RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques.-20) in the unamplified group have a p-value below 10e-20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e-20 < p < 10e-10) in the unamplified group have a p-value below 10e-10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics.We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p < 10eWe conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used. Microarray technology offers a high throughput approach to transcriptional profiling on a genome wide scale. However, the relatively large amount of starting material required for standard hybridization has limited its full potential. In complex biological systems such as the nervous system, the utility of this approach is complicated by the fact that even in anatomically discrete regions, many divergent cell types are intermingled. It is often desirable to investigate gene expression profiles of distinct cell types and although laser microdissection provides a solution to the problem of tissue procurement, the small amount of RNA that can be harvested has precluded a straightforward combination of both technologies. This limitation is compounded by the need of replication essential for statistical analysis.Another scenario in which the lack of sufficient tissue availability has been challenging is the correlation of the phenotype in individual experimental animals with comprehensive gene expression profiles. For example, so far it has not been possible to correlate the inter-individual behavioural variability in animal models of chronic pain with the corresponding correlates in gene expression in the principal anatomical components of the pain pathway as such structures in individual animals do not yield sufficient amounts of RNA for standard hybridization protocols.To overcome these issues, increasingly sophisticated approaches to RNA amplification from small tissue samples have been developed for use with microarrays. These fall principally into two categories. One is based on PCR and is characterized by an exponential increase in copy number while the other is based on the T7-polymerase in-vitro transcription (IVT) to achieve a linear amplification of targets. For maximum fidelity, linearity of target amplification is desirable. Thus, substantial work has focused on exploring the T7 linear techniques -4, in paThese observations are useful indicators of protocol validity; though, the ultimate fidelity criterion is the ability to maintain differential expression between different tissues or under varying experimental conditions. Some previous studies have reported a 50% drop in significant changes in gene expression using RNA amplification , suggestIn this study, we critically appraise the suitability and merits of transcript amplification from small biological samples for microarray expression profiling using the Affymetrix small sample protocol II. We confirm previous findings on the reproducibility and fidelity of the protocol. We present compelling evidence for the 3' bias introduced by RNA amplification. We show how distortions in intensity may be reflected in the expression ratios from biologically different samples. Importantly, we explore the effect of distortions in expression ratios on their statistical significance.OneRA (one round amplification) protocol and the small sample protocol as the TwoRA (two rounds amplification) protocol. TwoRA and OneRA samples from three different biological groups were used in this study: the DRG  group, the SN  group and the SA  group. In each group, there are 4 replicates from the TwoRA and 3 replicates from the OneRA MSA as well as the mean residual sum of squares MSE were calculated. The median of the MSA was higher than the median of the MSE (given in parenthesis) in all biological groups: DRG 0.050 (0.023), SN 0.062 (0.016), SA 0.068.We used an ANOVA approach to confirm that the variability between protocol groups is greater than that among replicates within each group. In particular, a one-way, two-levels ANOVA analysis was performed for each gene separately, with 3 measurements from the OneRA (level 1) and 4 measurements from the TwoRA (level 2). First, the between group mean sum of squares To test whether protocol variability is significantly greater than the residual variability, we derived p-values from the F-values (MSA/MSE) for each gene (using the upper tail of an F-distribution with 1 and 3 + 4 – 2 degrees of freedom). In fact, the p-values were far from uniformly distributed. Storey suggests the following estimate of the proportion of hypotheses from the null using p-values: the fraction of p-values above the median p-value m, divided by (1-m) [38]. This results in the following estimate of the proportion of genes with significantly higher amplification variability: DRG 47%, SN 50%, SA 41%. That is, in all cases at least 40% of genes show differences between protocols, which are not explained by variability within replicates.log2IN) – obtained by subtracting the log2 intensity in the OneRA from that of the TwoRA – with the probesets 3' locations on corresponding targets (see methods for a description of how these locations were obtained) . Bias arises from greater 3' representation of targets during TwoRA while 5' regions are often lost. This causes amplified RNA to be shorter than normal. Previously published work explored the increase in the 3'/5' signal ratios only from control genes  to prove that such effect occurred),4,7. In In a separate but related analysis, probesets whose absolute Δlog2IN values were greater than 2 were reviewed for their 3' location distribution Figure . This isInterestingly, with both populations of deviant probesets Figure  &3b, theThe ultimate aim of microarrays is the identification of differential expression. Thus, a good amplification protocol should faithfully maintain expression ratios. To verify this, we cross-compared expression ratios from biologically distinct tissue samples treated with the OneRA and the TwoRA protocols.activating transcription factor 3 .RNA processing and hybridization followed the Affymetrix methodology. In brief, the Affymetrix standard protocol  in the . The AffScanned images were first inspected for quality control (QC) using a variety of built-in QC tools from the Bioconductor package  of R, thFor further analysis, probesets 3' locations were obtained by downloading the MOE430a probe tab files made available by the Affymetrix online support [39]. A probeset location was considered equal to the 3' distance of the probe that is most distal from the 3' end of the corresponding target within the set.To test for differential expression, two statistical methods were used. The bayesian adjusted t-statistics from the linear models for Micoarray data (limma) package ,15 and tID participated in experiment design, performed all numerical analysis and produced the manuscript. LW has had an essential input into the statistical analysis of the data and contributed in writing up the manuscript. CAO contributed advice on the analysis and critically reviewed the manuscript. MK designed and implemented the experimental work, participated in the analysis and the drafting of the manuscript. All authors read and approved the final manuscript.


Persistence is a key factor for long-term blood pressure control, which is of high prognostic importance for patients at increased cardiovascular risk. Here we present the results of a post-marketing survey including 4769 hypertensive patients treated with irbesartan in 886 general practices in Switzerland. The goal of this survey was to evaluate the tolerance and the blood pressure lowering effect of irbesartan as well as the factors affecting persistence in a large unselected population.Prospective observational survey conducted in general practices in all regions of Switzerland. Previously untreated and uncontrolled pre-treated patients were started with a daily dose of 150 mg irbesartan and followed up to 6 months.After an observation time slightly exceeding 4 months, the average reduction in systolic and diastolic blood pressure was 20  -19.6 to -20.7 mmHg) and 12 mmHg (95% CI -11.4 to -12.1 mmHg), respectively. At this time, 26% of patients had a blood pressure < 140/90 mmHg and 60% had a diastolic blood pressure < 90 mmHg. The drug was well tolerated with an incidence of adverse events  of 8.0%. In this survey more than 80% of patients were still on irbesartan at 4 month. The most important factors predictive of persistence were the tolerability profile and the ability to achieve a blood pressure target ≤ 140/90 mmHg before visit 2. Patients who switched from a fixed combination treatment tended to discontinue irbesartan more often whereas those who abandoned the previous treatment because of cough (a class side effect of ACE-Inhibitors) were more persistent with irbesartan.The results of this survey confirm that irbesartan is effective, well tolerated and well accepted by patients, as indicated by the good persistence. This post-marketing survey also emphasizes the importance of the tolerability profile and of achieving an early control of blood pressure as positive predictors of persistence. Angiotensin II receptor antagonists (AIIRA) have demonstrated excellent efficacy in patients with hypertension ,2, heartHowever, the main message of the published guidelines remains that a normalization of arterial blood pressure in hypertensive patients is the key objective to the prevention of cardiovascular events, especially in high-risk categories, where stricter therapeutic targets and aggressive strategies to reach them have been proposed. Yet, achieving normalization of blood pressure (BP) remains a difficult task, or, in the words of the JNC-7 report : "The moThe inadequate persistence with therapy, i.e. the frequent switch or discontinuation of the prescribed therapy has been recognized as a frequent cause of treatment failure . PersistThe aim of this open prospective observational survey with 4769 hypertensive patients treated in general practices in Switzerland, was to evaluate the persistence with irbesartan in real-life settings over a period of about one year and to investigate factors affecting either positively or negatively the persistence. In addition, the tolerability profile and the effect of irbesartan on BP control were assessed.This prospective observational survey was conducted in general practices in all regions of Switzerland from October 1997 to March 1999 i.e. shortly after the launch of irbesartan in the country. The general practitioners (GPs) were asked to document their daily routine in the treatment of hypertensive patients with irbesartan; 1390 physicians were contacted, 1045 included patients and 886 documented the treatment. They were recruited by the field forces of the sponsors of the study i.e. BMS and Sanofi-Synthelabo Switzerland. GPs filled in a baseline visit (Visit 1) form for every treated patient, and could report up to five follow up visits in the following 3–6 months (End Form). They were asked to document two arterial blood pressure measurements at every visit and to report concomitant antihypertensive medication and adverse events, as well as changes or discontinuation of treatment. At the end of the observation period, GPs reported if their patients were continuing the treatment with irbesartan, alone or in combination with other antihypertensive drugs. About 1 year after baseline, patients with ongoing treatment with irbesartan received a Compliance Form Patient, where they were asked how irbesartan was tolerated  and how many times weekly they took irbesartan in the preceding 3–4 weeks; a further question was how many tablets  were taken daily. Their GPs received a Compliance Form MD, to report the last blood pressure measurement and if and when the patient had discontinued the treatment. The various forms were designed by external consultants. The data were collected by mail using self-addressed envelopes and processed by the consultants. Queries for incomplete forms were done by the sponsor representatives. No standard validated questionnaire was used. In Switzerland, this type of survey does not need to be submitted to an ethical committee.All patients with newly diagnosed hypertension, or with treated hypertension requiring a change in medication according to the GP, were considered for the survey. No standardized definition was used but physicians considered a BP >140/90 mmHg as hypertension. There were neither demographic nor clinical exclusion criteria. The only condition to participate was that patients should not have been pre-treated with irbesartan. Treatment was started with irbesartan 150 qd. Thereafter, physicians were free to change the antihypertensive therapy at any time during the follow-up based on their individual therapeutic goals .Values are presented as mean +/- sd. The statistical significance of between-group differences was computed using the 2-sided Chi-square test, the Mann-Whitney-U test or ANOVA methods as appropriate. For multivariate correlations, a logistic regression analysis with a dichotomous dependent variable was used . To support the results of the logistic regression models, Cox regression models with cumulative survival functions were further computed.As shown in Figure Table More that 90% of the Efficacy Sample patients received irbesartan 150 mg qd, as a monotherapy or in combination with other antihypertensives. 