Previous findings on relationships between infertility, infertility therapies, and autism spectrum disorders (ASD) have been inconsistent. The goals of this study are first, to briefly review this evidence and second, to examine infertility and its treatments in association with having a child with ASD in newly analyzed data. In review, we identified 14 studies published as of May 2013 investigating infertility and/or its treatments and ASD. Overall, prior results showed little support for a strong association, though some increases in risk with specific treatments were found; many limitations were noted. In new analyses of the CHildhood Autism Risk from Genetics and the Environment (CHARGE) population-based study, cases with autism spectrum disorder  and controls confirmed to have typical development (n = 388) were compared with regard to frequencies of infertility diagnoses and treatments overall and by type. Infertility diagnoses and treatments were also grouped to explore potential underlying pathways. Logistic regression was used to obtain crude and adjusted odds ratios overall and, in secondary analyses, stratified by maternal age (≥35 years) and diagnostic subgroups. No differences in infertility, infertility treatments, or hypothesized underlying pathways were found between cases and controls in crude or adjusted analyses. Numbers were small for rarer therapies and in subgroup analyses; thus the potential for modest associations in specific subsets cannot be ruled out. However, converging evidence from this and other studies suggests that assisted reproductive technology is not a strong independent risk factor for ASD. Recommendations for future studies of this topic are provided. Autism spectrum disorders (ASD) are developmental conditions characterized by restrictive, repetitive behaviors and deficits in communication and social interaction. Though genetic factors are known to be involved in the etiology of ASD, research demonstrates that environmental factors play a crucial role as well ,2. For eA number of studies have investigated maternal infertility, typically defined as failure to get pregnant after 12 months or more of unprotected intercourse without success , and usein vitro fertilization (IVF) [Although some reports have suggested an increased risk of ASD or, more broadly, developmental delays with use of infertility treatments, types of therapies assessed and definitions used vary by study ,20,21,22on (IVF) ,22, and on (IVF) ,18; one on (IVF) , while aon (IVF) ,25,26,27on (IVF) ,26,27. Oon (IVF) ,18,28 haThe underlying causes of potential associations between infertility treatments and ASD have not been carefully examined, but may include influences of the medications or procedures themselves, of underlying infertility etiology, or the integrity of the utero-placental unit in the resulting pregnancies. For instance, reproductive hormone imbalances are central to certain types of infertility, and are indications for treatments such as the use of ovulation induction drugs. A link between hormonal factors and ASD has been suggested through hypotheses about fetal testosterone  and evidThe evidence to date is inadequate for drawing conclusions about the relationship between infertility, its treatments, and ASD. In particular, there is a need for studies the utilize rigorous methods for diagnostic and exposure confirmation, adjust appropriately for confounders, and provide information on a wide range of specific infertility diagnoses and treatments. To address these gaps, we sought to examine whether different types of infertility and treatments for infertility were associated with ASD in a population-based case-control study with clinically confirmed diagnoses. We also examined potential underlying pathways in exploratory analyses. Based on prior work, we hypothesized that associations with ASD would differ according to type of treatment, and that those related to hormonal and inflammatory factors would show increased risk in association with ASD. Because our study could not address all questions and all limitations of prior studies on this topic, such as confounding by indication and examination of effects of very rare treatments and diagnoses on ASD risk, in addition to reviewing the literature and conducting our own analyses, we also provide recommendations and novel frameworks for future studies of infertility and its treatments in association with ASD.Participants in this study are part of CHildhood Autism Risk from Genetics and the Environment (CHARGE), an on-going, large, population-based case-control study drawn from several regions of California; the details of the study have been previously described . BrieflyInformation on fertility therapies was collected through a telephone administered interview with the mother, known as the Environmental Exposures Questionnaire (EEQ), through prenatal and infertility clinic medical record abstraction, and through a supplemental telephone interview related solely to infertility issues, used when medical records were not available but infertility or treatments were reported. Thus, both medical record and self-reported information were used to define the exposures of infertility and its treatments in primary analyses; among those defined as exposed, 71% had infertility noted in medical records and 81% had infertility treatments noted.All interviews were conducted in English or Spanish by trained personnel. For self-reported information, participants were asked: “Before you became pregnant with (CHILD), was there was there a period of 12 months or more when you had regular intercourse without using any method to prevent pregnancy and did not become pregnant?” Self-reported infertility was defined according to endorsement of this question. Participants were also asked: “Have you or your partner used any procedure or medication to help you get pregnant with (CHILD)?”, with detailed probes about different types of therapies, surgeries, and