The Angiotensin Converting Enzyme (ACE) insertion/deletion (I/D) polymorphism has received much attention in pharmacogenetic research because observed variations in response to ACE inhibitors might be associated with this polymorphism. Pharmacogenetic testing raises the hope to individualise ACE inhibitor therapy in order to optimise its effectiveness and to reduce adverse effects for genetically different subgroups. However, the extent of its effect modification in patients treated with ACE inhibitors remains inconclusive. Therefore our objective is to quantify the effect modification of the insertion/deletion polymorphism of the angiotensin converting enzyme gene on any surrogate and clinically relevant parameters in patients with cardiovascular diseases, diabetes, renal transplantation and/or renal failure.Systematic Review. We will perform literature searches in six electronic databases to identify randomised controlled trials comparing the effectiveness and occurrence of adverse events of ACE inhibitor therapy against placebo or any active treatment stratified by the I/D gene polymorphism. In addition, authors of trials, experts in pharmacogenetics and pharmaceutical companies will be contacted for further published or unpublished data. Hand searching will be accomplished by reviewing the reference lists of all included studies. The methodological quality of included papers will be assessed. Data analyses will be performed in clinically and methodologically cogent subgroups. The results of the quantitative assessment will be pooled statistically where appropriate to produce an estimate of the differences in the effect of ACE inhibitors observed between the three ACE genotypes.This protocol describes a strategy to quantify the effect modification of the ACE polymorphism on ACE inhibitors in relevant clinical domains using meta-epidemiological research methods. The results may provide evidence for the usefulness of pharmacogenetic testing for individualised ACE inhibitor therapy. Research in genetics and genome sequencing has led to a better understanding of the molecular genetic mechanisms and to the detection of inter-individual genetic differences, so-called polymorphisms, which may have a functional consequence on the response to drugs. Pharmacogenetic tests provide information to better predict and prevent therapeutic failures and adverse drug reaction and raise the hope for an individualised pharmacotherapy -3. AlthoThe ACE polymorphism identified in 1990 by Rigat and co-workers  is one oTherefore, our objective is to systematically review all randomised controlled trials that assessed to what extent the insertion/deletion polymorphism of the angiotensin converting enzyme gene influences the effect and adverse events of angiotensin converting enzyme inhibitors on any surrogate and clinically relevant parameters in patients with cardiovascular disease, diabetes, renal transplantation and/or renal failure.We will perform literature searches in (Pre-) MEDLINE , EMBASE , Biosis , the Cochrane Controlled Trials Register  and the Science citation index. A preliminary literature search in Medline has been carried out to estimate the range of relevant literature. Out of the citations of the pilot searches (172 citations) we identified articles that met our inclusion criteria. Keywords of these articles were used to refine our search strategies. In collaboration with an information specialist we designed the final search strategies for the six databases avoiding any language restrictions Click here for file


Contrast-induced nephropathy is an important cause of acute renal failure. We assess the efficacy of acetylcysteine for prevention of contrast-induced nephropathy among patients undergoing intravascular angiography.We conducted a systematic review and meta-analysis of randomized controlled trials comparing prophylactic acetylcysteine plus hydration versus hydration alone in patients undergoing intravascular angiography. Studies were identified by searching MEDLINE, EMBASE, and CENTRAL databases. Our main outcome measures were the risk of contrast-induced nephropathy and the difference in serum creatinine between acetylcysteine and control groups at 48 h.higher incidence in six of the latter studies). The pooled odds ratio for contrast-induced nephropathy with acetylcysteine relative to control was 0.54  and the pooled estimate of difference in 48-h serum creatinine for acetylcysteine relative to control was -7.2 μmol/L . These pooled values need to be interpreted cautiously because of the heterogeneity across studies, and due to evidence of publication bias. Meta-regression suggested that the heterogeneity might be partially explained by whether the angiography was performed electively or as emergency.Fourteen studies involving 1261 patients were identified and included for analysis, and findings were heterogeneous across studies. Acetylcysteine was associated with a significantly reduced incidence of contrast-induced nephropathy in five studies, and no difference in the other nine (with a trend toward a These findings indicate that published studies of acetylcysteine for prevention of contrast-induced nephropathy yield inconsistent results. The efficacy of acetylcysteine will remain uncertain unless a large well-designed multi-center trial is performed. Contrast-induced nephropathy is a leading cause for acquired acute reductions in kidney function ,2. DespiThe pathophysiology of contrast-induced nephropathy remains incompletely understood. However, current evidence suggests that contrast media induce prolonged vasoconstriction and medullary ischemia coupled with generation of free radicals and oxidative injury to tubular cells -11.Acetylcysteine, a thiol-containing anti-oxidant, has been hypothesized to prevent contrast-induced nephropathy. The potential benefit of acetylcysteine is believed to be mediated by its properties as a scavenger of free-radical species and by increasing the synthesis of nitric oxide, a potent vasodilator, in response to ischemic or other toxic injury in the kidney . Given tWe identified published randomized controlled trials of acetylcysteine for prevention of contrast-induced nephropathy during intravascular angiography using both electronic and manual search strategies. We supplemented this by scanning the reference lists of all identified articles, reviewing selected conference proceedings, and by contacting experts in the field. All languages and types of publications were considered eligible. The comprehensive literature search was initially performed in April 2003 and updated in June 2004 to identify any potential new studies that may have appeared.MEDLINE , EMBASE  and CENTRAL  databases were searched via OVID using an approach recommended for systematic reviews of randomized trials . PubMed Two individuals (SMB and WAG) independently evaluated identified articles for eligibility on the basis of four inclusion criteria: 1) study design , 2) target population (patients undergoing intravascular angiography), 3) intervention  and 4) outcome .Two reviewers (SMB and WAG) independently extracted data from all primary studies fulfilling eligibility criteria. Any discrepancies in extracted data were resolved by consensus. Data extracted included identifying information, focus of the study, details of study protocol and demographic data. The primary outcome measures were the incidence of contrast-induced nephropathy and change in serum creatinine. The secondary outcome measure was requirement for renal replacement therapy. Authors of the studies were contacted for additional information when applicable.et al [Two reviewers (SMB and WAG) independently assessed methodological quality of individual studies. Any disagreements were resolved by consensus. Items used to assess study quality were methods of randomization, any blinding, use of a placebo, reporting of losses to follow-up or missing outcome assessments, and evidence of important baseline differences between the groups -18. An oet al .The presence of heterogeneity can compromise the interpretation and validity of meta-analyses and can result from significant differences in methodology, study populations, interventions, outcomes, or chance . A priorData from all of the selected randomized controlled trials were combined to estimate the pooled odds ratio (OR) with 95% confidence intervals (CIs) using a random-effects model as described by Der Simonian and Laird ,21. The A total of 66 unique citations were identified by our initial search strategy Figure . After tAll the randomized controlled trials were published in the years 2002 through 2004. Tables The definition of contrast-induced nephropathy was variable across studies. Four studies defined contrast-induced nephropathy as a > 44.2 μmol/L increase in serum creatinine from baseline ,29,32,37The reported incidence of contrast-induced nephropathy was variable across studies. Table Table Meta-regression was performed to assess a number of clinical and study quality factors that may have led to heterogeneity across studies. Interestingly, these analyses suggest that the heterogeneity may be partially explained by whether the angiography procedures were performed electively or as emergency, because studies where all enrolled patients were undergoing elective procedures had significantly lower odds ratios than did studies where emergency cases were included .Other meta-regression analyses demonstrated that the heterogeneity could not be accounted for by differences in patient age , baseline serum creatinine , volume of contrast media  or diabetes mellitus . Likewise, heterogeneity was not accounted for by differences in study quality including use of blinding , concealment of randomization , use of placebo  or overall Jadad score .There was some evidence to suggest possible publication bias according to Begg's test  and a trend with Egger's test . Figure Our meta-analysis of 14 peer-reviewed studies of patients undergoing intravascular angiography may lead some to conclude that the administration of acetylcysteine causes a reduced incidence of contrast-induced nephropathy. However, such a conclusion may be premature based on data published to date because our systematic review reveals considerable heterogeneity of findings across trials. Furthermore, our meta-analysis of post-treatment creatinine values does not reveal any truly meaningful difference in serum creatinine levels at 48 h between the acetylcysteine and control groups. Finally, insufficient data are available to allow inferences to be drawn about the efficacy of acetylcysteine on clinically meaningful endpoints such as dialysis, length of hospitalization or mortality.et al that recently received considerable attention, and that rather firmly concluded that acetylcysteine is beneficial [et al, and more directly address the relevance of this heterogeneity in the overall interpretation of study results. Two other meta-analyses have also recently been published, and similarly concluded that acetylcysteine is beneficial; however, these studies also failed to adequately address the issue of the considerable heterogeneity across studies [This meta-analysis has several features that distinguish it from a similar meta-analysis by Birck neficial . First, neficial . Fourth, studies ,42. Coll studies . Many wi studies ,45 make The presence of heterogeneity and/or publication bias can compromise the interpretation of meta-analyses and result in erroneous and potentially misleading conclusions ,43. A stdefinitely efficacious based on data published to date. Our firm conclusion based on this meta-analysis of published trails is that although the data seem quite promising, the efficacy of acetylcysteine has not been definitively proven.There are many parallels between the intravenous magnesium story and our meta-analysis findings for acetylcysteine. The marked heterogeneity of findings across studies, and the finding of funnel plot asymmetry (indicating possible publication bias), ought to be viewed as strong cautionary points against making firm conclusions about the efficacy of acetylcysteine. And while it is true that acetylcysteine is inexpensive, easy to use and has a favorable side-effect profile, it is probably premature to conclude scientifically that it is To isolate potential sources of heterogeneity we