Reduced placental perfusion predisposes to the maternal syndrome pre-eclampsia characterized by systemically reduced perfusion. Considerable data support the role of angiogenic factors in the development of the maternal syndrome. Hypoxia-inducible factor (HIF-1) mediates the cellular responses to hypoxia e.g. by promoting angiogenesis.HIF1A gene, are associated with pre-eclampsia. We studied 108 women with pre-eclampsia in their first pregnancy, and 101 controls with normotensive pregnancies. Pre-eclampsia was defined as a blood pressure level of at least 140/90 mmHg in a woman who was normotensive before 20 weeks of gestation, and proteinuria at least of 0.3 g per 24-hour urine collection. The patients and controls were genotyped for variations in the exon 12 of HIF1A gene by sequencingHere we studied whether two single nucleotide sequence variants, c.1744 C>T that changes residue 582 of HIF-1α from proline to serine (P582S) and c.1762 G>A that changes residue 588 of HIF-1α from alanine to threonine (A588T) in the exon 12 of the The frequencies of the c.1744 C>T and c.1762G>A sequence variants were not significantly different between women with pre-eclamptic first pregnancies and women with normotensive pregnancies. In addition, two synonymous variants (c.1740G>A and c.1800A>T) were detected at comparable levels in the two groups. All variants were identified in the heterozygous form.HIF1A gene were not associated with pre-eclampsia in the Finnish population.The sequence variants in the exon 12 of the  Pre-eclampsia, a pregnancy-specific vascular disorder, complicates 3% of pregnancies, and it may threaten the survival of both mother and baby . The onsAbnormalities in the angiogenic balance have been proposed to as having a major role in the molecular cascade causing maternal endothelial dysfunction and systemically reduced perfusion in pre-eclampsia . HypoxiaHIF1A gene cause amino acid substitution within (rs11549465) or near (rs11549467) the minimal N-terminal transactivation domain (N-TAD) within the ODD that mediates interactions with the von Hippel-Lindau protein, which in turn targets HIF1-α for degradation  blood pressure (BP) level at least 140/90 mmHg, and 2) proteinuria at least 0.3 g per 24-hour urine collection . The bloThe blood samples were collected between January 1997 and April 1998 from women who, according to discharge records of the Helsinki University Central Hospital, had had severe pre-eclampsia between 1988 and 1998. The control samples were collected from women with uncomplicated pregnancies who gave birth at the Helsinki University Central Hospital. Patients and controls are of Finnish (Caucasian) origin. This study has been approved by the local ethical review committee and an informed consent was obtained from all the study subjects. The clinical characteristics of the patients are presented in Table HIF1A gene was performed using forward and reverse primers 5'-CAGAAGCAAAGAACCCAT-3' (F) and 5'-TCAAGAATTTGCGTTAG-3' (R), described by Resar et al. (2005) [2, 50 mM KCl and 0.1% Triton X-100, 10 nmol of each nucleotide (dNTP) and 0.75 U Dynazyme polymerase-enzyme . Polymerase chain reaction conditions were as follows: 4 min at 94°C followed by 35 cycles of denaturation step: 30 s at 94°C; annealing step: 30 s at 49°C; elongation step: 30 s at 72°C; and final extension for 10 min at 72°C terminated the reaction after final annealing. Amplifications were performed in a DNA 2720 Thermal Cycler .Genomic DNA was extracted from 10 ml of peripheral blood using a phenol-chlorophorm method. PCR amplification of the exon 12 of the . (2005) . The reaSequencing of the PCR amplicons were performed using Big Dye Terminator kit (version 3.1) supplied by Applied Biosystems . The reactions were run on an ABI 3730 capillary sequencer according to the manufacturer's instructions.. Our sample size has 80% power to detect a relative risk 2.2 for the rs11549465 variant, and a relative risk 2.7 for the rs11549467 variant in the HIF1A gene at a significance level α = 0.05. Statistical analyses were performed using GraphPad Prism 4 . The association between the polymorphisms and pre-eclampsia was estimated by comparing the frequencies in the two study groups. The statistical significance of the different proportions was measured using Fisher's exact test. Parametric and non-parametric tests were used as appropriate for the continuous clinical data. Differences were considered as statistically significant for P values <0.05. To determine if genotype frequencies deviate from the the Hardy-Weinberg Equilibrium (HWE) a Χ2 test was performed .The minor allele frequencies of the r rs11549465 and rs11549467 are 0.092 and 0.018, respectively, in the CEPH population (Utah residents with ancestry from northern and western Europe) HIF1A gene and the flanking exon-intron boundaries of HIF1A four exonic sequence variants were detected. All the variants in both patients and controls were detected in a heterozygous state. All four SNP genotype frequencies in both patients and controls were in agreement with Hardy-Weinberg equilibrium (p > 0.05.)As a result of sequencing exon12 of the HIF1A gene.No statistical differences were shown when comparing the frequencies of the non-synonymous sequence variants, rs11549465 and rs11549467 between patients and controls. Table In addition to two non-synonymous sequence variants, we found two synonymous sequence variants Figure . VariantHIF1A gene was not different between women with pre-eclamptic first pregnancies and controls with normotensive pregnancies. We also found two synonymous sequence variants in the exon 12 of HIF1A gene: rs34005929  and variant c.1800 A>T (Thr600Thr) which has not previously been reported in public databases.In this study performed in the genetically homogenous Finnish population, we found that the allele distribution of the two non-synonymous sequence variants, rs11549465  and rs11549467 , in the exon 12 of the HIF1A an interesting gene in contributing the maternal response to reduced placental perfusion.HIF-1α is known to play role in the normal development and pathology of placenta as a regulator of responses to hypoxia. Accumulation of HIF-1α protein in the pre-eclamptic placentas occurs as a consequence of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation due to proteosomal dysfunction . OverexpPre-eclampsia originates in the placenta but the target organ is maternal endothelium. Endothelial dysfunction, a central feature in pre-eclampsia, has been suggested to be a part of a more generalized inflammatory reaction . Innate HIF1A is one of the genes of importance in the pathways mediating the response to ischemia. The rs11549465 sequence variant was more common in patients who presented with stable exertional angina rather than acute myocardial infarction [HIF1A could reduce plaque neovascularization and the risk of intraplaque hemorrhage and of subsequent acute myocardial infarction [Pre-eclampsia increases the risk of future ischemic heart disease . This cofarction . These afarction .HIF1A gene in different tissues as well as, the effect of these variants in angiogenesis are inconclusive. The rs11549465 and rs11549467 variants of the HIF1A gene have been shown to have enhanced transcription activities in in vitro studies under both normoxic and hypoxic conditions [LDH-5, VEGF, GLUT1) was not increased in the presence of the rs11549465 variant in non-small cell lung cancer samples studied by immunohistochemistry [The results of the functional studies of the sequence variants in the nditions ,27. Rs11nditions , and in hemistry . In the hemistry .HIF1A gene are associated with the pre-eclamptic phenotype.To the best of our knowledge this is the first study to investigate whether the two non-synonymous sequence variants that lie within or near the N-TAD within the ODD in the exon 12 of the maternal These sequence variants are interesting because polymorphisms that reduce the activity of HIF-1α as a transcriptional activator could underlie pre-eclampsia by causing inadequate placental vascularisation in the early pregnancy which later leads to hypoxia in the placenta. We are not aware of studies comparing the frequency of these sequence variants between the pre-eclamptic placentas and placentas from normotensive pregancies, or studies measuring the HIF-1α levels in placentas heterozygous of homozygous for these sequence variants.HIF1A on the pathogenesis of maternal and placental pre-eclampsia. Pre-eclampsia involves complex interaction between placentation, vascular function and maternal metabolism. Thus, research and recognition of genes, such as HIF1A, and thereby biochemical components critical for such interactions increase our understanding of the molecular mechanisms leading from cellular hypoxia to metabolic changes.This retrospective study has several limitations. First, we did not have fetal DNA samples. Consequently, we were neither able to study the effect of fetal/placental genotype nor the maternal fetal genotype interaction. The other limitation is inadequate statistical power to detect small genotypic effects. Under a dominant model, the sample size provides 80% power to detect a genotype relative risk over 2.2, which is much higher than the risk conferred by the majority of susceptibility genes detected to date for complex disorders. For detection of small genotypic effects studies in larger sample sets are warranted. Further studies are also warranted to elucidate effects of the functional polymorphisms in HIF1A gene are not associated with pre-eclampsia in the Finnish population. The study has inadequate statistical power to detect small genotypic effects. Therefore, this gene should be assessed in bigger studies. Further studies are also warranted to elucidate possible fetal genotypic effects.Our data suggest that the sequence variants in the exon 12 of the SA and HL participated in the study design. MK conducted the sequencing. SH, MK and HL analysed the results. SH, MK, and HL wrote the first draft, and all authors the final version of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