medications for both male and female treatments. Endorsement of any of these treatments was defined as any self-reported infertility treatment , and reviewed all records received for presence of infertility diagnoses and treatments. The majority of the study group  had medical records available. For women who disclosed fertility problems and/or treatments either on their list of medical providers or in their interview responses, we also requested records from infertility specialists. An obstetrician/gynecologist (CW) reviewed prenatal medical records and medical records from specialists, and contacted participants to resolve discrepancies between responses in interviews and recorded exposures in (or omitted from) medical records if inconsistencies arose; however, the majority of women with both sources of exposure status had consistent information. i.e., OID without IVF or ART procedures noted); (3) Inflammatory group: underlying conditions thought to arise from or be associated with inflammation: cervical and tubal factor infertility, endometriosis, and typical treatments for these conditions—IVF and use of injectable “heavy duty” OIDs; and finally, (4) Male-factor group: male-factor infertility and treatments only without female issues—male surgeries and medications, sperm washing, sperm aspiration procedures, intrauterine insemination (IUI), or intracytoplasmic sperm injection (ICSI). For both the pathway variable and the grouped infertility diagnosis variable, for any individuals with multiple treatments or diagnoses, we classified according to the most invasive issue to obtain mutually exclusive categories.Infertility treatments were categorized as use of any type (binary variable) and individually by specific treatment: ART, use of ovulation—inducing drugs (OID), artificial insemination (AI), female surgical procedures, or male only procedures. Infertility diagnosis was also examined as any diagnosis, as well as by the following specific diagnoses: endometriosis, tubal factor, uterine factor, male factor, diminished ovarian reserve, ovulatory dysfunction, poly-cystic ovarian syndrome (PCOS), and unexplained infertility. Infertility diagnoses were also grouped into the following categories for analysis: (1) uterine factor infertility, fibroids, and ovarian cysts ; (2) PCOS, anovulation, irregular periods ; (3) cervical or tubal factor infertility, endometriosis (the “inflammatory infertility” group); and (4) male factor infertility and factors related only to sperm . In exploratory analyses, we created groups based on potential underlying pathways that might link ASD to either treatments or infertility diagnoses and problems noted in medical records. If medical records were not available, we made assumptions based on the type of treatment given as to the underlying indication. The pathway variable for these analyses was defined using four categories: (1) Structural group: uterine factor infertility, fibroids, ovarian cysts, and the surgical procedures used to repair them; (2) Hormonal group: PCOS, anovulation, irregular periods; and treatments of hormones or medications to influence ovulation or sex steroid hormone levels  infertility, (2) infertility therapies; and (3) hypothesized pathways. Analyses of infertility compared those with any infertility to those without. Due to the low prevalence of individual infertility diagnoses, we used the grouped infertility variable to examine different types of infertility; in these analyses, indicator variables were created for each of the 4 infertility groups described above, as well as one for unexplained or other infertility for individuals not fitting within any other grouping; the referent group was again those with no infertility. Parallel analyses were conducted for infertility therapies, comparing those with any infertility to those without. Analyses of individual infertility therapies with sufficient numbers compared those with that particular treatment to those without it (but who may have received others). The pathway analysis used those with no treatments or infertility as the referent group, with indicator variables for the other categories. vs. TD, and their 95% confidence intervals. We used logistic regression, and examined adjustment for the following potential confounders , based on a priori knowledge: maternal and paternal age (continuous variables), maternal race/ethnicity , maternal education (4 categories), insurance payment for delivery (public vs. private), birth order , pre-pregnancy smoking status (yes/no for regular smoking prior to pregnancy), and pre-pregnancy BMI (5 categories). Assessments of individual fertility therapies also considered further adjustment for use of other therapies (binary indicator). In selection of covariates, we examined variance inflation factors and correlation of covariates to avoid multicollinearity. All models also adjusted for the study matching factors . For all regression models, we report the associations in odds ratios for ASD In further analyses, we adjusted for potential biases arising from differential participation rates using weighted conditional logistic regression in Proc Survey Logistic. The weights were proportional to the inverse-probability of participation  to enhance generalizability to the source population. In secondary analyses, we examined associations among mothers aged 35 or older at delivery, and by diagnostic subgroup (autistic disorder and broader ASD). In sensitivity analyses, as our exposures were defined according to both self report and medical record sources, we compared results of exposures classified according to each of these sources of information in isolation. These analyses included 537 ASD cases and 381 TD controls. Parents of children with an ASD were slightly older than TD control parents, and case mothers were slightly more likely to have had a history of smoking; other demographic and lifestyle factors were similar between the groups . Nine percent of both ASD cases