performed a meta-regression analysis exploring several clinical and study quality factors. There was no evidence of association between effect size and baseline serum creatinine, volume of contrast media, or diabetes mellitus, all independently identified risk factors for development of contrast-induced nephropathy ,53. HoweFunnel plot asymmetry is often interpreted to indicate publication bias. However, it is important to consider that this asymmetry may also be due to other sources of bias that deserve further examination. In particular, fundamental disparities in study design, inconsistencies in methodological quality and differences in the definition of primary outcomes may have contributed to funnel plot asymmetry. Our meta-regression analysis explored the potential role of several study quality factors, and none were identified as statistically significant predictors of apparent acetylcysteine efficacy across trials. Nonetheless, it is quite possible that other unmeasured study quality factors may have contributed to biased results and accompanying funnel plot asymmetry.Contrast-induced nephropathy continues to be an active subject matter for clinical investigation ,56. A deAll of the above leads us to conclude that while acetylcysteine appears to be safe and inexpensive, its efficacy for the prevention of contrast-induced nephropathy remains unproven. The results of the trials that we reviewed to date should be viewed as early promising evidence of benefit, and suggest that it is now perhaps reasonable to use acetylcysteine in routine care because of its relative ease of use and safety. However, its true efficacy will remain uncertain unless a definitive well-designed multi-center trial is performed. Such a clinical trial will be most relevant if it addresses a priori clinically meaningful endpoints of renal insufficiency, rather than surrogate endpoints based on changes in creatinine levels alone, and further considers stratification on hypothesized important subgroups that may benefit such as those with a low ejection fraction .The authors declare that they have no competing interests.SMB developed the study protocol, conducted literature search, screened articles for eligibility, extracted data, analyzed data, interpreted results, wrote and revised the manuscript. WAG contributed to protocol development, screened articles for eligibility, extracted data, analyzed data, interpreted results, and provided critique of successive drafts of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currently most studies focus on the genes coding for proteins that regulate blood pressure as their physiological role makes them prime suspects.The present study examines how polymorphisms of the insertion/deletion (I/D) ACE and M235T AGT genes account for presence and severity of hypertension, and embeds the data in a meta-analysis of relevant studies.The I/D polymorphisms of the ACE and M235T polymorphisms of the AGT genes were determined by RFLP (restriction fragment length polymorphism) and restriction analysis in 638 hypertensive patients and 720 normotensive local blood donors in Weisswasser, Germany. Severity of hypertension was estimated by the number of antihypertensive drugs used.No difference was observed in the allele frequencies and genotype distributions of ACE gene polymorphisms between the two groups, whereas AGT TT homozygotes were more frequent in controls . This became significant (p = 0.035) in women only. AGT TT genotype was associated with a 48% decrease in the risk of having hypertension , and this risk decreased more significantly in women . The meta-analysis showed a pooled odds ratio for hypertension of 1.21  in Caucasians. No correlation was found between severity of hypertension and a specific genotype.The ACE I/D polymorphism does not contribute to the presence and severity of essential hypertension, while the AGT M235T TT genotype confers a significantly decreased risk for the development of hypertension in the population studied here. This contrasts to the findings of meta-analyses, whereby the T allele is associated with increased risk for hypertension. Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currentThe Renin-Angiotensin System (RAS) has a central role in regulating blood pressure and sodium homeaostasis. Genes encoding components of RAS, including angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensinogen II type-1 receptor (AGTR1), and renin, have been extensively investigated as genetic determinants of essential hypertension . PolymorIn 1992, the M235T AGT TT polymorphism was first reported to be associated with hypertension . This fiThe inconsistent results might be explained in part by the genetic and environmental heterogeneity among different ethnic groups . On the The present study investigates the relationship between variants of the I/D ACE gene and M235T AGT gene, and the presence and severity of essential hypertension in a large homogeneous German population. The effect of a combination of ACE and AGT gene polymorphisms on hypertension was also examined.The design of the study followed the guidelines proposed by Cooper et al , and the2 or dialysis), 15 were due to diabetic nephropathy, 5 to chronic pyelonephritis, 1 to chronic glomerulonephritis, 1 light chain deposits, 1 polycystic kidney disease, and 14 of unknown cause (no biopsy obtained). The severity of hypertension was estimated based on the number of antihypertensive medications used, a surrogate marker for the severity of hypertension[This cross-sectional study comprised a total of 1358 individuals from Weisswasser, a county town of 25,000 in Saxony, Germany. After giving informed consent, 720 normotensive subjects were selected from local blood donors and 638 hypertensive patients from the local renal care center. All hypertensive patients included in the study had been diagnosed as suffering from primary hypertension by the attending consultants on first contact with the clinic. Hypertensives were defined as those who received at least one antihypertensive medication. At the time of blood sampling, 34.2% were diabetic , and 65.1% were suffering from kidney disease. Of the 37 patients with K/DOQI stage 5  and restriction analysis were used to determine the frequencies of the I/D polymorphisms of the ACE gene, and homo-/ heterozygoty of the M235T AGT gene,21. ACE AGT M235T polymorphism was studied by first amplifying a 104 bp long fragment of the AGT gene using the following primer sequences: sense- 5'CCGTTTGTGCAGGGCCTGGCTCTCT3', and antisense: 5'CAGGGTGCTGTCCACACTGGACCCC3'.Tth 111I.The M -> T point mutation at position 235 creates a detection site for the restriction enzyme t test for continuous data and Pearson's χ2 test for categorical data. Allele frequencies were calculated with the gene-counting method. χ2 test was used for assessment of the Hardy-Weinberg equilibrium for the distribution of genotypes. Odds ratios were calculated with a 95% confidence interval. A P < .05 was considered significant.Statistical analysis was carried out using SPSS personal computer statistical package . Demographic characteristics were compared by 2 test using P-value < 0.05. The Mantel-Haenszel odds ratios were calculated by applying both fixed effect model and random effect model in case of heterogeneity.A meta-analysis was performed using Review Manager 4.2 (The Cochrane Collaboration) to further examine the association of the AGT M235T gene polymorphisms with essential hypertension in Caucasians. A systematic literature search in PubMed Medline for articles published between April 2002 and June 2004 was carried out using the following MESH-headings: "angiotensinogen/genetics", "hypertension/genetics", "blood pressure/genetics", and "adult". The search was limited to articles published in English and studies on Caucasian human subjects. Only 2 studies were left after strict examination according to the exclusion criteria listed in . A totalDemographic data are summarized in Table AGT M/T genotyping was successful in 637 hypertensives and 720 normotensives, and ACE I/D genotype was analyzed in 636 hypertensive and 719 control subjects. The roles of age and gender in the association between hypertension and ACE and AGT gene polymorphisms were examined by comparing the effects of ACE and AGT genotypes for hypertension in men and women, young and elderly subjects respectively. "Young" was defined as "age < 50 years old", and "elderly" was characterized as "age ≥ 50 years old".No differences were observed in ACE allele and genotype frequency distribution between hypertensives and controls with respect to gender and age, and no deviations from Hardy-Weinberg equilibrium were observed in any of subgroups P > 0.1). ACE genotypes I/I, I/D and D/D were of almost identical frequency within both groups  compared with those with MM genotype Figure . The stuThe effect of eight combinations  on hypertension was examined. No statistically significant association was observed between any combination above and hypertension in all subjects combined. Nonetheless, in women, both genotypes of TT, DD/ID and TT, II/ID were significantly associated with lower prevalence of hypertension  and a more significant association was found in women , compared to men. This is in stark contrast to findings from previous studies, including two German datasets [AGT 235 T-allele and/or TT genotype significantly increased the risk for essential hypertension in Caucasians: odds ratio T vs. M was 1.20 (95% CI: 1.11 to 1.29) [Surprisingly, the cross-sectional study presented here shows a higher prevalence of the T/T M235T AGT gene in the control group compared to the hypertensive group. AGT TT genotype was associated with a datasets ,32, and datasets ,22,47, wto 1.29) , odds rato 1.29)  includedto 1.29) AGT T allele among normotensives was higher than that among hypertensives (0.36 vs. 0.33). This was inconsistent with one previous meta-analysis in Caucasians, which showed that among controls, the mean allele frequency for the AGT T allele was 0.41 (95% CI: 0.34 to 0.48), and among cases, increased to 0.45 (95% CI: 0.38 to 0.52) [AGT T allele among hypertensives (0.33) was outside the lower border of the 95% confidence interval (0.38 to 0.52) reported in [Although no significant difference was observed in AGT T allele frequency distribution between hypertensives and controls with respect to age and gender, the frequency of the to 0.52) . In the orted in . This maThe AGT genotype distribution is not in Hardy-Weinberg equilibrium (P < 0.001) while no deviations from Hardy-Weinberg equilibrium are observed on the ACE genotype distribution in the same population. This may be explained by a shift toward a higher frequency of MT individuals (62.4%) instead of TT individuals (3.7%) in this specific population.The study population presented here contains a large proportion (65%) of patients with renal disease. While selection of participants based on patient records excluded those patients that had symptoms suggesting the diagnosis of secondary hypertension at first contact, the possibility remains that at least a part of the study population suffers from renal rather than essential hypertension. It should be noted, however, that the majority of studies included in the presented meta-analysis does not give specific information regarding renal function of hypertensives, and the largest study  is populAnother possible explanation is that the AGT T allele frequency may decrease with age, which was reported from the United Arab Emirates . The preA number of studies -54 examiThe effect of a combination of RAAS genes' polymorphisms on blood pressure has been investigated in the participants to the Olivetti Heart Study , in whicAlthough in most surveys the prevalence of hypertension appears to be equal in women and men , sex-speIn addition to the factor declared above, the result may be influenced by the study design and the composition of the sample population. The study design itself may influence the results. As mentioned above, this study was a cross-sectional study where subjects were assessed at a single time, and at that time most of the controls were younger than 50 years old. Animal studies have shown that hypertension genes may be activated for only certain periods during the life history of an organism . Hence, The population can be described as static population with a mixed Germanic-Slavonic background. Due to the location  and political and historical setting , the population may be assumed as homogeneous. Several recent studies -61 have In the meantime, it should be noted that the present study was carried out on an unusually large population . Given Despite the limitations mentioned, this cross-sectional study does not support the notion that the ACE I/D polymorphism contributes to the prevalence and severity of essential hypertension. However, the M235T TT genotype of AGT gene was detected to confer a significantly decreased risk for the prevalence of hypertension in women from this particular population. Despite the large sample size, the present study fails to revise the odds ratio in a meta-analysis of a total of 25 studies on the association between the AGT M235T polymorphisms and hypertension in caucasians. This observation may reflect a very specific local inheritance pattern of the AGT genotypes. If this holds true, studies aiming at drug development based on genomic traits must be scrutinized rigorously as therapeutic recommendations may be valid for selected subpopulations only.The author(s) declare that they have no competing interests.M.N. designed and initiated the study, analyzed the samples and wrote part of the manuscript. A-L.Z. and M.L. did the statistical analysis. A.M. devised the concept for meta-analysis and wrote the manuscript. L.P. created the figures. M.N. and A.M. should be considered as joint co- authors.The pre-publication history for this paper can be accessed here:


Raised activity of the renin-angiotensin system (RAS) may both amplify inflammatory and free radical responses and decrease tissue metabolic efficiency and thus enhance cerebral injury in the preterm infant. The angiotensin-converting enzyme (ACE) DD genotype is associated with raised ACE and RAS activity as well as potentially adverse stimuli such as inflammation. The DD genotype has been associated with neurological impairments in the elderly, and thus may be also associated with poorer motor or cognitive development amongst children born preterm prematurely.The association of DD genotype with developmental progress amongst 176 Caucasian children born at less than 33 weeks gestation  was examined at 2 and 5 1/2 years of age. Measured neuro-cognitive outcomes were cranial ultrasound abnormalities, cerebral palsy, disability, Griffiths Developmental Quotient [DQ] at 2 yrs, and General Cognitive Ability [British Ability Scales-11] and motor performance [ABC Movement], both performed at 5 1/2 yrs. All outcomes were correlated with ACE genotype.The DD genotype was not associated with lower developmental quotients even after accounting for important social variables.These data do not support either a role for ACE in the development of cognitive or motor function in surviving infants born preterm or inhibition of ACE as a neuroprotective therapy. Delight over recent survival gains for the very premature infant has been tempered by the frequent presence of cerebral injury and developmental impairment. One quarter of those born before 26 weeks postmenstrual age (at least 11 weeks premature) show evidence of severe cerebral injury including cognitive dysfunction by 30 months of age .Three factors seem to play important roles in the aetiology of preterm cerebral injury. Firstly, exposure to inflammatory stimuli is associated with white matter injury and cerebral palsy in the preterm . SecondlCandidate systems that might influence motor or cognitive outcome after premature birth are likely to be those which affect these responses. The human renin-angiotensin systems may be such a system. Angiotensin converting enzyme (ACE), a key component of the circulating (or endocrine) renin-angiotensin system (RAS), cleaves angiotensin I to yield the potent vasoconstrictor angiotensin II. In addition, ACE degrades vasodilator kinins. In these ways, endocrine RAS plays an important role in circulatory homeostasis. However, local RAS also exist in diverse human tissues including lung, myocardium, vasculature, lymphocyte and brain tissue. These are powerful regulators of mitochondrial respiration and whole-cell metabolism  and exerA common variant of the human ACE gene provides a tool to determine if ACE activity does influence developmental progress after preterm birth. The presence  rather than the absence  of a 284-base-pair fragment in the human ACE gene is associated with lower ACE activity in organs including both circulating inflammatory cells  and the The study was approved by the ethical committees of Southmead Hospital and United Bristol Health Care Trust. Parental consent was obtained for participation in neurodevelopmental follow-up   . BrieflyThe Griffiths Mental Development Scales, used to assess motor and cognitive performance, was performed at 2 years corrected age . The GriA psychologist performed the Griffiths Scales of Mental Development and a second psychologist performed the British Ability Scales (second edition) (BAS). The ABC Movement tests were performed by a trained research nurse. All assessments were blind to the child's neonatal course and subsequent progress.DNA was extracted from the Guthrie card blood spots (newborn metabolic screening cards). ACE genotype was determined using 3-primer PCR amplification . Primer An estimate of sample size suggested that 144 patients would be needed for this study. The assumptions made for this calculation were that DD genotype infants had a mean DQ of 92.5 (1/2 SD below the norm) compared to a mean DQ of 100 in the ID+II group, assumed typical genotype distributions, and a significance of 0.05 with 80% power.Data were stored in SPPS v9.0 for Windows. Lymphocyte  and tissp = 0.047) (table p = 0.65).Guthrie cards were located for 230 of 308 children. After exclusion of non-Caucasians and, at random, 1 child of any identical twin pairs (based on genotypes and gender) 176 babies with ACE genotype formed the study population  with follow-up data at 2 years. 122 of these also had follow-up at 5 1/2 years. The ACE genotype distribution was 49 [27.8%] DD, 73 [41.5%] ID, 54 [30.7%] II, demonstrated Hardy-Weinberg equilibrium, and was similar to that observed in the newborn term population from the same region of the UK  II). Baseline characteristics were independent of genotype, except that fewer individuals of DD genotype were from twin births . Thus the effect of any one polymorphism, with a relatively minor effect, may be swamped in the newborn infant by other protective mechanisms.The lack of any association between ACE genotype and scores of developmental progress was also surprising because we have demonstrated an association between DD genotype and markers of poor cardio-respiratory instability in the perinatal period in this patient group . This asWe cannot support an association of ACE genotype with cognitive or motor development in survivors born preterm or, thus, the use of RAS inhibition as a neuroprotective agent in the preterm. Given the current lack of understanding of the mechanisms leading to cerebral injury and subsequent impairment – particularly of higher function – in such patients, further genetic association studies of other candidate genes are warranted.ACE, angiotensin-1 converting enzyme; DQ developmental quotient, BAS, British ability scales (second edition); GCA, general cognitive ability; RAS, renin angiotensin system; PCR, polymerase chain rection.The author(s) declare that they have no competing interests.DH, HM, AW, NM conceived the study and its design and wrote the manuscript. DH, DD and SD performed data collection, DNA extraction and PCR and participated in analysis of the data with SH and HM. NM reviewed all cranial imaging. All authors participated in the writing of the manuscript and approved the final manuscript.


We reviewed articles using computational RF dosimetry to compare the Specific Anthropomorphic Mannequin (SAM) to anatomically correct models of the human head. Published conclusions based on such comparisons have varied widely. We looked for reasons that might cause apparently similar comparisons to produce dissimilar results. We also looked at the information needed to adequately compare the results of computational RF dosimetry studies. We concluded studies were not comparable because of differences in definitions, models, and methodology. Therefore we propose a protocol, developed by an IEEE standards group, as an initial step in alleviating this problem. The protocol calls for a benchmark validation study comparing the SAM phantom to two anatomically correct models of the human head. It also establishes common definitions and reporting requirements that will increase the comparability of all computational RF dosimetry studies of the human head. Cell phone safety remains a topic of broad public concern that attracts frequent media attention. This attention is focused on two areas of scientific controversy concerning cell phone safety. The first area is that of non-thermal biological effects. The existence of these effects is an important open question, but it is not the topic of this paper. However, if these effects exist, their manifestation will certainly be related to the amount of RF energy deposited in the tissue – RF dosimetry . The secThe phantom head that is now the world-wide standard for compliance testing is the Specific Anthropomorphic Mannequin (SAM). SAM was developed by members of IEEE Standards Coordinating Committee 34, SubCommittee 2, Working Group 1 (SCC34/SC2/WG1). This working group was created to develop recommended practices for determining SAR in the head via measurement techniques , and Fedth-percentile anthropometric data corresponding to the adult male head as tabulated by the US Army [SAM is a lossless plastic shell and ear spacer. Because current technology does not allow reliable measurement of SAR in small complex structures, like a simulated pinna, SCC34/SC2 chose to use a lossless ear spacer on SAM to maximize the energy reaching the head and minimize measurement uncertainty. SAM's dimensions were taken from the 90 US Army . The SAMA primary design goal for SAM was that, "SAM shall produce a conservative SAR for a significant majority of persons during normal use of wireless handsets" . To testThe groups headed by Gandhi and Kuster are not the only ones pursuing computational RF dosimetry using anatomically correct models of the human head -25. Not The first, and the most significant of these problems, is the treatment of the external ear (pinna). Specifically, the problem is determining whether the pinna may, or may not, be considered as part of the 1- or 10-gram SAR averaging volumes. When considering SAR averaging volumes the head and the pinna should be viewed as mutually exclusive, in-other-words the pinna is not part of the head but it is attached to the head. Some investigators have chosen to treat the pinna in accordance with IEEE Std C95.1-1999  and the Other investigators have treated the pinna in accordance with draft revision IEEE Std C95.1-200X. This draft standard expands the definition of extremity to include the pinna, which makes the pinna subject to a higher spatial peak SAR, see Table When comparing published results it is often difficult, or impossible, to determine whether head tissue SAR values are based on averaging volumes that include or exclude the pinna. In fact, some papers make no mention of how the pinna was treated. Although head tissue SAR is the major focus of attention, papers that consider the pinna as an extremity can not simply ignore its existence, the pinna must still meet the higher spatial peak SAR for extremities.Another part of the problem dealing with the treatment of the pinna is simply determining what tissue constitutes the pinna. The IEEE defines the pinna as, the largely cartilaginous projecting portion of the outer ear consisting of the helix, lobule, and anti-helix . UnfortuThe second problem area is the lack of common models. The only computer models that are common to all the computational RF dosimetry studies are the SAM and the Visible Human Male. The anatomic data for the Visible Human Male originated at the National Institutes of Health but many groups and individuals lent a hand in converting it into a computational model. While a few investigators have different