There is now good evidence that antiplatelet agents  prevent pre-eclampsia, a leading cause of morbidity and mortality for pregnant women and their babies. A Cochrane Review identified moderate, but clinically important, reductions in the relative risks of pre-eclampsia (19%), preterm birth (7%) and perinatal mortality (16%) in women allocated antiplatelets, rather than placebo or no antiplatelet.Uncertainty remains, however, about whether some women (in terms of risk) benefit more than others, what dose of aspirin is best and when in pregnancy treatment should ideally start. Rather than undertake new trials, the best way to answer these questions is to utilise existing individual patient data from women enrolled in each trial.Systematic review with meta-analysis based on individual patient data. This involves the central collection, validation and re-analysis of thoroughly checked data from individual women in all the available randomised trials.The objective is to confirm that antiplatelet agents, given during pregnancy, will reduce the incidence of pre-eclampsia. The review will then determine the size of this effect, and whether antiplatelets delay the onset of pre-eclampsia or its impact on important outcomes for women and their babies. It will also explore whether the effect of antiplatelets differs by womens' risk profile; when commenced during pregnancy; and/or by dose.The PARIS  Collaboration has been formed to undertake the review. This will be the first individual patient data review in the perinatal field. Final results should be available by 2006–7. World-wide, over half a million women die each year of pregnancy related causes with 99% of these occurring in low resource countries ,2. An esHigh blood pressure is common during pregnancy: approximately one in ten women will have their blood pressure recorded as above normal at some point before delivery . For womAlthough the outcome for most women is good, pre-eclampsia and eclampsia (the rare occurrence of seizures superimposed on the syndrome of pre-eclampsia) are major causes of maternal mortality. In low resource countries pre-eclampsia and eclampsia account for 10–15% of maternal deaths  whilst iPre-eclampsia is a multi-factorial condition. Although its aetiology remains unclear, there have been significant advances in the understanding of the pathophysiology of the disorder. The primary lesion is thought to be deficient trophoblast invasion of the maternal spiral arteries in the second trimester, leading to underperfusion of the uteroplacental circulation and placental ischaemia . The resIt is further hypothesised that the effect of antiplatelets may be different if treatment is started before placental implantation is complete. . If thisAlthough it is known that pre-eclampsia is a multi-system disorder, the relationship between the placental pathology and maternal endothelial response is not fully understood. Numerous maternal factors can predispose to the disorder, such as previous pre-eclampsia, diabetes, renal disease, chronic hypertension or other risk factors . The synThe effects of antiplatelet agents were first evaluated in small randomised trials, which reported striking reductions in the risk of hypertension and proteinuria -29. ThesThe updated Cochrane Review identified 51 trials with over 36,500 pregnant women evaluating antiplatelet agents, principally low dose aspirin, for the prevention of pre-eclampsia . Nine ofThe aim is now to extend this review based on aggregate data, to utilise the available data for every individual woman in each trial to help address these remaining questions. The use of individual data for each woman will allow for more powerful and flexible analysis of both subgroups and outcomes [These effects are much smaller than had initially been hoped for but, nevertheless, potentially they have considerable public health importance. The conclusion from this aggregate data review was that for most low-risk women large numbers of women would need to be treated with antiplatelets agents to prevent one episode of either pre-eclampsia or perinatal death. Whether there are specific high risk sub-groups of pregnant women for whom there might be greater benefits, remains unclear, as does the best time to initiate treatment, and at what dose. outcomes ,34. This• Many studies were excluded because publications did not report sufficient information to allow them to be included in the review.• The published aggregate data is variable in the completeness of outcome reporting and in definitions used between trials.• The aggregate data meta-analyses were restricted to analysing outcomes for complete trials. As many trials included women with a wide range of risk profiles at trial entry, trials could only be defined by average values. Such aggregated outcomes such as 'proportion of women developing pre-eclampsia' conceal a range of severity of disorder and so it was not possible to explore fully whether the effectiveness of antiplatelet therapy differed according to risk.• It was also difficult to make precise recommendations about when to start treatment. While most trials could generally be classified as starting treatment at earlier or later in gestational ages, there was a wide within-study variation that could not be explored with aggregate data analyses.• There is a markedly skewed distribution of effect estimates for each trial around the summary effect on pre-eclampsia, with more small positive trials than small negative trials. This suggests the possibilities of publication bias or that the differences in the characteristics of the women enrolled in small and large studies has an important influence on the effects of antiplatelet agents.• Obtaining individual patient data from previously excluded trials will allow assessment of whether these trials are eligible for inclusion, both in terms of available outcomes and methodological quality. This will potentially increase the power and scope of the analyses.• Trial level information obtained by direct discussion with the trialists enables clarification of the definitions and measurements used. Data for measured, but previously unreported, outcomes can also often be obtained and trialists, who are part of a Collaborative group, may be able to provide missing outcome data. Furthermore, we will be able to apply common definitions across trials based on each woman's baseline and clinical data. For example, where possible, we will define pre-eclampsia based on a set of uniform criteria.• Subgroup analyses will be performed for a number of different risk factors using individual patient's specific risk data, rather than using the aggregated risk profile of all women enrolled in a particular trial.• By obtaining gestational age at randomisation for each individual woman, more precise patient-based analyses can be performed exploring whether gestation at treatment commencement alters the effectiveness of antiplatelets.• Formation of a Collaborative group, is likely to lead to a more complete identification of all relevant trials, including those previously unpublished. This may help overcome the potential for publication bias. The patient-level data will also allow us to explore whether there were important differences in the characteristics of women enrolled in small and large trials.The objective of this review is to confirm that antiplatelet agents, given during pregnancy, reduce the incidence of pre-eclampsia. The review will then determine the size of this effect, and whether antiplatelets reduce the severity of pre-eclampsia and/or its impact on important outcomes for women and their babies. It will also explore whether the effect of antiplatelets differs by womens' risk profile, when commenced during pregnancy, and/or by dose.The main questions to be addressed in this review are:• Do antiplatelet agents, primarily low dose aspirin, have clinically important benefits for women at risk of developing pre-eclampsia and their babies?Investigation of this hypothesis will also explore whether the treatment effect differs, in a clinically meaningful way, between women with different risk factors such as those with a history of early onset pre-eclampsia, renal disease, diabetes, chronic hypertension, or autoimmune disease.• Does the planned dose of aspirin affect outcome in terms of preventing or delaying the onset of pre-eclampsia or other adverse outcomes, such as preterm birth or perinatal death?• Do the effects of aspirin differ according to gestation at onset of treatment?The Cochrane Controlled Trials Register and hand searching of relevant journals. Trialists will be asked if they know of any further studies. [See The search strategy to identify potentially eligible studies will include a search of the register of trials developed and maintained by the Cochrane Collaboration Pregnancy and Childbirth Review Group. Details of how this register is maintained are available elsewhere ,36, but ies. See  for the The inclusion and exclusion criteria for the types of study designs, participants, interventions and data completeness to be included in the review are listed below. Each potentially eligible study will be assessed independently by two members of the Secretariat, unblinded to the trial's identity. Any differences of opinion regarding the assessment of the inclusion criteria will be resolved by discussion between the two assessors. If differences cannot be resolved, a third member of the Secretariat will be asked to assess the study. If individual patient data are unavailable from an eligible trial, the trial will remain included in the review and aggregate data will be used.Studies will be included in the review if they were randomised trials. Quasi-random study designs, such as those using alternate allocation, will be excluded. The level of allocation concealment within each trial will be assessed according to the criteria outlined in the Cochrane Handbook , and claParticipants will be pregnant women at risk of developing pre-eclampsia. Women who started treatment postpartum will be excluded, as will those who already have a diagnosis of pre-eclampsia at trial entry .The interventions will be any comparisons of an antiplatelet agent (such as low dose aspirin or dipyridamole), or any combination of antiplatelet agents, compared with placebo or no antiplatelet agent. This is regardless of dose, mode of administration and irrespective of whether the antiplatelet is in combination with another drug. Trials that assessed only physiological outcomes following a short duration of intended therapy will be excluded.The main analyses will include all trials that fulfill the previous inclusion criteria, regardless of completeness of follow-up. Sensitivity analyses will be undertaken to assess the effect of the inclusion of data from trials where only small numbers of enrolled participants have available outcome data. The threshold for an acceptable level of data completeness may vary by outcome. For example, for long-term follow-up of women and children, data may be included if follow-up was less than 80% provided that substantive bias between the groups was unlikely. Other outcomes from each trial may only be included in the analysis if available for 80% or more of women.The individual patient data provided by the Collaborators will be de-identified, re-coded as required and stored in a custom-designed Microsoft ACCESS database. It will not include