and TD controls had used at least one type of infertility treatment for the index birth. Numbers were small for rarer types of therapies, but overall, frequencies were remarkably similar between the ASD and TD groups . The ASDIn adjusted analyses comparing ASD cases to TD controls, fertility therapies and infertility continued to show no association with odds of ASD . OverallIn subgroup analyses among mothers of advanced age (n = 237) and by diagnostic subgroup (367 autistic disorder and 170 broader ASD), results were very similar, and again non-significant for any associations with infertility and infertility treatments . However, it should be noted that numbers were small within these groups; only 27 cases used any fertility therapies among the advanced maternal age subgroup, with numbers for individual types of therapies around 10 or fewer. Likewise, sensitivity analyses utilizing only self-reported information, or only information from medical records, also did not demonstrate any significant associations.modest associations with rarer therapies or conditions. These topics should therefore be further explored in very large studies with standardized outcome ascertainment and rigorous exposure information.The results of this case-control study do not provide evidence for an association between fertility therapies and autism spectrum disorders. We examined a number of different types of therapies and conditions underlying the infertility being treated, and overall found remarkable similarity between ASD cases and typically developing controls. However, due to the low power to detect subtler effects in our study, we cannot exclude the potential for A major strength of this study, and an improvement over a number of prior studies examining infertility and/or its treatments in association with ASD, is the confirmation of both case status and exposures through rigorous, gold standard measures. All children included in these analyses were evaluated at the UC Davis MIND Institute for diagnostic confirmation, and detailed interviews were conducted and medical records abstracted (in the majority of the study group) for exposure information. In contrast, none of the prior studies examining these factors have confirmed case and comparison group status at this level of detail. We also had information on a full range of infertility diagnoses and treatments, which is lacking in other studies. Our estimates of frequency of use of a wide range of therapies according to ASD status thus fill a needed gap in the literature. Despite using retrospective reporting, as had been previously utilized in a number of prior investigations, we also collected medical records in a large majority of the group for confirmation. We also carried out a thorough confounder identification strategy, whereas many of the prior studies of infertility treatments and ASD failed to adjust for even basic sociodemographic risk factors ,22,25,27However, a number of limitations in our work should be noted. Despite a sample size of nearly 1,000 mother-child pairs, our study was limited by the relatively rare exposures, leading to small numbers in many categories. Thus, while we had sufficient power to detect odds ratios of at least 1.75 for treatments and diagnoses with prevalence over 5%, power was reduced to detect associations for specific therapy types with infrequent use. To date, only Hvitjorn and colleagues  have hadConsistent with our results, the majority of prior work suggests that use of assisted technologies does not increase risk of adverse child outcomes . A handful of studies have suggested increased risks of autism, or developmental delay, cerebral palsy, and imprinting disorders with use of ART ,19,22. HFor other types of infertility treatments, there is limited information on associations with ASD specifically. A handful of prior studies have suggested associations with ovulation drugs or medications  ,18,28, sThe Nurses’ investigation also saw an association with AI, which the CHARGE study did not replicate; however, the source of infertility treatment information in the Nurses’ study was not as rigorous as in the current study. We did find increased odds ratios for male treatments in our study; though non-significant, use of AI is sometimes indicated for male factors. Again, Grether and colleagues’ study found an association with IUI and ASD only among multiple births but not in singletons, providing mixed results. Given that Hvitjorn and colleagues’ definition of OID included use with and without AI, future studies should also investigate AI in association with ASD, both in singleton and multiple births.Our analyses of these infertility treatments considered adjustment for a number of potential confounders. Prior studies examining potential effects of infertility treatments have adjusted for or stratified on multiple births in attempt to assess the effect of the treatments on the various outcomes studied, not mediated by multiplicity. For comparison to this work, we examined exposures stratified by singleton and multiple births and found that results did not differ . However, conditioning on a downstream consequence of exposure can introduce bias. Another example is adjustment for birth weight, a common flaw in studies of prenatal exposures; again, we did not include birth weight in our multivariable models for this reason. Future large studies may benefit from the use of more sophisticated statistical methods, such as marginal structural models (MSMs) ,38, to di.e., those on the order of OR = 1.5 or less), given the number of exposed cases in each of the pathway groups. Continued investigation of such pathways and groupings as conducted here may be useful in learning more about potential underlying mechanisms.Infertility treatments have been hypothesized to influence ASD through a number of mechanisms, including hormonal influences of the medications, effects of invasive procedures on DNA methylation or other direct effects