69% of the Efficacy Sample patients received a constant monotherapy and 5.6% a constant polytherapy. In the Efficacy Sample, the mean reduction of systolic blood pressure (SBP) from baseline to the last visit was 20.2 ± 19.5 mmHg (p < 0.001). For the diastolic blood pressure (DBP), the mean reduction was 11.7 ± 11.3 mmHg (p < 0.001). Figure Since GPs did not receive specific instructions about therapeutic goals in this survey, various therapeutic targets were used to evaluate the success in controlling blood pressure Figure . As showAdverse events were reported for 383 patients (8.0% of the AE-Sample), more often by older patients  and by pre-treated patients . Yet, in the majority of patients (90.7%), irbesartan was well tolerated according to GPs. Tolerance was reported as poor only for 131 patients (2.7%). Adverse events led to discontinuation of irbesartan in 343 cases (7.4%). The most frequent side effects are listed in Table 3829 patients out of 4639 continued the treatment with irbesartan after the last visit . The main reasons for discontinuation of the remaining 810 patients were the occurrence of adverse events and an insufficient efficacy  who returned the Compliance Form Patient reported an irbesartan intake of 6–7 times per week, i.e. more than 80% of the prescribed doses. All subgroups scored around 90%  entered the trial after a failed experience with other antihypertensive drugs. Irbesartan proved to be the drug inducing more persistence in a comparative study with newly-diagnosed patients ; therefoIn this survey, an attempt was made to obtain information on drug adherence using simplified questionnaires addressed to the patients and physicians. Unfortunately, only a small proportion of these questionnaires were filled by the participants. There is therefore a high probability of bias towards highly compliant patients. Moreover, questionnaires are known to overestimate drug adherence. Hence it is not surprising that good compliance with the dosing schedule was reported by more than 90% of the patients after about one year treatment, meaning that this fraction of the patients reported to have taken irbesartan at least 6 times a week in the preceding 3–4 weeks. Yet, the results of more than 1000 questionnaires confirm previous observations such as the lower compliance in men and the absence of effect of age. Indeed, Degoulet at al have also reported thatmale sex is a variable significantly associated with an increased dropout rate in hypertensive patients attending a hypertension clinic . Of inteIn conclusion, this survey confirms that irbesartan is a safe and effective antihypertensive drug in clinical practice. A high persistence with irbesartan was found in this program which is likely related to the consistent reductions in blood pressure and the good tolerability profile. This post-marketing survey has also tend to confirm in a large population of patients that achieving an early control of blood pressure may be a positive predictor of persistence. Based on this observation one could encourage physicians to start the antihypertensive therapy more aggressively using higher doses of well tolerated drugs such as angiotensin II receptor antagonists or fixed low-dose combinations in order to improve blood pressure control as well as long-term persistence.All authors had a consultant agreement with both Bristol Myers Squibb and Sanofi-Synthelabo. These companies supported the study financially and monitored the study.MB has written the paper, BH, PG and BW have contributed to the conception of the study, to the analysis of the data and to the redaction of the paper.The pre-publication history for this paper can be accessed here:


Utilizing a linkage resource for association analysis requires consideration both of the marker data used and correlations among relatives in pedigrees. We previously developed a method for association testing in pedigrees. We applied our method to 50 replicates of microsatellite data surrounding five genes involved in high-density lipoprotein (HDL) in the Genetic Analysis Workshop 13 (GAW13) simulated data and examined association with HDL as well as linkage disequilibrium (LD) between markers.Although no association was intentionally simulated, we found significant evidence of weak LD between microsatellite markers (flanking/~5 cM from the genes), in some but not all replicates. This level of LD compared well to that observed in the real GAW13 Framingham data. Only one region had sufficient replicates to assess power, and this was low (12.5–20.8%). More power was attained using all individuals and accounting for relationships, compared with one independent individual/pedigree, although this was not significant due to small sample sizes. Not accounting for relatedness inflated statistical significance (p < 0.0001).A correction for dependence is necessary in association studies to avoid an inflation of significance probabilities. Our results further illustrate that use of microsatellite marker data is not an effective approach for association testing. Association and linkage disequilibrium analyses are tools that are often used to fine-map and refine promising linkage findings. These tools are particularly effective because they increase power to detect genes with small effects . Many peThere is great potential for the utilization of pedigree resources for association analyses. Our group and others have developed methodologies to enable association analyses to be performed in extended pedigrees ,3. If muFor each simulation, our program uses a gene-drop to generate null genotype configurations for a given set of individuals in pedigrees. The gene-drop requires the assignment of genotypes to the pedigree founders, based on allele frequencies estimated from the study population. This is followed by the use of Mendelian inheritance probabilities to determine the genotypes of all descendants. The resulting genotype configuration on each pedigree represents a possible configuration under the null hypothesis of no association between allele and disease. Using the simulated genotypes for individuals for whom real data are available and the true phenotype data, the statistic of interest is calculated . The resulting statistic is from the null distribution because it was derived from data under the null hypothesis of no association. This procedure is repeated many times, creating an empirical null distribution. The real genotype data for the same individuals and real phenotype data are then used to calculate the observed statistic. This observed statistic is then compared with the empirical distribution to determine significance. The method can be applied to pedigrees of any structure and size and to any statistic of interest.Association analyses are based on either testing the true disease-causing variant itself, or alleles at a marker in linkage disequilibrium with the true variant. Since linkage disequilibrium (LD) generally extends over very short distances  and sinThe Genetic Analysis Workshop 13 (GAW13) simulated data were modeled on the real GAW13 Framingham Heart Study data, as a genomic search with average resolution of approximately 9.5 cM, and hence no LD, or association, was intentionally simulated. To investigate the value, if any, of using linkage microsatellite marker data for association studies, we chose to analyze the simulated high-density lipoprotein (HDL) phenotype (dichotomized to the two extreme quartiles: high versus low quartile) and microsatellite markers either flanking or within approximately 5 cM of the true location of five genes involved in determination of baseline simulated HDL, as indicated in the 'answers'.Here we report the results of our empirical simulation method using a chi-squared association analysis for genotype data in the GAW13 simulated data. Although no association was intentionally included in the GAW13 simulated data, we report the underlying linkage disequilibrium (LD) between microsatellite markers and the ability of our empirical association method to detect power and false-positive signals. As a comparison we also report the LD in the Framingham data for the same location as one of the simulated baseline HDL genes.The first 50 replicates of the simulated GAW13 data with missing genotype and phenotype values were utilized in this study. There were 330 families, ranging in size from 7 to 84 members, in each of the 50 replicates studied.Eleven baseline genes  contribute to the HDL phenotype. We chose to study five of these genes , selected to represent a spectrum of percent contribution to the HDL trait , BMI (using mean height and weight across the study period), smoking (ever/never), alcohol (ever/never), and fasting glucose (ever/never > 126 mg/dl).GAW13_HDL(PED)). Second, we used our own linear regression equation to adjust HDL (LR_HDL) using the above listed covariates, where for each replicate we performed a linear regression with these covariates as independent variables to determine the regression coefficients. Third, we performed an 'independent' (IND) analysis by selecting only the first individual from each family with data and also using the GAW13 'answers' (GAW13_HDL(IND)).We considered the analysis of the HDL data in three different ways. First, we adjusted the maximum HDL value using the generating equation as provided in the 'answers'  and for the haplotype frequencies under H0 (no association) were used to calculate the raw disequilibrium, from which D', the proportion of maximum possible disequilibrium, was determined. Significance was determined by the EH program.The estimating haplotype (EH) program  was usedTo compare results of the simulated data to the GAW13 Framingham data, D' for the three most common alleles at the first four markers on chromosome 4, corresponding to the exact position of the four markers surrounding gene b22 in the simulated data set, were analyzed.It should be noted that the simulated data were generated to contain linkage to baseline HDL genes, but not LD or association with particular gene variants in the candidate genes. However, linkage and association differ only in the fact that the former is a phenomenon of loci and the latter of alleles at loci. In replicates where LD exists across markers, the simulated linkage creates a scenario equivalent to allelic heterogeneity for association. While certainly not ideal, allelic heterogeneity is a reasonable model for real data in which multiple common variants within a gene may increase disease susceptibility.GAW13_HDL(PED) with an analysis where we ignored genealogy and included all individuals (ALL) with data in the pedigrees without