any patient identifying information such as names or addresses. Electronic data will be located on a secure, password protected network server. Copies of hardcopy data will be stored in locked filing cabinets until converted into electronic format, and will then be securely destroyed. Only authorised personnel will have access to the data. All data will be securely stored and archived according to the policies of the major funder, the Australian National Health and Medical Research Council.The data will be checked with respect to range, internal consistency, consistency with published reports and missing items. Trial details such as randomisation methods, and dose and timing of the interventions will be cross-checked against any published reports, trial protocols and data collection sheets. Integrity of the randomisation process will be examined by reviewing the chronological randomisation sequence and pattern of assignment, as well as the balance of prognostic factors across treatment groups (taking into account stratification factors). Inconsistencies or missing data will be discussed with the individual trialists and attempts will be made to resolve any problems by consensus. Each trial will be analysed individually, and the resulting analyses and trial data will be sent to the trialists for verification.There has been extensive consultation with the PARIS Collaborative Group regarding what data items to collect for each woman in the analyses. The following section contains the list of data items requested from trialists, which has been compiled following this consultation. More detailed definitions for the data items listed below can be found in Tables 1 informed consent2 dates trial opened and closed to accrual3 total number of women randomised4 treatments used in each arm of the trial5 intended duration of treatments6 definitions of key outcomes used in the trial7 method of random allocation8 stratification factors used9 methods of allocation concealment1 unique identifier for the enrolled woman, coded for anonymity2 date of randomisation3 gestational age at randomisation, or best estimate of expected date of delivery or last menstrual period4 woman's date of birth or age5 any previous pregnancy6 blood pressure 7 presence of proteinuria8 presence of oedema9 risk factors – multiple pregnancy, autoimmune disease, renal disease, diabetes, chronic hypertension, previous gestational hypertension or pre-eclampsia/eclampsia (including early onset disease), family history of pregnancy-related hypertensive disorders, previous fetal growth restriction, previous perinatal death, abnormal uterine artery Doppler flow and abnormalities on other diagnostic tests1 hypertension during pregnancy 2 proteinuria during pregnancy (including date and/or gestation at onset)3 oedema during pregnancy4 pre-eclampsia5 drug treatment for pre-eclampsia or hypertension6 severe maternal morbidity 7 onset of labour (spontaneous or induced or pre-labour caesarean section)8 mode of delivery 9 antepartum haemorrhage 10 postpartum haemorrhage and/or estimated blood loss at delivery11 maternal mortality1 gestational age at birth and/or date of birth2 birthweight3 gender4 small for gestation age 5 miscarriage or stillbirth: date and/or gestational age at death/loss6 neonatal or infant death: date and/or age at death7 admission to special care baby unit or neonatal intensive care unit8 use of assisted ventilation9 number of days in hospital or date of hospital discharge10 neonatal bleeding 11 child growth and development This section contains a summary of the planned analyses. The full, detailed analysis plan will be discussed and agreed upon by the Collaborators before any data have been analysed.2 statistic will also be used to assess consistency of results.Analysis will aim to be of all women ever randomised and will be based on intention to treat. In the main analyses a two stage approach will be taken. Outcomes will be analysed in their original trial and then these separate results will be combined to give an overall measure of effect. A fixed effect model will be used and the assumption of homogeneity of treatment effects will be tested using the chi squared test. The IThe main analyses comparing the effect of antiplatelet agents with placebo or no antiplatelet agents will be undertaken for all outcomes listed below, for the woman and any fetus/baby. The planned sub-group and sensitivity analyses will be restricted to the designated main outcomes listed as follows:• pre-eclampsia• pregnancy loss / neonatal death• pre-term birth• small for gestational age infant• pregnancy with serious adverse outcome• early onset pre-eclampsia• maternal death• severe maternal morbidity• antepartum haemorrhage• placental abruption• induction of labour• Caesarean section delivery• postpartum haemorrhage• gestation at delivery• infant admission to special care or neonatal intensive care unit• infant required assisted ventilation• neonatal bleeding• infant deathThe planned sub-group analyses will be restricted to the designated main outcomes unless there are clear indications for expanding the analyses further.The effect of antiplatelet therapy may vary across the trials in the meta-analysis because they have used different agents in different ways. To explore this further, analyses are planned whereby trials will be grouped according to the agent used and by dose. These analyses will focus on the main outcomes. Trials will be classified into subsets based on the following:Trials will be grouped by the type of antiplatelet agent  given as the active treatment.In trials that used aspirin alone, trials will be grouped by planned aspirin dose .One of the strengths of individual patient data reviews is that it allows us to assess eligibility and outcome using individual women's characteristics. Subgroup analyses will explore whether any particular risk factors act as effect modifiers. That is, are there any particular types of women who benefit more or less from antiplatelet agents? These analyses will take account each individual woman's own characteristics, rather than relying on summary measures of the 'average' risk profile of all participants in an individual trial.Analyses will be undertaken to explore whether there are any particular types of women who benefit more or less from antiplatelet agents based on the following criteria:Women normotensive at trial entry:previous hypertensive disorders of pregnancy: previous early onset (≤33 weeks + 6 days gestation) pre-eclampsia or eclampsia / previous pre-eclampsia / previous gestational hypertension / no previous hypertensive disorders of pregnancy / no previous pregnancy but family history of hypertensive disorders of pregnancy / no previous pregnancy and no family history of hypertensive disorders of pregnancy- diabetes: pre-existing diabetes at enrolment / no pre-existing diabetes at enrolment- renal disease: pre-existing renal disease / no pre-existing renal disease- autoimmune disease: autoimmune disease / no autoimmune disease- multiple pregnancy: multiple pregnancy / singleton pregnancy- maternal age: <20 years / 20–35 years / >35 years. Maternal age may also be analysed as a continuous variable.- diagnostic test results: abnormal uterine artery Doppler scan / other diagnostic test abnormalities / no abnormal diagnostic test results- previous SGA: previous small for gestational age infant / previous infant not small for gestational age / no previous infant- primigravida: first pregnancy with no other risk factors / first pregnancy with one or more risk factors / second or subsequent pregnancy with one or more risk factors / second or subsequent pregnancy with no risk factors- Women with hypertension at trial entry:hypertension: gestational hypertension / chronic hypertension- To determine whether antiplatelet agents are differentially effective if given earlier in pregnancy and to determine the magnitude of any difference, gestational age at randomisation will be primarily analysed as a continuous variable in regression analyses. However, a subgroup analysis with women classified according to the following categories may also be performed: <16 weeks, 16–19 completed weeks, 20–23 completed weeks, 24–27 completed weeks, ≥28 weeks gestation. If numbers are insufficient for any category, categories will be combined.a. To assess whether results are robust to the inclusion or exclusion of particular types of trials or patients, the following sensitivity analyses will be conducted:• exclusion of trials that did not use a placebo for the control group• exclusion of trials of small size• exclusion of poor quality trials b. To assess whether results are robust to different methods of analysis or different definitions of pre-eclampsia ,39, the • comparison of analyses using random effects and fixed effect models• comparison of analyses using individual patient data (IPD) only with analyses using individual patient data and aggregate data where IPD unavailable• comparison of analyses using different definitions of pre-eclampsiaThese sensitivity analyses will be carried out for the main outcomes.Depending on what data are available, the level of heterogeneity encountered and available time a one-stage modeling approach may also be undertaken to further explore important key outcomes as appropriate.Participants in the individual trials have previously given informed consent to participate in their respective trial. The data for this project are to be used for the purpose for which they were originally collected and are available through an agreement between all trialists of the PARIS Collaboration. These trialists remain the custodians of their original individual trial data at all times. Data are provided on the stipulation that all trials have received ethical clearances from their relevant bodies.Membership of the PARIS Collaboration will be representative(s) from each of the trials contributing data to the review with an accompanying project coordination and data management structure as described in this section.The membership and responsibilities of each of these management groups is as follows:1 (co-chair), L Duley2 (co-chair), L Askie1 (project coordinator), M Showell1 (project administrator), B Farrell2, L Stewart3, M Clarke4, J King5, C Roberts.1 The first six members act as the Secretariat.The Steering Groupwill be responsible for project management decisions and will meet approximately 4–6 times per year, usually via teleconference. Membership: D Henderson-Smart1 Centre for Perinatal Health Services Research, University of Sydney, Australia;2 Resource Centre for Randomised Trials, University of Oxford, Oxford, UK;3 Medical Research Council Clinical Trials Unit, London, UK;4 UK Cochrane Centre, Oxford, UK;5 Royal Women's Hospital, Melbourne, Australia.ad hoc meetings. Co-chairs of the Advisory Group: C Redman, C Roberts.The aim of the Advisory Group is to facilitate representative input from the Collaborative Group to the Steering Group. Membership of the Advisory Group will include people who have contributed to the trials included in the project and other international experts. Each trial that recruited over 1000 women will be invited to have a representative on the Advisory Group. This group will not have regular meetings, but may be consulted from time to time by means of email or teleconference, and may have occasional All potentially eligible trialists will be contacted