ACE are associated with the plasma ACE concentration. The ACE is associated with the angiogenesis of ovarian endothelium in vitro as well as steroidogenesis and follicular growth in cattle. Since ACE induces a high blood supply and hypersteroidogenesis in the ovary, it may be associated with polycystic ovary syndrome (PCOS) which exhibits hyperplasia, hypervascularity of the ovarian theca interna and stroma, as well as disorderd steroidogenesis. Therefore, we hypothesized that the ACE plays some roles in the human ovary. To investigate whether the ACE I/D polymorphisms are associated with the steroidogenesis disorder in PCOS and contribute to the susceptibility of PCOS in Chinese women, we designed a case-controlled association study in 582 individuals.I/D polymorphisms of ACE I/D polymorphisms were assessed in 582 reproductive-age women. Genotyping and frequency of ACE I/D polymorphisms were obtained by PCR amplification that was performed on genomic DNA isolated from blood leucocytes. Results were analyzed in respect to clinical test results.The P = 0.458). However, there were significant differences in the concentrations of testosterone among three genotypes both in the PCOS patients and controls . Differences were also found between these groups with distinct genotypes: DD versus II and DI versus II in the PCOS patients as well as DD versus DI and DD versus II in the controls. There were significant differences in the ratio of LH/FSH among three genotypes in the patients (P = 0.01). However, there were no statistical differences in the BMI, AAM, E2 concentrations and other serum hormone concentrations among the three genotypes both in the PCOS patients and controls.The frequencies of the D allele and the genotypic distributions  in the women with PCOS did not differ from those in controls (ACE I/D polymorphisms were not associated with the pathogenesis of PCOS. However, the polymorphisms were associated with the steroidogenesis in the ovary. The observation indicated that the ACE I/D polymorphisms were not the key etiological factor, which in stead may be associated with the aggravated clinical manifestations of PCOS.The  ACE gene, is one of the components of RAS and can be expressed in multiple tissues including ovaries [ACE gene, the so-called ACE I/D polymorphism. Thus researchers use the I/D polymorphism as a valid marker for studying the associations between the ACE gene polymorphism(s) and pathophysiological conditions.There is evidence to indicate that the RAS may influence oocyte maturation, ovulation and steroidogenesis as well as formation of corpus luteum through complex interactions with other systems . ACE, en ovaries . In addi ovaries  and the  ovaries ,5, stero ovaries , and fol ovaries  in cattl ovaries  demonstrPCOS, present in 5%–10% of reproductive-age women, is a constellation of hyperandrogenism, hyperinsulinism ,10, mensACE genotype is associated with the abnormal levels of hormones in PCOS patients, we designed a case-control experiment including 346 PCOS patients and 236 non-PCOS individuals as controls.Patients with PCOS not only have the clinical characteristics of oligomenorrhoea, hirsutism, infertility and hyperinsulinemia but also exhibit obesity, hypertension, dyslipidemia, and an increased pro-thrombotic state ,11. TheyA total of 582 women were studied. 346 of them were patients with PCOS and 236 were non-PCOS women as controls. The controls were selected by excluding the diagnosis of PCOS according to the 2003 Rotterdam Criteria and exhibiting normal menstrual cycles (<32 days). All the individuals in the control group were without obesity, hirsutism (the F-G hirsutism score ≥ 7), acne, or overmuch sebum. All the women recruited for the study were from the Chinese Han ethnic group. The study was approved by Medical School of Nanjing University, and informed consent was obtained from every study participant.Patients with PCOS were diagnosed by the 2003 Rotterdam Criteria . The Rot®-100 as a medium . For each subject, the presence  or absence  of the 287-bp Alu repeat in intron 16 of the ACE gene was determined by measuring the size of DNA fragments after polymerase chain reaction (PCR) amplification. The PCR conditions were as described by Brigitte Rigat et al. [Genomic DNA was extracted from venous blood samples using Chelext et al. , using aACE alleles and genotypes among the patients and controls were obtained by direct enumeration based on the PCR results, and the deviation from Hardy-Weinberg equilibrium was evaluated using the χ2-test. Genotypic distributions between the patients and controls were compared by χ2-test of the 2 × 3 tables. The analysis was performed using the SAS system software . The results of serum hormone levels were reported as MEANS ± SD. The regression and correlation tests were used to analyze the association of age or BMI and the concentrations of the hormone. Differences in serum hormone levels among different genotypic individuals were assessed by using one way analysis of variance (ANOVA). Tukey-test was used for further analysis of the differences among the three genotypes, which was performed using the SAS system software. P < 0.05 was considered statistically different.The frequencies of ACE genotypic distributions were 28.32% (DD), 46.24% (ID), 25.43 (II) in the patients, and 32.63% (DD), 45.34% (ID), 22.03% (II) in the controls, respectively. In both the patient and control groups the genotypic distributions were in agreement with Hardy-Weinberg equilibrium . No significant difference was observed in the frequency of the genotypic distributions between the patients with PCOS and the non-PCOS controls (P = 0.458)  (Table P = 0.01). In contrast, no statistical differences in the concentration of estradiol (E2) were observed among the three genotypes in both patients and the controls  Table . Furtherls Table . We founACE gene had no influence on the occurrence of PCOS. No significant differences were observed in the distribution of the genotypes between the patients and the controls. Regardless of the criterion used for assessing patients of PCOS  morphology on ultrasound, and clinical or laboratory evidence of hyperandrogenism), no differences were observed among the three genotypes. These results suggested that the D allele of the ACE gene was not a major etiological factor for PCOS. Our current finding is contrary to the observation reported by Yunxia Cao et al [In this case-control study the D allele of the ao et al ,19 in a ao et al . We trieao et al .P < 0.05 respectively). The DD genotype was associated with a higher level of ACE than either the ID or II genotype [ACE gene may directly or indirectly contribute to the expression of the testosterone in the ovaries. There can be a number of explanations for the phenomenon: firstly, the ACE is associated with the angiogenesis of ovarian endothelium in vitro [Although there were no significant differences in the distributions of the genotypes between the patients and the controls, we observed significant differences in the concentrations of testosterone among the three genotypes both in the patients and the controls Table . By furtgenotype , which iin vitro . Althougin vitro ,22 and tin vitro . If the in vitro  and fromin vitro ,26. The ACE I/D polymorphisms is only one of the co-factors that affects the etiopathogenisis of PCOS by interacting with other factors. The I/D genotype is more susceptible to hyperandrogenism.An interesting finding was that the total T concentrations between the I/D and D/D genotypes in the controls were different, whereas in the PCOS patients such concentrations between the I/D and I/I genotypes were different. One interpretation of this finding is that the I/D is a susceptible genotype in the population of hyperandrogenism. In the controls, the I/D and I/I genotypes demonstrate no differences in the concentrations of T. However, according to the prior studies, these two genotypes have different concentrations of ACE. Two possibilities may account for this observation: 1) the concentration of T is not associated with ACE; 2) ACE is not the key factor that influences the concentration of T. However, in the PCOS patients the total T concentrations between the I/D and I/I genotypes were different, which cannot be explained if ACE is not associated with the concentration of T. The most probable reason may be that the ACE gene was not associated with the pathogenesis of PCOS. However, the polymorphisms were associated with the steroidogenesis in the ovary. This suggests that although the I/D polymorphisms in the ACE gene were not the key etiological factor, it may be associated with the aggravated clinical manifestations of PCOS.The I/D polymorphisms in the I/D Polymorphism: Insertion/Deletion Polymorphism; ACE: Angiotensin-converting Enzyme; RAS: the renin-angiotensin system; PCOS: Polycystic Ovary Syndrome; LH: luteinizing hormone; FSH: follicle-stimulating hormone; E2: estradiol; BMI: body mass index (body weight in kilograms divided by square of height in meters); AAM: age at menarche; T: testosterone; P: progesterone; RIA: radio-immunity assay; PCR: polymerase chain reaction; PCO: polycystic ovarian; Ang II: Angiotensin II.The authors declare that they have no competing interests.JS, HF, FL and CY carried out DNA extraction and the molecular genetics experiments. JS, LY, YW performed the statistical analysis and drafted the manuscript. YC, XW, HF and SH participated in sample collection. LY participated in the designing of the study and helped to carry out the molecular genetics experiments. YW and YC conceived and directed the study, and helped to draft the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Preeclampsia is a major cause of maternal morbidity and mortality worldwide. Despite decades of research into the condition, the ability of clinicians to predict preeclampsia prior to the onset of symptoms has not improved significantly. In this review, we will examine the pathophysiology underlying preeclampsia and will look at potential biomarkers for early prediction and diagnosis. In addition, we will explore potential future areas of research into the condition. Preeclampsia is a multisystem disorder of pregnancy, which complicates 3%-5% of pregnancies in the western world. It is a major cause of maternal morbidity and mortality worldwide. The cardinal clinical features of the condition are hypertension and proteinuria occurring after 20 weeks gestation in women who were not previously known to be hypertensive. Other signs and symptoms include edema and headache, and in severe cases, the condition is associated with seizures (eclampsia), liver, and kidney dysfunction as well as clotting abnormalities, Adult Respiratory Distress Syndrome and fetal growth restriction (FGR) .The cause of preeclampsia remains unknown, and the only known cure is delivery of the fetus and placenta. Various agents have been evaluated to see whether they influence the development of preeclampsia. Aspirin has been studied extensively; it was summarized in a Cochrane review by Despite decades of research into the condition, predicting which women are at increased risk of developing preeclampsia remains problematic. Identifying “at-risk” women is an important aim; because modern obstetric care places emphasis upon the primary care setting for expectant women, a marker which identified high-risk women would allow for closer supervision in secondary care. Such a marker would also facilitate recruitment for trials of potential therapeutic agents, for accurate diagnosis, and for timely intervention whenever problems develop. Furthermore, predicting preeclampsia in women with underlying conditions such as diabetes and chronic hypertension would be of great clinical value.Clinicians have traditionally relied on maternal risk factors, such as increased maternal age, family history, and preexisting diseases, for determining which women are at increased risk. The problem with using these risk factors is that millions of women worldwide have these risk factors but do not develop preeclampsia. Moreover, the majority of them are nonmodifiable. There have been many screening tests evaluated in the literature over the years for predicting preeclampsia; these have been comprehensively reviewed in a World Health Organization publication  Figure . A furthThe imaging technique that has so far been most widely used for predicting preeclampsia has been uteroplacental Doppler ultrasound. Impaired placental perfusion, one of the hallmarks of preeclampsia, can be assessed by measuring flow waveform ratios or by detecting diastolic notching of the uterine arcuate vessels. Uterine Doppler studies can, however, be inconsistent: different types of machine, different gestational age at assessment, and different definitions of abnormal flow velocity waveforms make accurate comparison of studies difficult. A large systematic review of 43 studies involving 40,000 patients  found thAttention has therefore turned in recent years towards identifying maternal markers of placental dysfunction which are raised in women who go on to develop