any correction for relatedness. A paired t-test for the average of the -ln(p) across markers over the 50 replicates was used to compare the results.In all analyses we compared the highest and lowest quartiles of the HDL phenotype of interest. We used the chi-squared statistic for independence considering both dominant and recessive models as our statistic of interest. For each analysis 10,000 simulations were generated to create the empirical null distribution. To illustrate the necessity to correct for relatedness, we compared the results from p < 0.01) for each of the three analysis types, indicating a power estimate for each. Two regions (surrounding b14 and b18) had sufficient replicates without LD (p > 0.5) to assess false-positive findings. For these regions we show the proportion of replicates without LD for which significant associations were found (p < 0.01).We report the number of replicates showing LD at various levels of significance for each gene region. Only one region surrounding gene b22 had sufficient replicates with significant LD to assess power. For this region we also show the percentage of the replicates with significant LD for which a significant association was found (LR_HDL) and classic chi-square analyses (for GAW13_HDL(IND)) were performed using STATA 6.0 . Fisher's exact p-values are reported for the GAW13_HDL(IND) analyses, where appropriate.All regression analyses (for IND) analysis the average sample size per replicate with data was 328.7 individuals and ranged between 324 and 330 individuals. For all other analyses using all individuals with available data, the average sample size per replicate was 1672.6 individuals and ranged between 1627 and 1715. More than 95% of families within the 50 replicates had two or more members per family with data.For the 'independent' (GAW13_HDL(PED) with the uncorrected GAW13_HDL(ALL) showed extreme statistical significance , suggesting that not accounting for correlations among family members substantially underestimates the variance and inflates the significance, as expected.A comparison of the empirical method using p < 0.05) was present in at least one replicate , values of D' ranging from 9.7 to 47.6 were observed. These values compared well to those found for the simulated data (D' = 9.4–31.8).p < 0.01) was found using the various analyses. Power was low for all comparisons and ranged from 12.5–20.8%. Power was higher for analyses that corrected for covariates and that used pedigree data , rather than 'independent' data , although these differences are not statistically significant due to a small sample size (n = 24). However, 20.8 % is significantly different from 0.05 (type I error rate) as assessed by binomial distribution theory with n = 24 (p = 0.006).As the region containing b22 was the only one with significant LD present in sufficient replicates (24/50), we chose to further study power of the association analyses for only this region. Table p > 0.5). Two regions surrounding genes b14 and b18 had sufficient replicates  to assess false-positive findings. Results are shown in Table The number of false-positive results was assessed in gene regions for which sufficient replicates were available and for which LD was not evident . In fact, the frequency of detectable LD in replicates correlated reasonably well with available marker density  of the regions did not exhibit a high frequency of LD over the 50 replicates. Furthermore, as pointed out above, even for the one region with significant LD, power was low. These results could be extrapolated to association analyses in the real GAW13 Framingham data where LD was comparable and where small effect genes are expected. This indicates caution in the interpretation of any positive association findings in those data.Microsatellite markers are not an ideal marker type for association testing. We selected the nearest two to four markers for each gene (each either flanking or within ~5 cM of the gene of interest). However, 5 cM represents a large distance between markers and true gene variants for association testing. Testing single-nucleotide polymorphism (SNP) variants within the gene, which are more likely to be in strong LD with disease-causing variants, would have been a more powerful situation.A limitation of our analysis is that not all alleles were analyzed. Only the three highest frequency alleles for each marker were used to both increase the chance of an allele being observed sufficiently for association testing and to reduce multiple testing problems. This modified allele system is not biased for investigating LD, since alleles were not grouped according to their respective disequilibria. Further, we used the same allele definition for LD estimation and association, thus they are directly relevant to one another. We may, however, have missed positive association findings by not testing all alleles. A further limitation is that these data were not simulated to specifically contain association, and our power will be limited by the situation equivalent to allelic heterogeneity, which has been shown to reduce power in association-based analyses . In addiIn conclusion, our results indicate that the empirical approach makes efficient use of data collected from extended pedigrees. Further, this study illustrates the lack of power available by using linkage microsatellite markers, and strongly cautions against doing so for association testing.


Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model.The Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant.Cystitis was generated in uvrY mutant in the cystitis model.This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the  The rapid adjustment of bacteria to new conditions largely relies on two-component systems (TCS). In the prototypic model an environmental stimulus interacts with the N-terminus of a sensor protein, leading to autophosphorylation of a specific histidine residue using ATP. The sensor then acts as a kinase and transmits the phosphate group to a conserved aspartate residue on a cognate response regulator protein. This normally enables the regulator to control the transcription of a certain set of genes by sequence specific DNA binding.Escherichia coli genome has identified more than 30 TCS [Escherichia coli sensor proteins, ArcB, EvgS, TorS and BarA, have two extra domains, D1 and H2, besides the H1 transmitter domain and are thus denoted as 'tripartite' [Escherichia coli tripartite sensor BarA  forms a TCS with the UvrY response regulator protein [csrB and csrC RNAs [barA or uvrY genes in Escherichia coli, as a consequence, has drastic effects on the ability of the bacteria to successfully grow in a competition depending on the carbon source of the medium [Escherichia coli and Salmonella enterica also cause other phenotypic effects, including changes in motility, adhesion and biofilm formation [Sequencing of the n 30 TCS . Four Espartite' . The Esc protein ,8. UvrY srC RNAs -11, whicsrC RNAs . The CsrsrC RNAs . Deletioe medium . Mutatioormation ,15-19.Salmonella enterica, ExpS-ExpA in Erwinia carotovora, BarA-VarA in Vibrio cholerae and GacS-GacA in Pseudomonas spp, clearly diminishes virulence of the bacteria [Pseudomonas GacS-GacA system for infections in either animals, nematodes, insects or plants [csrB/csrC regulatory RNAs as well as the genes encoding the HilA and HilC transcriptional regulators in Salmonella [Deletions of orthologous systems in other bacteria; BarA-SirA in bacteria ,21. The r plants . The undr plants , but thelmonella ,23,24.Salmonella enterica [via the sensor or not. Considering the fact that the system is activated in bacterial monocultures, we earlier proposed that a stimulus reflects the energy/growth status of the cell [The nature of a physiological stimulus acting directly on the sensor is still elusive. Bile and intestinal short fatty acids have a clear effect on this two-component system in enterica ,26, alththe cell .Escherichia coli is the causative agent in more than 80% of the uncomplicated human urinary tract infections [Macaque fascularis monkeys express the digalactoside receptors to which the P fimbriated uropathogenic Escherichia coli strains bind and thus provide a suitable animal model system to study a cystitis [fections . Macaquecystitis . Here weDS17 wild type and the AKP168 uvrY mutant. Urine samples were collected at day 2, 5, 7, 13 and 15 to determine the colony forming units (CFU) and the relative number of wild type and mutant bacteria. Both monkeys exhibited typical symptoms of an acute cystitis as measured by bacteruria and an increased level of leukocyte markers in the urine . The growth varied significantly between the individual urines but there was no significant difference in growth between the mutant and the wild type in any of the three urines . Our premise that the initial composition of the individual urine determines the competition profile agrees well with this reproducibility. The observed fluctuations after day 1–2 are thus unlikely to be explained by random processes or by spontaneous mutations in either the AKP168 or the wild type strain creating a growth advantage in stationary phase, GASP mutants [uvrY mutants in the urine cultures was the result of additional weakening mutations, we demonstrated that mutants taken at day 7 from the different urine competitions could successfully outcompete the wild type strain in a competition in LB  complemented with LB. In these competitions where there is a constant excess of gluconeogenic carbon sources in the medium, the e Figure , similare Figure . Thus, tuvrY mutant has an impaired ability to efficiently metabolize the different nutrient components in the urine. This also emphasizes that an optimal growth rate is critical for bacteria during an infection. In analogy with our results, it is interesting to note that long-term growth of Pseudomonas strains in gluconeogenic media selects for strains with inactivating mutations in the orthologous GacS-GacA system, unlike when the same strains are tested in an infection model (Arabidopsis) [Although our model system cannot mimic the exact conditions in the bladder, such as the continuous addition of fresh urine, the data suggest that a idopsis) . It wouluvrY mutant observed in the in vivo cystitis model. Earlier studies have demonstrated effects of the BarA-UvrY TCS and the downstream Csr system on adhesion, aggregation and biofilm formation [We cannot exclude that other factors under the control of this TCS contributes to the lowered fitness of the ormation ,15-19, auvrY mutant in the cystitis model as urine consists of several different carbon sources and as the prevalent carbon sources will change over time. This also agrees well with the observed higher fitness of the uvrY mutant in competitions in purely gluconeogenic media as the uvrY mutant is predisposed for gluconeogenesis. In conclusion, the ability to grow quickly and benefit from the available carbon and energy sources is crucial for virulence. During infection there may be different carbon sources available and we believe that the ability to know when to metabolize them is in part conferred by the BarA-UvrY two-component system.This paper demonstrates that the BarA-UvrY system is a determinant for virulence in a monkey cystitis model. Based on different competition profiles we propose that the BarA-UvrY TCS is important for the bacteria to efficiently switch between different carbon sources present in urine. This would provide a plausible explanation to the reduced fitness of the Macaque fascularis monkeys were infected by inoculation of a bacterial suspension (1 ml of 108 bacteria/ml), containing equal amounts of DS17 wild type and the isogenic uvrY mutant AKP168, into the bladder using a catheter (the bladder void volume is approximately 150 ml). The monkeys had not been exposed to the DS17 strain earlier. Bacteria had been taken from fresh colony forming agar (CFA) plates  and resuspended in phosphate buffered saline (PBS). Inocula were directly analyzed on selective plates to assure that the level of mutant was close to 50 %. Samples from monkeys were collected by suprapubic bladder aspiration, using a syringe with vacutainer, at the time points indicated until two successive negative cultures were obtained  in 80 × 70 × 170 cm  cages, two in each, and were handled by professional personnel according to SMI regulations. Experiments were pre-approved by the local ethics committee in Stockholm.Two adult female Escherichia coli strain DS17 [AKP168 derivative [uvrY gene disrupted by a kanamycin resistance cassette. The AKP168 mutant exhibited no measurable difference in growth relative the parental DS17 strain in a range of different media, as measured by optical density and CFU [In this work we employed the uropathogenic ain DS17  and the rivative , with th and CFU .Growth was normally conducted in a 20 ml volume in 100 ml Erlenmeyer glass flasks at 37°C in a shaker with 200 rpm. Urine collected from healthy human male donors was centrifuged at 4000 rpm and sterile filtered through a 0.45 μM CA-filter . Urine-LB was prepared by mixing four parts of sterile filtered urine with one part of 5 × LB .In growth curve experiments each strain was grown as an over night culture in LB  started directly from a -80°C vial), washed twice in PBS, inoculated 1:100 in 20 ml urine. 