models the only ones that can be compared across all the published results are SAM and the Visible Human. This also means that the only repeatable comparison that can be made is between the SAM and the Visible Human. It seems obvious that one can neither prove nor disprove that SAM produces a SAR greater than the maximum local SAR induced in humans for a significant majority of persons during normal use of wireless handsets, when there is only one anatomically correct model available for comparison.Although not a major problem, it is still true that dielectric properties and names of tissue types in anatomically correct models have varied between investigators.Of course the head model is only half of any computational RF dosimetry study, the model of the RF source is the other half. The only common source that has been used in several published studies is a dipole ,28-30. SThe third problem is that of inconsistent positioning of the model cell phone relative to the head model. Simulated SAR in near-field situations is mainly a function of the geometry of the RF current density distribution on the source model and its geometric separation from the lossy head tissue . When thUsual practice is to align a monopole cell phone antenna with the FDTD grid to avoid the stairstep effect. The head model is then rotated to the correct position relative to the cell phone. After rotation, the voxelized model must be remeshed to align the voxels with the FDTD grid. This is not a trivial task and algorithms to perform remeshing are constantly being improved.The authors have noted some unintended artifacts in voxelized models after remeshing. The first of these is grooving. Figure The jagged edges caused by grooving are not limited to surface features. Figure Grooving has not been observed with all FDTD software and even when it has been seen it has not occurred with all models. Researchers should routinely examine their models after rotation to insure grooving is not a problem.All FDTD programs must, of necessity, perform their calculations on voxelized models. However some programs use CAD models which are only converted to voxelized format after all rotation has been done. These programs avoid most coordinate transformation problem but they are not infallible. They must still convert smoothly undulating biological surfaces into rectilinear voxels. Figure Because the FDTD method calculates the electric fields at the voxel edges, the X, Y and Z-directed power components associated with a voxel are defined in different spatial locations. These components must be combined to calculate SAR in the voxel. There are three possible approaches to calculate the SAR: the 3-, 6-, and 12-field components approaches. The 12-field components approach is the most complicated but it is also the most accurate and the most appropriate from the mathematical point of view . The 12-After the SAR in every voxel is determined multiple voxels must be combined to compute the 1- or 10-gram SAR spatial averaging volumes. These normally cubic volumes become difficult to construct at the surface of a model or when the volume is constrained to a particular tissue type. The particular algorithm used to construct these volumes can influence the resultant 1- or 10-gram SAR values. However, the actual algorithm used to construct the spatial averaging volumes is usually not reported.net) are related byWhen the cell phone model is placed next to the SAM or anatomically correct model, it changes the cell phone's antenna feed-point impedance. The antenna feed-point impedance (Z), feed-point current (I) and net input power . This working group was created to develop recommended practices for determining SAR in the head via computational techniques . This stThe protocol has two parts; a benchmark validation study; and a set of common definitions, models, and reporting requirements. The benchmark validation study is underway with fifteen participants. All participants should finish the study by mid-2004 and the results should be published by early 2005. Hopefully the common definitions, models, and reporting requirements will be used in future investigations making comparison of results easier.The protocol asks all participants to report peak spatial SAR for averaging volumes that both include and exclude the pinna. The voxels comprising the pinna in the provided anatomic models are flagged so all participants will conform to one definition of the pinna. The pinna voxels are flagged by prefixing the standard tissue type with pinna-; such as pinna-skin, pinna-cartilage, and pinna-fat. The electrical properties of the flagged pinna voxels are unchanged. The IEEE Std 1528 definition for the pinna was followed and the choice of each flagged voxel was confirmed by an Ear-Nose-Throat surgeon.The Benchmark Validation Study calls for each participant to run twelve simulations: three head models, at two frequencies (835 and 1900 MHz), and in two cell phone positions (touch and tilted). The models are SAM, the Visible Human, and a seven year old Japanese male . Each moFor the two anatomically correct models, the tissue names and properties in the header file were made consistent with the definitions found on the Italian National Research Council, Institute for Applied Physics web site .Although they are not part of the benchmark validation study, SCC34/SC2/WG2 plans on releasing several new anatomically correct models in the next few months to expand the population of models available for study.A generic cell phone is described for use in all benchmark validation studies, see Figure The reference points, necessary for positioning the cell phone relative to the anatomically correct model, are also contained in the header file for each model.To aid comparison of results from all the participants, a common coordinate system was defined with origin at the acoustic output of the cell phone, see Figure The FDTD technique is called for by the P1529 draft . FDTD waThe participants are asked to report the following FDTD data for all simulations: The boundary conditions used and the minimum distance between the model and the boundary of the computational space. The time step size and the number of time steps used. The grid (voxel) size and whether the grid was homogeneous or graded. To calculate the SAR in each voxel the protocol recommends the 12-field components approach. All 1- or 10-gram spatial averaging volumes are to be constructed in accordance with IEEE C95.3, Annex E.The participants are asked to report the following SAR data for all simulations: The peak spatial SAR, both 1 g and 10 g averages, for all tissue (head plus pinna), head only, and pinna only averaging volumes and the location of the averaging cubes. The peak point value SAR and its location. A color coded SAR distribution for both 1 g and 10 g averages, in the ZY plane.For a realistic study it would be ideal to simulate the real world situation. The question that remains is: "Does a real-world cell phone keep the power or the feed-point current constant when placed next to different human beings with different head shapes and head sizes"? Unfortunately there is not a definitive answer to this question. The behavior of a mobile phone depends on the system design and the power amplifier circuits. A detailed discussion for real-world mobile telephones has to be addressed by future projects with more realistic mobile phones models for numerical simulations.For now it is important to scale the calculated SAR values to net input power and feed-point current and to present both results. The behavior of a real-world mobile phone is within the SAR range of scaling to the net input power and scaling to the feed-point current. For human health and safety considerations a worst case approach is desirable. Until further knowledge on the behavior of a real-world cell phone is available, the scaling producing the worst case result  must be taken into account.The current version of IEEE Std C95.1  does notInvestigators should inspect all models after rotation to be sure they are free of artifacts caused by meshing along the new coordinate axes. If necessary, artifacts should be manually corrected before running the simulation. Blindly accepting the output of meshing algorithms can lead to errors. All relevant data and assumptions for the computational RF dosimetry study, as discussed in section 2 "Problem areas", must be reported in such detail that the reader is able to compare the results to other studies. The names and electrical properties for all anatomically correct models should comply with those shown on the Italian National Research Council, Institute for Applied Physics web site.To facilitate broad based comparisons, new anatomically correct models should be placed in the public domain or made available for a modest fee. The number of anatomically correct models suitable for electromagnetic modelling and widely available for comparison to the SAM is still low. Because the SAM is intended to represent a significant majority of persons during normal use of wireless handsets, comparison to a large variety of anatomically correct models is desirable. It is the hope of IEEE SCC34/SC2/WG2 that consistent results in the benchmark validation will show that, by adhering to some common definitions and procedures, FDTD studies from different investigators using different anatomically correct models are comparable.Protocol for the Computational Comparison of the SAM Phantom to Anatomically Correct Models of the Human Head and this manuscript. WK, present chairman of IEEE SCC34/SC2/WG2, wrote approximately 25% of the manuscript, developed and simulated the phone model and supplied several of the figures. Both authors read and approved the final manuscript.BB, past chairman of IEEE SCC34/SC2/WG2, drafted the


The genes encoding proteins in the thrombomodulin-protein C pathway are promising candidate genes for stroke susceptibility because of their importance in thrombosis regulation and inflammatory response. Several published studies have shown that the Ala455Val thrombomodulin polymorphism is associated with ischemic heart disease, but none has examined the association with stroke. Using data from the Stroke Prevention in Young Women Study, we sought to determine the association between the Ala455Val thrombomodulin polymorphism and the occurrence of ischemic stroke in young women.All 59 hospitals in the greater Baltimore-Washington area participated in a population-based case-control study of stroke in young women. We compared 141 cases of first ischemic stroke (44% black) among women 15 to 44 years of age with 210 control subjects (35% black) who were identified by random digit dialing and frequency matched to the cases by age and geographical region of residence. Data on historical risk factors were collected by standardized interview. Genotyping of the thrombomodulin Ala455Val polymorphism was performed by pyrosequencing.The A allele (frequency = 0.85) was associated with stroke under the recessive model. After adjustment for age, race, cigarette smoking, hypertension, and diabetes, the AA genotype, compared with the AV and VV genotypes combined, was significantly associated with stroke . The AA genotype was more common among black than white control subjects (81% versus 68%) but there was no significant interaction between the risk genotype and race (adjusted odds ratio 2.7 for blacks and 1.6 for whites). A secondary analysis removing all probable (n = 16) and possible (n = 15) cardioembolic strokes demonstrated an increased association .Among women aged 15 to 44 years, the AA genotype is more prevalent among blacks than whites and is associated with increased risk of early onset ischemic stroke. Removing strokes potentially related to cardioembolic phenomena increased this association. Further studies are needed to determine whether this polymorphism is functionally related to thrombomodulin expression or whether the association is due to population stratification or linkage to a nearby functional polymorphism. Thrombosis is a dynamic balance between factors that promote clot formation, antithrombotic mechanisms, and fibrinolysis. Central to this balance is the thrombomodulin-protein C antithrombotic mechanism. Thrombomodulin forms a 1:1 complex with thrombin on the vascular endothelium, thereby inhibiting the