of the procedure/treatment, impaired egg quality, influences of the underlying infertility, or simply through associations with downstream consequences of the treatment  ,18,41. WRecommendations for future studies: It is evident that very large studies will be needed to fully elucidate the potential effects of individual infertility treatments and underlying diagnoses on risk of ASD. Besides simply increasing numbers, future studies may also benefit from the following suggestions. Rigorous diagnostic and exposure confirmation methods and definitions are required to avoid misclassification and bias. In addition to studying infertility diagnoses and treatments, investigating related factors, such as sub-fertility and time to conception may help to shed light on whether infertility itself is associated with ASD. Models testing the effect of infertility treatments among those with infertility will also be useful in determining independent effects of treatments versus the indication for treatment. Further, making use of hypothesized underlying pathways, such as those proposed here, may help to determine whether groups of diagnoses and treatments point to a likely etiologic role. Finally, analyses can be improved by avoiding adjustment for downstream effects of exposures and rather considering mediators, as described above, through use of one or another type of mediation analysis , all of which require thorough adjustment for confounders of the intermediate-by-outcome association. strong increases in risk of ASD. However, the known risks associated with infertility therapies, such as prematurity, low birth weight infants and multiple births, remain as concerns associated with use of these therapies, and evidence suggests the need for continued long-term follow-up of children conceived using these procedures [Our work and that of others highlights the need for very large studies in order to fully address the topic of infertility and its treatments in association with ASD. Overall, the evidence to date suggests that women using infertility therapies do not need to be concerned about ocedures . Women u


Metabolic syndrome (MeS) is being recognized as a risk factor for insulin resistance and cardiovascular disease. The present study was aimed to find out the possible association between 45-bp I/D polymorphism of uncoupling protein 2 (UCP2) and MeS.DNA was extracted from peripheral blood of 151 subjects with and 149 subjects without MeS. 45-bp I/D variant of UCP2 was detected using polymerase chain reaction (PCR).Our finding showed that 45-bp I/D polymorphism was associated with protection against MeS . The I allele decreased the risk of MeS  in comparison with D allele.In conclusion, our result suggests that 45-bp I/D polymorphism is associated with the risk of MeS, which remains to be cleared. Metabolic syndrome (MeS) is described as the combination of clinical disorders that increase the risk for obesity , insulin resistance, glucose intolerance, dyslipidemia, non-alcoholic fatty liver disease and cardiovascular diseases including atherosclerosis, stroke and hypertension,2. The pThe dysregulation of uncoupling proteins (UCPs), which translocate protons into the mitochondrial matrix resulting in heat generation without ATP synthesis, may conThis case-control study was done on 151 patients with and 149 without MeS which we used in previous studies as descrPosition of the 45-bp I/D polymorphism of UCP2 is shown schematically in Figure The differences between the categorical and contentious data were assessed by chi-square and independent sample t-tests, respectively. The association between genotypes and metabolic syndrome was assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CI) from logistic regression analyses. P-value less than 0.05 were considered statistically significant. The data analysis was performed using the SPSS18 software. According to our findings, sample power was calculated by using STATA 10 software.A total of 300 subjects including 151 MeS patients and 149 subjects without MeS were recruited in the study. The demographic and biochemical characteristics of the study participants are shown in Table The genotype distribution of 45-bp I/D polymorphism of UCP2 in subjects with and without MeS were in Hardy Weinberg equilibrium (HWE) .In the present study, we analyzed the possible association 45-bp I/D polymorphism of UCP2 gene and MeS in a sample of Iranian population. The UCP2 45-bp I/D was associated with MeS, so that the frequency distribution of I/D genotype as well as I allele were significantly lower in cases than that of controls. Metabolic syndrome is a combination of risk factors for cardiovascular disease (CVD) and T2DM. These factors include hyperglycemia, hypertension, dyslipidemia (high level of triglyceride and low HDL-cholesterol), and obesity ,27. PrevSome studies have found no associations between UCP2 45-bp I/D polymorphism and obesity, resting energy expenditure, BMI and insulin secretion-32. ThouOguzkan-Balci et al. have fouPapazoglou et al. have fouCrispim et al. investigIt has been reported that UCP2 I/D heterozygous decreased the risk of end-stage renal disease (ESRD). MitchelIt has been proposed that impaired adipose tissue expression of UCP2 may play a role in the pathophysiology of obesity. To dateOne of the limitations of the present study is relatively small sample size. Consequently, analysis according to MeS components was not possible.In conclusion, our findings showed that the 45-bp I/D polymorphism of UCP2 was associated with decreased risk of MeS. Larger studies with different ethnicity are required to validate our findings.No competing financial interests.MH designed the study, supervised the study, analyzed the data and drafting the manuscript. HR collected the data and carried out the laboratory studies. MAK collected the data, and reviewed the manuscript. MT analyzed the data and reviewed the manuscript. All authors read and approved the final manuscript.