and invited to become members of the Collaborative Group. The corresponding author for each study will be contacted in the first instance. If there is no response, the associated statistician, data manager and/or other authors will be contacted. This process will be updated annually for the duration of the project, to ensure that new trialists are offered the opportunity to join the project and contribute their data.The project will be coordinated from the Centre for Perinatal Health Services Research (CPHSR), University of Sydney, NSW, Australia. The coordination centre will be responsible for the daily management of the project including correspondence, newsletter production, maintaining current trialist contact information and meeting/teleconference organisation.The data management centre, based at the UK Cochrane Centre, will be responsible for the receipt, storage, and analysis of project data as directed by the Collaborative Group via the Steering Group.All members of the Collaboration, including the Steering Group, the Advisory Group, and representatives of each participating trial, will be invited to attend regular Collaborators' meetings. These meetings will be scheduled, where possible, to coincide with the biennial International Society for the Study of Hypertension Pregnancy (ISSHP) congresses. The meetings will be designed to allow maximum input from the participating trialists into the design, conduct, analysis and reporting of the project's results. The final Collaborators' meeting, at which the results will be presented for discussion, is scheduled for 2006 in Oxford, UK. The discussion at this meeting will provide the basis for the paper publication.The National Health and Medical Research Council (NHMRC) of Australia have provided funding for the project through the Centre for Perinatal Health Services Research, University of Sydney. These funds are: a three year project grant (ID: 253636) for the overall project administration base in Sydney, and a Sidney Sax Public Health Postdoctoral Fellowship (ID: 245521), based in Oxford and Sydney. Additional support is being provided by the Resource Centre for Randomised Trials and the UK Cochrane Centre, located in Oxford, UK, and the Medical Research Council Clinical Trials Unit in London, UK.The results of the project's analyses will be presented to, and discussed with, the Collaborative Group before publication. The aim of publication will be presentation of the results, rather than their interpretation. The main manuscript will be prepared by the Secretariat, and then circulated to the Steering and Advisory Groups for comment and revision. The revised draft paper then will be circulated to all members of the Collaborative Group for comment before publication. All publications using these data will be authored in the name of the PARIS Collaboration, as follows: Perinatal Antiplatelet Review of International Studies (PARIS) Collaboration.Despite good evidence that antiplatelet agents  reduce the incidence of pre-eclampsia and its consequences, such as preterm birth and perinatal mortality, uncertainty remains regarding whether some women (in terms of risk) benefit more than others, when in pregnancy treatment should ideally start, and whether treatment effectiveness is dependent on antiplatelet dose.The best way to answer these questions is to utilise existing individual patient data from all women enrolled in trials that have addressed this question. This approach has been described as the 'gold standard' of systematic review methodology as it allows for more powerful and flexible analysis of both subgroups and outcomes.The PARIS Collaboration has been formed to undertake a systematic review of all available trials, with meta-analysis based on individual patient data, to answer these important clinical questions. This will be the first individual patient data review in the perinatal field. Provision of data by the participating Collaborators commenced in 2004, and results will be ready for presentation in 2006. Following consultation and discussion with the Collaborative Group, the main publication is expected in early 2007.The author(s) declare that they have no competing interests.All authors, the named members of the PARIS Collaboration Steering Group, contributed to the development of the protocol, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:List of eligible trials text document listing potentially eligible trialsClick here for fileSuggested coding sheet table listing variables and suggested codingClick here for fileData provision form form to collect trial level data and data provision proceduresClick here for file


Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.We have generated several hundred lines of zebrafish  A screen for tumour suppressor genes in zebrafish has identified several genes encoding ribosomal proteins, indicating that these genes might be an unappreciated class of cancer genes The third group of RP mutant lines included five lines that were not tumor prone. These lines, with mutations in L3, L24, LP1, S12, and S15, were indistinguishable from controls in tumor incidence and spectrum . We also analyzed a total of 65 RP heterozygotes with grossly visible tumors (including the fish represented in (NF2a) of two distinct zebrafish genes that are highly homologous to the mammalian neurofibromatosis type 2 gene (NF2). The insertion abrogates expression of this gene in homozygous mutant embryos  at approximately six-week intervals between eight and 14 mo of age. As shown in Dominant mutations that predispose vertebrates to cancer can be activated oncogenes, recessive tumor suppressors, or haploinsufficient tumor suppressors . SeveralIn mice, a tumor cell line has been described in which one copy of an RP gene is deleted and the other copy has suffered a mutation that may contribute to tumorigenesis . To ruleIn yeast, a decrease in the amount of at least some RP genes results in a reduction in the amount of the corresponding ribosomal subunit and a reduction in the number of assembled ribosomes . To deteIn this study, we have found that heterozygous mutations in 11 different ribosomal protein genes predispose zebrafish to cancer, predominantly to zMPNSTs, but also to other rare tumor types. All of these mutations reduce RP gene expression, indicating that these 11 genes are not oncogenes. Moreover, in the tumors we examined, the wild-type allele appeared to be present and did not contain point mutations; thus these genes are not recessive tumor suppressors. Rather, our findings suggest that these 11 genes are haploinsufficient tumor suppressor genes; that is, reducing their activities by about a factor of two increases the likelihood of cancer. These findings raise two important, unanswered questions: first, how do these mutations lead to cancer, and second, do similar mutations cause cancer in humans?S7, S8, S15a, S18, S29, L7, L13, L23a, L35, L36, and L36a, predispose to cancer suggests that a function shared by RPs underlies their role in this phenotype. However, not all RP genes were cancer genes: S12, S15, L3, L24, and LP1 heterozygotes appeared normal. This raises the possibility that the oncogenic RP genes could conceivably share some novel biological function independent of their role in the ribosome and that inhibition of this function leads to tumor formation. Individual RPs have been implicated in a wide variety of biological functions, including cell cycle progression, apoptosis, and DNA damage responses  are associated with a mutation of rpS19 , and thirpS19 . It has rpS19 , but it While these examples from mouse and human are consistent with the idea that mutations in individual RP genes might contribute to tumorigenesis in mammals, they have seemed to be unusual examples, rather than suggesting that RP genes in general might be potential cancer genes. Our study suggests for the first time, we believe, that this is a general property of many RP genes. The possibility that a reduction in ribosome levels might be oncogenic in mammals is further supported by the fact that mutations in DKC1, a pseudouridine synthase that is required for rRNA processing and for properly functioning ribosomes, cause dyskeratosis congenita, a disease characterized by both premature aging and increased tumor susceptibility .rp mutations will prove to increase the incidence of tumors in humans as they do in zebrafish. If so, it may be advantageous to devise diagnostic strategies based on ribosomal protein levels or on a function that these proteins share, for example, in translation, rather than on the analysis of such a large number of individual genes.If RP genes frequently cause human cancers, it is not at all certain that their role would have been detected. Even a deliberate search for their involvement in human cancers would be difficult because there are so many (80) RP genes. This plethora of genes, the fact that it is hard to know which tumor type(s) to examine for RP mutations, and the fact that the mutations might lie in regulatory elements rather than protein-coding regions of the genes would make such a search difficult. Nonetheless, given the high degree of conservation of biological mechanisms among vertebrates, it seems likely that NF2a is a tumor suppressor gene in this system demonstrate the power of large-scale, forward-genetic screens in the zebrafish to identify new disease susceptibility genes.In summary, by examining aging populations of mutant lines of fish with defects in embryonic essential genes, we identified a novel group of cancer genes. The ability to identify cancer genes by screening populations of fish heterozyogous for recessive embryonic mutations and the reassuring finding that The insertional mutagenesis screen was carried out as previously described . Stocks Adult fish were euthanized in ice water and fixed within 30 min in Bouin's solution, embedded in paraffin, and sectioned as previously described .DNA was prepared from tumor tissue or tail tissue isolated from fish prior to fixation for histology. PCR was conducted with one primer complementary to proviral sequence and two primers complementary to sequences on either side of the insertion for the appropriate mutation. Primer sequences were as follows: hi10: 10gen5 (5′-CAGCACAGATTCTTGAAAGCGCC-3′), 10gen3 (5′- GCATATGTAGCATCTCGAAGGTCC-3′), and NU3X (5′- TGATCTCGAGCCAAACCTACAGGTGGGGTC-3′); hi258: 258A5a (5′-GGTACGTCTGTGCTTATGTTTGTGTC-3′), 258A3a (5′-TCTCAAGACTTCATCCATTCATAATTCTGC-3′), and NU3X; hi1974: 1974c1 (5′-CTACACCACAGGTATCTCAAGGG-3′), 1974c1est3 (5′-CCACCACGGACTCTTATTGTGTG-3′), and IPL3 (5′-TGATCTCGAGTTCCTTGGGAGGGTCTCCTC-3′).rpL36a: 10rt5 (5′-CAACCATGGTAAACGTACCGAAG-3′) and 10RTR (5′-CACAAAAGAAGCACTTGGCCCAGC-3′); rpL35: 258RTF2 (5′-GCTGCTTCCAAGCTCTCAAAAATCC-3′) and 258RTR (5′-TGCCTTGACGGCGAACTTGCGAATG-3′); rpS8: 1974RTF1 (5′-TCTCAAGGGATAACTGGCACA-3′) and 1974RTR1 (5′-GAACTCCAGTTCTTTGCCCTC-3′); actin: actinF (5′-CATCAGCATGGCTTCTGCTCTGTATGG-3′) and actinR (5′-GACTTGTCAGTGTACAGAGACACCCT-3′). For visualization of 18S and 28S RNA, two embryo equivalents of RNA were electrophoresed through a nondenaturing agarose gel containing 0.5 μg/ml ethidium bromide. For detection of beta actin RNA, four embryo equivalents of RNA were electrophoresed through a 7.5% formaldehyde/MOPS-buffered agarose gel, blotted to Hybond N+ , and hybridized with a random primed beta actin probe.RNA was prepared from mutant and wild-type embryos using Trizol reagent . For RT-PCR, serial dilutions of first strand cDNA were amplified for 30 (hi1974) or 35 (hi10 and hi258) cycles using the following primers for the genes indicated: Figure S1rpl36 locus, which has its own scale bar.The genomic sequence of part of each of these genes is represented as exonic (boxed) and promoter or intronic (line). White boxes represent 5′ UTR while shaded boxes represent coding exons. Where no white boxes are shown, the location of the 5′ UTR and beginning of the coding region has not been determined relative to the part of the locus shown here. In all cases, at least one coding exon  is downstream of the region of the gene represented here. The position and orientation of the proviruses are shown above each genomic sequence. All drawings are to the scale of the top scale bar, except the (62 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes discussed in this paper are L13 (AY561516), L23a (AY561517), L24 (AY099532), L3 (AY561514), L35 (AF506205), L36 (AY561518), L36a (AY099511), L7 (AY561515), LP1 (AY561519), NF2a (AY561520), S12 (AY561510), S15 (AY561511), S15a (AY561512), S18 (AY099517), S29 (AY561513), S7 (AY561508), and S8 (AY561509).The GenBank (