procoagulant actions of thrombin and converting protein C to activated protein C -5. TheseTHBD) maps to chromosome 20p11.2, contains a single exon and no introns, and spans 4 kb . The thrombomodulin protein is expressed primarily on the luminal surface of vascular endothelial cells and consists of 557 amino acids (aa) : an N-terminal lectin-like module (aa 1–154), a hydrophobic region (aa 155–222), six epidermal growth factor (EGF)-like modules (aa 223–462), a serine and threonine rich region (aa 463–497), a single transmembrane segment (aa 498–521), and a short cytoplasmic tail (aa 522–557) [The thrombomodulin gene  is a population-based case-control study that was initiated to examine risk factors for ischemic stroke in young women. In that study the term "population-based" means that cases and their comparison group were identified from the same defined population. The study area included all of Maryland (except the far Western panhandle), Washington DC, and the southern portions of both Pennsylvania and Delaware. Cases were female patients 15 to 44 years of age with a first cerebral infarction as identified by discharge surveillance at 59 regional hospitals and through direct referral by regional neurologists. The methods for discharge surveillance, chart abstraction, and case adjudication have been described previously ,13,14. TTHBD genotyping at the Ala455Val polymorphism for 141 cases and 210 control subjects. This included all case and control samples that were available at that time. Genotyping was performed blinded to case-control status. Genomic DNA was extracted from stored peripheral blood lymphocytes by using standard protocols . The THBD Ala455Val polymorphism was determined by pyrosequencing. The single-nucleotide polymorphism region of the gene was amplified by polymerase chain reaction (PCR) with the use of published primers [We performed  primers ,16. The THBD Ala455Val polymorphism and stroke: age, race, current cigarette smoking, hypertension, diabetes mellitus, history of angina or myocardial infarction (angina/MI), use of oral contraceptive pills (OCP) or hormone replacement therapy (HRT), sickle cell disease, and sickle cell trait. Age, race, current cigarette smoking status, use of OCP or HRT was determined by subject reports . Hypertension and diabetes mellitus, sickle cell disease or sickle cell trait were determined by asking study participants (or a proxy) if a physician had ever told them that they had the condition.We assessed the following potential confounders of the association between the alleles of the We compared means by t tests and proportions by χ2 tests. The probability values presented are based on two-sided tests. Because of the low frequency of the V455 allele, we compared the frequency of the combined AV/VV genotype between cases and controls. Adjusted odds ratios derived from logistic regression were used to determine whether the presence of the Ala455Val test allele was associated with an increased risk for stroke after differences in age, race, current cigarette smoking, hypertension, and diabetes mellitus were controlled for. Additional analyses included: 1). adding ischemic heart disease (angina/MI) into the logistic regression model; 2). evaluation for interactions between genotype and OCP/HRT 3). an analysis excluding sickle cell trait, and 4). an analysis excluding cardioembolic strokes.Characteristics by case-control status are described in Table The distribution of genotypes was in Hardy Weinberg equilibrium for the pooled set of cases and controls, both in total and by race.Among control subjects, the prevalence of the AA genotype was 81% (59/73) for blacks and 68% (93/137) for whites. The relationship between the Ala455Val genotypes and selected stroke risk factors in control subjects is summarized in Table Table The strength of association between the AA genotype and stroke remained unchanged including history of angina or myocardial infarction in the logistic regression model . Neither OCP/HRT use, nor sickle cell trait demonstrated an interaction with genotype and additional adjustment for these factors did not alter the association between the AA genotype and stroke.Among the 141 stroke patients, 70 (50%) had a least 1 probable cause, 30 (21%) had no probable cause but a least one possible cause, and 41 (29%) were indeterminate. Table A secondary analysis removing all probable (n = 16) or possible (n = 15) cardioembolic strokes was performed using the same adjusted model including age, race, smoking, hypertension, and diabetes. An increased association between non-cardioembolic stroke and the AA genotype was demonstrated .THBD Ala455Val polymorphism, the prevalence of the AA genotype among our control population was similar to that previously reported for the Atherosclerosis Risk in Communities (ARIC) Study population [In our study of the pulation . Our respulation . VasculaTHBD Ala455Val polymorphism and coronary artery disease (CAD) have yielded conflicting results. A Swedish case-control study found the alanine allele was associated with CAD [THBD Ala455Val polymorphism and CAD [THBD Ala455Val polymorphism, ours included, is due to population-stratification bias, a functionally neutral polymorphism that serves as a marker for a nearby functional mutation (linkage disequilibrium), or the true existence of different associations in the different study populations.Several recent studies evaluating the with CAD . In contwith CAD . A Briti and CAD . Consist and CAD , we obsePopulation-stratification bias is due to confounding by population admixture . An unidTHBD Ala455Val locus may be in linkage disequilibrium with an unobserved "high-risk" susceptibility locus. Linkage disequilibrium is a function of the history of the population, and thus true associations can occur in one population and not another.The THBD Ala455Val polymorphism, thereby defining a susceptibility locus for the disease. An important criterion for a true susceptibility locus is that the polymorphism is associated with a change in protein expression or function. The THBD Ala455Val polymorphism has not been associated with variation in soluble thrombomodulin concentrations [Our results are also consistent with a causal association between stroke and the trations , but soltrations ,8,20,21.trations . Furthertrations .Thrombomodulin has not previously been examined as a candidate gene for stroke susceptibility. We found that among women aged 15 to 44 years, the AA genotype is more prevalent among blacks than whites and is associated with increased risk of early-onset ischemic stroke. Removing strokes potentially related to cardioembolic phenomena increased this association. Further studies are needed to determine whether this association is due to population stratification, linkage to a nearby functional polymorphism, or variation in thrombomodulin expression or function.The author(s) declare that they have no competing interests.All authors certify that they participated in the conceptual design of this work, the analysis of the data, and the writing of the manuscript to take public responsibility for it. All authors reviewed the final version of the manuscript and approve it for publication. J.W.C., S.J.K., B.D.M., M.A.W. and R.F.M. participated in the writing of the initial draft. M.G., K.K.S., W.H.G. and S.C.R. participated in the genotyping. J.W.C., S.J.K., B.D.M., and L.J.R. participated in the data analysis. All authors provided critiques of the final manuscript.Dr. Cole's effort on this project was supported in part by an American Academy of Neurology Clinical Research Training Fellowship, by the National Institutes of Health Research Training in the Epidemiology of Aging (Grant T32-AG00262-04), and by the Department of Veterans Affairs, Baltimore, Office of Research and Development, Medical Research Service, and Stroke Research Enhancement Award Program. Dr. Kittner was supported in part by the Department of Veterans Affairs, Baltimore, Office of Research and Development, Medical Research Service, Geriatrics Research, Education and Clinical Center, and Stroke Research Enhancement Award Program; a Cooperative Agreement with the Division of Adult and Community Health, Centers for Disease Control and Prevention; the National Institute of Neurological Disorders and Stroke and the NIH Office of Research on Women's Health; the National Institute on Aging Pepper Center Grant P60 12583; and the University of Maryland General Clinical Research Center (Grant M01 RR 165001), General Clinical Research Centers Program, National Center for Research Resources, NIH.The pre-publication history for this paper can be accessed here:


Periconceptional use of vitamin supplements containing folic acid reduces the risk of a neural tube defect (NTD). In November 1998, food fortification with folic acid was mandated in Canada, as a public health strategy to increase the folic acid intake of all women of childbearing age. We undertook a comprehensive population based study in Newfoundland to assess the benefits and possible adverse effects of this intervention.This study was carried out in women aged 19–44 years and in seniors from November 1997 to March 1998, and from November 2000 to March 2001. The evaluation was comprised of four components: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements.12 deficiencies, and no evidence of improved folate status masking haematological manifestations of vitamin B12 deficiency. The proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased from 17% to 28%.The annual rates of NTDs in Newfoundland varied greatly between 1976 and 1997, with a mean rate of 3.40 per 1,000 births. There was no significant change in the average rates between 1991–93 and 1994–97 . The rates of NTDs fell by 78% (95% CI 65%–86%) after the implementation of folic acid fortification, from an average of 4.36 per 1,000 births during 1991–1997 to 0.96 per 1,000 births during 1998–2001 . The average dietary intake of folic acid due to fortification was 70 μg/day in women aged 19–44 years and 74 μg/day in seniors. There were significant increases in serum and RBC folate levels for women and seniors after mandatory fortification. Among seniors, there were no significant changes in indices typical of vitamin BBased on these findings, mandatory food fortification in Canada should continue at the current levels. Public education regarding folic acid supplement use by women of childbearing age should also continue. Neural tube defects (NTDs) are birth defects resulting from the failure of neural tube closure during early development of the human embryo. The 1997 Canadian national NTD birth prevalence was 0.75 per 1,000 births (live births and stillbirths), down from 1.16 per 1,000 in 1989 -4. HistoEvidence from a number of studies has demonstrated that periconceptional use of vitamin supplements containing folic acid reduces the risk of NTDs -8. AlthoBecause of concern that public education campaigns alone would not be effective in achieving optimal periconceptional folic acid intake for the majority of women, food fortification with folic acid was proposed as a strategy to ensure that all women of childbearing age increase their dietary intake of this vitamin. In November 1998, Health Canada mandated fortification of white flour and enriched pasta and cornmeal with folic acid . Since d12 deficiency, a condition that affects 10–15% of the population over age 60 years [12 deficiency, thus delaying diagnosis and treatment of the condition while its attendant neurologic manifestations progress. Seniors may be at particular risk since the incidence of vitamin B12 deficiency increases with age.The question of whether folic acid fortification of grain products poses any serious health risk has been controversial. The main concern has been the potential masking of vitamin B60 years ,15. IncrWe therefore undertook a comprehensive population based study to evaluate the effectiveness of the public health strategy of food fortification with folic acid and to determine possible adverse effects resulting from fortification.This evaluative study was designed as a population based study and included four components as follows: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements. The latter three components of the study were carried out in two phases; the first phase took place prior