preeclampsia. In this review, we will look at novel biomarkers which have been used in the prediction of preeclampsia and explore potential future areas of investigation.Although the cause of preeclampsia remains elusive, the origin of the condition is recognized as lying in the placenta. This is known to be the case because preeclampsia occurs only in the presence of pregnancy, it resolves after delivery of the placenta and it can occur in the absence of a viable fetus, for example, in molar pregnancies. Placental development is a closely regulated process which is essential for normal fetal development and for maintaining a successful pregnancy. Blood supply to the placenta is via the spiral arteries, which, in turn, are branches of the uterine arteries. Early in normal pregnancy, the cytotrophoblastic cells of the developing placenta invade the uterine wall, disrupting the endothelium and tunica media of the spiral arteries. The vascular wall of the spiral arteries is remodeled; this, in turn, leads to a transformation of the spiral arteries from low-flow, highly resistant vessels into the high-flow, low-resistance vessels which are vital for normal placental development.There are thought to be two stages to cytotrophoblastic invasion: the first involves invasion of the decidual segments of the spiral arteries, at around 10-12 weeks gestation; the second involves invasion of the myometrial segments at 15-16 weeks . In preeAs research in the field of preeclampsia progresses, much of the attention in recent years has been focused on peptides related to angiogenesis. Angiogenesis, the development of new blood vessels from existing endothelium, is essential for normal placental development. Two of the angiogenic growth factors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are thought to contribute to normal trophoblastic proliferation and implantation , and it Soluble FLT-1  is a secreted splice variant of FLT-1. It binds to and neutralizes the angiogenic actions of VEGF and PlGF  and is tβ1 and β3 (Another peptide that has been implicated in the pathogenesis of preeclampsia is soluble endoglin (sEng). Endoglin, a coreceptor for transforming growth factors 1 and β3 , is high1 and β3 . In norm1 and β3 . Consist1 and β3 . A promi1 and β3 . Rana etPlacental protein 13 (PP-13) is a 32-kDa dimer protein, one of a group of proteins which are known to be highly expressed in the placenta. It has been prepared in recombinant form and is thought to be involved in placental implantation and maternal vascular remodeling . During Pregnancy-associated plasma protein A, (PAPP-A) is a large highly glycosylated protein complex produced by the developing trophoblast , which iInsulin resistance has long been implicated in the pathogenesis of preeclampsia. Carbohydrate metabolism is known to be altered in women with preeclampsia, whereas fasting insulin levels have also been shown to be elevated prior to the onset of disease . Furtherβ cells, and, following initially increased insulin sensitivity, there follows a progressive increase in insulin resistance throughout the second and third trimesters (Normal pregnancy is characterized by increased insulin secretion by the pancreatic imesters . Sex-horimesters  and in pimesters . One stuimesters . In contimesters . This reimesters , althougAdiponectin, an adipocyte-derived cytokine involved in carbohydrate and fat metabolism, is another protein whose levels are inversely correlated with insulin resistance. High concentrations of adiponectin have been shown to be protective against the development of type 2 diabetes , and serAPOE gene on chromosome 19 has 3 common alleles, encoding 3 plasma ApoE isoforms, e2, e3, and e4. ApoE e4 is a known risk factor for familial Alzheimer's disease, whereas both e2 and e4 isoforms have been associated with abnormally high triglyceride and VLDL levels (One of the mechanisms by which preeclampsia has been postulated to develop is via abnormal lipid metabolism associated with oxidative stress. Women with preeclampsia have an abnormal lipid profile, with elevated concentrations of triglyceride-rich lipoproteins, which may contribute to endothelial dysfunction . ApolipoL levels . It has L levels . The rolMany studies have been reported using inhibin A and activin A as predictors of preeclampsia. Both are glycoproteins, are members of the transforming growth factor β family, and during pregnancy, are largely released by the fetoplacental unit. Inhibin A has an important endocrine role in the negative feedback of gonadotrophins, whereas activin A is thought to have activity in various biological tissues . In normThe cause of these rises in activin A and inhibin A is not yet fully understood; and whether these hormones have a role in the etiology of preeclampsia is not yet known. Further prospective studies, including measurement of urinary inhibin A, are clearly required.A number of gene polymorphisms have been found to be associated with the risk of developing preeclampsia. A recent well-powered study into the genetics of preeclampsia, however, did not confirm significant associations between a single nucleotide polymorphisms in candidate genes and preeclampsia . Recent Further advances are expected from proteomic research. Proteomics has been defined as “knowledge of the structure, function, and expression of all proteins in the biochemical or biological contexts of all organisms” . CompariIt appears likely that the development of preeclampsia involves several different pathophysiological mechanisms, as evidenced by the diversity of the peptides that have been studied to date. Current research is driven by pathways that are known to be involved in the development of the condition, and one advantage of proteomics-based research is that many of these mechanisms can be integrated. In addition, since proteomics is a “hypothesis-free” research tool, it is likely that this line of investigation will open new avenues for potential biomarker discovery and for new diagnostic and preventative measures.Metabolomics is a further area that has the potential to contribute significantly to future research in preeclampsia. In complement to studies examining the human genome and proteome, metabolomics can be defined as a “systematic study of the unique chemical fingerprints that specific cellular processes leave behind” . In commAlthough improvements in obstetric and neonatal care have led to a reduction in morbidity and mortality from preeclampsia, our ability to predict the condition has not improved significantly. We currently rely on “secondary prevention” of preeclampsia: women who have previously had the disease are closely monitored throughout pregnancy. The majority of women who develop preeclampsia, however, are only diagnosed once they have developed the full-blown manifestations of the condition, by which time treatment options are limited. Rather than being a separate condition, preeclampsia has been previously described as the extreme end of a maternal systemic response engendered by pregnancy itself . We haveAn increased understanding of the molecular mechanisms underlying preeclampsia has led to several potential areas of investigation. In addition to studies mentioned above, novel biomarkers such as urine orosomucoid, an acute phase protein, show promise in early prediction of preeclampsia . To date


C-reactive protein is a well established marker of inflammation and has been used to predict future cardiovascular disease. It is still controversial if it plays an active role in the development of cardiovascular disease. Recently, polymorphisms in the gene for HNF1α have been linked to the levels of C-reactive protein and coronary artery disease.We investigated the association of the rs2259816 polymorphism in the HNF1A gene with the circulating level of C-reactive protein and the hazard of coronary artery disease in the LURIC Study cohort.Compared to CC homozygotes, the level of C-reactive protein was decreased in carriers of at least one A-allele. Each A-allele decreased CRP by approximately 15%. The odds ratio for coronary artery disease was only very slightly increased in carriers of the A-allele and this association did not reach statistical significance.In the LURIC Study cohort the A-allele of rs2259816 is associated with decreased CRP but not with coronary artery disease. C-reactive protein (CRP) is a well established biochemical marker of inflammation and has been used to predict future cardiovascular disease -3. As itA recent meta-analysis has shown that associations of CRP with ischemic vascular disease depend considerably on conventional risk factors and other markers of inflammation making a causal role of CRP in the development of CAD also unlikely .Genetic factors have been estimated to have a great influence on the variance in plasma CRP level -20. A nuRecently, polymorphisms in the HNF1A gene  have been linked to the levels of C-reactive protein and coronary artery disease -36. ThisIn our study we attempted to confirm the reported association of rs2259816 to CRP and CAD in the LURIC Study cohort.Ludwigshafen Risk and Cardiovascular Health (LURIC) study includes consecutive white patients hospitalized for coronary angiography between June 1997 and May 2001. A detailed description of LURIC has been published [The ublished . The stuCAD has been defined angiographically using the maximum luminal narrowing estimated by visual analysis. CAD was defined as the presence of a visible luminal narrowing (> 20% stenosis) in at least one of 15 coronary segments according to a classification of the American Heart Association . IndividAmong the 3113 individuals in whom rs2259816 genotypes were available, 665 (21.4%) had no angiographic CAD while 2448 (78.6%) had CAD.Diabetes mellitus was diagnosed according to the criteria of the American Diabetes Association . FurtherFasting blood samples were obtained by venipuncture in the early morning. 