APOE) ε2/ε3/ε4 polymorphism, which also is a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death. Here, we test the hypothesis that the APOE polymorphism may modulate the cholesterol-raising effect of coffee.The response of serum cholesterol to diet may be affected by the apolipoprotein E  and women (78%) aged 29–65 years, took part in a study with four intervention periods: 1 and 3) a coffee free period of three weeks, 2 and 4) 600 mL coffee/day for four weeks.APOE ε2 positive individuals had significantly lower total cholesterol concentration at baseline , but the cholesterol-raising effect of coffee was not influenced significantly by APOE allele carrier status.APOE ε 2 allele is associated with lower serum cholesterol concentration. However, the APOE polymorphism does not seem to influence the cholesterol-raising effect of coffee.The  APOE gene sequence results in the 3 common alleles , which can produce 6 different genotypes . The ε2, ε3 and ε4 alleles encode three distinct forms of apoE  and have approximate frequencies of 8%, 77%, and 15%, respectively, in white populations [APOE ε2 allele display lower levels of plasma cholesterol compared with individuals carrying the APOE ε3 allele, whereas individuals with the APOE ε4 allele show higher levels of plasma cholesterol, especially LDL-cholesterol [APOE ε3/ε4 and ε4/ε4 genotypes absorb cholesterol effectively and have higher non-fasting serum triglyceride values than ε4 negative individuals [APOE gene is shown to be a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death [APOE ε4 allele. The APOE ε4 allele is also considered a strong risk factor for Alzheimer's disease [Apolipoprotein E (apoE) is a structural component of triglyceride-rich lipoproteins, chylomicrons, very-low-density lipoproteins (VLDL), and high-density-lipoproteins (HDL) . VariatiHD death , but theHD death . Both thHD death  and the HD death  suggest  disease -16.APOE ε2/ε3/e4 polymorphism.The serum cholesterol-raising effect of coffee is due to the diterpenes kahweol and cafestol . EarlierThe study was organised as a prospective, controlled study with four consecutive trial periods. The first and third periods were 3 weeks of total coffee abstention. The second and fourth period consisted of 4 weeks with the subjects consuming 600 mL filter brewed coffee/day.The main outcome or effect variable was total serum cholesterol and the effect was assessed as the difference between the measurements at the beginning and the end of the coffee free periods (coffee abstention) and the difference between measurements at the beginning and at the end of the four weeks of coffee consumption Figure . Trial dParticipants were recruited by advertising in Gothenburg's major newspaper. Inclusion criteria were age-range 30–65 years, free from clinically recognized chronic diseases, such as cardiovascular diseases, cancer, renal disorders, liver disease and diabetes mellitus. The participants were required to be free from anti-epileptic or cholesterol lowering drugs, and had been using coffee on a regular basis for at least five years and were currently non-smokers (at least for the last six months).During the coffee drinking periods the participants were instructed to drink about 600 mL filter brewed coffee/day (4 cups), according to standardised measures. The coffee was provided to guarantee that they were all exposed to the same brand and quality of filter brewed coffee. All participants also got the same kind of standardised coffee filter and measuring spoon.The coffee filters used were of the brand Euro-Shopper, made by Indupa N.V., Zaventem, Belgium. Divergence from the 4 cups was reported. The participants were allowed to drink tea and other caffeine containing beverages during the coffee-free periods.Non-fasting blood samples were drawn at inclusion and at three, seven, ten and fourteen weeks after start of the study. Prior to analysis, prepared serum was stored at -70°C.2) was recorded once during the study. Blood pressure was recorded by manual device and EKG and heart rate were recorded at all five visits.The blood samples were analysed for blood lipids  (Lp(a)) and urate in serum. Serum cholesterol and triglycerides were determined by an enzymatic procedure on a Hitachi 917 analyzer. HDL cholesterol was determined enzymatically after precipitation of VLDL, LDL and chylomicrones by α-cyklodextrinsulphate and dextransulphate. Determination of Lp(a) was done by the method Tint Elize Lp(a) of Biopool International. Serum urate was analysed by Hitachi 917 autoanalyser. Body Mass Index , but not in the ε2-positive group  and coffee consumption prior to the study (Table Coffee consumption resulted in a significant cholesterol increase in the εThe dietary survey did not reveal any important changes during the four intervention periods . Coffee APOE genotypes differ in the absorption efficiency of cholesterol from the intestine, in the synthesis rates of cholesterol and bile acids, and in the production of LDL apoprotein B [APOE e2/e3/e4 polymorphism [APOE polymorphism [APOE ε4 allele. Here, we investigate for the first time the possible influence of the APOE polymorphism on the cholesterol-raising effect of filtered coffee.Subjects with different rotein B ,26. Thismorphism ,28. One APOE ε4-positive individuals did not differ significantly from ε4-negative individuals with regard to baseline cholesterol concentration or coffee-induced changes in cholesterol concentration. However, we confirm that ε2-positive individuals display significantly lower cholesterol levels at baseline than ε2-negative individuals. A tendency was seen that ε2-positive individuals might be partly protected from the cholesterol increasing effect of coffee consumption. This was, however, only seen in the first trial period and will require further investigations. In conclusion, the APOE ε2/ε3/ε4 polymorphism is not a strong modulator of the cholesterol-increasing effect of coffee. Other genes should be discussed and further investigation is needed to see if there is a genetic factor in the cholesterol-raising effect of coffee.


Drosophila and C. elegans.A high-throughput method for genotyping by mapping InDels. This method has been used to create fragment-length polymorphism maps for  Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both  For humans and model organisms, such as worms and flies, the availability of high-density sequence polymorphism maps greatly facilitates the rapid mapping and cloning of genes -3. Key aDrosophila melanogaster [The most direct approach for SNP detection is sequencing of a PCR product spanning the polymorphism, but this is too costly and labor intense for high-throughput genotyping. For this reason, several different strategies and methods have been developed in order to detect SNPs more efficiently. In general, assays can be grouped into strategies, where the nature of the SNP is determined by directly analyzing the primary PCR product and those that require a secondary assay performed on the primary amplification product -6. An imnogaster .Caenorhabditis elegans and Drosophila are used regularly in genotyping projects [C. elegans. However, this approach is labor intensive and requires specialized chemistry and equipment. Using DNA microarrays, large numbers of SNPs can be analyzed in parallel, but the number of individuals that can be analyzed is low because of the high cost per chip [Of the methods that detect the SNP in a secondary assay, restriction fragment length polymorphism (RFLP) analysis are very popular . For thiprojects ,3,11. Hoprojects . Here, aprojects . Swan anprojects  have devper chip ,16.Besides SNPs, short tandem repeats (STRs) or microsatellites represent another class of sequence polymorphisms used for genotyping -21. STRset al. [Drosophila are of the InDel type. Also, two independent studies in C. elegans found that InDels constitute between 25% and 28% of all polymorphisms [Drosophila [C. elegans [Interestingly, a significant proportion of the currently available polymorphisms are caused by small insertions or deletions (InDels). Weber et al.  identifiet al.  who founorphisms ,14. In a elegans ).To take full advantage of this class of small InDel polymorphisms, we have developed a strategy that allows us to detect most, if not all, InDels by analyzing the lengths of primary PCR products on a capillary sequencer at single base-pair resolution. We call these assays fragment length polymorphism (FLP) assays. Importantly, this approach can easily be automated on standard robotic pipetting platforms as it involves a simple PCR reaction setup. Furthermore, allele calling is performed automatically using the Applied Biosystems GeneMapper software commonly used for genotyping STRs .C. elegans (one every 0.9 megabase-pair (Mbp)) and 54 FLP assays at 4 cM resolution for the Drosophila autosomes. This set of FLP assays allows us to rapidly map mutations to small chromosomal subregions with a minimum of manual input. Furthermore, we provide a list of predicted InDels for which additional assays can be readily designed in the chromosomal subregion of interest. Those non-validated FLPs enhance the resolution of the map by a factor of 5.6 and 17.9, respectively.To demonstrate the feasibility of this strategy, we have validated 112 evenly spaced FLP assays at 3 centimorgan (cM) resolution in C. elegans or Drosophila.We show the usefulness of this approach by identifying novel alleles of previously characterized genes. In summary, we have taken advantage of a publicly available dataset to adapt a technology widely used for STR analysis to genetic mapping. Thanks to the complete automation of genotyping, this approach is considerably faster, more reliable and cheaper than previously used mapping strategies in To detect a FLP, the region of interest is amplified in a standard PCR reaction with one fluorescently labeled primer, and the PCR products are directly analyzed on a capillary sequencer. Fragment sizes are determined automatically relative to an internal size standard with AppliedBiosystem's GeneMapper software . The software then allocates fragment sizes to previously calibrated genotypes.Taq polymerase has the tendency to catalyze the addition of adenosine (A) to the 3' end of PCR products. This activity could make it difficult to achieve the single base-pair resolution required to assay all available InDels and may hamper allele-calling . However2R090 and 231 samples homo- and heterozygous for the C. elegans Bristol and Hawaii backgrounds, respectively, for the 1-bp InDel ZH5-16. 