to the introduction of mandatory fortification, from November 1997 to March 1998 and the second phase occurred from November 2000 to March 2001, after two years of implementation of mandatory fortification.This study was undertaken in Newfoundland because of the historically high rates of NTDs in the province, and because of strong interest in the health community in this initiative. Newfoundland and Labrador, with a population of approximately 500,000, has about 5,000 births annually. An urban (St. John's) and rural  location in the province were chosen as the sites for this study. Data collected from these sites were compared between Phase I (November 1997 to March 1998) and Phase II (November 2000 to March 2001). Table In order to examine temporal changes in the rates of NTDs in Newfoundland, data were compiled from the Newfoundland and Labrador Medical Genetics Program from 1976 to 2001. This Program ascertains cases of NTDs annually and maintains an NTD database. The database has recorded cases of NTD since 1976. Cases are identified in the following ways: provincial live birth and stillbirth notification forms, maternal-fetal medicine referrals (only one tertiary care unit in the province), and letters sent to all medical records departments of all provincial hospitals requesting data on cases assigned ICD-9/10 codes associated with NTDs or terminations for NTDs. These multiple sources are utilized to ensure complete ascertainment. NTD cases include anencephaly, spina bifida and encephalocele diagnosed in live births, stillbirths  and fetuses from pregnancies terminated  after a prenatal diagnosis of an NTD.For the knowledge assessment component of the study, women between the ages of 19 and 44 years were recruited through a random telephone survey. In the initial telephone survey, women were asked about their use of vitamin supplements and knowledge of the importance of folic acid for reducing the risk of NTDs or for fetal development. Women who completed the initial telephone survey were subsequently screened for their eligibility for dietary and blood assessments. Women who were not taking supplements containing folic acid and not pregnant were eligible to participate. This sampling procedure for Phase I and Phase II resulted in a response rate of 59.7% and 65.4%, respectively, with no difference between urban and rural response rates. A total of 233 women were recruited into Phase I and 204 women were recruited in Phase II, who completed components II, III and IV of the study.12 deficiency or anaemia and not taking vitamin B12 or supplements containing folic acid, were eligible for dietary and blood sample assessments. A total of 202 seniors were recruited in Phase I and 186 were recruited in Phase II .Seniors were recruited in the same manner as the samples of women, but were drawn only from St. John's, Newfoundland. Seniors aged 65 years or over, not diagnosed with vitamin BIn order to determine intakes of naturally occurring folate  pre and post fortification, and dietary intakes of folic acid (the synthetic form of the vitamin) post fortification, a Willett food frequency dietary questionnaire  was admi12 status in Phase I and Phase II. Laboratory tests for complete blood count (CBC), red blood cell (RBC) folate, serum folate, creatinine, vitamin B12, plasma homocysteine (HCY) and methylmalonic acid (MMA) were conducted at the laboratories of the Health Care Corporation of St. John's.The women and senior participants were also asked to provide a sample of blood in order to determine blood folate and vitamin Bpermitted in Canada as of December 1996 [Rate of NTDs was defined as the number of above described NTD cases, divided by the total number of live births, stillbirths, and pregnancy terminations for an NTD (termed as "births" hereafter). First we examined the temporal trend in annual rates of NTDs from 1976 to 2001 using 3-year moving average rates, then we focused on comparison of the NTD data for the most recent 11 years, identified as pre-supplementation (1991–1993), pre-fortification (1994–1997) and post-fortification (1998–2001). We regard the year 1997 as a transition period, or partial fortification period, since fortification of white flour and enriched pasta and cornmeal was ber 1996 . Thus weMean daily intakes of naturally occurring folate were calculated for women aged 19–44 years and for seniors in Phase I and Phase II. Also, for Phase II, average daily intakes of folic acid from fortified foods were calculated.12were skewed. Values were therefore log transformed to give an approximate normal distribution for estimation of geometric mean and confidence intervals. Unless otherwise stated, all laboratory values presented in this paper are geometric means and 95% confidence intervals (CI). Differences in the frequency of high or low results based on reference values were tested by Pearson chi-square statistics.Data from the blood analyses were tested for normality with the Komogorov-Smirnov test, and differences between groups were tested using the non-parametric Mann-Whitney U test. The distributions of plasma MMA, plasma HCY, serum folate, RBC folate and serum vitamin BAll data for this study were entered into SPSS  Rel. 10.0 after the end of each phase. Data from the dietary interviews were analyzed using Epi-Info (Version 6.04d), while the laboratory data and the data about knowledge and use of supplements were analyzed using SPSS.There were 617 ascertained cases of NTD among live births, stillbirths and pregnancies terminated for an NTD in Newfoundland over the 26 year period. The annual rates of NTDs in the province varied greatly over time, with the lowest rate of 2.18 per 1,000 births in 1989, and the highest rate of 5.92 per 1,000 births in 1995. The average rate of NTDs between 1976 and 1997 was 3.40 per 1,000 births. A dramatic drop is seen in 1997, in which the rate of NTDs was 2.20 per 1,000 births, down from 5.49 per 1,000 births in the previous year. The decreasing trend continued after 1998 . It is worthwhile to note that there has been no significant increase in the proportion of NTDs from terminated pregnancies since 1994.There was no statistically significant change in the average daily intake of naturally occurring folate among either women aged 19–44 years or seniors between Phase I and Phase II . Seniors generally had dietary folate intake slightly higher than women of childbearing age. In Phase I, the average daily intake of naturally occurring folate was 306 μg/day for seniors and 262 μg/day for women aged 19–44 years, while in Phase II, the average daily intake of naturally occurring folate was 290 μg/day for seniors and 248 μg/day for women aged 19–44 years.The implementation of mandatory fortification resulted in an average additional dietary intake of 70 μg/day of folic acid in women aged 19–44, and 74 μg/day of folic acid among seniors. It is noteworthy that for the women the average daily folic acid intake due to food fortification was less than the approximately 100 μg that was previously predicted for women of childbearing age. The maximum dietary intake of folic acid due to fortification for an individual woman was 235 μg/day, and for an individual senior was 219 μg/day.Serum folate and RBC folate increased significantly from Phase I to Phase II in both women aged 19–44 years and seniors (p < 0.001). For both age groups, there was a corresponding decrease in mean plasma HCY levels Tables  and 4. T12 levels in women aged 19–44 and seniors  was 18.8% prior to fortification and following fortification this proportion declined to 11.8% (p = 0.032) (data not shown). A statistically significant increase in mean plasma MMA levels was observed in women subjects (p = 0.008) but not in seniors (p = 0.229) . The proportion of women taking a vitamin supplement containing folic acid increased substantially between the two time periods . Information about folic acid dosage was not collected.The results of a number of studies have led to the conclusion that periconceptional folic acid supplementation reduces the risk of NTDs -8. AmongIn March 1996 the US Food and Drug Administration (FDA) announced that it would permit addition of folic acid to enriched flour and other enriched cereal grain products, and that this addition would be mandatory as of January 1998. The level of fortification was set at 0.14 mg folic acid per 100 g of cereal grain product. It was determined that at this level of fortification, the intake of folate  for the target and the general population would be kept below 1,000 μg/day, which was deemed to be the safe upper limit. This level of fortification was estimated to increase the average daily intake of folic acid in women of childbearing age by about 100 μg . SubsequOur results show a highly significant drop in the rate of NTDs in Newfoundland, taking into account all identified affected pregnancies . The 78% (95% CI 65%–86%) reduction in the NTD rate after implementation of fortification is greater than the 18%–22% reduction predicted at current levels of fortification ,28, and The large reduction in the rate of NTDs in Newfoundland may be due, at least in part, to the fact that Newfoundland had higher background rates of NTDs. This population may be more sensitive to the influence of folic acid. In a large-scale public health campaign in northern and southern China, periconceptional use of 400 μg/day folic acid supplements was associated with a reduction in NTD risk of 79% for women in northern China, where the baseline NTD rate was high and similar to that observed in Newfoundland. A lower risk reduction of 41% was observed in the southern region where the pre-campaign NTD rate was much lower .The 65% increase in the proportion of women taking vitamin supplements containing folic acid, from 17% in Phase I to 28% in Phase II, suggests that an increasing trend in folic acid supplementation may have played a role in the declining NTD rate in Newfoundland. In this study it was not possible to determine the individual contribution of supplementation and fortification to the trend in NTDs.The annual rate of NTDs in the pre-fortification period (1994–97) did not differ significantly from that of the pre-supplementation period (1991–93); this is true whether 1997 is excluded or included in the pre-fortification period. The increase in the rates of NTDs in 1995 and 1996 appears random and largely unexplainable. The changes in the NTD rates between 1994–1996 and 1991–1993 and between 1995–1997 and 1991–1993 were not statistically significant. In addition, our data do not show an obvious increase in the proportion of NTDs in terminated pregnancies during 1994 and 1996 (data available upon request).The questionnaire used in this dietary assessment was a modified Willett questionnaire , adminisThe daily intake of naturally occurring folate among women aged 19–44 years in this study (average 248 μg/day in the Phase II sample) was similar to values found in other studies of women's diet ,35. For The results of this study provide strong evidence of improved blood folate status in women aged 19–44 years following mandatory fortification with folic acid. Women showed evidence of increased levels of serum and RBC folate and decreased levels of plasma HCY. These results are consistent with an earlier study examining the effect of fortification in the Framingham offspring study cohort .Mandatory food fortification with folic acid has resulted in improvements in folate indices in seniors. Both mean serum folate and mean RBC folate increased following folic acid fortification  in seniors post fortification in our study. Thus, these results show no evidence of a deterioration in vitamin B12 status among seniors. Furthermore, there is no evidence of improved folate status resulting in masking of the haematological manifestations of vitamin B12 deficiency among seniors as a group. There was no evidence of deteriorating vitamin B12 status among young women participants based on vitamin B12 measurements. The upward trend in plasma MMA levels and higher proportion of abnormal values among young women is being further evaluated. It is unlikely that this is a direct effect of folic acid fortification and this observation is not consistent with any known effects of folic acid on vitamin B12 status.There was a decline in the proportion of seniors with low