'Sensitive' C-reactive protein was measured by immunonephelometry on a Behring Nephelometer II  after completion of the patient recruitment in 2001 in samples stored at -80°C. In the C-reactive protein assay used, the limit of detection for C-reactive protein is 0.17 mg/L; it is linear up to 500 mg/L. The lowest and the highest C-reactive protein concentrations encountered in this study were 0.17 and 269 mg/L, respectively. Blood glucose was determined enzymatically using the hexokinase/glucose-6-phosphate dehydrogenase method . Lipoproteins were separated by a combined ultracentrifugation-precipitation method (β-quantification) . Cholest®). Primer and probe sets were designed and manufactured using Applied Biosystem's 'Assay-by-Design' custom service.Genomic DNA was prepared from EDTA anticoagulated peripheral blood by using a common salting-out procedure. The C/A polymorphism rs2259816 was genotyped by a 5' exonuclease assay . CRP was not normally distributed and had to be logarithmically transformed before performing ANOVA. Co-variables were used as indicated.In models assuming a co-dominant (additive) effect of the alleles, genotypes were coded as 0, 1, and 2, respectively, and genotypes were either treated as interval-scaled or categorical variables, the most frequent genotype being considered as the reference category in the latter case. When assuming a dominant effect, the genotype CC was coded as 0, and the combined remaining ones were coded as 1. When assuming a recessive effect, the genotypes CC and AC were coded as 0, genotype AA as 1. Further, the estimated marginal means of the dependent variables along with their 95% confident intervals (CI) are reported in the ANOVA procedures and the least significant difference t-test was used for post hoc comparisons. Estimated marginal means are not observed means; rather they represent predicted means estimated at the co-variables held at their respective actual means. Finally, we analyzed the association between rs2259816 genotypes and angiographic CAD in an analogous fashion by logistic regression. Multivariable adjustment was carried out in two steps, first for sex and age, and then, in addition, for cardiovascular risk factors  was used.We included 3113 subjects in the current analysis. Compared to the control group without CAD, patients with angiographic CAD were significantly older; current or past smoking, diabetes mellitus, and hypertension were more prevalent. CAD patients had higher systolic blood pressure, higher fasting glucose, higher triglycerides and lower HDL-C. Crude LDL-C concentrations were higher in controls, due to the fact that 57.1% of CAD patients were treated with lipid-lowering drugs compared to 18.3% of controls. When we accounted for the use of lipid-lowering drugs, age, and gender, however, LDL-C was significantly higher in CAD patients (adjusted mean 118 mg/dl) than in controls (adjusted mean 113 mg/dl). Body mass index and diastolic blood pressure were similar between patients and controls  were included as main effects; statistical adjustments were made for age, sex and cardiovascular risk factors, namely body mass index, diabetes mellitus, hypertension, smoking, LDL-C, HDL-C and TG . The circulating plasma concentration decreased from 3.88 mg/l (3.63 - 4.16) in CC homozygotes to 3.43 mg/l (3.21 - 3.65) in heterozygotes and to 2.95 mg/l (2.64 - 3.31) in AA homozygotes. As statins also lower CRP we repeated our analysis including only patients not receiving lipid lowering therapy but this yielded basically the same results (data not shown).Compared to CC homozygotes, carriers of the A-allele had significantly lower plasma concentration of CRP -1α. Several polymorphisms in this gene have been linked to lower CRP ,34. We aThese findings are in line with Grammer et al. who didn't find an association of polymorphisms in the CRP gene itself and CAD in LURIC  and suppNevertheless, we think that HNF-1α might play an important role as a link between metabolic and inflammatory pathways. As such it could be involved in the pathogenesis of atherosclerosis and further work is required to define the role of HNF-1α more exactly.In the LURIC Study cohort the A-allele of the rs2259816 polymorphism in the HNF1A gene is associated with decreased CRP but not with coronary artery disease. These results support the view that CRP does not play an active role in the development of CAD.The authors declare that they have no competing interests.MEK performed the statistical analysis and drafted the manuscript. TBG assisted in the statistical analysis and helped to draft the manuscript. WR performed the genotyping. MW conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/157/prepub


SREBF-2 1784G>C (rs2228314) and SCAP 2386A>G, are related to the progression of coronary atherosclerosis and the risk of pre-hospital sudden cardiac death (SCD).Disturbed cellular cholesterol homeostasis may lead to accumulation of cholesterol in human atheroma plaques. Cellular cholesterol homeostasis is controlled by the sterol regulatory element-binding transcription factor 2 (SREBF-2) and the SREBF cleavage-activating protein (SCAP). We investigated whole genome expression in a series of human atherosclerotic samples from different vascular territories and studied whether the non-synonymous coding variants in the interacting domains of two genes, SREBF-2 and SCAP genotypes.Whole genome expression profiling was completed in twenty vascular samples from carotid, aortic and femoral atherosclerotic plaques and six control samples from internal mammary arteries. Three hundred sudden pre-hospital deaths of middle-aged (33–69 years) Caucasian Finnish men were subjected to detailed autopsy in the Helsinki Sudden Death Study. Coronary narrowing and areas of coronary wall covered with fatty streaks or fibrotic, calcified or complicated lesions were measured and related to the SREBF-2 in atherosclerotic carotid plaques (types IV-V), but not in the aorta or femoral arteries (p = NS for both), as compared with the histologically confirmed non-atherosclerotic tissues. In logistic regression analysis, a significant interaction between the SREBF-2 1784G>C and the SCAP 2386A>G genotype was observed on the risk of SCD (p = 0.046). Men with the SREBF-2 C allele and the SCAP G allele had a significantly increased risk of SCD , compared to SCAP AA homologous subjects carrying the SREBF-2 C allele. Furthermore, similar trends for having complicated lesions and for the occurrence of thrombosis were found, although the results were not statistically significant.Whole genome expression profiling showed a significant (p = 0.02) down-regulation of SREBF-2 1784G>C and SCAP 2386A>G) in the cholesterol homeostasis regulating SREBF-SCAP pathway may contribute to SCD in early middle-aged men.The results suggest that the allelic variants ( Cholesterol is an essential component of cellular membranes. Cellular cholesterol homeostasis is controlled by sterol regulatory element-binding transcription factors (SREBFs), which are transcription factors that modulate the transcription of genes involved in lipid and cholesterol metabolism. In sterol-depleted cells, SREBF cleavage activating protein (SCAP) forms a complex with SREBF and assists in its transportation to the Golgi apparatus where it can be processed by two specific proteases and then release the amino-terminal transcription-activation domain of the SREBF. The active form of SREBF can then travel to the nucleus where it binds to the promoters of target genes . The SREAs regulatory factors of cellular cholesterol metabolism, SREBF-2 as well as SCAP together with low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase are assumed to be down-regulated in human atherosclerotic plaques due to the cholesterol overload. However, we were able to find only one publication quantitatively comparing the expression of SREBF-2 mRNA in human atherosclerotic plaques with nearby macroscopically intact carotid artery tissue . This anSREBF-2 and SCAP locus might affect the progression of atherosclerosis. SREBF-2 is encoded by a gene  on human chromosome 22q13 [Given the central role of the SREBF-SCAP pathway in the regulation of cholesterol, the variation in the me 22q13 . A commome 22q13  and fromme 22q13 . This pome 22q13 .SCAP gene is located at chromosome 3p21.3 with 23 exons [SCAP gene has an exonic polymorphism located at exon 16  and leading to isoleucine-to-valine substitution (I796V) [SCAP is a membrane-bound protein containing two distinct domains: a sterol-sensing domain which serves as a cell cholesterol sensor and a carboxy-terminal domain with multiple WD repeats which mediates protein-protein interaction . The hum23 exons . The SCA (I796V) . This po (I796V) , but not (I796V)  and prav (I796V) .SREBF-2 1784G>C and SCAP 2386A>G at present. The combination of SREBF-2 1784G>C and SCAP 2386A>G has an effect on LDL cholesterol levels among familial hypercholesterolemia females [SCAP 2386A>G genotypes were found to modify the association between SREBF-2 1784G>C and myocardial infarction (MI) in men [There are two publications about the interaction effect of  females . InteresSREBF-2 and SCAP in human atherosclerotic tissues and compared them in different arterial beds, i.e. the carotid, aorta and femoral region, to methodologically accurately confirmed normal arterial tissue. We also related the variations 1784G>C and 2386A>G in the interacting domains of the SREBF-2 and SCAP genes to the areas of the different types of atherosclerotic lesions in the coronary arteries and to the risk of sudden cardiac death (SCD) and acute myocardial infarction (AMI) in an autopsy series of 300 Finnish men included in the Helsinki Sudden Death Study (HSDS).Therefore, we investigated the expression of The vascular sample series consists of 20 samples from the carotid artery, the femoral artery and the abdominal aorta . Six control samples were taken from internal mammary arteries . The study was approved by the Ethics Committee of Tampere University Hospital, and the study subjects gave their informed consent. The samples were taken from patients subjected to open vascular surgical procedures at the Division of Vascular Surgery, Tampere University Hospital. The vascular samples were histologically classified according to the recommendations of the American Heart Association .The fresh tissue samples were soaked in RNALater solution  and isolated with Trizol reagent  and the RNAEasy Kit . The concentration and quality of RNA were evaluated spectrophotometrically with Agilent . Over 23,000 known genes and gene candidates were analyzed using Sentrix Human-8 Expression BeadChips, according to manufacturer's instructions . In brief, 200 ng aliquot of total RNA from each sample was amplified to cDNA. In vitro transcription reaction of cDNA to cRNA was performed overnight (14 h) including biotin-11-dUTP for labeling the cRNA product. Each sample cRNA (1500 ng) was hybridized to Illumina's Sentrix Human-8 Expression BeadChip arrays. Hybridized biotinylated cRNA was detected with 1 μg/ml Cyanine3-streptavidine . BeadChips were scanned with the Illumina BeadArray Reader. The method has been described in more detail previously .Sequence homologues for the SCAP and SREBF-2 proteins were obtained by PSI-BLAST . The bioThe HSDS was launched to study the lifestyle and genetic factors predisposing to sudden death in Finnish middle-aged men who lived in Helsinki and its surroundings. The present autopsy series was collected during 1991–1992 (n = 300) at the Department of Forensic Medicine, University of Helsinki. The cause of death was cardiac in 39% (n = 117), other diseases in 21% (n = 63) and suicides or accidents in 40% (n = 120) of the subjects. The study was approved by the Ethics Committee of the Department of Forensic Medicine, University of Helsinki.At autopsy, the proximal parts of the left anterior descending coronary artery (LAD), right coronary artery (RCA) and left circumflex coronary artery (LCX) were collected for analysis. The definition of atherosclerotic lesions was based on the protocols of two international studies: the International Atherosclerosis Project, Standard Operating Protocol 1962  and the At autopsy, coronary angiography was performed using vulcanising liquid silicone rubber . The proCoronary thrombosis and myocardial infarction were recorded at autopsy, and the presence of MI was confirmed by nitro blue tetrazolium staining and by a histological examination of the myocardium. The presence of neutrophil granulocytes was considered diagnostic of an AMI. Thrombosis was defined by a reddish clot attached to the coronary wall if the clot could not be detached with saline flushing.A spouse, a relative or a close friend of the deceased could be interviewed in 147 cases. The questionnaire included a review of risk factors including hypertension, diabetes, past and recent smoking, drinking habits and previous illnesses. On the basis of these interviews, men were classified as smokers or non-smokers. Ex-smokers were included in the category of smokers for statistical analysis. Average daily alcohol consumption of the deceased was calculated based on the information given by the persons interviewed. Based on questions concerning previous illnesses, 50 men had suffered from hypertension and 22 from diabetes.SCAP 2386A>G genotyping was based on PCR amplification, restriction enzyme analysis and DNA electrophoresis. The DNA samples were amplified by PCR, using the primers 5'-TTGTGCTGCGCGGCCACCTCA-3' and 5'-AGGAGGAAAGGGCAGCCGCAC-3'. PCR was performed in a volume of 50 μl. Cycle conditions were 94°C for 4 min, then 28 cycles of 94°C for 1 min, 64°C for 1 min and 72°C for 1 min, with a final extension step of 5 min at 72°C in a PTC-225 thermal cycler . 