2R090 exhibits both stuttering and A addition and hence is especially difficult to resolve . The genotype was correctly and automatically assigned by GeneMapper in all 423 assays. Thus, automated genotyping based on FLPs is sensitive down to single base-pair resolution and is extremely robust. The accuracy of FLP mapping is comparable to other methods such as TaqMan , minisein 2,000 ), and pyin 2,000 ).C. elegans, genetic experiments are performed almost exclusively in the background of the standard wild-type strain N2 (C. elegans variety Bristol) [C. elegans, variety Hawaii) has proved extremely useful [et al. [C. elegans were validated to date . Most failures to detect a FLP are probably due to original sequencing errors. The average distance between neighboring FLP assays is about 0.9 Mbp. This physical distance corresponds to about 3 cM, assuming 300 kb per map unit, and encompasses between 100 and a maximum of 500 genes  . For geny useful . When coy useful . Startin [et al. , 112 FLPe Figure . The cons Figure . The lenDrosophila FLP map, a set of 54 FLP assays (12 to 17 per arm of the two major autosomes) was validated from the list of polymorphisms identified by Berger et al. [C. elegans, the confirmation rate of the predicted Drosophila InDels was 80% (n = 30). Furthermore, another 509 InDels are predicted at 248 sites for which an assay can be established to discriminate between EP and FRT strains . The validated Drosophila FLP assays were evenly spaced on the genetic map with an average distance between neighboring assays of about 4 cM, corresponding to an average resolution of 1.77 Mbp on the physical map encompassing 95,55 Mbp [To establish a 5,55 Mbp ,28. TakiDrosophila FLP assays are in part derived from a set of InDels of size difference 7 bp or more . Howeveleotides , so far C. elegans FLP map, we mapped three previously characterized mutations on chromosome II that exhibit diverse phenotypes. Those were the centrally located let-23(sy1) allele that causes an 80% penetrant vulvaless phenotype [rol-1(e91) in the middle of the left chromosome arm, which causes the animals to roll around their body axis [unc-52(e444) mutation located at the right end of the chromosome, which results in a paralyzed phenotype [1 cross-progeny was selected (F1 self-progeny would exhibit a mutant phenotype). Finally, mutant self-progeny was isolated in the F2 generation and used for genotyping  with one centrally located FLP assay per chromosome  mutation showed linkage to the X chromosome of N2 in addition to chromosome II. This pseudo-linkage could be due to a suppressor of the Rol phenotype present on the CB4856 X chromosome. In a second step, 48 F2 animals for each mutation were analyzed with eight FLP assays along chromosome 2  phenotype, and staining of the adherens junctions with the MH27 antibody showed defects in the formation of the vulval torroid rings , indicating that the zh41 mutation in the lin-11 open reading frame (ORF) is responsible for the vulval phenotype.To demonstrate the usefulness of the henotype , rol-1 and by genotyping existing SNPs in the candidate interval . In manyC. elegans allows us to rapidly map a mutation down to a small region containing, on average, 200 candidate genes by crossing a mutant strain to CB4856 and analyzing 48 F2 animals with 300 to 500 PCR reactions.In summary, FLP mapping in C. elegans, zebrafish (Danio rerio) or Arabidopsis genetics, Drosophila strains are very heterogeneous and of ill-defined origin [Drosophila resembles human genetics in that standard inbred lines do not exist and the genotypes of the parental lines have to be determined first. As genome-wide polymorphism databases for reference strains are available [yw strains for the whole set, four FRT-Minute or FRT-cell-lethal strains at the relevant autosomal arms [2R017, Figure Drosophila strains .In contrast to the well defined genetic backgrounds used for d origin ,9,11. Invailable ,11, a limal arms , as wellVI.29 and EP0755 we recovered three types of recombinant chromosomes: recombinants with a crossover proximal or distal to the mutation, respectively, and double-crossovers . Furthermore, data from this and other FLP mappings in this region allowed us to further refine the genetic map . This kind of experimental data is helpful to space new FLP assays more evenly on the genetic map should the available map turn out to be inexact.In a genetic screen devised to isolate genes that regulate growth and are situated on chromosome 2R, we found a complementation group characterized by a mild overgrowth phenotype . Second, we genotype recombinants with SNPs available in the region of interest and resolve them by RFLP, sequencing or DHPLC ,9. Thirdeletions ,38.  the resC. elegans or Drosophila. For example, 7.3% of the Arabidopsis sequence polymorphisms are InDels [Arabidopsis, C. elegans or Drosophila FLPs.It seems likely that in most organisms the frequency of polymorphisms caused by InDels is in the same range as found in humans, e InDels . Thus, FC. elegans was done according to standard procedures described in [C. elegans alleles used were: LG I: lin-11(zh41), lin-11(n389); LG II: rol-1(e91), let-23(sy1), unc-52(e444). Drosophila strains and the genetic screen have been described previously [Culturing and crossing of ribed in . C. eleglin-11n38; LG II: eviously ,40-42.2, 0.45% NP-40, 0.45% Tween-20, 100 μg/ml freshly added proteinase K) and incubated for 60 min at 65°C followed by heat-inactivation of proteinase K at 95°C for 10 min. Before PCR, 90 μl double-distilled H2O (ddH2O) was added to obtain a total volume of 100 μl per lysate.Adult worms were collected in 10 μl lysis buffer  Debris was allowed to settle for about 5 min, and 50 μl of each supernatant were transferred into a 96-well PCR plate. The reactions were incubated in a thermo-cycler for 30 min at 37°C and finally for 10 min at 95°C to heat-inactivate proteinase K. Before PCR amplification, the crude DNA extracts were diluted 20-fold to reduce the concentration of proteins that might be harmful for the capillary sequencer.DNA from recombinant flies was extracted in bulk by squishing flies through mechanical force in a vibration mill (Retsch MM30) programmed to shake for 20 sec at 20 strokes per second . Single 2, 1x PCR reaction buffer, 0.25 U EuroTaq polymerase (Euroclone). PCR reaction setup was done in 96-well plates using a Tecan Genesis pipetting robot with disposable tips. PCR was carried out in two MJR thermo-cyclers that are integrated into the robot. The current setup allows for the sequential processing of six 96-well plates at a time. Cycling parameters were 2 min 95°C, 20 sec 95°C, 20 sec 61°C (-0.5°C for each cycle), 45 sec 72°C (for 10 cycles) followed by 24 cycles of 20 sec 95°C, 20 sec 56°C, 45 sec 72°C and a 10 min 72°C final extension. Following PCR, reactions were diluted 1:100 in water, and 2 μl diluted PCR products were mixed with 10 μl HiDi formamide containing 0.025 μl LIZ500 size standard (Applied Biosystems). This dilution before analysis on the capillary sequencer is necessary to reduce signal intensity because too strong signals compromise data analysis. In addition, sample dilution reduces the risk of damaging the capillaries with proteins or lipids present in the crude lysates. The dilution was done with standard tips using the Tecan Genesis pipetting station. Carryover of fragments was prohibited by a simple wash step with H2O. Fragments were analyzed on an ABI3730 capillary sequencer using POP7 polymer according to standard procedures. Data were analyzed using AppliedBiosystems GeneMapper software and raw data were treated further with Microsoft Excel.Diluted single-worm lysates (2 μl samples) or single fly extracts were added to 23 μl PCR reaction mix. Final concentrations in the PCR reaction were: 0.4 μM forward/reverse primer, 0.2 mM dNTPs, 2 mM MgClThe following additional data are available with the online version of this article. Additional data file C. elegans mapping results are given in Additional data files C. elegans and Drosophila, respectively. Additional data file Drosophila chromosome 2. Additional non-validated FLPs can be found in Additional data files C. elegans) and Additional data file Drosophila).Further General information on fly geneticsClick here for additional data fileProof-of-principle for chromosomal linkagewith 3 known mutations on chromosome 2. Assays used to assess linkagewere ZH1-01, ZH2-01, ZH3-05a, ZH4-03, ZH5-01 and ZHX-02Click here for additional data filelet-23 to its subchromosomal region (C. elegans)Mapping of Click here for additional data filerol-1 to its subchromosomal region (C. elegans)Mapping of Click here for additional data fileunc-52 to its subchromosomal region (C. elegans)Mapping of Click here for additional data fileC. elegans FLP mapping flow chartClick here for additional data fileDrosophila FLP mapping flow chartClick here for additional data fileElectropherograms demonstrating the accuracy of allele-callingClick here for additional data fileC. elegansTables of primer and sequence data of experimentally verified FLP assays in Click here for additional data fileDrosophilaTables of primer and sequence data of experimentally verified FLP assays in Click here for additional data fileDrosophila chromosome 2A table of the refined genetic distances for FLP assays on the right arm of Click here for additional data fileC. elegans InDels LGI)Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs (Click here for additional data file