1 ml samples were withdrawn and the optical density was measured at 600 nm at the indicated time points with a spectrophotometer.600 = 0.15–0.30. Subsequently, cultures were mixed 1:1 to a 20 ml volume. Samples of 100 μl were withdrawn at the indicated time intervals and subjected to appropriate dilution in PBS. Dilutions were added to Luria-Bertani plates, with or without kanamycin (10 μg/ml), together with 5 to 6 glass beads (6 mm diameter) . Plates were gently agitated for 20 seconds to evenly spread the bacteria over the plate, after which the beads were removed. The relative level of mutant was calculated by dividing the number of colonies growing on the selective plate with the number of colonies growing on the non-selective plate.In competition experiments each strain was grown as an over night culture in LB , washed twice in PBS, inoculated 1:100 in urine, urine-LB, or plain LB, and grown as an individual culture to AHT, AKP and KJ carried out the experimental work. RM provided expertise for the primate experiments. All authors have been involved in the design and planning of the experiments and the drafting of the manuscript. OM coordinated the study. All authors read and approved the final manuscript.


The World Health Organization estimates that malaria kills an African child every 30 seconds. Many of these children die because they develop severe anemia (a deficiency of red blood cells). As many as 5 million cases of severe malarial anemia occur in African children every year, and 13% of these cases are fatal. Turning the statistics around, more than half of young children in African countries where malaria is endemic (constantly present) are anemic. Nutritional deficiencies and various infections account for some of this disease burden, but malaria is one of the most important factors contributing to anemia.The malaria parasite destroys red blood cells  as part of its life cycle, releasing hemoglobin (Hb)—an iron-containing protein that carries oxygen around the body—into the circulation. Free Hb can cause oxidant stress, which is itself associated with anemia in malaria. An important modulator of such stress is a serum protein called haptoglobin (Hp), which captures Hb during hemolysis.1Hp allele make only Hp1-1, a homodimeric protein. People with two copies of the2Hp allele (the2/2Hp genotype) make Hp2-2, a large circular polymer, and those with one copy of each allele make the linear polymer Hp1-2 in addition to these two forms. The functional properties of the three Hp forms are somewhat different. In particular, Hp2-2 binds Hb much less tightly than the other forms. Sarah Atkinson and her colleagues reasoned, therefore, that the2/2Hp genotype might be a risk factor for anemia in children in malaria-endemic areas. To test their hypothesis, they measured Hb levels in Gambian children at the start and end of the malaria season, and now report in a new study that, as predicted, the2/2Hp genotype is associated with seasonal childhood anemia in this population.Hp exists in three molecular forms that are genetically determined by two variants  of a single gene. People who have two copies of theHp genotypes, assessed their blood Hb and serum Hp concentrations and iron status, and determined whether they were infected with malaria parasites. These variables were re-measured at the end of the malaria season. In addition, the researchers determined two other genetic polymorphisms that might influence Hb levels over the malaria season—anHb variant that causes sickle cell anemia(HbS), andglucose-6 phosphate dehydrogenase (G6PD) gene variants associated with hemolytic anemia.Most cases of malaria in The Gambia occur between September and December, so the researchers recruited 780 children aged two to six years from ten Gambian villages in July 2001, determined theirHp genotype. This univariate (single) analysis included 671 children—a few children were not included because of incomplete data. Baseline hb levels were not affected byHp genotype, but the average drop in hb was 8.9 g/l in the 17% of children with the2/2Hp genotype compared with only 5.1 g/l in children with the other genotypes. By contrast, the magnitude of the drop in Hb levels over the season was not affected byHbS orG6PD genotype—two other genetic traits that affect the red blood cells. Because multiple factors influence Hb concentrations , the researchers also did a multiple regression analysis of their data to test the effect of all such factors on Hb levels at the end of the malaria season. There were 565 children who had data complete enough for this more detailed analysis, and, once again, theHp genotype emerged as a risk factor for anemia, even after adjusting for other factors that affect Hb levels.Atkinson and her colleagues first analyzed their study population in terms of theirHp genotype and seasonal childhood anemia may reflect the reduced ability of the Hp2-2 polymer to scavenge free Hb and its bound iron after malaria-induced hemolysis. They also discuss why2Hp, a potentially detrimental allele, should be common in The Gambia, where malaria is endemic.2Hp arose from1Hp about 2 million years ago, and its subsequent spread across the world seems to have been driven by a strong genetic pressure, such as exposure to a life-threatening disease. The authors suggest that malaria may be one of the diseases that helped to select for the2Hp allele; it is possible that the2Hp allele may provide protection from life-threatening malaria, albeit at the expense of impaired hematological recovery from mild and asymptomatic malaria. In a related Perspective , Stephen Rogerson expands on the possible mechanisms of Hp-related anemia, and considers what the wider health implications of this study might be.Atkinson and her colleagues suggest that the association between


Sipa1 . The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) within the human SIPA1 gene are associated with metastasis and other disease characteristics in breast cancer.There is growing evidence that heritable genetic variation modulates metastatic efficiency. Our previous work using a mouse mammary tumor model has shown that metastatic efficiency is modulated by the GTPase-activating protein encoded by n = 300) consisted of randomly selected non-Hispanic Caucasian breast cancer patients identified from a larger population-based series. Genomic DNA was extracted from peripheral leukocytes. Three previously described SNPs within SIPA1 (one within the promoter [-313G>A] and two exonic [545C>T and 2760G>A]) were characterized using SNP-specific PCR.The study population , and the variant 545C>T was associated with estrogen receptor negative tumors (P = 0.0012) and with progesterone negative tumors (P = 0.0339). Associations were identified between haplotypes defined by the three SNPs and disease progression. Haplotype 3 defined by variants -313G>A and 2760G>A was associated with positive lymph node involvement (P = 0.0051), and haplotype 4 defined by variant 545C>T was associated with estrogen receptor and progesterone receptor negative status .The variant 2760G>A and the -313G>A allele were associated with lymph node involvement (SIPA1 germline polymorphisms are associated with aggressive disease behavior in the cohort examined. If these results hold true in other populations, then knowledge of SIPA1 SNP genotypes could potentially enhance current staging protocols.Our findings imply that  Breast cancer is a major public health concern among Western female populations. In 2004 breast cancer was the most common form of malignancy diagnosed in females in the USA and the second leading cause of cancer mortality in women . The greBRCA1 and BRCA2, with specific germline mutations being demonstrated to be associated with various indicators of poor outcome  or FAM [5-(&6)-carboxyfluorescein]) specific for wild-type and variant alleles of each of three SIPA1 SNPs, respectively. Sequences of PCR primers and fluorogenic probes are given in Table lsewhere . Each prSIPA1 SNP genotypes were determined by the fluorescence ratio of the nucleotide-specific fluorogenic probes.Reaction mixtures consisted of 300 nmol/l of each oligonucleotide primer, 100 nmol/l fluorogenic probes, 8 ng template DNA, and 2× TaqMan Universal PCR Master Mix (Applied Biosystems) in a total volume of 10 μl. The amplification reactions were performed in a MJ Research DNA Engine thermocycler  with two initial hold steps  and 40 cycles of a two-step PCR . The fluorescence intensity of each sample was measured post-PCR in an ABI Prism 7700 sequence detection system (Applied Biosystems), and t test to compare means for continuous variables and Wilcoxon's sum rank test to compare medians. Variables that were not normally distributed such as tumor size were log transformed. χ2 test or Fisher's exact tests were used to test for differences between categorical variables and testing for Hardy-Weinberg equilibrium. Unconditional logistic regression adjusting for multivariate covariates such as age at diagnosis was used to estimate the adjusted odds ratios (ORs). Lewontin's D prime (D') and correlation coefficient (r2) were calculated as two measures of linkage disequlibrium. Using the E-M algorithm  and cases patients regional and metastatic disease [n = 154]). The data in Table P = 0.0042). Those with localized disease had a smaller mean tumor size . In addition, the group with localized disease had a higher frequency of well differentiated tumors (P < 0.0001). No difference in the distribution of estrogen receptor (ER)-positive tumors and a marginal statistical significant difference in progesterone receptor (PR)-positive tumor status were observed between the two study groups. No statistically significant associations were observed between stage of the disease and other epidemiologic risk factors such as alcohol use, smoking, age at menarche, menopause status, parity, estrogen use, and family history of breast, ovarian, or prostate cancer among first-degree relatives.Descriptive data on breast cancer patients included in the study are summarized in Table The allele frequencies for the three SNPs in all breast cancer patients included in the study were 0.359 for SNP -313G>A, 0.556 for SNP 545C>T, and 0.144 for SNP 2760G>A. For none of the SNPs did the distribution deviate from Hardy-Weinberg equilibrium. All three SNPs were in pairwise linkage disequlibrium, as indicated by the parameter D', with D' values of 0.94 between rs931127 and rs3741378, 0.85 between rs931127 and rs746429, and 0.86 between rs3741378 and rs746429.P = 0.0139). Second, 545C>T was associated with ER-negative tumors, with 15.3% of those with CC genotype being ER negative as compared with 34.8% among those with CT or TT genotype (P = 0.0006); it was also associated with PR-negative tumors, with 27.8% of those with CC genotype being PR negative as compared with 42.0% among those with CT or TT genotype (P = 0.0350). This indicated that the presence of the T allele is associated with a number of indicators of a more aggressive disease process. Third, the frequency distribution of genotypes for the 2760G>A SNP revealed a significant association between the presence of the AA or GA genotype and advanced disease and the presence of positive lymph nodes at diagnosis. The combined frequency of homozygote variant AA and heterozygote GA genotypes was significantly higher in those individuals with nonlocalized, advanced disease than in individuals in whom the tumor remained confined to the breast . A similar pattern was observed with respect to lymph node involvement, in which the combined frequency of homozygote variants (AA) and heterozygotes (GA) was higher in those individuals with metastasis in one or more axillary lymph nodes than in those with no nodal involvement . These findings indicate that the variant A allele is associated with a more aggressive disease process.In a univariate analysis between the distribution of the genotypes and tumor characteristics, we observed the following. First, -313G>A SNP genotypes were significantly associated with the presence of positive lymph nodes at diagnosis; 33.3% of those with GG genotype had positive lymph nodes as compared with 52.7% among those with GA or AA genotype (P = 0.0083). This implies that an individual with the GA or AA genotype was 2.15 times more likely to have positive lymph nodes than was an individual with GG genotype. The multivariable OR of the -545C>T allele and ER and PR status were 2.90  and 1.86 , respectively. Similarly, for the -2760G>A SNP the multivariate OR of the presence of positive lymph nodes was 2.05 . This implies that an individual with the GA or AA genotype was 2.05 times more likely to have positive lymph nodes than was an individual with the GG genotype.The genotype frequencies for each of the three SNPs in the study population by tumor characteristics are presented in Table P = 0.0051) compared with haplotype 1. Thus, women with the estimated haplotype 3 were 2.20 times more likely to present with positive lymph nodes than were women with haplotype 1. In the analysis of ER status and haplotypes, the haplotype specific OR for haplotype 4 was 2.85  compared with haplotype 1. Thus, women with the estimated haplotype 4 were 2.85 times more likely to present with an ER-negative tumor than were women with haplotype 1. In the analysis of PR status and haplotypes, the haplotype-specific OR for haplotype 4 was 2.16  compared with haplotype 1. Thus, women with the estimated haplotype 4 were 2.16 times more likely to present with a PR-negative tumor than were women with haplotype 1.We investigated the association between haplotypes defined by these three SNPs and the presence of positive lymph nodes, and ER and PR status. There were a total of seven haplotypes that were imputed from the data on 260 patients with complete genotypes on all three SNPs. However, four of those haplotypes accounted for more than 98% of the observations. Table SIPA1 SNPs and indicators of aggressive breast cancer in this population. Specifically, we report a number of particularly interesting findings regarding nodal status at the time of diagnosis and tumor sex hormone receptor status; these findings suggest that SIPA1 germline variation contributes to metastatic potential in breast cancer. With regard to lymph node involvement, the variant -313G>A and 2760G>A alleles were strongly associated with axillary node involvement at the time of diagnosis on univariate analysis. Indeed, the apparent effect of these SNPs on the frequency of metastasis that was evident upon univariate analysis was complemented by the outcome of multivariate and haplotype frequency estimation. Multivariate analysis of genotype frequencies showed that possession of either of the -313G>A or 2760G>A variant alleles was associated with a significantly increased likelihood of nodal involvement. Similarly, haplotype analyses demonstrated that the haplotype consisting of the wild-type allele of 545C>T and the variant alleles of the two other studied SNPs . However, it has become increasingly accepted that hereditary factors influence all stages of tumor pathogenesis in apparently sporadic cancers. These heritable defects will be of low penetrance, but hypothetically they could affect any part of the neoplastic cascade, including modulation of metastatic efficiency. Evaluation of quantitative trait loci in inbred mouse models has proven a particularly powerful means of studying the effects of this type of genetic variation. The present study is an additional example of how mouse quantitative trait locus data, when used in an appropriate manner, can help to unravel the complexities of hereditary influences in sporadic human cancer [The importance of these data is heightened when one considers the outcome of our linkage and functional studies of pulation ,13,20, cn cancer .SIPA1 modulates metastatic efficiency is currently unclear, it is worthwhile to speculate on how observations of SIPA1 functionality in mice and humans may relate to the process of tumor progression and characterization. Specifically, tumor development in a background of germline-encoded SIPA1 dysfunction may enhance the ability of tumor cells to escape the primary lesion through alteration in cell morphology/polarity and/or by weakening the strength of intercellular contacts. This argument gains further potency when one considers the negative regulatory effect of SIPA1 on Rap1, and that Rap1 has been implicated in the maintenance of epithelial polarization [Although the specific mechanism by which rization  as well rization . FurtherSIPA1 has been shown to have GTPase activity that is specific for Rap1 and Rap2, both of which are members of the Ras family of GTPases, and are involved in regulating cell proliferation, differentiation, and adhesion [SIPA1 appears to play a prominent role in regulating cell adhesion, and transient expression of this gene in HeLa cells induces cellular rounding up and detachment from the culture surface [1-integrin, and thus regulates the interactions between these molecules and other cellular and extracellular matrix proteins involved in the process of adhesion [SIPA1 negatively regulates integrin-mediated cell adhesion via Rap1 GAP activity [SIPA1 interacts with the cytoskeletal-anchoring protein AF6 through a common protein-protein interaction motif known as a 'PDZ domain' [adhesion . Indeed, surface . It appeadhesion -28. It hactivity . Specifi domain' .SIPA1 has been shown to have potent effects on cellular proliferation. For example, SIPA1-deficient mice develop a spectrum of symptoms resembling those seen in human myelodysplastic and myeloproliferative disorders [SIPA1 are associated with enhanced metastatic efficiency in primary breast carcinoma.In addition to the effects on intercellular adhesion, disruption of isorders ,30. ThisSIPA1 germline variation in modulating tumor progression efficiency, the most obvious of which is to replicate the associations seen here in other populations. It is also vital to resequence various elements of the SIPA1 gene to attempt to identify novel polymorphisms, the most critical of which is that encoding the PDZ protein-protein interaction domain – a region that is critical to SIPA1 functionality [SIPA1 functionality. This hypothesis is somewhat supported when one considers that a haplotype containing the 2760G>A variant allele is associated with more aggressive disease at diagnosis, as measured by positive lymph node involvement. Conversely, it may well be that the 2760G>A SNP is in linkage disequilibrium with a nearby yet uncharacterized SNP with a more obvious functional effect. Indeed, one could postulate that the associations observed in the present study are in fact a consequence of another gene in linkage disequilibrium with SIPA1, because the functional significance (if any) of each of the polymorphisms has not been assessed. However, we argue that this is unlikely because we investigated genes in the chromosomal regions surrounding Sipa1 in the mouse and found no data supporting the hypothesis that surrounding genes are involved in modulating metastatic efficiency [Sipa1 functionality in the mouse and the fact that human haplotype blocks are probably significantly smaller than in inbred mice, leads to the conclusion that the observed associations are a result of SIPA1 polymorphism.However, further experiments will be required to confirm the role of ionality . This enficiency . This, iSIPA1 variation in isolation is probably too low to allow for the development of an assay that would possess sufficient sensitivity and specificity to determine metastatic propensity accurately. However, it is plausible that a panel of polymorphisms derived from a number of metastasis 'susceptibility' genes would possess the power and accuracy to enable the development of a clinically relevant prognostic assay, which will necessitate the identification of further metastasis efficiency modifier genes.The implications of this work are potentially far reaching in that it may well prove possible to assess metastatic potential through genotyping polymorphisms in germline DNA. The ability to evaluate the risk for metastatic disease from readily available tissues such as blood could be a powerful means to augment currently available means of assessing prognosis in breast cancer. However, we do acknowledge that the effect of germline SIPA1 germline polymorphisms are associated with aggressive disease behavior in the present cohort. If the results of this study hold true in other populations, then the potential for this to have a positive impact on current breast cancer staging and treatment protocols may be great. For example, staging of primary breast cancer in cases in which there is no palpable evidence of nodal metastasis typically requires axillary dissection to assess nodal status. However, controversy exists as to the actual benefit of nodal sampling in those patients with small tumors because of the morbidity frequently associated with this procedure. For example, the incidence of axillary node relapse in patients with stage T1a tumors (tumor >0.1 cm but not >0.5 cm in greatest dimension) treated without axillary dissection was 2% [SIPA1 polymorphisms could be used as an additional element of the staging process that might allow axillary node-negative patients with small primary tumors  to be classed as being at increased risk for development of metastasis. In turn, this could then become a powerful influence on whether to initiate adjuvant therapy in patients who would not currently be considered for this type of treatment.Our results imply that n was 2% . It is pCI = confidence interval; D' = Lewontin's D prime; ER = estrogen receptor; NCBI = National Center for Biotechnology Information; OR = odds ratio; PCR = polymerase chain reaction; PR = progesterone receptor; SNP = single nucleotide polymorphism.The authors declare that they have no competing interests.NC was responsible for genotyping polymorphisms, contributed to study design, and drafted the manuscript. The University of California Irvine investigators played several roles in this report. HA-C contributed to the study design, the recruitment of subjects and the interviewing and biospecimen collection, and drafting and editing of the manuscript. AZ contributed to the study design, recruitment of subjects and data analysis, and drafting and editing of the manuscript. DP contributed to study design, biospecimen processing, and drafting and editing of the manuscript. JH participated in data processing and analysis, and editing of the manuscript. KH participated in study design, was responsible for supervising laboratory work, and was involved in manuscript preparation. All authors read and approved the final manuscript.


To tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.By using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival  and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.Validation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years . In multivariable analysis the classifier is an independent predictor for local recurrence.Our findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy. Breast-conserving therapy (BCT) is a well-established treatment modality for early (stages I and II) breast cancer. The treatment consists of complete surgical excision of the tumor followed by whole breast irradiation. In multiple randomized trials comparing BCT with mastectomy, their equality in overall survival has been shown -7. A larIdentifying patients at high risk for local recurrence in advance and individualizing treatment in these patients  or a primary mastectomy) is desirable. Several risk factors for local recurrence have been recognized -17. MargPrevious studies have shown the ability to predict distant-metastasis-free and overall survival in breast cancer with the use of microarray analysis -25. MechPrevious analyses of gene expressions patterns in a series of 295 early-stage breast cancer patients have identified gene expression signatures that powerfully predict the risk of distant metastasis and mortality ,23,26,27n = 161) were selected. BCT consisted of a wide local excision and axillary lymph node dissection followed by whole breast irradiation ; 144 patients received a boost to a median dose of 15 Gy, 98 patients received a low boost (14 to 18 Gy), and 46 patients received a higher boost (20 to 26 Gy). The boost technique was delivered using 192I (iridium) implantation (88 patients), electrons (31 patients) or photons (25 patients). Pathological margins were assessed as free of tumor in 134 patients, focal involvement by invasive carcinoma only in 3 patients, focal involvement by ductal carcinoma in situ (DCIS) only in 5 patients, and focal involvement by both DCIS and invasive carcinoma in 4 patients; in 8 patients there was more than focal involvement of the margins by DCIS, or the degree of margin involvement could not be determined with certainty. For four patients there was invasive carcinoma at the margin, but the extent of involvement could not be determined with certainty, and for three patients this was the case for both invasive carcinoma and a DCIS component. Of all 27 patients with any extent of involved margins, 2 did not receive a boost, 6 received a low boost and 19 a high boost of radiotherapy. Follow-up assessment was at least yearly, including mammography. Seventeen patients developed a local recurrence; the resection margins of the primary tumor had been free of any tumor for 15 of these 17 patients. One patient who later developed a local recurrence had focal involvement of the resection margins with invasive carcinoma, and one patient had focal involvement with DCIS. The 10-year local-recurrence-free percentage was 85% .This analysis is based on previously reported gene expression profiles of tumors from a series of 295 stage I and II breast cancer patients treated at the Netherlands Cancer Institute between 1984 and 1995 . All patOn the basis of the microarray identification number, patients were alternately assigned to the training set  or to the validation set. This was done separately for locally recurring and locally tumor-free patients, thereby creating almost equal-sized sets with balancing for local recurrence.Patient characteristics for both the training and validation series are equally divided and are shown in Table RNA isolation, labeling of complementary RNA, and hybridization to 25,000-element oliogonucleotide microarrays, and measurement of expression ratios were performed as described previously .The microarray data for analysis of the 70-gene prognosis profile were obtained with the use of the same 25,000-element oligonucleotide array; the correlation coefficient of the expression of these 70 genes with the good prognosis profile was calculated as reported previously . The twoThe entire expression database for all 295 patients is publicly available online , as are The first method we used for predicting local recurrence is PAM : class prediction as described by Tibshirani and colleagues . This meSubramanian and colleagues  have desThe following previously established gene expression profiles were used to find a classifier for local recurrence after BCT:1. The wound-response gene expression signature is a characteristic pattern of expression of a set of genes that have been identified as being induced or repressed in response to bovine serum, a surrogate wound environment ,34. This2. The 70-gene prognosis profile was originally trained to predict metastasis within 5 years ('poor prognosis' group), or no metastasis within 5 years ('good prognosis' group) .in vitro experiment in which epithelial cells are exposed to hypoxia [3. The hypoxia profile is a gene expression classifier from an  hypoxia .First, these gene expression profiles were used in their original form and tested for their value in predicting local recurrence after BCT in the complete set of 161 patients.Second, we optimized these profiles by using the clinical data on local recurrence.Using the training data set, we calculated for each of the three profiles the average pattern of expression of the genes that comprised each profile, for patients who developed a local recurrence . For all patients in the training and validation set we then calculated the non-centralized Pearson correlation to each of the three average patterns of expression . A correlation value between -1 and +1 is thereby derived for all patients and indicates the similarity in gene expression between an individual patient (tumor) and the average gene expression of tumors from patients in the training set who developed a local recurrence.For each of the three signatures a plot of these correlation values , SPSS 13.0®  and Microsoft Excel® (Microsoft Corporation) were used. The PAM analysis was performed with version 1.2 (November 2003) [All patients who developed a local recurrence were considered; 16 patients developed a local recurrence as a first event and in 1 patient local recurrence occurred concurrently with distant metastases. Local-recurrence-free percentages were calculated with the Kaplan–Meier method (log-rank test for comparison). All Kaplan–Meier curves are available as supplementary data in Additional file 5. Comparison of categorical variables between groups was performed with a χer 2003) ,35.Additional material and methods are available described in Additional file 9.In a previously reported series of 295 patients with stage I and II breast cancer diagnosed at age less than 53 years, 161 had been treated with BCT . Of these 161 patients, 17 developed a local recurrence. From the series of 161 patients, a training and validation set were selected as described in the Materials and methods section, distributing local recurrences equally. This resulted in a balanced distribution of year of treatment and major risk factors for local recurrence between the two groups as well ; the median follow-up time for the patients in the entire training set was 7.9 years. Six out of nine local recurrence patients had received a boost .In the validation set, 8 out of 80 patients had developed a local recurrence at a median interval of 3.72 years (range 1.2 to 8.4 years); the median follow-up time for all patients in the validation set was 7.6 years. Seven out of these eight patients had received a boost .We used the PAM algorithm with the training data set to search for gene expression profiles predictive of local recurrence, starting with a gene list that consisted of the top 5,000 most variably expressed genes with technically well-measured expression, represented by microarrays . The performance of this approach, as evaluated by cross-validation, seemed to be poor: only 4 out of 9 local recurrences and 50 of the 72 non-local recurrences could be predicted accurately in the training set at this threshold (using a profile based on 684 genes). The use of fewer genes resulted in more accurate prediction for non-local-recurrence patients, but less accuracy for local-recurrence patients. In the validation series only 1 out of 8 local recurrences and 65 out of 72 non-local recurrences were predicted correctly .We used the GSEA  software to analyze whether or not gene sets defined on the basis of biological processes or pathways were significantly correlated with local recurrence. We first ran the analysis on the full probe set . We than ran the analysis on the same selection of genes used for the PAM analysis . Neither the full probe set nor the top 5,000 probe set revealed any gene set to be significantly upregulated or downregulated in samples from tumors that recurred locally, after correction for multiple testing.We have previously shown that three distinct gene expression signatures can each be used to subdivide breast carcinomas into two groups that are associated, respectively, with good and poor distant-metastasis-free and overall survival: a '70-gene' prognosis profile ,23, a woin vitro model for wound healing and can be used to classify tumors as having an 'activated wound-response signature' or a 'quiescent wound-response signature' [The wound-response signature was derived from an gnature' ,30. In tex vivo model in which epithelial cells are exposed to hypoxia [The hypoxia-response profile is a gene expression classifier derived from an  hypoxia . In thisp = 0.04; log-rank) of predicting local recurrence (13% versus 19% at 10 years for quiescent versus activated).Of these three signatures, only the wound-response signature showed some evidence  failed and the previously identified gene expression signatures could not convincingly predict the risk of a local recurrence, we used genes from the original profiles and constructed a new classifier based on the relationship of their expression patterns to clinical outcome observed in our study .Each of these three signatures was originally defined for purposes other than the prediction of local recurrence, but we hypothesized that the optimization of each signature's pattern threshold by training on local recurrence data might improve their performance for this new purpose.Applying the method described in the Materials and methods section, a correlation value of 0.2364 was calculated as a cutoff point for the 70-gene expression profile as a predictor of local recurrence, resulting in one misclassified patient with local recurrence. The recurrence risks at 10 years were 7% and 28% for the low-risk and high-risk groups, respectively.p = 0.34; log-rank). The sensitivity was 63% (5 out of 8) and the specificity 50% (36 out of 72). At 10 years the recurrence risk was 13% versus 14% for the low-risk group versus the high-risk group. Furthermore, more than half the patients were assigned by this classifier to the high-risk group (41/80).In the