The exceptional progress in obstetrics, especially in the second half of the 20th century, creates a series of dilemmas, without having yet the answer to old, basic questions, such as premature delivery and prevention of preeclampsia. Sexual liberty and the delay of the first pregnancy are responsible for the drop in natality rate and the increasing demand for assisted reproductive technologies. Their success is marked, though, by a drastic increase in the multiple birth rate and its complications. The advancing maternal age brings along a number of cardiovascular pathologies that endanger not only the present pregnancy, but also the long distance health prognosis. Plus, there is a marked risk for foetal chromosomal anomalies, which explains the need and the importance of prenatal diagnosis. Probably the newest branch in materno–foetal medicine, it offers many answers and solutions, but there are still a lot of question marks. In conclusion, the progress is necessary, is expected, but it should always be filtered by time and experience. Obstetrics, a science born at the same time with human kind, having been crucially assimilated within its development and progress, has slowly evolved through thousands of years and today, we like to think that we have reached a point that comes close to the apex of our pyramid of knowledge. A peak where there are no longer uncertainties, where we do not longer have materno–foetal mortality and morbidity statistics, where human reproduction is a risk free phenomenon. Reality unfortunately, begs to differ.The road of obstetrics may begin with Hippocrates, the one who assumed that birth is triggered by the foetus who ‘becomes agitated and thus breaks the membranes’, may continue with Leonardo da Vinci who, during the Renaissance, struggles to imagine the intrauterine development of the foetus, noting that it ‘has a daily growth inside the womb that surpasses its growth after birth’.All great humanists, regardless of the period in time in which they lived and thought, were preoccupied by the decryption of the intrauterine growth mystery. What the twentieth century and especially its second half brings new, is the concern to treat and to prevent complications associated to pregnancy and birth. The development of molecular biology and genetics, the understanding of physiological mechanisms and their pathological alterations, the spectacular progress of computer technology with its potential applications in medicine, have cleared up for good the mystery of emergence of the human race, which was transformed from a myth into a science with numerous extensions and overspecializations, a science where the assistance of birth, a hundred years ago the basis of the trade, is now of secondary importance. Thus, at this moment, it is more appropriately to replace the term ‘obstetrics’ with the one ‘maternofoetal medicine’, more comprehensive and including both participants of this inseparable partnership.The peak of the pyramid is still far. Even though outnumbered hypotheses were issued, many theories were made, a great deal of studies were elaborated, thousands of articles and papers were written, the statistical numbers are tough and show that the percentage of premature births is still high, almost unchanged in the last decades and the impairments caused by them constitute a great social, moral and economic burden, even in the developed countries with high medical standards. To be more precise in this matter, it must be noted that in 2005 in the United States, 12.7% of all births were premature, understanding by this a gestational age of less than 37 finished weeks or 259 days. 2.03% happened before 37 weeks while 9.1% happened between 34 and 36 weeks . In compMultiple pregnancy constitutes a true epidemic of the last decade, having been undoubtedly a result of clinical success of assisted reproduction techniques. In natural conditions, the incidence of twin pregnancies is 1 to 80 and that of triple pregnancies is 1 to 8000, according to Hellin's law. In practice though, in the United States, in 2002 twin pregnancies made up 4% of all births and triple pregnancies, 3% . The incA sensible question is why there is an increased appeal to the assisted reproduction services. The way of life in particular of population in developed countries, emancipation of women, sexual liberation, effective hormonal contraception, planned delay of maternity in order to favour professional success, imbalance in the institution of family, all these have contributed to the decrease of natality in particular by the aging of gametes and the rise in endometriosis and pelvic inflammatory disease incidence. It is difficult to asses the prevalence of infertility prior to the sixties, when it was regarded as a sporadic infliction. Though we know that in 1982, 11% of female American population that sought to become pregnant had fecundity issues, in 2002 this percentage rose to 15% . AccordiThe advanced maternal age may also generate other complications. First, women over 40 years of age tend to associate a series of pathologies like gestational diabetes, obesity, chronic hypertension, especially renal hypertension. The incidence of preeclampsia in this category of women is high, in particular if it is their first pregnancy. Maternal age represents an independent significant risk factor for the development of preeclampsia  [Although it is one of the first complications of pregnancy that has been isolated, identified and thoroughly described by obstetrics classics, in the age of genetic diagnosis in maternal blood, in utero therapy and stem cells, preeclampsia still constitutes an unresolved problem, a major source of maternal mortality and morbidity for which there are yet few efficient therapeutic resources, affecting between 10 to 20% of all pregnancies. Namely a ‘disease of theories’, which illustrates the frailty of the ground on which its physiopathological vision has been built, having as a result a poor efficiency of treatment, its definition criteria have little been changed since its first description.  