vitamin BWe have documented the rate of NTDs among live births, stillbirths and terminated pregnancies known to have an NTD. It was not possible to include NTDs that may have occurred in pregnancies that resulted in a spontaneous abortion or a termination that occurred for reasons other than a congenital anomaly.12 status. On the other hand, the fact that we observed such marked improvements in blood folate status leads us to conclude that there was a real increase in exposure to folic acid through fortification, over the study period.This study, and other studies of fortification in Canada, are limited by the fact that there was no precise date when exposure to food fortified with folic acid began. The addition of folic acid to white flour and enriched pasta and cornmeal was permitted as of December 1996. Industry was switching to folic acid-containing enrichment premixes, especially towards the end of 1997, in anticipation of both US requirements for fortification as of January 1, 1998, and Canadian plans to implement mandatory fortification. Although this requirement did not come into force in Canada until late 1998, the Phase I (November 1997 to March 1998) subjects of our study may have consumed at least some food fortified with folic acid. This would result in an underestimate of improvements in blood folate status due to fortification, and might lead us to miss adverse effects on vitamin BAnother limitation of this study is the possible underestimation of folic acid intake due to fortification. Our calculations were based on the assumption that manufacturers are fortifying flour at the required level. It has been suggested that allowance for "overages" is resulting in higher amounts in the affected products . Also, fWe initially selected a random sample of subjects through random digit dialling, and asked eligible respondents for voluntary participation in the study. The reasonable level of response for the dietary questionnaire and blood sampling among rural and urban women aged 19–44 years suggests that with caution, we can generalize the results to all Newfoundland women of childbearing age. However, these findings may not be representative of the rest of Canada because of population differences in factors such as genetic background and dietary behaviour. These differences may also affect the generalizability of the NTD trend.The sample response rate for the dietary questionnaire and blood sampling in seniors was approximately 45% both in Phase I and in Phase II. Many of the refusals to participate were due to illness of the eligible person. Furthermore, seniors residing in long term care settings were not included. Thus our sample population of seniors may be healthier than the general population age 65 and over in the province.12 status based on biochemical and haematological indices.The implementation of food fortification with folic acid has been accompanied by a marked decrease (78%) in the rate of NTDs in Newfoundland. The blood folate status of women aged 19–44 years improved following mandatory fortification. There is no evidence of adverse effects of the current levels of fortification on individuals aged 65 years and older. Specifically, there is no evidence to suggest an adverse effect of folic acid fortification on detection of abnormalities in vitamin BBased on these findings, mandatory food fortification with folic acid should continue in Canada at the current levels. Over the time period of this study, the proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased. It was not possible to determine the magnitude of the separate contributions of fortification and supplementation to the decline in NTDs. Therefore, we recommend that public health efforts to promote awareness of the importance of folic acid supplementation among women of childbearing age continue. Ongoing surveillance of NTDs in Newfoundland and other parts of Canada is necessary to determine if the decline in NTD rate is maintained, and to enable further evaluation of prevention strategies. National surveillance of congenital anomalies including NTDs is a critical public health function that should be strengthened where necessary.We look forward to the results of a current epidemiologic study, funded by the Canadian Institutes of Health Research (CIHR), of NTDs in 7 Canadian provinces between 1993 and 2002. There is also research into the relationship between increased folic acid consumption and reduced risk of other congenital anomalies, cardiovascular disease and cancer -45. As tThe authors declare that they have no competing interests.CBC, complete blood countCI, confidence intervalCIHR, Canadian Institutes of Health ResearchFDA, Food and Drug AdministrationHCY, homocysteineMCV, mean corpuscular volumeMMA, methylmalonic acidNTD, neural tube defectRBC, red blood cellSPSS, Statistical Package for Social SciencesSL and CM oversaw the whole study and drafted the manuscript. SL carried out the analysis of NTD rate and statistical analysis. RW, LL, KSO and HS designed the study and carried out the data collection and the dietary assessment. ER, AL and VP participated in the design and carried out the blood analysis. MC carried out the collection of NTD data. All authors read, revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is genuine need to develop interventional treatment options for management of lung tumors. Radiofrequency ablation (RFA) is one such alternative being promoted to treat lung tumors recently. Larger studies should help define RFA's further development. Furthermore fluorodeoxyglucose positron emission tomography (PET) has been reported to be an accurate indicator of treatment response in variety of tumors. This study focuses on the evaluating the feasibility of RFA and usefulness of PET scan in lung tumors after RFA procedure.Between November 1999 and May 2002, 50 patients with primary or metastasis pulmonary tumors underwent RFA procedure. The electrode was guided to the target areas using computerized tomography (CT). Tumors smaller than 3.5 cm were given single RFA, while tumors larger than 3.5 cm received RFA to multiple sites. Maximum 4 lesions or 6 target areas were treated during one operating procedure. Whole body and/or lung PET images were acquired; identical site CT images and chest X-ray were taken 1 week before and after RFA.Of the 50 patients, 17 had single lesions while rest had multiple lesions. Tumors smaller than 3.5 cm were completely dissipated after RFA. In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated. While CT showed that tumor image became larger 1 to 2 weeks after RFA procedure. PET demonstrated tumor destruction in 70% cases, compared to 38% in CT.The present study shows RFA to be safe and effective treatment option for lung tumors. PET is superior to CT in evaluation the effectiveness of RFA treatment shortly after the procedure. Lung cancer continues to be the leading cause of cancer deaths in United States . The oveRadiofrequency ablation (RFA), is an imaging-guided percutaneous ablative procedure, that has been suggested to be an effective treatment option for patients with non-small cell lung cancer (NSCLC) and metastatic disease who are not suitable candidates for surgery ,4. GuidePET has been reported to be an accurate indicator of treatment response in variety of tumors -17. HoweBetween November 1999 and May 2002, 50 patients with either primary or metastatic lung tumors were enrolled in to a prospective single group trial. Patient characteristics are detailed in Table Patients received a chest X-ray and CT for preoperative evaluation and a repeat scan after RFA procedure. A PET scan was performed one week after the treatment.The Radiofrequency ablation was carried out using RF-2000 generator and related software purchased from Radio Therapeutics Corporation, USA; PET imaging was done using an Advance 2 Scanner .Patients received general anesthesia along with local infiltration of Lidocaine. The electrodes were directed to target areas during RFA procedure using CT scan. The initial power applied was 50 W, which was subsequently increased to a maximum 90 W over several minutes. RFA continued for 5 to 15 min until roll off was achieved, which continued for 2 min to stop. Tumors smaller than 3.5 cm were given full heating energy only once, while tumors larger than 3.5 cm received multiple RFA to different areas. Maximum 4 lesions or 6 target areas were treated during one procedure.18F-FDG 296 MBq – 440 MBq (8 mCi -12 mCi) intravenously. After another period of rest lasting for 45–60 min, the whole body and/or lung images was acquired by PET scanner. PET was also acquired at 5–8 bed positions, typically from the base of skull to the mid thigh, which was identical to the CT protocol used in the present study. The complications of the treatment are detailed in table One to two weeks after the procedure and a 4 hour fast, patients were taken for PET scan. They were made to rest for 15 min, and then received After RFA procedure a number of complications were seen. Fever and/or congested pneumonia were commonest complications seen in 32% of patients; however, they were cured in a week with antibiotics treatment. Pneumothorax occurred during procedures in 18% and the patients were treated with aspiration. Five of these had small pneumothorax that did not require and treatment. One patient had hemothorax which required intercostals drainage (ICD) which was removed 2 days later. These were no life threatening events or deaths.Post procedural PET demonstrated the effectiveness of RFA on lung tumors. Tumors smaller than 3.5 cm showed complete response after RFA Figure . In tumoThe Chest X-ray and CT showed that tumor image became larger 1 to 2 weeks after RFA procedure Figure . These mSince RFA ablates lung tumors directly and locally, marginal tissues surrounding the tumor are frequently partially damaged leading to occasional pneumonia. It is difficult for regular CT and/or chest X-rays to discriminate pathological-physiological tissue damage and fibrillation from the treatment effect of RFA. PET on the other hand provides information on functional and metabolic activity anatomically, and is the only available technique which can specifically diagnose tumors or necrosis after surgery and radiotherapy effectively [28]. Our experience too proves that PET is particularly superior to CT in its ability to evaluate the effectiveness of RFA treatment early after therapy.RFA is a relatively noninvasive, well-tolerated approach. It could destruct tumor completely within the effective diameter while avoiding the surgery, side effects of radiotherapy and toxicity of high dose chemotherapy. Our observations suggest that RFA can kill lung tumors smaller than 3.5 cm after a single RFA procedure. The effect of RFA appears to be limited within 3.5 cm diameter area with the current instruments. However, this also suggests that RFA may not damage the normal tissues surrounding the small tumors. The malignant lesions dissipated in 1 to 2 weeks, while the surrounding tissue stayed intact. While at this period regular chest CT and chest X-ray may show enlarged lesion images. This is in agreement with other reports. With improvements in technology, RFA in combination with other options may further reduce the morbidity and mortality of cancer deaths . Though Despite inherent deficiency of trial design our single group study clearly demonstrates that RFA can be an effective treatment option for lung tumors. Unlike other interventional techniques, RFA provide controlled regions of coagulation necrosis with a single application to an area with 3.5 cm diameter. RFA may cure small lung tumor, reduce tumor burden in larger lesions and may be combined with external beam radiation and/or systemic chemotherapy for further improvements. PET provides functional and metabolic activity anatomically and is particularly superior to CT in evaluation the effectiveness shortly after RFA procedure. Absence of follow-up information and randomization in the current study are two major fallacies which need to be addressed in subsequent studies.SJK is the leading physician and drafted the manuscript.RL, WL, HW, XZ, YM all participated in the study, patient management, literature search and preparation of manuscript.All authors have read and approved the final version of the manuscript.None declared.