validation set the separation could not be reproduced (p = 0.4). By employing this threshold on the validation set, 46 patients (58%) were classified as 'high risk' and 34 (42%) as low risk. At 10 years the recurrence risk was 13% versus 15% for low-risk versus high-risk, respectively. The sensitivity and specificity were 75% (6 out of 8) and 44% (32 out of 72), respectively.For the hypoxia-response gene expression signature, the threshold that misses one patient with local recurrence seemed to be 0.254. In the training set the estimated 10-year recurrence risks for the low-risk and high-risk groups as determined by using this threshold were 10% and 19%, respectively. The performance of this signature on the validation set was not significant . The predictive performance of the wound-response local recurrence signature on the validation set, using the threshold derived from the training set, is shown in Figure The wound-response signature was first identified as a pattern of changes in gene expression induced by exposing fibroblasts to serum. A threshold value of 0.3179 for the correlation with the wound-response signature (as derived from the 380 available genes) resulted in one missed local recurrence in the training set. With the use of the threshold value for classification, 30 patients (37%) were classified as high-risk, and 51 (63%) as low-risk. In the training set, the 10-year recurrence risk was 6% for the 'low-risk' group and 35% for the 'high-risk' group. With this threshold, in the validation set 26 patients (33%) were classified as high-risk, and 54 (67%) as low-risk. The 10-year recurrence risk in the validation set was 5% for the low-risk group, in contrast with 29% for the high-risk group (p = 0.01). Further results of the multivariable analysis are shown in Table To assess the independent prognostic value of the supervised wound-response signature, it was tested in a Cox regression model that also included known clinico-pathological risk factors for local recurrence, using the independent validation data set. The factors that independently predicted a local recurrence in multivariable analysis in the EORTC 'boost versus no boost' trial were tested together with the local recurrence classifiers . In the We have shown that gene expression profiling using DNA-microarray analysis can be used in risk stratification for local recurrence after BCT in breast cancer patients. We tested several different methods: standard supervised analysis , profiles that have previously been used to predict distant metastasis and survival, and a combination of both approaches by supervising these previously established predictive profiles with clinical data on local recurrence.p value of 0.0008 associated with the supervised wound signature. It is important to note that the gene set derived from an in vitro model, the wound response, but not the wound-response gene expression signature itself, was adapted to derive a gene expression pattern predictive of a local recurrence in patients who were treated with BCT.The supervised wound-response signature derived by this approach is the only profile that could predict a local recurrence after BCT. This new method begins with a biologically defined gene set and constructs a classifier by a supervised learning procedure from the expression pattern of the selected gene set, using clinical data. This thereby uses not only the genes themselves but also the expression levels for the different genes in the signature that are correlated with the clinical end point . The power of this signature in predicting local recurrence was highly significant, even if we conservatively take into account the fact that we tried a total of seven methods, by applying a Bonferroni correction to the Gene expression profiling has been successfully applied to distinguish molecular subtypes in breast cancer and to predict metastases and overall survival -23,25. MBecause the number of patients and the number of local recurrence events was low in this study, these results must be interpreted with caution. Although the hazard ratio for a local recurrence in the validation series is high (15), the confidence interval is wide . Given the potential contribution of this prognostic signature to improve decision making in planning the treatment of breast cancer and an improved understanding of the pathogenesis of local recurrence, an independent test of its predictive value is a high priority. Recently, in The Netherlands, a randomized clinical trial started, to compare the standard radiotherapy dose (50 Gy whole breast and 16 Gy boost to the tumor bed) with an additional boost dose (26 Gy). In this trial, fresh frozen tumor samples will be collected. This trial will allow prospective validation of the signature.We have presented a method of integrating biologically derived gene expression profiles and clinical data to build a classifier for local recurrence after breast-conserving therapy. Although numbers of events are small and the resulting confidence intervals are wide, it is the first classifier based on microarray analysis to specifically predict a local recurrence after breast-conserving therapy. In our series the classifier is the most powerful predictor and is independent of clinical and pathological variables.in situ; GSEA = gene set enrichment analysis; PAM = prediction analysis for microarrays.BCT = breast-conserving therapy; DCIS = ductal carcinoma The authors declare that they have no competing interests.DSAN conceived the method of building the gene expression profile specifically for local recurrence and performed the primary analysis. DSAN, BK, AAMH, JTAC, JBC, LFW, HJP, HB, POB, HYC and MJvdV analyzed and interpreted data. DSAN, AAMH and MJvdV wrote the paper. All authors read and approved the final manuscript.An Excel file containing all clinical data, including all labels for various classifiers , and the correlation values  for all patients.Click here for fileAn Excel file containing the expression data for the 70 genes for all patients  and validation set separately) and the calculated 70 genes local recurrence centroid data and correlation values for all patients to this centroid.Click here for fileAn Excel file containing the expression data for the hypoxia genes for all patients  and validation set separately) and the calculated hypoxia local recurrence centroid data and correlation values for all patients to this centroid.Click here for fileAn Excel file containing the expression data for the wound signature genes for all patients  and validation set separately) and the calculated wound signature local recurrence centroid data and correlation values for all patients to this centroid.Click here for fileAn Excel file containing all Kaplan–Meier curves for the original classification and the supervised classifications.Click here for fileA text file containing the expression data for the 5,000 most variable expressed genes on the array for all patients in the training set.Click here for fileA text file containing the expression data for the 5,000 most variable expressed genes on the array for all patients in the validation set.Click here for fileAn Excel file containing the results from the PAM analysis.Click here for fileA Word file containing supplementary additional details of material and methods.Click here for file


Patients with low back pain are frequently encountered in primary care. Although a specific diagnosis cannot be made for most patients, it is likely that sub-groups exist within the larger entity of nonspecific low back pain. One sub-group that has been identified is patients who respond rapidly to spinal manipulation. The purpose of this study was to examine the association between two factors (duration and distribution of symptoms) and prognosis following a spinal manipulation intervention.Data were taken from two previously published studies. Patients with low back pain underwent a standardized examination, including assessment of duration of the current symptoms in days, and the distal-most distribution of symptoms. Based on prior research, patients with symptoms of <16 days duration and no symptoms distal to the knee were considered to have a good prognosis following manipulation. All patients underwent up to two sessions of spinal manipulation treatment and a range of motion exercise. Oswestry disability scores were recorded before and after treatment. If ≥ 50% improvement on the Oswestry was achieved, the intervention was considered a success. Sensitivity, specificity, and positive likelihood ratio were calculated for the association of the two criteria with the outcome of the treatment.141 patients  years) participated. Mean pre- and post-treatment Oswestry scores were 41.9 (± 10.9) and 24.1 (± 14.2) respectively. Sixty-three subjects (45%) had successful treatment outcomes. The sensitivity of the two criteria was 0.56 , specificity was 0.92 , and the positive likelihood ratio was 7.2 .The results of this study demonstrate that two factors; symptom duration of less than 16 days, and no symptoms extending distal to the knee, were associated with a good outcome with spinal manipulation. Low back pain (LBP) is a common and costly condition in primary care practices. ManaginMany experts agree that sub-groups exist within the large category of patients diagnosed with "non-specific" LBP,5. The dOne proposed sub-group among patients with otherwise non-specific LBP consists of those who are likely to respond to spinal manipulation There isFlynn et al examinedThe results of these studies support the validity of these five criteria for identifying patients with LBP likely to benefit both immediately and in the long-term, from spinal manipulation. The pragmatism of applying these criteria for routine use in primary care, however, may be compromised by the requirements that the patient complete a questionnaire (the Fear Avoidance Beliefs Questionnaire), and thSubjects for this analysis were 141 patients with LBP who were participants in one of two previous studies. Seventy-one subjects were participants in the study used to develop the criteria for predicting success with manipulation, and 70 sSubjects were between 18–60 years of age with a primary complaint of LBP with or without referral into the lower extremity, and an Oswestry disability score of at least 30%. Patients with "red flags" for a serious spinal condition , signs consistent with nerve root compression , current pregnancy, or prior surgery to the lumbar spine or buttock were excluded.All subjects completed a series of self-report questionnaires and underwent a standardized physical examination prior to treatment. Subjects were asked to identify the date of onset of their LBP. Subjects also completed a body diagram to indicate the anatomical distribution of symptoms, a numeriAfter completing the baseline examination, all subjects received the same manipulation intervention protocol. Subjects were sent to physical therapy and received 1–2 treatment sessions within one week