centered around their respective means. Statistical inference was based on 90% Wald confidence intervals or two-sided p-values.All analyses were performed with SPSS version 11.5 . Average daily coffee intake was divided into four categories: 0 (non-drinkers), 1 to 375 mL (light drinkers), 376 to 813 mL (moderate drinkers), and 814 mL and over (heavy drinkers).  To evaluate biologic interaction (departures from additivity), a single composite variable with six joint-exposure categories was created; the COMT In a subgroup of 258 participants with urinary catecholamine measurements, we performed a supplemental linear regression analysis of urinary adrenaline and noradrenaline excretion in categories of coffee intake. Data on catecholamine excretion according to COMT genotype were not available.HH genotype were less likely to be diabetic and had lower levels of leisure-time physical activity than those with the other two genotypes; systolic blood pressure was marginally higher in the LL genotype.Baseline characteristics according to coffee intake are shown in In the supplemental analysis of urinary catecholamine excretion among 258 participants, the amount of adrenaline excreted was over two-fold in the heavy coffee intake category compared with non-drinkers (p = 0.008 for trend); for noradrenaline, the respective difference was 30% (p = 0.039 for trend).LL genotype (LL genotype with heavy drinkers in the other two genotypes was 3.25 (1.29–8.16). Further adjustment for systolic blood pressure, serum LDL and HDL cholesterol concentration, and diabetes minimally decreased the odds ratio estimate to 3.21 (1.22–8.42). In the combined HH or LH category, the adjusted odds ratios for light and moderate coffee drinkers were 1.45 (0.64–3.27) and 1.39 (0.67–2.88), respectively; the corresponding odds ratios in the LL genotype were 1.73 (0.68–4.42) and 1.31 (0.58–2.99), using the heavy drinkers with either the COMT HH or LH genotype as the reference category  but clearly increased in heavy drinkers with the COMT genotype . After acategory . AdjustmIn this population-based cohort of middle-aged men initially free from symptomatic CHD followed-up for an average of 13 years, we found a relationship between consumption of caffeine-containing coffee and the incidence of fatal or nonfatal CHD events that is highly dependent on COMT genotype. In men who were either homozygous for the high COMT activity allele or heterozygotes, heavy coffee intake did not increase the incidence of acute coronary events. In men who were homozygous for the low-activity COMT allele, however, heavy coffee intake was associated with a considerably higher incidence of acute coronary events. The relative excess in CHD incidence in the low-activity genotype was over two-fold among drinkers of more than 6.5 cups of coffee per day, after adjustment for age, smoking, family history of CHD, plasma vitamin C concentration, systolic blood pressure, serum LDL and HDL cholesterol concentration, and diabetes.We are not aware of any previous studies that have addressed the modification of the coffee–CHD relationship by COMT activity. Tens of studies investigating the relationship between coffee consumption and the incidence of CHD have been carried out, but the results remain inconsistent. Several large studies carried out in the United States have found no appreciable increase in CHD risk with increasing coffee consumption The findings have been in part dependent on the type of study: case–control studies have generally reported higher effect estimates than cohort studies Two recent studies have observed a U-shaped pattern between coffee consumption and CHD incidence Caffeine is metabolized by the polymorphic cytochrome P450 1A2 (CYP1A2) enzyme. Recently Cornelis and co-workers determined whether CYP1A2 genotype modifies the association between intake of caffeinated coffee and the risk of nonfatal myocardial infarction These findings led us to consider novel explanations behind the dose–response relationship between coffee consumption and CHD incidence. Coffee and caffeine have been shown to stimulate adrenomedullary secretion, resulting in raised levels of circulating catecholamines, adrenaline in particular S-adenosyl-homocysteine and consequent further inhibition of COMT activity Hence, we hypothesized that heavy intake of caffeine-containing coffee induces a “chemical stress” and that the effect of this stress response on CHD incidence is more evident in those whose catecholamine metabolism is slower than usual and the mechanisms of tolerance may be overwhelmed. The main enzyme responsible for the metabolism of circulating catecholamines is COMT Our study is limited by the small study cohort and the fact that only 78 CHD events were observed during an average follow-up of 13 years, resulting in imprecise effect estimates reflected as wide confidence intervals. Nevertheless, we were able to control for the most important potential confounding factors and still the difference in CHD incidence between the COMT activity categories among heavy coffee drinkers persisted. Residual confounding alone is unlikely to explain the remaining excess incidence. Another major limitation of our study is restriction of the study population to men only. Female hormones may influence catecholamine pharmacodynamics In conclusion, we suggest that circulating catecholamines play a role as mediators of the effect of heavy coffee intake on the risk of CHD events. The elevated risk may be more pronounced in or even restricted to those whose catecholamine metabolism is slower than usual. Further research is required to confirm or refute our hypothesis.