As the complexity of this disease is revealed step by step, the list of biological alterations that accompany preeclampsia grows accordingly. This does not seem to improve significantly the prognosis of these pregnancies but merely complicates the specialists' goal. In long term, a women who has developed early preeclampsia, has a high risk of cardiovascular disease and by that understanding hypertension, cardiac ischemic disease, stroke and cardiac death.This relative risk varies between 3.5 and 1.5, depending on studied population and criteria of definition , 10. ThIt is not clear to what extent these metabolic changes can be lowered or even be avoided completely by an effective therapy of the hypertension episode in pregnancy. It is well known that the origin of these changes lies in the early stage of pregnancy, in the period of trophoblastic invasion, while consequences appear much later. That's why, efforts are aimed at the prediction and prevention of this disease. A good screening test should be simple, certain, quick, cheap and reproducible and should allow medical intervention in order to prevent the disease or at least improve its prognosis.The first predictive test used was the ‘roll–over test’, which, although efficient, was proven to be outdated. Serum measurements of angiogenic factors such as VEGF or PIGF , angiogenic proteins, like soluble endogline (sEng) or fragments of VEGF type 1 receptor (s–Flt1), also known as fms–like tyrosine–kinase, prove to be more useful though hardly accessible. Thus, in the serum of women at risk of preeclampsia there is an increased ratio between s–Flt1 and sEng and a decreased ratio between VEGF and PlGF, which determine a diffuse endothelial lesion, with alteration in the capillary permeability . These cUterine artery Doppler ultrasonography is one of the most known prediction techniques. Through its simplicity, it has also been established as a screening technique although its flaws are many, having great inter–observer variations and not being considered appropriate for a screening test. Even with these in mind, a met analysis of 43 studies, which included 42000 women, revealed that a positive Doppler test increases a woman's risk from 2,5%  to 8 – 15%, while a negative test decreases this risk to 1,5% . TheoretSimilarly, although hyperuricaemia is frequent in preeclamptic women and there was a lasting idea that uric acid levels grow a few weeks before tensional values grow as well, a series of recent studies have proven that this test does not have a predictive value and therefore can not be used in screening .In these conditions, clinical routine evaluation, with systematic measurement of arterial pressure and look out for preeclampsia signs, has a predictive value similar to the most costly and newest diagnostic tests. That is, we are exactly where we left off.Another controversial area is the prenatal diagnosis, a completely new science, developed at the same time with ultrasonography and genetics. Clearly, it is the domain with the greatest progress and hopes for the future. If 30 years ago ultrasound served generally to confirm the position of foetus, its cardiac activity and at most, the diagnosis of brutish structural anomalies, now we track down discrete facial dimorphisms and the lack of corpus callosum formation. Technical progress has introduced the spirit of competition, imposed the re–evaluation of fine anatomy and embryology data with continuously up–to–date and new information in the field of biology and genetics. In this way, there has been an evolution to the point of prenatal detection of some pathologies that don't even have a known prognosis and there are not few the situations when we encounter couples who ask pregnancy termination because the foetus has an isolated great cisterna magna or a partial corpus callosum agenesis. There is also the reverse of the coin. The numerous serological and ultrasound tests confuse doctor and patient alike, the patient refusing thus any additional investigation guided by her faith and optimism. Amniocentesis, umbilical cord centesis, trophoblastic biopsy can constitute effective diagnosis means but at the same time as many risks for the evolution of pregnancy.Following the diagnosis, an in utero therapy has been developed. Starting with Harrison who in the eighties performed the first intervention of this kind for a foetus with diaphragmatic hernia, there is a shift to a more conservative approach. Of course that the defect may be repaired before birth, but this does not improve too much the prognosis of the foetus who is consequently exposed to prematurity. Thus, at this moment, only minimal repair surgeries are practiced, by means of foetoscopy or only under ultrasound guidance, in cases of low urinary tract obstruction, unilateral pleural effusion, diaphragmatic hernia or isolated ventriculomegaly.Still, matters are far from ending at this point. Prenatal diagnosis in the future years will require the simple drawing of maternal blood in which foetal cells and foetal DNA will be isolated and used for all necessary tests. At least, that's what we would like to believe. We do not know yet to what extent this will simplify matters or on the contrary become another gate to unknown realms, but we owe belief and support to progress, filtering though the discoveries through our past experience and through the test of time.