10% DMSO was included in PCR reaction. The PCR-amplified DNA fragment was incubated for 16 hours with 10 U of MslI (New England Biolabs). Electrophoresis was performed on a 3% agarose gel containing ethidium bromide. Genotype information was obtained for 269 men.DNA was extracted from frozen cardiac muscle samples. The SREBF-2 1784G>C genotyping, DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination, with the ABI Prism 7000 Sequence Detection System . PCR reaction containing genomic DNA, 2 × TaqMan universal PCR Master Mix, 900 nM of each primer and 200 nM of each probe was performed in 96-well plates according to standard protocol in a total volume of 25 μl. After cycling, end-point fluorescence was measured, and genotype calling was carried out by the allelic discrimination analysis module. Genotyping was successful in 271 men. As a means of quality control for genotyping, empty controls and random duplicate samples were used.For the SREBF-2 and SCAP genotypes as well as autopsy data were available in 247 cases; this autopsy cohort constituted the final study population. Data analysis for continuous variables was based on analysis of variance (ANOVA) and analysis of covariance (ANCOVA), in which the possible confounding effects of age, body mass index (BMI) and hypertension were taken into account by including them in the model as covariates. Categorical variables were compared with the χ2 test. Fisher's exact test was used to test the genotype frequencies under Hardy-Weinberg equilibrium. Non-normally distributed data concerning the areas of atherosclerotic changes were analysed after square-root transformation, but the results are displayed as crude data. We analysed the interaction between SREBF-2 1784G>C and SCAP 2386A>G genotypes using logistic regression. Logistic regression analysis was also used to determine odds ratios for SCD according to SREBF-2 1784G>C and SCAP 2386A>G genotype groups. The statistical analysis was performed by means of SPSS Version 14.0.1 . The level of significance was set at p < 0.05.The non-parametric Mann-Whitney U test was used for comparison of gene expression between atherosclerotic and control tissues. Complete data on SREBF-2 gene expression was reduced 1.5-, 1.1- and 1.3-fold in the carotid artery (n = 9), aorta (n = 7) and femoral artery (n = 4), respectively. Only the SREBF-2 expression level in the carotid artery was significantly lower in atherosclerotic tissue than in control tissue . The similar trend of the SCAP gene expression was also found in three arteries, although it did not reach statistically significant. When all atherosclerotic arterial samples (type IV-V plaques) were pooled, there were no differences in the mean gene expression of either SREBF-2 or SCAP in atherosclerotic tissue as compared with histologically confirmed healthy control samples (type 0).The The search by PSI-BLAST yielded 64 homologue sequences for SCAP and 62 sequences for SREBF-2, after the removal of duplicates and hypothetical proteins.The mutation position in SCAP is physicochemically conserved (the conserved property is hydrophobicity), and the frequencies of the physiochemically similar amino acids  are approximately equal. The disease-causing mutation studied here (I796V) is thus a conservative substitution. The mutation position in SREBF-2 is not at all conserved, and the following amino acids are found at this position: Y, V, T, S, R, Q, L, K, G, E and A. Q and E are the amino acids with the highest frequency of occurrence in the alignment. The mutations do not seem to have a direct structural effect on the proteins.SREBF-2 and SCAP genotypes as well as autopsy data, full risk factor data was available in 124 cases. The distributions of SREBF-2 1784G>C and SCAP 2386A>G genotypes were in accordance with the Hardy-Weinberg equilibrium . The characteristics of the study subjects according to SREBF-2 and SCAP genotype are shown in Table SREBF-2 genotype. Only SCAP 2386A>G GG carriers had a higher prevalence of hypertension than subjects with other genotypes. No significant differences existed in the occurrence of SCD, AMI and thrombosis between the SREBF-2 or SCAP genotypes . The percentage of men with the SREBF-2 C allele in combination with the SCAP G allele among SCD victims tended to be higher than that found in the non-SCD group (34.7% vs. 26.3%). Carriage of the SREBF-2 C allele and the SCAP G allele was associated  with an increased risk of SCD when compared with subjects carrying the SREBF-2 C allele and those with the SCAP AA genotype  with an increased risk of having complicated lesions when compared with subjects carrying the SREBF-2 C allele and the SCAP AA genotype. The combination of the SREBF-2 C allele and the SCAP G allele was present in 8 of the 20 men (40%) with coronary thrombosis, whereas the SREBF-2 C allele in combination with the SCAP AA genotype was present in 2 of the 20 men (10%) with coronary thrombosis , and this interaction is prevented by the variations, impairing the formation of the SCAP/SREBF-2 complex. However, because 3D structures of these proteins are not available, this must be interpreted with caution. According to the secondary structure predictions, the 1784G>C position in SREBF-2 is in α-helix. Equally high confidence values were obtained for the 2386A>G position in SCAP, but for a β-strand. The mutant sites are therefore on the ordered secondary structural elements and possibly on the surface of the proteins, which is supported by many predictors when the amino acid substitutions are considered to be tolerated. However, our study is epidemiological in nature and still need for empirical in silico proof for the interaction.Our results are in accordance with a previous study in which the in males . Our resSREBF-2 and SCAP mRNA expression study, and the HSDS samples were used for the association study between SREBF-2 1784G>C as well as SCAP 2386A>G and coronary atherosclerosis. The form of atherosclerosis in these two materials might be different, because they were collected from different vascular territories with different local haemodynamic conditions. Other limitations are related to the nature of autopsy studies: since the subjects had died suddenly and most of them had not seen a doctor nor had any blood samples taken prior to their death, we were unable to determine the lipid levels. We also did not measure the cholesterol and apolipoprotein levels in post-mortem samples. Furthermore, this study only enrolled Finnish men. We cannot ascertain whether the interactive effect of the SREBF-2 1784G>C and the SCAP 2386A>G variant would extend to other populations or women, as well.The limitations of our study include the two distinct sample materials we used: The vascular samples collected from elective patients subjected to open vascular surgical procedures were used for the SREBF-2 in atherosclerotic carotid plaques as compared with histologically confirmed normal tissues. We also showed a gene-gene interaction between the SREBF-2 1784G>C and the SCAP 2386A>G genotypes on the risk of developing SCD in middle-aged men. We conclude that SREBF-2 and SCAP may be implicated in the progression of atherosclerosis and the risk of developing SCD. Future structure-function and replication studies with large samples are required to improve the understanding of how SREBF-2 and SCAP function, in addition to explaining the results presented here.In summary, we demonstrated a down-regulated expression of The authors declare that they have no competing interests.YMF participated in the study design, performed the statistical analysis and drafted the manuscript. PJK participated in designing of the Helsinki Sudden Death Study (HSDS), in performing of autopsies and collecting of coronary samples, in performing of interviews of the next of kin, and revising the final draft of the manuscript. ML participated in vascular samples collection and isolation of RNA. EI, JM and OAK participated in the analysis and interpretation of the morphometric, coronary cast, the risk factor and the MI classification data of the HSDS. OJ and JPS performed surgical procedures for vascular samples. NO performed surgical procedures for vascular samples and revised the manuscript critically. JT and MV performed bioinformatics analysis. LK and JTS participated in executing of microarray lab work and pre-analyzing the results. RL participated in the study design. TL participated in the study design and revised the manuscript critically. All authors read and approved the final manuscript for publication.


HLA class II  alleles could provide further clues to the identity of the infection, since HLA molecules exhibit allotype-restricted binding of infection-derived antigenic peptides. We clustered >30 HLA-DPB1 alleles into six predicted peptide-binding supertypes , based on amino acid di-morphisms at positions 11 (G/L), 69 (E/K), and 84 (G/D) of the DPβ1 domain. We found that the DPβ11-69-84 supertype GEG (DP2), was 70% more frequent in BCP ALL , and 98% more frequent in cases diagnosed between 3 and 6 years (P<10−4), but not <3 or >6 years, than in controls. Only one of 21 possible DPB1 supergenotypes, GEG/GKG (DP2/DP4) was significantly more frequent in BCP ALL (P=0.00004) than controls. These results suggest that susceptibility to BCP ALL is associated with the DP2 supertype, which is predicted to bind peptides with positively charged, nonpolar aromatic residues at the P4 position, and hydrophobic residues at the P1 and P6 positions. Studies of peptide binding by DP2 alleles could help to identify infection(s) carrying these peptides.Childhood B-cell precursor (BCP) ALL is thought to be caused by a delayed immune response to an unidentified postnatal infection. An association between BCP ALL and  However, the P4 peptide-binding pocket, at position β69, also makes an important contribution to antibody and peptide-binding . We compared their frequencies in childhood BCP ALL, non-BCP leukaemia and solid tumours recruited as part of the Peptide binding by HLA class II allotypes, including DP, is the outcome of interactions between the amino acid side chains of the peptide and four major peptide-binding pockets  or non-BCP acute leukaemia. The non-BCP leukaemias were the sum of Pro-B ALL , T-ALL , and AML (n=208). Diagnostic immunophenotyping was carried out according to the protocol for UK Medical Research Council leukaemia trials (n=409) were also recruited as part of the UKCCS (n=864) born in Manchester UK between 1991 and 1997 were used as controls  were validated by the UKCCS data centre at the Epidemiology and Genetics Unit, University of York.Childhood leukaemia cases were recruited between 1992 and 98 as part of the UK Childhood Cancer Study . Leukaema trials . Childhohe UKCCS . UmbilicHLA-DPB1 molecular typing was carried out as previously described in detail . Supertype allele and genotype frequencies were compared in cases and controls using global and univariate statistical analysis. As discussed previously  used for supertype clustering was included in the analysis. Global case–control supertype frequencies were