Schizophrenia is a common psychiatric disease affecting about 1% of population. One major problem in the treatment is finding the right the drug for the right patients. However, pharmacogenetic results in psychiatry can seldom be replicated.5-HT2A) receptor gene, tryptophan hydroxylase 1 (TPH1) gene, and G-protein beta-3 subunit (GNB3) gene. We recruited 94 schizophrenia patients representing extremes in treatment response to typical neuroleptics: 43 were good responders and 51 were poor responders. The control group consisted of 392 healthy blood donors.We selected three candidate genes associated with serotonergic neurotransmission for the study: serotonin 2A , p = 0.005] or in the control population . TPH1 A779C C/A genotype was inversely associated with good treatment response when compared with non-responders  or with the controls , and a clearer association when compared with the controls .We do, in part, replicate the association between More findings on the consequences of functional polymorphisms for the role of serotonin in the development of brain and serotonergic neurotransmission are needed before more detailed hypotheses regarding susceptibility and outcome in schizophrenia can be formulated. The present results may highlight some of the biological mechanisms in different courses of schizophrenia between men and women. Schizophrenia is a devastating disease which affects about one percent of people world-wide . Recent The pharmacogenetics of schizophrenia has focused on studying dopamine and serotonin related genes . Several5-HT2A) receptor gene polymorphism is associated with treatment response to conventional antipsychotics  or in the control population  (Table TPH1 C/A genotype was less common in the responders' group compared with non-responders  or with the controls . In males, C/T genotype was more common in responders than in controls   reported a similar association in male patients but not in female patients. The study design was very similar in both studies. However, an obvious reason for these different results may be the different gender distribution in these studies. In the study by Joober et al. there were respectively 74.4% and 71.1% males in non-responders and responders. In the present sample, there were 56.9% and 39.5% males in these categories. One explanation for the gender differences detected may be that treatment-resistant women with schizophrenia constitute a severely ill subgroup within schizophrenia [We report an association between ophrenia .5-HT2A T102C polymorphism has been associated with a small schizophrenia increasing effect [In meta-analysis, g effect ,12. Beca5-HT2A T102C C-allele has been found to increase 5-HT2 receptor expression in human temporal cortex [The occurrence of l cortex . There il cortex .TPH1 A779C polymorphism and response to typical neuroleptics. We cannot present any conclusive reason why the treatment response was inversely associated with a heterozygous genotype. However, heterosis is a common feature which may occur in up to 50% of all gene associations [TPH1 A779C A-genotype has been associated with lowered serotonin metabolite 5-HIAA levels in the CSF [To our knowledge, this is the first report of an association between ciations . So far,ciations . The TPH the CSF , and on  the CSF . The rol the CSF .GNB3 polymorphism [Male heterozygous genotype carriers showed a better response to typical neuroleptics. So far heterosis has been reported by only one study, in which Korean male medical students were the more prone to seasonal variation the more often they carried heterozygous genotype of morphism . It shoumorphism . Howevermorphism  was founSerotonergic mechanisms may have a greater impact on medication response of schizophrenia than has so far been acknowledged. This approach needs more studies including different endophenotypes of schizophrenia regarding age at onset and medication response. These subgroups are likely to have different susceptibility and treatment response altering factors. The results of this study may give rise to further hypotheses regarding the role of gender in the course and outcome of schizophrenia.5-HT2A: serotonin 2A receptor