The purpose of this study was to replicate genetic factors associated with the susceptibility to Behçet's disease (BD). We conducted a two-stage candidate genes association and functional study, involving 477 BD patients and 1,334 normal controls of Chinese Han descent.LOC100129342, KIAA1529, CPVL, UBASH3B and UBAC2, were performed using TaqMan single nucleotide polymorphism (SNP) assays. Real-time PCR and luciferase reporter assay were performed to test the function of the identified promoter polymorphism. The main outcome measures were genotype frequencies and expression levels in BD patients.The genotyping of five candidate genes/loci, including UBAC2 , but not LOC100129342, KIAA1529, CPVL, UBASH3B was associated with the susceptibility to BD in Chinese Han. The fine-mapping association study of UBAC2 identified six risk SNPs for BD in the Chinese cohort; three of them were verified in validation study . Functional analysis showed that the risk T allele of the promoter polymorphism rs3825427 had a significantly lower promoter activity than the non-risk G allele (P = 0.002) and a decreased expression of UBAC2 transcript variant 1 in peripheral blood mononuclear cells (PBMCs) and skin of normal controls carrying the risk T allele than that in individuals with the G allele . The mRNA expression of UBAC2 transcript variant 1 was significantly decreased in PBMCs and skin of BD patients as compared with controls . The mRNA expression of UBAC2 transcript variant 2 was significantly increased in skin of BD patients as compared with controls (P = 0.004).The first-stage study results showed that UBAC2, and suggests that UBAC2 may be involved in the development of BD through its transcriptional modulation.This study replicates a predisposition gene to BD,  HLA-B51 has been found to be strongly associated with BD [HLA-B51 only accounts for 20% of the risk genes for this disease [HLA contributory loci/genes for BD [IL23R [CTLA4 [eNOS [SUMO4 [NOD2 [IL1A [SOD) [Behçet's disease (BD) is generally considered as a refractory multisystem disorder characterized by recurrent oral ulceration, genital ulceration, recurrent uveitis and multiple skin lesions ,2. BD pr with BD -7, it is disease . A wholes for BD . CandidaD [IL23R , CTLA4 [R [CTLA4 , eNOS [1A4 [eNOS , SUMO4 [S [SUMO4 ,14, NOD2O4 [NOD2 , IL1A [1D2 [IL1A , mangane1A [SOD)  and so o1A [SOD) .LOC100129342, KIAA1529, CPVL, UBASH3B and ubiquitin-associated domain containing 2 (UBAC2), among which the genetic association between UBAC2 and BD was lately confirmed and functional significance was tested by the same study group [UBAC2 was associated with BD in Han Chinese. Fine mapping and validation studies further confirmed UBAC2 as susceptibility gene for Behçet's disease. Further functional study showed that the risk T allele of rs3825427 in UBAC2 was associated with the decreased promoter activity and its mRNA expression. These genetic associations and functional studies suggest that UBAC2 is a risk gene for Behçet's disease.Fei and coworkers  conductedy group . As far Written informed consent was obtained from each participant, and this study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University and Zhongshan Ophthalmic Center, Sun Yat-sen University (Permit Number: 2009-201004).One hundred forty-seven BD patients and 951 normal controls enrolled in the first-stage were recruited from April 2005 to March 2008 in Southern China at Zhongshan Ophthalmic Center, Sun Yat-sen University. The replication cohort is comprised of 330 Han Chinese BD patients and 383 Han Chinese normal controls, which were recruited from April 2008 to March 2010 in Western China at the First Affiliated Hospital of Chongqing Medical University  using Chinese Han Beijing data to screen out tagSNPs including rs3825427, rs1927726, rs9517668, rs9554581, rs4636771, rs7332161, rs9517701, rs7325747, rs912129, rs4772190 and also included the reported BD associated SNP rs9513584 [We chose the enrolled 25 SNPs based on the following principle: we focused on the P-value. The call rate of SNP rs9517699 did not meet the quality standard in the replication study , so SNP rs9517699 was excluded in the replication analysis  on the Applied Biosystems 7500 Real-Time PCR System according to the manufacturer's instructions. All SNPs tested in this study had a genotyping success rate > 98% and accuracy > 99% as judged by random re-sequencing of 20% of samples in all subjects.Genomic DNA was extracted from the peripheral blood of patients and controls