compared using the CLUMP programme of χ2 statistic (T1) from a series of simulated case–control tables. In univariate analysis, cross-product odds ratios (ORs), and 95% confidence intervals were calculated from case–control supertype and genotype frequencies by the RERI program in the Linkage Utility Package, P-values for case–control supertype and genotype differences using Fisher's Exact test. Six supertypes require an uncorrected P-value <0.008, and 21 supergenotypes an uncorrected value <0.002 to achieve significance (P=0.05). No correction for the total number of classical DP alleles was applied. DPB1 and DQA1, or DQB1 alleles.HLA-DPB1 types for 982 cases of childhood leukaemia , not including childhood lymphoma (n=864) were used as controls. Male–female ratios were slightly higher in the leukaemia cases (1.22) than the solid tumours (1.14) and controls (1.01).The UKCCS is an epidemiological case–control study designed to test the role of environmental factors in the aetiology of childhood cancer and leukaemia . As partia cases . A mixedDPB1 alleles in the cases and controls could be clustered into six supertypes , DP2 (GEG), DP3 (LKD), DP4 (GKG), DP6 (LED), and DP8 (GED).The majority (90%) of >30 pertypes , consistn=895) and the newborn controls (n=864), we identified 14 DPβ69E alleles, of which four are DP2 (GEG), seven are DP6 (LED), and three are DP8 (GED). Of 15 DPβ69K alleles, six are DP4 (GKG), six are DP3 (LKD), and three are DP1 (GKD). In global χ2 analysis, the supertype frequency in the total leukaemia series was significantly different (P<10−6) from the controls (P=0.04). In univariate analysis, DP2 (GEG) : 1.6, 95%, CI, 1.2–2.0; 2 sided P=0.0002) and DP8 (GED)  were significantly more frequent in leukaemia cases than controls. DP6 (LED)  was only marginally significant without correction for six supertypes, while DP2 and DP8 were significant after correction.In the total leukaemia case series  and non-BCP acute leukaemia (n=208) revealed that DP supertypes in BCP ALL differed significantly from the newborn controls (P<10−6), but non-BCP leukaemia was only marginally significant (P=0.04) (DP2 (GEG)  and DP8 (GED)  were significantly more frequent, after correction for six supertypes, than controls. DP6 (LED) was not significant in BCP ALL, but was significant in non-BCP leukaemia . DP1 (GKD) was significantly less frequent, after correction, than controls in BCP ALL , but not in non-BCP leukaemia.Stratification of the leukaemias into BCP ALL ((P=0.04) . In univDP2 and DP8 raised the possibility of a chance finding. To test this, supertype frequencies in four BCP ALL case series were compared with controls: (1) cases included in our previous study ; (3) half of the cases in the previous study combined with the ‘new’ cases (n=343); (4) the ‘new’ cases (n=128) alone. DP2 and DP8 were significant in all four case series, though only DP2 remained significant after correction , but was not significant in BCP ALL diagnosed <3 or >6 years. DP4 was significantly increased in BCP ALL diagnosed <3 years, though not after correction. DP8 was not significant after correction, while DP1 protected from BCP ALL in all age groups.To determine the relationship between the age at diagnosis of BCP ALL and DPB1 genotypes, such as DPB1*0201/0202 can have a homozygous supergenotype (DP2/DP2:GEG/GEG) (DP2/DP4), was associated with a significantly increased risk (110%), after correction, of BCP ALL . DP2/DP4 was associated with an increased risk (130%) of BCP ALL arising between 3 and 6 years of age , but not <3 or >6 years of age. No DP2 supergenotypes were significant in non-BCP leukaemia or solid tumours. DP4/DP6 (GKG/LED) was significant in non-BCP leukaemia after correction , but not in BCP ALL or solid tumours. Homozygous DP1 (GKD/GKD) significantly protected, after correction, against BCP ALL . No other DPB1 supergenotypes were significant at any of the diagnostic ages.To determine which combination of supertype alleles was associated with BCP ALL, we compared the frequency of all 21 possible supergenotypes  in BCP ALL, non-BCP leukaemia, and solid tumours with newborn controls . Note thGEG/GEG) . Of the DP supertype associations could be explained by LD with HLA-DQ alleles, we analysed the co-occurrence of DP and DQ alleles in 451 BCP ALL cases, using POPGENE. We detected only one DP allele, 1601, in LD with DQ . Five BCP ALL cases (0.4%) typed for DPB1*1601, a frequency not significantly greater than in the controls, indicating that the DP-supertype results cannot be explained by LD between DP and DQ alleles.To test whether the β1 domain was associated with BCP ALL , 69 (P4 pocket), and 11 (P6 pocket) represents an expanded version of the scheme proposed by DP1 (GKD), DP2 (GEG), DP3 (LKD), DP4 (GKG), DP6 (LED), and DP8 (GED) since they broadly resemble those defined in the primed lymphocyte test (PLT) as DPw specificites . In a rare disease such as BCP ALL in which there are likely to be multiple aetiological factors, weak HLA associations potentially require hundreds of cases and controls to allow for correction for multiple testing. Supertype analysis, in which alleles are clustered according to common functional  properties, overcomes this problem. DPB1 alleles comprise combinatorial series of six variable regions (A-F) encoded by exon 2 , LED (DP6), GED (DP8)), and to compare these with three homologous β69K series (GKG (DP4), LKD (DP3), GKD (DP1)).The DP2 (GEG), a 98% increase in DP2-associated risk between 3 and 6 years of age, and a 130% increased risk associated with a single supergenotype, DP2/DP4. This association was not present in BCP ALL diagnosed <3 or >6 years of age, and leads us to conclude that the peak of BCP ALL . There is no evidence that childhood BCP ALL is caused by an oncogenic virus (DP2 suggests that binding of specific peptide(s) and T-cell activation are involved in causation, which is inconsistent with immune evasion by an oncogenic virus. The negative association of DP1 with BCP ALL may be due to the binding and recognition of TEL-AML1 peptide(s) in children with pre-ALL with this supertype, as discussed elsewhere (DPB1*0501-restricted (DP1) CD4+ T cell response  of 15 heterozygous supergenotypes (GEG/GKG) is associated with BCP ALL fits this model.The delayed response to infection hypothesis for BCP ALL  proposesozygotes , a recenDPB1*0201 peptide-binding data and molecular modelling (β84) and pocket 6 (β11) deep and hydrophilic, preferentially-binding hydrophobic and aromatic amino acids, notably phenylalanine (F), and tyrosine (Y)  are likely to bind to DP2.Using sine (Y) . This pr162D-N179 containing two overlapping T-cell epitopes, 163FHFEVFNFV171 and 165FEVFNFVPC173 that are restricted by DPB1*0401 (DP4), and DPB1*0201 (DP2) (DP2) and GKG (DP4), consistent with the association of BCP ALL with DP2/DP4. While this conclusion is speculative it points to a need for detailed sero-epidemiological studies of RSV in BCP ALL.In this context,


ERCC6 (NM_000124) gene. The risk allele also increased ERCC6 expression. ERCC6 is involved in DNA repair and mutations in ERCC6 cause Cockayne syndrome (CS). Amongst others, photosensitivity and pigmentary retinopathy are hallmarks of CS.Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the developed countries and is caused by both environmental and genetic factors. A recent study  reported an association between AMD and a single nucleotide polymorphism (SNP) (rs3793784) in the ERCC6 and AMD (2682 AMD cases and 3152 controls). We also measured ERCC6 mRNA levels in retinal pigment epithelium (RPE) cells of healthy and early AMD affected human donor eyes. Rs3793784 conferred a small increase in risk for late AMD in the Dutch population (The Rotterdam and AMRO-NL study), but this was not replicated in two non-European studies . In addition, the AMRO-NL study revealed no significant association for 9 other variants spanning ERCC6. Finally, we determined that ERCC6 expression in the human RPE did not depend on rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (P = 0.018).Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study) were used to analyse the genetic association between ERCC6 and AMD. Nevertheless, our findings on ERCC6 expression in the RPE suggest that ERCC6 may be functionally involved in AMD. Combining our data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 may be caused by diverse, small and heterogeneous genetic and/or environmental determinants.Our meta-analysis of four Caucasian cohorts does not replicate the reported association between SNPs in  Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the Western world. The prevalence of AMD rises sharply with age, affecting 4% of the population over the age of 60 and more than 10% of individuals older than 75 As a complex disease, AMD has environmental as well as genetic determinants.CFH) gene HTRA1 and ARMS2 genes CFH is the main inhibitor of the alternative pathway of the complement system, and thus plays an important role in the control of the innate immune system. Recently, associations with AMD were also found for other genes of the complement cascade  is of interest. They reported that a single nucleotide polymorphism , in the promoter region of the ERCC6 DNA repair gene was associated with AMD. The SNP was associated with AMD susceptibility, both independently and through interaction with a SNP (rs380390) in CFH.Indeed, the retina, and especially the RPE is exposed to high levels of oxidative stress ERCC6 SNP altered the expression level of the gene In addition, they found that the ERCC6 gene is involved in DNA repair, and loss of function mutations in this gene cause Cockayne syndrome (CS) Ercc6 in the mouse resulted in a mouse model for CS Ercc6 -/- mouse is hypersensitive to X rays, which confirmed that oxidative DNA damage is involved in CS retinal pathology.The ERCC6 promoter SNP rs3793784: C>G (c.-6530C>G) with AMD was analyzed in the population-based Rotterdam study and, in parallel, in the Dutch AMRO-NL case-control study. Replication was performed in two large non-European AMD case-control (Columbia University and Age-Related Eye Disease Study (AREDS)) studies. We subsequently investigated the potential modifying effect of CFH Y402H, LOC387715 A69S and smoking. Next, nine other ERCC6 (tag) SNPs were genotyped in 375 cases and 199 controls from the AMRO-NL study population. Finally, to examine a possible functional relationship, we determined both rs3793784 dependent and normal ERCC6 mRNA expression levels in healthy and early AMD affected human RPE isolated from human donor eyes.In order to further explore the role of oxidative DNA damage in AMD, the potential association of ERCC6 c.-6530C>G (rs3793784) with AMD was analyzed in the population-based Rotterdam Study and, in parallel, in the Dutch AMRO-NL case-control study. In the Rotterdam Study, 427 of 6418 persons were diagnosed with early AMD and 78 persons with late AMD at entry of the study. The mean follow-up time was 7.85 years. In this period, 1039 persons remained without AMD (“controls”), while 509 progressed to early AMD and 93 to late AMD (%). In the AMRO-NL study, 331 unrelated AMD patients and 170 controls were genotyped for rs3793784. Of those, 84 persons were diagnosed with early AMD and 247 persons with late AMD at entry of the study. Genotype frequencies were in Hardy-Weinberg Equilibrium in both studies. The population frequency of ERCC6 G allele was 0.43. Baseline characteristics stratified for the ERCC6 c.