Parallel computing is frequently used to speed up computationally expensive tasks in Bioinformatics.a) pair-wise sequence alignments that are used as a first step to multiple alignment account for most of the CPU time in DIALIGN. Since alignments of different sequence pairs are completely independent of each other, they can be distributed to multiple processors without any effect on the resulting output alignments. (b) For alignments of large genomic sequences, we use a heuristics by splitting up sequences into sub-sequences based on a previously introduced anchored alignment procedure. For our test sequences, this combined approach reduces the program running time of DIALIGN by up to 97%.Herein, a parallel version of the multi-alignment program DIALIGN is introduced. We propose two ways of dividing the program into independent sub-routines that can be run on different processors: (By distributing sub-routines to multiple processors, the running time of DIALIGN can be crucially improved. With these improvements, it is possible to apply the program in large-scale genomics and proteomics projects that were previously beyond its scope. Multiple sequence alignment continues to be an active field of research in Computational Biology and a number of novel approaches have been developed during the last years, see  for an oet al. evaluated the capability of alignment programs to detect conserved non-coding sites in genomic sequences. These authors conclude that DIALIGN can produce alignments with high coverage and sensitivity, as well as specificity to detect constrained sites [DIALIGN  is a vered sites .anchored alignment option [Though DIALIGN produces alignments of high quality, it is slower than alternative multi-alignment programs. Especially if large genomic sequences are to be aligned DIALIGN is far more time-consuming than the above mentioned specialized programs for genomic alignment. A recently introduced t option  can be ut option -21. Herechain of local fragment alignments with maximum total weight score is identified. A fragment or fragment alignment is defined as an un-gapped local pair-wise alignment, and the weight score of such a fragment is calculated based on a P-value i.e. on the probability of its random occurrence, see [For multiple alignment, the DIALIGN algorithm works as follows: in a first step, all respective optimal pair-wise alignments are carried out. This means that, for each pair of input sequences, a nce, see  for a dence, see .N input sequences, N × (N - 1)/2 pair-wise alignments are to be calculated; fragments contained in these pair-wise alignments are then used to build up a multiple alignment in greedy fashion. If the maximum sequence length is bounded by some constant, the time-complexity of this algorithm as a function of the number N of sequences is as follows: Performing all pair-wise alignments takes O(N2) time. During the greedy procedure, O(N) independent fragments can be included into the multiple alignment; additional fragments would be either inconsistent or already contained in the existing multiple alignment. Accepting a single fragment takes O(N2) time since for each accepted fragment, so-called consistency frontiers are to be updated. These frontiers are used to decide if subsequent fragments are consistent with previously accepted fragments. Thus, the worst-case time complexity of our multi-alignment algorithm is O(N3). Test runs with real data show, however, that the real time-complexity is something between quadratic and cubic, see [For a set of bic, see  for a derelative proportion of CPU time used for pair-wise alignments decreases with the number of input sequences, as can be expected from the above theoretical considerations.The current version of DIALIGN uses an efficient algorithm to update the consistency frontiers. This means that, although performing all pair-wise alignments has a lower theoretical time complexity than processing the fragments from these alignments in the greedy algorithm, for realistic data sets most of the CPU time is spent on the pair-wise alignments. For example, for a set of 20 protein sequences with an average length of 367 amino acid residues, as much as 97.4 % of the CPU time is used to perform the 20 × 19/2 = 190 pair-wise alignments. However, the N × (N - 1)/2 pair-wise alignments to the available processors in order to balance the work load. Here, we are using a greedy algorithm to find a satisfactory work-load distribution in reasonable time: we first assign a processor to the pair-wise alignment with the longest expected running time, then assign a processor to the second-largest pairwise alignment etc.The pair-wise alignments that are calculated as the first step of the multi-alignment procedure are completely independent of each other. Thus, it is obvious that the total program running time can be crucially reduced by running these procedures on parallel processors. Here, an important point is to distribute the work load evenly to the different processors in order to minimize the total program running time. To this end, our algorithm first estimates the running time for each pair-wise alignment as a function of the sequence length. As outlined in , the runanchored alignment procedure has recently been implemented [CHAOS to identify a chain of high-scoring local alignments for each pair of input sequences . To select a consistent sub-set of these local alignments, a greedy algorithm is used, see [The quadratic time complexity of the original DI ALIGN algorithm for pair-wise alignment is clearly not efficient enough to align large genomic sequences. To improve the running time of DIALIGN for long sequences, an lemented ,24. For sed, see  for detatrie data structure to identify pairs of segments with a user-defined upper bound on the number of mismatches per segment pair. It defines a local alignment as a chain of such gap-free local alignments that are located within a certain distance from each other. Finally, the algorithm returns an optimal chain of such local alignments. After the consistency check, these alignments are used by our algorithm to define anchor points in order to narrow down the search space for the final pair-wise alignment procedure that is performed by DIALIGN as explained in [CHAOS uses a ained in .x is assigned to a residue y through one of the anchor points, this means that x is the only residue that can be aligned with y in the final output alignment. Whether or not x and y will be aligned depends on the degree of local sequence similarity that DIALIGN detects. Moreover, all residues to the left of x can be aligned only with residues to the left of y and vice versa, see Figure non-anchored alignment, then the result of the anchored alignment procedure will necessarily be the same as for the non-anchored procedure. In particular, this is the case if all anchor points are part of the optimal non-anchored alignment.To be precise, an anchor point is a pair of segments, one segment from each of the two input sequences; this way each position in the first segment is assigned to the respective position in the second segment. If a residue cutting positions to split the input sequences into smaller sub-sequences, and we reduce the program running time for DIALIGN by aligning these sub-sequences independently on multiple processors. This procedure is related to Stoye's well-known divide-and-conquer approach to multiple alignment [In the present study, we use selected anchor points as lignment  and to tlignment .non-additive weighting function w for segment pairs (fragments). If a large fragment f is split into two smaller sub-fragments f1, f2, the sum w(f1) + w(f2) is, in general, lower than the original weight w(f), see [It should be mentioned that, unlike the above outlined anchoring procedure, distributing sub-alignments to multiple processors may well affect the resulting output alignments, even if the selected cut positions are consistent with the optimal alignment. No matter how well our anchoring positions are chosen, we can generally not expect the optimal alignments of our sub-sequences to coincide with the optimal alignment of the original input sequences The reason for this behaviour is that DIALIGN uses a (f), see . As demoTo reduce the total running time of the alignment procedure as far as possible, our algorithm distributes the sub-sequence alignments evenly to the available processors. At the same time, it minimizes the loss of alignment quality that may be caused by splitting the sequences into too many subsequences. To this end, we first identify a chain of anchor points using CHAOS. The program then divides the sequences at every anchor point, thereby producing a large set S1 of pairs of relatively small sub-sequences. The running time for each of the corresponding sub-alignments is estimated as described above. Using these estimates, the sub-sequences in S1 are concatenated in such a way that a set S2 of larger sub-sequences is obtained that can still be evenly distributed to the available processors. Here again, we use a greedy approach to assign processors to sub-alignments. As a result, our algorithm minimizes the program running time by balancing the work load among the processors while it maximizes the length of the aligned sub-sequences, thereby reducing the possible loss of alignment quality.) that is a Linux-SMP cluster with two Pentium III processors (650 MHz) and 1 GB main memory per node. The nodes are connected by a Myrinet 1.28 GBit/s switched LAN. The software was also compiled and tested on a Sun fire with 8 processors and on an ordinary Linux-based workstation pool that generally exists in every institute.We decided to use the message-passing interface (MPI) ,28 for oat al. [genomic sequences has recently been investigated by Pollard et al. [existing approach, we do not evaluate the quality of the produced output alignments; the ability of our software to produce biologically meaningful alignments under various conditions has been evaluated in the above cited papers. Herein, we compare the running time of our parallel software to the original serial version of the program.The performance of existing multi-alignment software has been evaluated in detail. All programs have been extensively tested by their authors; in addition several independent studies have been carried out using numerous sets of real and artificial benchmark data. The quality of multiple-protein-alignment programs including DIALIGN has been systematically studied by Thompson at al.  and by Lat al. . The abid et al. . Since tp of processors. Using 64 processors, the running time for these data sets can be reduced by 94.82%, 90.80% and 75.90%, respectively, compared to the serial running time. The observed differences in the relative speed-up are due to the different proportion of CPU time that is spent on the pairwise alignments, see [As a first test example, we aligned sets of proteins with 20, 55, and 100 sequences, respectively. The program distributed the pair-wise alignments to different processors as described in section. Table nts, see . The larnts, see ). Furthents, see .bp . These pairs of subsequences were concatenated to obtain a set S2 of larger sub-sequences that can be evenly distributed to the available processors.Next, we looked at the improvement of program running time that can be achieved for large genomic sequences using the algorithm described in section. As a test example, we used a set of three syntenic genomic sequences from mouse, rat and human. Each of these sequences is around 1 MB in length. The program CHAOS identified a total of 15,818 anchor points; 4,294 for human/mouse, 4,072 for human/rat and 7,452 for mouse/rat. Most of these anchor points were consistent with each other, only 121 out of the 15,818 anchor points had to be discarded because of consistency problems [Using CHAOS anchor points, the serial version of our program took 267,574 s = 74 h 19 m 34 s to compute the multiple alignment of our input sequences on a single processor of our cluster. We estimate that, without the anchoring option, the original DIALIGN program would have taken around three weeks to align these sequences. By contrast, the parallel version of the program with 64 processors took only 6,583 s = 1 h 49 m 43 s to align the same sequence set, corresponding to a running time improvement by 97.5 %. With a speed-up from more than three days to less than two hours, DIALIGN can now compute long-range multiple alignments of genomic sequences that were, until recently, far beyond its scope.Göttingen Bioinformatics Compute Server (GO-BICS) at . The program code is available on request.The program will be available online through MS parallelized DIALIGN, installed it on the Kepler cluster, performed the test runs and wrote parts of the manuscript. KN and MK participated in the design of the project and manuscript preparation. BM supervised the project and wrote most of the manuscript. All authors read and approved the final manuscript.