The ACE gene contains a polymorphism consisting of either the presence  or absence  of a 287 bp alu repeat in intron 16. The D allele is associated with increased ACE activity in both tissue and plasma. The DD genotype is associated with risk of developing ARDS and mortality. The frequency of the D allele is higher in patients with pulmonary fibrosis, sarcoidosis and berylliosis. The role of this polymorphism has not been studied in the development of BPD in the premature newborn.ACE I/D genotype was determined in 245  mechanically ventilated infants weighing less than 1250 grams at birth and compared to outcome (death and/or development of BPD).2 on 28 days or 36 weeks PCA). Secondary outcomes  similarly were not influenced by the ACE ID polymorphism.The incidence of the D allele in the study population was 0.58. Eighty-eight (35.9%) infants were homozygous DD, 107 (43.7%) were heterozygous ID and 50 (20.4%) were homozygous II. There were no significant differences between genotype groups with respect to ethnic origin, birth weight, gestation, or gender. There was no effect of the ACE I/D polymorphism on mortality or development of BPD (OThe ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams. Prematurely born infants who require mechanical ventilation (MV) frequently develop chronic lung disease known as bronchopulmonary dysplasia (BPD). Oxidant injury, mechanical disruption of the lung, inflammation and subsequent failure of lung development are considered the major mechanisms in the pathogenesis of BPD. The development of BPD is characterized by an initial acute inflammatory component followed by variable degrees of lung fibrosis and failure of alveolar septation, both of which ultimately impair the development of the immature lung -5. The iThere is increasing evidence to support the role for the activation of the renin-angiotensin system (RAS) system during ALI. In ARDS there is an increase in bronchoalveolar lavage and a concomitant decrease in circulating angiotensin converting enzyme (ACE) activity,7. This There is significant variation in circulating ACE activity among individuals, which may be genetically determined. The human ACE gene is located on chromosome 17 (17q23) and contains a polymorphism consisting of either the presence  or absence  of a 287 bp alu repeat in intron 16. The delBecause of the relationship between inflammation and activation of the RAS and the association with lung fibrosis and adverse outcome in ARDS, the ACE l/D polymorphism may modify risk for the development of chronic lung disease or death in mechanically ventilated VLBW infants. The purpose of this study was to determine if there is a relationship between the ACE I/D polymorphism and respiratory outcomes of death or the development of BPD, in mechanically ventilated very low birth weight (VLBW) infants.Genomic DNA used for this case controlled study was extracted from archival tracheal aspirate (TA) pellets 223 patients) or blood (22 patients) collected prospectively as part of an ongoing study of genetic factors in the development of complications of prematurity. The TAs that were used as a source of genomic DNA were originally collected as part of long term longitudinal studies examining cytokine concentrations and the development of CLD[ patientsCultures for genital mycoplasmas were performed on samples collected in the first few days of life. Clinical and outcome data were abstracted from the clinical record and included information on respiratory outcome, survival and development of complications of prematurity.Isolation of total DNA from blood or TA pellets was performed using the QIAmp DNA Mini kits™ . Briefly, TA pellets were suspended in 200 μl of sterile phosphate buffered saline by vigorous vortexing, then digested in proteinase K and applied to silica gel spin columns. Columns were washed in the manufacturer's supplied buffers and the total DNA was eluted in 200 μl elution buffer. Blood (200 μl) was extracted similarly to the TA pellets.2 1.5 mM, KCl 50 mM, 5%DMSO, Triton X-100 0.1%, 200 micromolar each of dNTPs, 10 pmol each primer, 1–2 U of Taq polymerase, and 1 microliter of DNA. DNA was denatured at 95°C for 5–10 min, followed by 30 cycles of denaturation at 94° for 60 sec, annealing at 67 for 60 sec, and extension at 72° for 2 minutes, with a final extension at 72° for 7 min. PCR products were separated on 2% agarose gel containing 0.5-microgram/ml ethidium bromide. After gel electrophoresis the bands were visualized by UV-transillumination. The PCR product is a 190 bp fragment in the absence of the insertion (D genotype) and a 490 bp fragment in the presence of the insertion (I genotype).ACE I/D polymorphism was performed by microplate PCR method as described previously. . Tracheal aspirate cultures obtained during the first few days of life grew Ureaplasma urealyticum (Uu) on at least one occasion from 74 of 217 infants cultured (34%) and Mycoplasma hominis (Mh) from 29 (13%). One hundred and fifty one (67%) infants were oxygen dependent at 28 days and 55 (25%) were oxygen dependent at 36 weeks PCA. There were 39 (16%) patients who died  during their initial hospitalization (24 before 28 days of age and 15 after 28 days).Two hundred and forty five (245) patients had complete clinical information on respiratory outcome and genomic DNA available for genotyping. Mean gestational age and birth weight of the study population was 26.4 ± 0.1 weeks and 869 ± 12 grams respectively. One hundred and ninety-four (79%) infants were African-American, 47 (19 %) were Caucasian and 4 (2%) were Hispanic. Male: female ratio was 147:98. All patients required MV at birth and 228 (93%) infants were treated with exogenous surfactant therapy  and Caucasian infants (0.59) (p = 0.664). Fifty (20.4%) infants were homozygous II, 107 (43.7 %) were heterozygous ID and 88 (35.9%) were homozygous DD.Baseline clinical characteristics of birth weight, gestational age, race, gender, TA isolation of Mh, and the need for surfactant replacement were not different between genotype groups Table . IsolatiClinical characteristics of surviving infants who were or were not oxygen dependent at days are shown in Table Because the ACE I/D polymorphism may have different functional effects on plasma and tissue ACE activities in different ethnic groups, we analyzed the effects of ACE on the incidence of BPD separately for Caucasian and African-American infants. (The effects of the ACE I/D polymorphism on the incidence of BPD and other outcomes for the combined group can be found in the online data supplement). Table The effects of ACE genotype on outcome in ventilated African-American infants less than 1250 grams are shown in Table Since birth weight and gestation are the primary determinants of adverse outcome, analysis was repeated in both infants greater than 750 grams and infants ≤ 750 grams. There was no significant effect of ACE genotype when this subgroup analysis was performed (data not shown).The RAS is activated during lung injury and plays a role in several pathological processes. In addition to the pulmonary endothelium, respiratory epithelium also possesses significant ACE activity. Fas-induced alveolar epithelial cell apoptosis is dependent on local AT-II production and interaction with it receptor.,11 FurthThe frequency of the D allele in our study population was not different than reported in our local population or for other groups,24,31. TIn our study population, the ACE ID polymorphism was not associated with an altered risk for the development of BPD, oxygen dependency at 28 days, death  or composite outcomes (BPD or death) suggesting that factors other than genetic variation at this locus contribute to these outcomes. This is in contrast to that seen in adults with ARDS or other lung disorders such as pulmonary fibrosis, sarcoidosis and berylliosis where the D allele is associated with disease -21.1 +915 G/C SNPs on the development of BPD[Indeed genetic factors may not have a large influence in a disease (BPD) where maturity at birth has such an overwhelming influence. Our previous study in this population failed to find any association of the tumor necrosis factor-α-308 G/A, MCP-1-2518 A/G or transforming growth factor-βnt of BPD AdditionUreaplasma urealyticum colonization in these infants. Chorioamnionitis, which is highly correlated with isolation of Uu, is a risk factor for PVL.[The finding of increased incidence of PVL in Caucasian infants with the II genotype may be due more to the greatly increased incidence of  for PVL. Because Our study has several limitations. The ACE I/D polymorphism has only been demonstrated to be functional in Caucasians. Data linThe observations of this retrospective case controlled study are also limited somewhat by selection bias. Because only infants who were mechanically ventilated were included, the true impact of this polymorphism on the complications of prematurity may be underestimated. Since this was a retrospective study using stored material no attempt was made to correlate phenotype with genotype (ie TA ACE measurements).Lung injury and the subsequent maladaptive repair process that leads to the development of BPD is complex with a great many factors that interplay to determine outcome. It is very likely that other genetic play a role in determining the risk of poor outcome. Polymorphisms in cytokines, their receptors, bacterial pattern recognition molecules, surfactant proteins, and heme oxygenase-1 are known to alter the course of other pulmonary diseases and should be examined as to their potential role in the development of BPD in the premature infant.The ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams.The author(s) declare that they have no competing interests.RJB conceived and organized the study, prepared the manuscript and performed the statistical analyses. KY performed the genotyping, and assisted with manuscript preparation. JL oversaw collection tracheal aspirates, assisted with recruitment of subjects into the original studies, and assisted with editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Contains Tables showing baseline clinical characteristics of African American and Caucasian infants separately by Genotype groupClick here for file