geneTPH1: tryptophan hydroxylase 1 geneGNB3: G-protein beta-3 subunit geneThe author(s) declare that they have no competing interests.SA: data management and statistical analyses, collecting the clinical material, writing the first draft of the manuscriptOK: data management and statistical analyses, collecting the clinical material, writing the first draft of the manuscript AI: collection of clinical materialRR: planning and performing the genotypingsTL: planning and organising the blood collection, DNA isolation and genotypingEL: statistical analyses, coordinating the clinical studyIn addition, all authors participated in composing and editing the article as well as interpreting the results. All authors have given their final approval of the version to be published.The pre-publication history for this paper can be accessed here:


Fgf10 and Fgfr2b mutant mice, as well as human studies, suggest that FGF10/FGFR2b signaling may play an essential, nonredundant role during embryonic SMG development. To address this question, we have analyzed the SMG phenotype in Fgf10 and Fgfr2b heterozygous and null mutant mice. In addition, although previous studies suggest that the FGF10/FGFR2b and FGF8/FGFR2c signaling pathways are functionally interrelated, little is known about the functional relationship between these two pathways during SMG development. We have designed in vivo and in vitro experiments to address this question.Analyses of Fgf10 and Fgfr2b heterozygous mutant and null mice and demonstrate dose-dependent SMG phenotypic differences. Hypoplastic SMGs are seen in Fgf10 and Fgfr2b heterozygotes whereas SMG aplasia is seen in Fgf10 and Fgfr2b null embryos. Complementary in vitro studies further indicate that FGF10/FGFR2b signaling regulates SMG epithelial branching and cell proliferation. To delineate the functional relationship between the FGF10/FGFR2b and FGF8/FGFR2c pathways, we compared the SMG phenotype in Fgfr2cΔ+//Fgf10+/- double heterozygous mice to that seen in wildtype, Fgf10+/- (Fgfr2c+/+/Fgf10+/-) and Fgfr2cΔ +/(Fgfr2cΔ+//Fgf10+/+) single heterozygous mutant littermates and demonstrate genotype-specific SMG phenotypes. In addition, exogenous FGF8 was able to rescue the abnormal SMG phenotype associated with abrogated FGFR2b signaling in vitro and restore branching to normal levels.We analyzed Fgf10 and Fgfr2b null mice. Though we were able to rescue the abnormal phenotype associated with reduced in vitro FGF10/FGFR2b signaling with exogenous FGF8 supplementation, our results indicate that the FGF10/FGFR2b and FGF8/FGFR2c are nonredundant signaling pathways essential for in vivo embryonic SMG development. What remains to be determined is the in vivo functional relationship between the FGF10/FGFR2b signal transduction pathway and other key signaling pathways, and how these pathways are integrated during embryonic SMG development to compose the functional epigenome.Our data indicates that FGF10/FGFR2b signaling is essential for the SMG epithelial branching and histodifferentiation, but not earliest initial bud formation. The functional presence of other endogenous signaling pathways could not prevent complete death of embryonic SMG cells in  PreBud Stage, SMG development begins as thickening of the oral epithelium adjacent to the tongue. During the Initial Bud Stage, this thickening grows down into mandibular arch mesenchyme to form the initial SMG bud. With continued epithelial proliferation and downgrowth, the SMG primordium becomes a solid, elongated epithelial stalk terminating in a bulb. Repeated end-bud branching results in the formation of a network of epithelial branches and terminal buds (the Pseudoglandular Stage). These epithelial branches and terminal buds hollow out by cell apoptosis during the Canalicular and Terminal Bud Stages, respectively, to form the ductal system and presumptive acini, with mucin protein being produced by the presumptive acini.Mouse submandibular gland (SMG) development is initiated with a thickening of the oral epithelium of the mandibular arch around embryonic day 11.5 (E11.5) and is best conceptualized in stages. In the Morphogenesis of complex organs such as the SMG is regulated