Miscarriage is a common event but is remarkably difficult to measure in epidemiological studies. Few large-scale population-based studies have been conducted in the UK.This was a population-based two-stage postal survey of reproductive histories of adult women living in the United Kingdom in 2001, sampled from the electronic electoral roll. In Stage 1 a short "screening" questionnaire was sent to over 60,000 randomly selected women in order to identify those aged 55 and under who had ever been pregnant or ever attempted to achieve a pregnancy, from whom a brief reproductive history was requested. Stage 2 involved a more lengthy questionnaire requesting detailed information on every pregnancy (and fertility problems), and questions relating to socio-demographic, behavioural and other factors for the most recent pregnancy in order to examine risk factors for miscarriage. Data on stillbirth, multiple birth and maternal age are compared to national data in order to assess response bias.The response rate was 49% for Stage 1 and 73% for the more targeted Stage 2. A total of 26,050 questionnaires were returned in Stage 1. Of the 17,748 women who were eligible on the grounds of age, 27% reported that they had never been pregnant and had never attempted to conceive a child. The remaining 13,035 women reported a total of 30,661 pregnancies. Comparison of key reproductive indicators  with national statistics showed that the data look remarkably similar to the general population.This study has enabled the assembly of a large population-based dataset of women's reproductive histories which appears unbiased compared to the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility. Despite improvements in obstetric care in the UK over the past fifty years, it is estimated that around one in five pregnancies will end in miscarriage  ,2. The pAlthough many large-scale population-based studies of miscarriage risk have been conducted elsewhere -10, relaStudies of miscarriage have tended to be clinical-based, and are thus subject to selection bias. For example, gestations are later among miscarriages reaching hospital-based clinics. Many miscarriages are managed at home, and some are not reported to a clinician. Not only is miscarriage hard to measure, and different clinical sources rarely see the full range of cases, but reported risks of miscarriage tend to be pregnancy-rather than woman-based: estimates of risk tend to relate to the proportion of pregnancies ending in miscarriage, and there are very few studies examining the risk of experiencing one, two or more miscarriages, or the chances of conceiving following a miscarriage . Large pAn increasing number of couples are also seeking help for problems achieving a pregnancy. Although it is estimated that up to 15% couples experience such problems , few popWe now report on a large UK population-based survey of reproductive health, the National Women's Health Study. The study design was developed from several other large epidemiological surveys of reproductive outcome which showed that a postal method could be used to obtain full reproductive histories from large study populations ,14,17,18Eurodirect [This was a population-based cross-sectional postal survey of reproductive histories of adult women living in the United Kingdom in 2001, designed to enable the construction of a retrospective population-based reproductive cohort and a case-control study of risk factors for miscarriage. A sample of women was randomly selected from electronic electoral registers for England, Wales, Scotland and Northern Ireland held by the company rodirect . All UK Eurodirect based on forename, whereby the sampling frame was restricted to women thought likely to be aged 55 and under on the basis of their name. This process was based on empirical data relating to birth certificates going back to the beginning of the 20th century, from which it could be calculated that, for example, those named "Elsie" are likely to be aged over 55, and those named "Kylie" under 55 years. Predictions are further refined by examination of combinations of names within a household (a "Jane" married to or living with an Alfred likely to be older than a "Jane" married to or living with a "Darren") and length of residency (e.g. someone registered to vote at the same address for 12 years has to be over 30). We requested a random sample of 61,000 women likely to be aged 55 and under . After removing those known to be under age 18 at study , the final sample consisted of 60,814 women.In order to reduce possible biases associated with memory, we aimed for a sample aged 55 years and under at survey. Date of birth is not, however, routinely recorded on the electoral register. To avoid unnecessary mailing and expense, we therefore made use of a probabilistic process offered by The study received approval from the Trent Multi-Centre Research Ethics Committee and the Ethics Committee of the London School of Hygiene & Tropical Medicine.The postal survey had two stages. Stage one consisted of a single-page "screening" questionnaire which asked for details of all pregnancies experienced by study participants, as well as periods of infertility and infertility treatment. This form was sent to the whole sample and included "opt-out" boxes to be ticked if the recipient had never been pregnant and had never attempted to have children, and/or was over age 55, and/or did not wish to take part. The second stage of the study consisted of a longer postal questionnaire which was sent to all those responding to Stage 1 who had ever been pregnant or who reported ever attempting to conceive and who agreed to be re-contacted. Excluded from this second stage were women who had had one or more termination for non-medical reasons (i.e. for reasons other than that a defect had been identified in the fetus or that continuing the pregnancy would put the mother at risk) and no other pregnancies. The Stage 2 questionnaire requested more general detail about the women ; detailed information on all pregnancies ; plus socio-demographic and behavioural details relating to the most recent pregnancy. These details included questions relating to weight at start of pregnancy, nausea, smoking, coffee and alcohol consumption, diet, vitamin intake, ill health, air travel, sexual intercourse, occupation and stress levels. The most recent pregnancy was selected to minimise biases related to recall, and since it could be at the start, middle or end of the reproductive careers of these women whose ages at survey ranged from 18 to 55 years potential biases relating to ending reproductive careers on a "success" were not expected to be large. For those whose most recent pregnancy had ended in miscarriage , brief information relating to clinical management of miscarriage and the advice given was also requested. Permission to access clinical notes relating to outcomes reported in the questionnaire, and to contact the women for further study if needed, was also requested. In order to increase the number of cases for the case-control analysis of risk factors for miscarriage, women who had had a miscarriage recently (since 1995) but whose last pregnancy was not a miscarriage were sent a third questionnaire. This was a shortened version of the Stage 2 questionnaire, containing only those questions relating to biological, socio-demographic and behavioural details of the most recent pregnancy, but now requesting these details in relation to the most recent miscarriage. Such women then had two pregnancies in case-control analyses and standard errors were computed using a robust method based on the "sandwich estimate" to account for this statistically.A free telephone helpline was run throughout the study, to answer queries and refer on to other organizations for professional help, if appropriate, and this was well used.all first registered births, if live. No formal statistical comparisons of maternal age were made, partly because the numbers were so large that slight, non-meaningful, nuances in the data would give a statististically significant result, and render the comparison meaningless, and partly because the average ages in the general population, though comparable, were expected to be similar but slightly older in the general population data owing to the fact that the data related to births within marriage only. Births where the date of birth or maternal age were not known were excluded from all comparisons with population data.All analyses in this paper were performed using Stata statistical software . To inveThe response to the first stage of the study is summarised in Table 12,695 women aged under 55 at survey had been pregnant at least once. These 12,695 women, whose average age at survey was 40.5 years, had started their reproductive careers from 1963 to 2002, 75% having their first pregnancy in 1980 or later  women ever attempting to have children (successfully or unsuccessfully) agreed to participate in the second stage of the study. Of these 596 (5%) were not sent a Stage 2 questionnaire, 212 because they had only ever had one or more termination of pregnancy for non-medical reasons, and 384 because their Stage 1 form arrived back after mailing had ended. A total of 10,828 women were thus sent a second stage questionnaire. The response to this second stage was high (73%), though 2% of women had decided that they no longer wished to participate  birth was remarkably similar to national data for both Stage 1 and Stage 2 data Table . ExactlyUsing a novel method, the National Women's Health Study has enabled a large UK population-based dataset to be assembled, comprising full reproductive histories, including any history of infertility, for 13,035 women, 12,695 of whom had conceived 30,661 pregnancies. We have obtained further detailed information for 7,702 of these women , including fertility diagnoses for both male and female partner (if appropriate), and lifestyle and behavioural risk factors for the most recent pregnancy. Seventy-five percent of these women consented to their medical notes being accessed in relation to information reported in the questionnaire, and 90% agreed to be contacted again, thus providing the means to carry out a population-based cohort study of these women at some time in the future.UK population-based data, collected at government level by England & Wales, Scotland and Northern Ireland, relate to registered births (live and still) and terminations of pregnancy, with Scotland also routinely collecting maternity data on hospital deliveries at any gestation. The National Women's Health Study goes one step further than this, providing the whole reproductive picture. Rather than being a pregnancy-based, cross-sectional survey, the data collected for each woman covers the complete spectrum of reproductive outcomes from infertility problems through miscarriage, ectopic pregnancies and terminations , to live and stillbirths, and does not rely on legal definitions for inclusion in the dataset. Furthermore, unlike most epidemiological studies of adverse reproductive outcome such as miscarriage, the data source is not clinical , but relates to women selected randomly from the UK electoral register. And for outcomes such as infertility no other data currently exist to enable estimation of how many pregnancies in the population as a whole result from fertility treatment.The study does rely on maternal recall and this could be a source of bias. Studies of self-reported reproductive history and exposures relating to reproductive events have, however, found maternal recall to have acceptably high reliability, and to be little affected by time from event -26.In terms of the key reproductive indicators of stillbirth, multiple delivery rates and maternal age at first birth, the data look remarkably similar to the general population. We therefore feel confident that response was unlikely to be related to adverse reproductive outcome. Indeed, the average age at survey of around 40 years, coupled with average ages at first birth which are exactly as would be expected from general population data, could be seen to indicate that non-responders to the survey tended to concentrate among younger women who had not yet tested their fertility. In addition, we feel confident that those responding to the more detailed Stage 2 questionnaire are an unbiased sample of those responding to Stage 1. Both Stage 1 and Stage 2 data can thus can be considered unbiased with respect to reproduction, and representative of patterns among all women in the UK population who have ever tried to have children, hence prevalence estimates might be taken as unbiased estimates of hard-to-measure outcomes such as miscarriage and pregnancies conceived through assisted reproduction