before re-examination. Subjects received the manipulation intervention at each treatment session. The same manipulation technique was used for all subjects. The subject was supine. The physical therapist positioned the subject into side-bending, and then rotated the subject in the opposite direction. A quick thrust to the pelvis was delivered by the therapist in a posterior and inferior direction  were categorized as likely to have a good prognosis following the manipulation intervention. Subjects with 1 or 0 criteria present were categorized as likely to have a poor prognosis.All subjects were categorized on two criteria from the baseline examination. The body diagram was used to determine if the subject's symptoms extended distal to the knee or not. The categorization of symptom distribution using body diagrams has been found to be highly reliable. The subWe first examined the relationship between the two criteria rule and the original five-criteria rule for predicting success. Subjects with at least 4 out of 5 of the original criteria present were categorized as having a good prognosis with manipulation, while those with three or fewer were categorized as having a poor prognosis. Using the five-criteria rule as the reference standard, we calculated the accuracy of the two criteria as the percentage of times the two-criteria categorization agreed with the five-criteria categorization. We also calculated the sensitivity, specificity, and likelihood ratios with associated 95% confidence interval (CI).OSW - Final OSW) /Initial OSW* 100%. If the percent improvement was ≥ 50%, the intervention was considered a success.The outcome of the manipulation intervention was determined from the change on the OSW occurring between the baseline and post-treatment examinations. The percent improvement on the OSW was calculated as . Specificity was calculated as the percentage of subjects with a non-successful outcome who were categorized with a poor prognosis based on the presence of only 1 or 0 criterion . The positive likelihood ratio was calculated as (sensitivity / (1-specificity)) and represents the increase in odds favoring a successful outcome when both criteria are present.The baseline characteristics of the 141 subjects are listed in table The mean baseline and one-week OSW scores were 41.9 (± 10.9) and 24.1 (± 14.2) respectively. The mean percent change on the OSW was 41.2% (± 33.9%). Sixty-three subjects 45%) experienced 50% or greater improvement on the OSW and were categorized as successful with the treatment (mean change 73.8% (± 15.4%)), and 55% were categorized as unsuccessful (mean change 14.8% (± 17.9%)). Subjects with both criteria present experienced significantly greater change on the OSW, and were more likely to be categorized as a successful manipulation outcome than subjects with one or zero criteria present  can be easily assessed in a primary care setting. The two pragmatic criteria also showed a high level of accuracy in relation to judgments based on the original five criteria . Judgments from the two criteria showed a strong predictive relationship with the clinical outcome. Although the two criteria rule did sacrifice some accuracy related to the original five criteria rule, it appears that sufficient accuracy is maintained with a substantial increase in ease of use. The original five criteria rule has been validated in a randomized clinical trial. This twCurrent clinical practice guidelines and expert recommendations generally support a "stepped care" approach for management of patients with LBP in whichIn this study, 29% of subjects fit the two criteria and were designated as having a good prognosis with manipulation, suggesting this sub-group of patients may not be inconsequential. The high specificity (0.92) and positive likelihood ratio (7.2) indicate that patients with both criteria present should be referred for a manipulation intervention based on the high likelihood of rapid success. The sensitivity (0.56) and negative likelihood ratio (0.48) associated with this two-criteria rule were only moderate, indicating a relatively high potential for false negative results . Given the safety of manipulation in the lumbar spine, this fiThe exclusion criteria used in this study should be taken into account when considering the clinical implications of the results. In particular it is important to note that patients with signs of nerve root compression, low levels of disability, or prior surgery to the low back were excluded. The likely response of these patients to a manipulation intervention cannot be determined from these results. This study used one manipulation technique. It cannot be ascertained if the same criteria would apply to a different technique, however the lack of specificity associated with manipulation techniques,35 suggeIndividuals with "non-specific" LBP are not a homogenous group, and different sub-groups of patients are likely to preferentially respond to different therapeutic management strategies. One sub-group consists of those patients with a good prognosis following spinal manipulation intervention. The results of this study demonstrate an association between two factors; symptom duration of less than 16 days, and no symptoms extending distal to the knee, and outcome of a manipulation intervention.The author(s) declare that they have no competing interests.JMF participated in the design and statistical analysis of this study. JDC participated in the design and performance of this study. TWF participated in the conceptual development and performance of this study.The pre-publication history for this paper can be accessed here:


The author discusses a new study reporting the birth weight of patients carrying a mutation in either of two closely related genes associated with maturity-onset diabetes of the young, testing the hypothesis that the primary defect caused by these genes results in decreased insulin secretion. Type 2 diabetes is a very common metabolic disease and its incidence is increasing rapidly worldwide . DespiteEpidemiologic studies have shown that genetic variation plays a very important part in determining the risk of developing diabetes. Currently available data suggest that for the vast majority of patients with T2DM, the genetic risk is determined by a variable combination of an unknown number of common genetic variants. Any of these variants alone is neither sufficient nor necessary for diabetes to occur. Genetic analysis of T2DM has yielded only a few such genetic variants that are consistently associated with risk of disease. The study of monogenic disorders of glucose metabolism may shed light on potential mechanisms for the common form of T2DM.PLoS Medicine, Pearson et al. report the birth weight of patients carrying a mutation in either of two closely related genes associated with the MODY syndrome, HNF4A and HNF1A, testing the hypothesis that the primary defect caused by these genes results in decreased insulin secretion [To date, mutations in more than ten genes have been associated with different forms of monogenic diabetes. Maturity-onset diabetes of the young (MODY), an autosomal dominant disease with onset of diabetes typically before the age of 25 years, can be caused by mutations in more than six genes . NeonataBirth weight is largely determined by fetal insulin secretion. Defective insulin secretion, such as is found in patients with neonatal diabetes, is associated with low birth weight . In contHNF1A and HNF4A are associated with insulinopenic diabetes in young adults [HNF1A and HNF4A are thought to interact with each other, affecting each other's expression level in the beta-cell [HNF1A and HNF4A mutations are phenotypically similar. It was expected, therefore, that defects in either gene would affect fetal growth in the same way.Since both g adults ,11, it weta-cell . PatientHNF4A mutations were associated with increased fetal weight in patients, whereas HNF1A mutations did not appear to affect fetal weight at all. This increase in fetal growth found in HNF4A mutation carriers appears to be a direct result of the fetal genotype and is not significantly altered by maternal genotype. Similar weight gain was seen in fetuses carrying an HNF4A mutation, regardless of whether the mutation was inherited from the father or the mother. Furthermore, this mutation-associated increase in birth weight is also seen when genetically discordant siblings are compared, negating the importance of intrauterine environment in determining fetal weight in this setting.Surprisingly, Pearson et al. found that HNF4A mutations had transient neonatal hypoglycemia, and in three of these elevated insulin levels were documented. In contrast, none of the normal siblings or the patients carrying an HNF1A gene mutation had neonatal hypoglycemia. This unexpected finding was confirmed in a mouse model of beta-cell-specific HNF4A depletion, which resulted in intrauterine and postnatal hyperinsulinism, with hypoglycemia in the postnatal period.To determine if this increase in birth weight is indeed caused by fetal hyperinsulinism, Pearson et al. reviewed patient records, and determined that eight out of 54 of the patients with HNF4A mutations cause increased insulin secretion in the fetal and neonatal period, resulting in increased birth weight and neonatal hypoglycemia. Over time, the beta-cell function decreases, and insulinopenic diabetes develops, usually during the third decade of life.Thus, it appears that MODY-associated ABCC8), a major component of the beta-cell ATP-regulated potassium channel, result in a similar phenotype: hyperinsulinemic hypoglycemia with macrosomia in infancy, decreased glucose-stimulated insulin secretion in adolescence and young adulthood, and frank diabetes during middle age [HNF4A mutations recapitulate this series of events by modulating the expression of either of the two genes that make up the KATP channel (ABCC8 and KCNJ11), Pearson et al. determined the expression of these genes in islets isolated from their mutant mice, but found no evidence of abnormal expression levels.This is not the first example of fetal hyperinsulinism developing into insulinopenic diabetes later in life. Huopio et al. report that dominant inactivating mutations in the sulfonylurea receptor  promoter of HNF4A have been associated with polygenic T2DM. Could the scenario described by Pearson et al. be a common pathogenic mechanism for T2DM? Future studies are likely to address these important questions.Genetic data that have accumulated over the last ten years suggest that T2DM is a genetically heterogeneous disease in which either or both of these two scenarios could be important in any given individual. The current study demonstrates that a third scenario, in which a primary beta-cell defect results in increased insulin secretion, and thus increased fetal growth, long-term beta-cell damage, and late-onset insulin deficiency, may also be important in the pathogenesis of diabetes in some patients. It is not known how the same