-6530C>G genotype are presented in Table S1.The potential association of ERCC6 c.6530C>G allele is summarized in Table S2. In the Rotterdam Study, we found a significant association between ERCC6 and late AMD in the incident analyses. Although not statistically significant, a similar trend was seen in the prevalent analyses. In contrast, analysis of the AMRO-NL study showed no significantly increased disease OR for persons carrying potential ERCC6 risk alleles. To determine the risk for the general Dutch population, we combined data from the Rotterdam Study and the AMRO-NL study population. This yielded a borderline significant increased risk of late AMD for persons homozygous for rs3793784 . Analyzing subtypes of late AMD separately  did not yield statistical significant results: Table S2. We also analyzed the interaction of ERCC6 c.-6530C>G with three known prominent AMD risk factors in the largest combined population study available to us (The Dutch Rotterdam and AMRO-NL studies). As assessed by calculation of the synergy index (SI) no significant interaction between the ERCC6 variant and smoking was found . Neither did we find a significant SI for the interaction with CFH Y402H  and LOC387715 A69S . These results implied that these risk factors did not modify the relation of ERCC6 with AMD.The risk of AMD for the Finally, rs3793784 was also screened in two non-European study populations (Columbia and AREDS). The results from the genotype analysis of these populations are shown in ERCC6 SNPs that span and tag the entire ERCC6 gene. The LD plot and the distinct haplotype blocks for the nine selected SNPs, as generated by Haploview ERCC6 gene. Corresponding LD scores (D' and R2) for each selected marker of the ERCC6 gene with a MAF>10% are also given (Table S3).We next selected nine other ERCC6 gene expression, with the risk allele (G) showing higher expression ERCC6 SNP in DNA derived from the retina of 23 well characterized healthy old and early AMD affected donor eyes. We next determined ERCC6 mRNA expression in laser dissected RPE cells from the same eyes, since this cell type occupies a central position in AMD pathology. Late AMD was not considered for RPE expression studies because of the lack of suitable cell material. There were no significant differences in age distribution between each genotype group. The results are presented in ERCC6 expression levels. A two way ANOVA was used to statistically test the effect of genotype and AMD status on ERCC6 expression. The ANOVA did not detect an independent significant effect for rs3793784 genotype on ERCC6 expression (P = 0.293). In addition, the combined effect of genotype and AMD status on ERCC6 expression was not significant either (interactionP = 0.531). However, the ANOVA did demonstrate a significant effect of AMD status on ERCC6 expression, independent of the rs3793784 genotype . Interestingly, the expression of ERCC6 in healthy RPE was nearly twice as high as in early AMD affected RPE  of European-American descent. In total, this study consisted of 2682 AMD cases and 3152 ethnically matched controls.We genotyped the rs3793784 and AMD in all study populations.Contrary to the findings of the initial association study by Tuo et al. (2006) ERCC6 rs3793784 have a higher risk of AMD, and if these persons also die earlier, it could explain the discrepancy between prevalent and incident analyses. However, in the Rotterdam Study, the ERCC6 variant was not associated with increased mortality (not shown); in addition, there was no unequal age-related genotype distribution (not shown). Thus, the small difference between the incident and prevalent risks in the population based cohort probably occurred by chance due to relatively small patient sample sizes. In the AMRO-NL case-control study, association between the rs3793784 SNP and AMD was not found. The combined data of the two Dutch populations (the Rotterdam Study and AMRO-NL study) still showed a marginally increased risk of homozygous ERCC6 carriers for late AMD. Since this effect was largely caused by one sub-population only (incident cases and controls from the Rotterdam study), we chose to include two additional study populations. Replication of the association in two non-European study populations clearly failed: In both the Columbia University and AREDS data, no association was present between rs3793784 and AMD.We only detected a significant risk in the incident analysis of the Rotterdam Study, but not in the prevalent analysis. This prompted us to specifically analyze whether selective mortality may be involved: persons with two copies of ERCC6 SNP rs3793784 essentially in the same AREDS cohort and did find a statistically significant association. How can this discrepancy be explained? One possible explanation is the large difference in the number of AREDS participants screened: Tuo et al. CFH Y402H or any of the other two major AMD risk factors: smoking or the LOC387715 A69S SNP. Overall, the possible weak association in a single Dutch sub-population and the negative association in three large replication cohorts, imply that the ERCC6 C-6530>G polymorphism has a minor role, if any, in the pathogenesis of AMD. This conclusion is supported by a recent study by Chen and coworkers, who conducted a genome-wide association scan for AMD in 2157 cases and 1150 controls. Amongst other, they evaluated evidence for involvement of previously associated SNPs and found no association between the ERCC6 SNP rs3793784 and AMD Our negative association findings in the AREDS population are of particular interest: Tuo et al. ERCC6 -6530C>G, is a contributor to the potential AMD risk observed. In order to further elucidate the putative role of this gene in AMD we tested nine other common ERCC6 variants in the AMRO-NL study. One of these SNPs was in perfect LD with the previously associated SNP, rs2229760. None of the selected SNPs showed a significant association with early or late AMD. These results strengthen the conclusion described above and suggest that common SNPs in the ERCC6 gene confer no or little risk for AMD.Finally, it is of course possible that another genetic variant or a variant in linkage disequilibrium (LD) with ERCC6 expression level: the risk allele (G) resulted in 2–3 times higher ERCC6 expression than the C variant ERCC6 mRNA expression in the human RPE, which occupies a central position in AMD pathology. In this cell type, we could not reproduce the paradoxical finding of Tuo and co-workers ERCC6 RPE expression levels were significantly reduced in early AMD affected eyes, independent of rs3793784 genotype.Tuo et al. (2006) found that rs3793784, located in the untranslated 5′ flanking region of the gene, influenced ERCC6 in the RPE of early AMD affected eyes, suggests that ERCC6 is involved in AMD. It is plausible that this involvement relates to oxidative stress and aging. Functional studies have implicated oxidative stress in the RPE in the aetiology of AMD ERCC6 plays a role in repair of (oxidative) DNA damage. Loss of function mutations in ERCC6 cause the autosomal recessive disorder Cockayne syndrome, which includes many severe physical and neurologic features, with premature aging and retinopathy as hallmarks of the disease. These observations led to the hypothesis that (oxidative) DNA damage and insufficient DNA repair contribute to aging pathology, including AMD leukocytes of AMD patients and controls: Wozniak and co-workers found that AMD patients had higher levels of endogenous oxidative DNA damage and a lower repair capacity of DNA lesions The significantly reduced expression of ERCC6 in the RPE. In CS patients both ERCC6 alleles carry loss of function mutations ERCC6 in the RPE may well contribute to the development of AMD.We found that early AMD affected eyes had an almost 50% reduced expression of cause of the reduced ERCC6 transcription in RPE of early AMD affected eyes is not clear. However, given its implication in the multifactorial disorder AMD, and our inability to associate common ERCC6 SNPs with AMD, we hypothesize that the genetic involvement of ERCC6 in AMD, if any, may reside in (combination of) rare SNPs. Alternatively, the reduction in ERCC6 transcription levels in relation to AMD may also be due to other genetic factors or to environmental insults, such as prolonged smoking.The ERCC6 and AMD. We did not find a significant interaction between ERCC6 SNP rs3793784 and either smoking, CFH or LOC387715. Using RPE from human donor eyes, we also could not confirm rs3793784 genotype dependent expression of ERCC6.In summary, our study did not confirm a consistent relation between common genetic variation in ERCC6 in human RPE affected by early AMD, regardless of the rs3793784 genotype. The latter finding suggests that ERCC6 may be functionally implicated in the aetiology of AMD. Combining our data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 expression may be caused by diverse, small and heterogeneous genetic and/or environmental determinants.We did find significantly lower expression levels of All studies were approved by the Ethics Committees of the Academic Medical Center Amsterdam, the Erasmus Medical Center Rotterdam, the Institutional Review Board of Columbia University and the Age-Related Eye Disease Study (AREDS) Access Committee. All studies followed the tenets of the Declaration of Helsinki. All participants provided signed informed consent for participation in the study, retrieval of medical records, and use of blood and DNA for AMD research.Altogether, we utilized one prospective, population-based study and three case-control studies consisting of 2682 AMD cases and 3152 ethnically- and age-matched control subjects.ERCC6 SNP rs3793784 was analyzed in the Rotterdam Study and, in parallel, in the AMRO-NL case-control study population. Next, replication was carried out in two non-European study populations (Columbia University and AREDS). Finally, nine ERCC6 (tag) SNPs were genotyped in the AMRO-NL study population.Genetic association of the In The Rotterdam Study all inhabitants of 55 years or older living in a suburb of Rotterdam, the Netherlands, were invited to participate The AMRO-NL study population consisted of 375 unrelated AMD patients and 199 controls. All subjects were Caucasian and recruited from the Netherlands Institute of Neuroscience Amsterdam and Erasmus University Medical Centre Rotterdam (Amsterdam-Rotterdam-NL (AMRO-NL)), through newsletters, via patient organizations, and nursing home visits. Controls were aged 65 years and older, and were unaffected spouses or non-related acquaintances of cases, or individuals who attended the ophthalmology department for reasons other than retinal pathology.the Columbia University study population, consisted of 368 unrelated individuals with AMD and 368 unrelated controls of European American descent, recruited at Columbia University  as previously described Cases and controls from The AREDS study population was initially conceived as a long-term multicenter, prospective study of the clinical course of AMD and age-related cataract. Briefly, patients over the age of 55 years who self-reported to participating eye clinics between 1992 and 1996 were considered for AREDS, and eligibility was based on AMD symptoms and retinal photographs. A total of 1,699 patients were selected and enrolled in the clinical trial, all were put into one of five AMD severity categories, and then randomized into intervention and non-intervention groups. In this sample set, there were 929 AMD cases and 217 patients, free of AMD, acted as controls. The full details and results of the original AREDS study are reported elsewhere Study subjects from the Rotterdam, AMRO-NL and Columbia University study populations underwent ophthalmic examination and fundus photography covering a 35° field centered on the macula after pupil dilatation at each visit . Signs of AMD in those study populations were graded according to (a modification of) the international classification and grading system for AMD ERCC6 c.