techniques. Such data will be invaluable as population-based reference data for epidemiological studies of reproduction.In addition to both pregnancy-and woman-based population prevalence estimates, further papers to follow include reports of case-control analyses of behavioural and lifestyle risk factors for miscarriage.In summary, we have assembled a large population-based dataset of women's reproductive histories which appears representative of the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility.None declaredNM and PD initiated the research and participated in protocol design, data collection, analysis and writing the paper. SP participated in data collection and analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Echocardiography has the ability to noninvasively explore hemodynamic variables during pharmacologic or exercise stress test in patients with heart failure. In this review, we detail some important potential applications of stress echocardiography in patients with heart failure. In patients with coronary artery disease and chronic LV dysfunction, dobutamine stress echocardiography is able to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Exercise testing, in combination with echocardiographic monitoring, is a method of obtaining accurate information in the assessment of functional capacity and prognosis. Functional mitral regurgitation is a common finding in patients with dilated and ischaemic cardiomyopathy and stress echocardiography in the form of exercise or pharmacologic protocols can be useful to evaluate the behaviour of mitral regurgitation. It is clinical useful to search the presence of contractile reserve in non ischemic dilated cardiomyopathy such as to screen or monitor the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents. Moreover, in patients with suspected diastolic heart failure and normal systolic function, exercise echocardiography could be able to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Finally, in the aortic stenosis dobutamine echocardiography can distinguish severe from non-severe stenosis in patients with low transvalvular gradients and depressed left ventricular function. The identification of viable hibernating myocardium in patients with coronary artery disease and chronic left ventricular (LV) dysfunction is, up to today, the most common use of stress echocardiography in patients with heart failure. However, to search viable myocardium or the presence of contractile reserve is only one of plugs of the physiopathologic puzzle in a failing heart Figure  and 2. ITherefore, the present review will detail some important potential applications of stress echocardiography in patients with heart failure in the evaluation of the different clinical and physiopathologic aspects of heart failure syndrome.The most common cause of heart failure in the Western world is coronary artery disease, accounting for up to 60% of cases . In patiAfter brief episodes of coronary occlusion and reflow a reversible global LV dysfunction can occur. This phenomenon was called myocardial stunning . It is cHibernating myocardium" was first termed by Rahimtoola to indicate the state of reversible dysfunctional myocardium, which was considered to be the result of a state of persistently impaired myocardial function at rest, caused by reduced coronary blood flow, and which could be partially or completely restored to normal either by improving blood flow or reducing oxygen demand [The term "n demand .Echocardiography can detect viable myocardium during infusion of drugs which have ability to elicit an enhanced contractile response by recruiting contractile proteins. At least two drugs have these proprieties: the dobutamine, a synthetic β1 agonist with additional α1- and β2-stimulating properties and the enoximone that inhibits cyclic adenosine monophosphate-specific phosphosdiesterase ,7. RoutiAn initial evaluation of end diastolic wall thickness of akinetic segments with resting echocardiography can be used as an initial screening technique for assessment of viability. Indeed, akinetic regions with an end diastolic wall thickness <6 mm do not contain viable myocardium and do not improve in function after revascularization . HoweverDuring stress echocardiography is possible to observe four response patterns based on regional wall function: normal, ischemic, viable and necrotic. In the normal response, a segment is normokinetic at rest and normal or hyperkinetic during stress. In the ischemic response, a segment worsens its function during stress from normokinesis to dyssynergy. In the necrotic response, a segment akinesia remains akinetic during stress. In the viability response, a segment with resting dysfunction improves during stress. During pharmacologic stress, a viable response at low dose can be followed by ischemic response at high dose (biphasic response). This biphasic response is suggestive of viability and ischemia, with jeopardized myocardium fed by a critically stenosed coronary artery . A restiregional function after revascularization was 84% and 81% respectively [The overall sensitivity and specificity of dobutamine echocardiography for predicting recovery of ectively . In a stectively . On the Sensitivity of dobutamine echocardiography may be affected by several factors such as the severe reduction of myocardial blood flow that can preclude the contractile response, the premature interruption of dobutamine infusion, resting tachycardia that may renders the myocardium ischemic and dobutamine can only augment ischemia . On the global myocardial function after revascularization. At this purpose there is a relation between improvement in left ventricular ejection fraction (LVEF) and the number of segments with contractile reserve, indicating that extent of jeopardized but viable myocardium determine the magnitude of improvement of LV function after revascularization. Usually a level of ≥ 4 viable segments, which corresponds an improvement in wall motion score index >0.25 (about 20% of left ventricle), is advised as a cutoff value to predict improvement of LVEF [The main clinical issue to search the myocardial viability is that patients with evidence of hibernating myocardium who do not undergo revascularization have poor prognosis with high incidence of cardiac events . In cont of LVEF . Thus, p of LVEF   to calculate the regugitant volume and the effective regurgitant orifice area (EROA), the LV volumes to assess the myocardial contractility and the tricuspid regurgitant jet velocity to measure the sPAP that is an useful index of the haemodynamic burden of MR.First, in symptomatic patients with LV dysfunction and a clinical picture suspicious for new or worsening MR, but not evident at resting echo examination, exercise echocardiography can demonstrate a worsening of MR which helps to correlate this scenario with the patient's symptoms  unmask patients at high risk of poor outcome. The cardiac mortality rate of medically treated patients with dynamic MR during exercise is 39% at 20 months which represents excess mortality in patients in functional class II or III . While in the ischemic cardiomyopathy the search of myocardial viability is focused to find the presence of reversible segmental myocardial dysfunction and its possible effect on global systolic LV recovery after revascularization, in DCM the primary end point is to evaluate the presence of residual global contractile reserve. Both dobutamine and exercise testing have been used in the study patients with DCM, but there is a clear predominance for the use of dobutamine test. The doses of dobutamine utilized vary from investigators, but safety in its use in this population has been documented in doses as high as 40 μg/kg per minute. In the interpretation of results both wall motion score index and the LV volume to derive LVEF must be calculated.LV systolic function at the time of diagnosis has been proposed to be the strongest predictor of survival in DCM, but now the presence of contractile reserve recognised by dobutamine echocardiography seems to be the best marker of good prognosis in patients with severe LV dysfunction at rest ,38. PatiStress echocardiography may also help in the identification of patients in the initial phase of cardiomyopathy overt normal resting echocardiographic parameters. Both dobutamine and exercise have to be used to screen for the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents .The prevalence of diastolic heart failure in the community is now to be at least as high as that reported in previous studies of hospitalised patients; almost half of all patients with heart failure have diastolic heart failure . The terResting echocardiography is most useful in the assessment of LV size, LVEF and the use of Doppler-derived indices of diastolic function has impact on the identification of diastolic dysfunction. However, to determine whether an abnormality of diastolic function is the cause of the patient's symptoms, we need to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Therefore, the stress echocardiography, in particular exercise echocardiography could be useful in dyspnoeic patients with apparently normal LV function to unmask the presence of diastolic dysfunction (signs of elevated LV filling pressure) during exercise as cause of symptoms.Patients with diastolic heart failure, as well as those with diastolic dysfunction and little or no congestion, exhibit exercise intolerance for several reasons. First, an elevated LV diastolic and pulmonary venous pressure during exercise causes reduction in lung compliance, which increases work of breathing and evokes the symptom of dyspnoea . Second,All these aspects can be assessed during exercise echocardiography Table . In partStress echocardiography with dobutamine infusion is particularly useful in clinical decision making in patients with aortic stenosis with LV dysfunction and low transvalvular gradients. In this group of patients, an important clinical question rises: is the low gradient a consequence of low cardiac output due to a severe aortic stenosis which has led to LV dysfunction or is the low gradient a consequence of LV dysfunction is unrelated to aortic stenosis and the aortic stenosis is an incidental finding?It is well known that the transvalvular gradients are flow-depentent parameters so that they are influenced by LV function. The aortic valve area can be accurately determined by Doppler echocardiography with continuity equation and that correlate well with Gorlin formula . HoweverTherefore, the assessment of valve area does not solve the diagnostic dilemma in these patients, because we cannot distinguish between severe fixed from flow-dependent (relative) aortic stenosis. Thus, it is important to perform pharmacological manoeuvres to increase cardiac output so that valve area can be calculated at higher flow rate. Dobutamine stress echocardiography until 20 γ/kg/min with concomitant evaluation of cardiac output, aortic valve area and gradients, is a useful and reliable test to distinguish between severe fixed from relative aortic stenosis in presence of low gradient and LV dysfunction. On the basis on test results, it is possible to distinguish 3 groups of patients  Figure : 1. Pati2 rather than an increase of ≥ 0.3 cm2 from baseline alone [When interpreting the results of a dobutamine study in these patients to rule out or confirm definitively the presence of a severe fixed aortic stenosis, it is advisable to use an absolute cut-off value of the aortic valve area at peak of dobutamine >1 cmne alone ,50.Beyond the identification of viable hibernating myocardium, stress echocardiography is particular useful in patients with systolic and diastolic heart failure to assess the different physiopathologic component of heart failure syndrome and can aid to an appropriate clinical decision making.LV: left ventricularMR: mitral regurgitationLVEF: left ventricular ejection fractionDCM: dilated cardiomyopathyTAPSE: Tricuspid annular plane systolic excursionsPAP: Systolic pulmonary artery pressureThe manuscript is not under consideration elsewhere and the data presented have not been previously published. All authors have read and approved the manuscript. No financial support was received for this study. The content of this manuscript is not associated with any financial interest or other relations that could lead to a conflict of interest.Concerning the authorship, the listed authors have contributed as follows to the manuscript:EA and MP: 1) conception, design, analysis and interpretation of data, 2) drafting of the manuscript and 3) final approval of the manuscriptMO and AM: 1) critical revision of the manuscript for important intellectual content, and 3) final approval of the manuscript.