by the functional integration of parallel and broadly related signaling pathways which regulate cell proliferation, apoptosis and histodifferentiation -6. To unFgfr1, Fgfr2 and Fgfr3 genes generates isoforms which differentially bind specific FGF ligands . Tiss ligands ,10. Liga ligands ,9,11].Functional studies have demonstrated that embryonic SMG epithelial cell proliferation, branching morphogenesis, and histodifferentation are regulated through growth factor, cytokine, and transcription factor-mediated signaling pathways, including EGF, TGF-β, Shh, FGFs, and Eda ,6,12-21.Fgfr2b-/- null mice die at birth due to lung insufficiency and exhibit severe dysmorphic organs, including agenesis or dysplasia of the lungs, mammary glands, pancreas, thyroid, teeth, and limbs  decrease in branching morphogenesis compared to controls  decrease in cell proliferation was seen in the presence of 10 ng/ml FGFR2b-Fc chimera compared to control . Mutant mice heterozygous for either Fgfr2c (Fgfr2cΔ+//Fgf10+/+) or Fgf10 (Fgfr2c+/+/Fgf10+/-) exhibit hypoplastic SMGs compared to WT (Fgfr2c+/+/Fgf10+/+) littermates  in Fgf10-/- mice. This result suggests that FGFR2b signaling is essential to maintain bud 4 and to induce the other mammary placodes whereas another FGFR2b ligand (probably FGF7) acts redundantly with FGF10 to maintain the mammary gland placode. Similarly, differences in pancreatic development were observed between Fgf10 and Fgfr2b null mice  were sacrificed on day 13 and day 14 of gestation, and embryos were dissected in cold PBS and staged according to Theiler . E13 . This method has been successfully used to interrupt FGFR2b, FGFR2c, FGFR1b and FGFR1c signaling during embryonic SMG development ,15,21. WThe cell proliferation index was determined as previously described . Paired Cell proliferation was quantitated as the ratio of PCNA-positive epithelial cells/total epithelial cells. The mean cell proliferation index was determined per section and the mean cell proliferation index of the three sections/explant was determined for each treatment. The data was arcsin transformed and the means ratios compared by t-test.Paired E13 SMG primordia were cultured in 10 ng/ml FGFR2b-Fc chimera for an initial period of 3 hrs and then each pair was cultured in FGFR2b-Fc or FGFR2b-Fc + 500 ng/ml FGF8  for 3 days. This FGF8 peptide concentration was determined to induce a significant increase in branching morphogenesis (data not shown). A concurrent control experiment was conducted as an internal control to verify that FGFR2b-Fc chimera supplementation interrupted branching morphogenesis; these controls consisted of E13 primordia cultured in 10 ng/ml FGFR2b-Fc chimera or 10 ng/ml IgG-Fc for 3 days. The explants were collected and mean Spooner ratios determined and compared as described above. Four explants per treatment were analyzed.ALSG-aplasia of lacrimal and salivary glandBSA-bovine serum albumenCONT-controlFGF-Fibroblast growth factorFGFR2b-FGF receptor 2-IIIbFGFR2c-FGF receptor 2-IIIcERK-p44/p42 mitogen activating kinases (ERK-1/2)MAPK-mitogen-activating protein kinasesP13K-phosphatidylinositol 3-kinasePBS-phosphate-buffered salinePKC-protein kinase CPLC-γ-phospholipase C γ1-/--Fgfr2b-/-R2bR2c-Fgfr2cRT-PCR-reverse transcriptase-polymerase chain reactionSMG-submandibular salivary glandWT-wildtypeFgf10 and Fgfr2b mutant mice and harvested and genotyped the embryos. SB assisted in the analyses of mutant mouse histopathology. MKH generated the Fgfr2cΔ+//Fgf10 +/- single and double mutant mice, harvested and genotyped these embryos, and assisted in the analysis of their histopatholgy. MM participated in the design and coordination of this study, assisted in the analysis of histopathology, performed the statistical analysis, and helped draft the manuscript. All authors read and approved the final manuscript.TJ designed and coordinated the study, was involved in aspects of all experiments, and drafted the manuscript. GA prepared the histological sections for this study and performed some of the morphology and cell proliferation experiments. DW participated in morphological analyses and culture experiments and generated all figures. FGS generated the