-6530C>G), rs1061170 (CFH Y402H) and rs10490924 (LOC387715 A69S) were done with the Taqman assay . Genotyping of the nine ERCC6 (tag) SNPs was performed using an Illumina GoldenGate assay on a BeadStation 500 GX .Standard DNA techniques were applied as described elsewhere ERCC6 gene. SNP data were used from the Centre d'Étude du Polymorphisme Humain (CEPH) population (Utah residents with ancestry from northern and western Europe) by use of the International HapMAP Project. Available at: http://www.hapmap.org/, NCBI build 36, dbSNP b126; Accessed January 7, 2009. SNP selection was based on criteria like functional relevance, minor allele frequency (MAF)>10%, coverage of the main linkage disequilibrium (LD) blocks and tagging of the most common haplotypes. Tag SNPs were selected by use of Tagger, an option of Haploview 2r>0.8).Nine SNPs were selected to span and tag the entire Studies on human eye tissue were carried out in accordance with the Declaration of Helsinki on the use of human material for research. Donor eyes were obtained from the Corneabank Amsterdam. The method of selection of eyes for this study was essentially described elsewhere ERCC6 rs3793784, located in the untranslated 5′ flanking region of the gene, influenced ERCC6 expression level: the G allele resulted in 2–3 times higher ERCC6 expression than the C variant Tuo et al. (2005) showed that ERCC6 mRNA expression in the RPE cells with real-time qPCR, in triplicate ERCC6 were normalized using the geo-mean of the expression of internal control genes RPLP0, PPIA, and EEF1a1P value of <0.05.We measured Characteristics of the Dutch participants were compared among those affected and non-affected with analysis of covariance for continuous variables, and with logistic regression analysis for discrete variables adjusting for age and sex.ERCC6, CFH and LOC387715 genotype distributions were tested using a Chi2 test.Hardy-Weinberg Equilibrium (HWE) of the In the Rotterdam Study, odds ratio's for prevalent AMD were estimated with logistic regression analyses, and relative risks for incident AMD were estimated with Cox proportional hazards analyses. Logistic regression was used in the AMRO-NL and the replication studies of Columbia University and AREDS to calculate odds ratios (ORs) and 95% confidence intervals (CI) in SPSS for windows  for risk of AMD with the major alleles as reference. For the Rotterdam Study and the AMRO-NL study population, ORs were adjusted for age and sex.ERCC6 c.-6530C>G on AMD was determined for smoking, CFH Y402H, and LOC387715 A69S contrasting late AMD with no AMD. Analyses were initially performed on separate data sets of the Rotterdam Study , the AMRO-NL case-control study , and subsequently conducted on combined data from the Netherlands (of population-based and case-control groups) and data of all population studies combined. Statistical significance for biological interaction was determined by calculating the synergy index (SI), which measures deviation from additivity of two risk factors Interaction with Table S1Baseline characteristics of the study populations.(0.05 MB DOC)Click here for additional data file.Table S2ERCC6 c.-6530C>G genotypes. Abbreviations: AMD, age-related macular degeneration; OR, odds ratio; HR, hazard ratio The ORs and HRs are estimates of the relative risk of AMD, and represent the risk of disease (AMD vs stage 0) in the genetic risk group divided by the risk of disease (AMD vs stage 0) in the nonrisk group (noncarriers).a adjusted for sex, age.Risk of age-related macular degeneration for (0.10 MB DOC)Click here for additional data file.Table S3ERCC6 gene. AMD  =  age- related macular degeneration; MAF  =  minor allele frequency. “A” indicates common allele, “a” minor allele. Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis (with the control group as reference group and respectively early and late AMD as outcome variable). Adjusted for age and sex.Odds Ratios (OR) and 95% Confidence Intervals (CI) of early and late age-related macular degeneration cases versus unrelated controls of the Amsterdam study population for single nucleotide polymorphisms in the (0.10 MB DOC)Click here for additional data file.Table S4ERCC6 gene expression in RPE from eye donors with early AMD or non-affected age-matched donors. Both raw expression values for the three selected RPE housekeeping genes  and ERCC6, as well as the resulting normalized expression values for ERCC6 are presented. AMD =  age- related macular degeneration. Normalization =  normalized to the geometric mean of three housekeeping genes  RT-PCR analysis of (0.06 MB DOC)Click here for additional data file.


The angiotensin-converting enzyme (ACE) gene in humans has an insertion-deletion (I/D) polymorphic state in intron 16 on chromosome 17q23. This polymorphism has been widely investigated in different populations due to its association with the renin-angiotensin system. However, similar studies for Arab populations are limited. This study addresses the distribution of the ACE gene polymorphism in three Arab populations .D allele among the three Arab populations, Egyptians (0.67), Jordanians (0.66) and Syrians (0.60), which is similar to those obtained from previous studies for Arab populations.The polymorphisms of ACE gene were investigated using polymerase chain reaction for detection of an I/D mutation. The results showed a high frequency of the ACE The relationship between ACE alleles and disease in these three Arab populations is still not known, but the present results clearly suggest that geographic origin should be carefully considered in the increasing number of studies on the association between ACE alleles and disease etiology. This study adds to the data showing the wide variation in the distribution of the ACE alleles in different populations and highlights that great care needs to be taken when interpreting clinical data on the association of the ACE alleles with different diseases. AluYa5 element inside intron 16 producing three genotypes  [D allele is related to increased activity of ACE in serum [DD genotype while the lowest was seen in the II genotype [Angiotensin-converting enzyme (ACE), a key enzyme of the rennin-angiotensin system, is localized in the kidney . The ACEozygote) . Althougin serum ,4. The hgenotype . Severalgenotype  and diabgenotype . Howevergenotype ,10. Moregenotype ,12.In this study we aim to investigate the distribution of ACE gene I/D polymorphism in three Arab populations . The three Arab populations have a mixed genetic background with an ethnic heterogeneity. Most of the three populations are of Mediterranean or Arabic origin that migrated from the Arabian Peninsula and surrounding areas.2, 10 mM Tris-HCl (pH 8.4) and Taq DNA polymerase (1.25 Units). The reaction were subjected to 32 cycles: an initial denaturation of 60 s at 94°C, 30 s denaturation at 94°C, 45 s at the annealing temperature 58°C, extension at 72°C for 45 s. Following the amplification cycles, a final extension was performed at 72°C for 10 min. For analysis, 20 μl of each sample was fractionated on a 2% agarose gel with 0.05 μg/ml ethidium bromide. PCR products were directly visualized using UV fluorescence. The homozygous individuals for the D allele (DD genotype) were identified by the presence of a single 190 bp PCR product. The homozygous for the I allele (II genotype) were identified by the presence of a single 490 bp PCR product. The heterozygous individuals (ID genotype) were identified by the presence of both 190 and 490 bp PCR products. Because the D allele in heterozygous samples is preferentially amplified, all samples that were typed initially as a DD genotype were reanalyzed using an insertion-specific primer pair, as reported by Lindpaintner et al. [I allele and no bands were detected for samples with DD genotype.The human population samples used for this study have been described previously and were available from previous studies . The samr et al. , except Statistical analysis was performed using SPSS version 15 statistical package for windows. Allele and genotype frequencies were calculated by direct counting; the Hardy-Weinberg equilibrium was assessed by an exact test provided by the Arlequin program .II genotype, 40 were heterozygous for the ID genotype and 56 were homozygous for the DD genotype, giving a D allelic frequency of 0.679. Among 52 Egyptians living in Sinai that were studied, 4 individuals were homozygous for the II genotype, 27 were heterozygous for the ID genotype and 21 were homozygous for the DD genotype, giving a D allelic frequency of 0.663. There was no significant statistical difference between the two Egyptian groups . The average frequency of the D allele among the two studied Egyptian groups was 0.674. As regards the studied individuals from Syria, we found that 9 individuals were homozygous for the II genotype, 38 were heterozygous for the ID genotype and 23 were homozygous for the DD genotype, giving a D allelic frequency of 0.600. As regards the Jordanians, we found that 13 individuals were homozygous for the II genotype, 15 were heterozygous for the ID genotype and 32 were homozygous for the DD genotype, giving a D allelic frequency of 0.658. Based on the allele frequencies, it is possible to predict the genotype frequency considering that they follow the Hardy-Weinberg equilibrium. This means that the frequencies have a binomial distribution according to the following equation: p2+2pq + q2 = 1, where p and q are the allelic frequencies of I and D, respectively, and p2, 2pq and q2 are the genotype frequencies of II, ID, and DD, respectively. No significant deviations from the Hardy-Weinberg equilibrium were observed except for Jordanians which probably represent a random statistical fluctuation. Table D allele in the populations analyzed here as well as selected populations from previous studies.As shown in Table D allele of the ACE gene  element later inserted at the locus, generating the I allele and creating an ancestral polymorphism in the pre-migration African human population. As human populations moved out of Africa during Paleolithic migrations 100,000 years ago [The worldwide distribution of the le Table  suggestsears ago , they caD allele represents the ancestral state of the ACE gene without an Alu repeat insertion in the region of intron 16 [Alu elements are most abundant, accounting for more than 10% of the human genome.Although the I/D polymorphism in the ACE gene implies either an insertion or a deletion, there is only an insertion event, and the ntron 16 . In factD allele among Egyptians, Jordanians and Syrians are comparable to those obtained from previous studies in other Arabs, add to the data indicating the wide variations observed in the frequency of the ACE alleles among the peoples of the world and highlights that great care needs to be taken when interpreting clinical data on the association of the ACE alleles with different diseases.In conclusion, the results of the distribution of the ACE I/D gene polymorphism obtained for the The authors declare that they have no competing interests.ASH designed the research project. ASH performed the experiments and statistical analysis. MAB contributed reagents/materials/analytic tools. AHS and MAB wrote the manuscript. All authors have read and approved the final manuscript.