using the QIAamp DNA Blood Mini Kit  according to the manufacturer's instructions. The Genotyping was determined by TaqMann = 8) and normal controls (n = 52) by Ficoll-Hypaque density-gradient centrifugation. Skin was obtained from the skin lesions of patients (n = 4) and the scrotum of normal controls (n = 22). Total RNA was isolated from skin and PBMCs of the patients or controls by using the QIAamp® RNA Blood Mini kit  or Qiagen RNeasy Fibrous Tissue Mini Kit  with treatment of Dnase I, according to the manufacturer's instructions and reversed into cDNA according to the Superscript protocol . To compare the mRNA expression of the two protein-coding transcript variants of UBAC2, Real-time Quantitative PCR was performed using the Applied Biosystems 7500 System (Applied Biosystems) with the following primers (UBAC2F:5'CCG GCT CCA GTG GGC TCT ACA3'/UBAC2R:5'GGG CGA GCA GGA GGG AGA GG3') for the UBAC2 transcript variant 1 to generate a 81 bp product and (UBAC2F: 5'TAGGAAGTCGTGGCGAGGGAGC3'/UBAC2R: 5'GCCTTGTCTGCTGAC CACCGCT3') for the UBAC2 transcript variant 2 to generate a 84 bp product. The β-actin (β-actinF: 5'GGA TGC AGA AGG AGA TCA CTG3'/β-actinR:5'CGA TCC ACA CGG AGT ACT TG3') was chosen as the internal reference gene to normalize UBAC2 expression. RT-PCR conditions were one cycle of 95°C for 10 minutes, followed by 40 cycles in which each cycle included 95°C for 15 sec, 60°C for 1 minute, and 95°C for 15 sec, 60°C for 1 minute, 95°C for 15 sec, 60°C for 15 sec.Anticoagulated blood samples were obtained using vacuum tubes with EDTA. PBMCs were prepared from venous blood of BD patients . This vector was then transiently transfected into HEK293 cells. Reporter plasmid was transfected to cells using Lipofectamine reagent . Transfection efficiency was standardized by cotransfecting with pRL-SV40 (Promega). Luciferase activity was determined after 24 hours incubation using a Luciferase Assay system . For each plasmid construct, three independent transfection experiments were performed, and each was done in triplicate.The whole genome synthesized promoter sequences of P >0.05). Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated by using using SPSS version 17.0  to estimate disease risk. Linkage disequilibrium (LD) was examined by using Haploview . To account for multiple testing, the Bonferroni correction was applied. Review Manager 4.2  was used to perform meta-analysis. The conditional logistic regression analysis  was performed to localize the effect and identify the number of independent effects.Hardy-Weinberg equilibrium (HWE) was tested using the chi-square test and no SNPs showed significant deviation from HWE (P >0.05).The clinical findings of the BD patients included in our study are shown in Table LOC100129342, KIAA1529, CPVL, UBASH3B and UBAC2, have been identified as risk factors for BD in Turkish. These five candidate genes/loci were not validated by other ethnic populations. We conducted a candidate gene association study to confirm the association result using 147 BD patients and 951 controls. Our results showed that the rs9513584 polymorphism inUBAC2 was associated with the susceptibility to BD   . Therefore, the multiple associations within the UBAC2 locus are not independent.The variant in Pc = 2.2 × 10-3, OR = 1.5, Second-stage Pc = 9.3 × 10-3, OR = 1.4, combined Pc = 6.9 × 10-6, OR = 1.5; rs9517668, First-stage Pc = 1.7 × 10-3, OR = 1.6, Second-stage Pc = 0.03, OR = 1.3, combined Pc = 3.3 × 10-4, OR = 1.4; rs9517701, First-stage Pc = 5.1 × 10-3, OR = 1.5, Second-stage Pc = 9.0 × 10-3, OR = 1.4, combined Pc = 2.9 × 10-5, OR = 1.4; respectively)  in rs3825427. The mRNA level of UBAC2 transcript variant 1 was found decreased in PBMCs and the skin of normal individuals with the TT genotype compared with those with the GG genotype  . Sequencing was performed to validate the result of synthesis and the result showed that only one variant was observed in the location of the rs3825427 polymorphism. The luciferase reporter expression was found to be decreased in the T allele as compared to cells carrying the G allele (P = 0.002)    as compared with wild-type mice [SNP rs3825427 in the Several possible limitations of the present study merit particular consideration. First, the patients enrolled in our study were recruited from the Eye Department. Because BD usually affects multiple systems, the patients enrolled in this study might, therefore, represent a separate disease population. Second, the patients enrolled in the replication study were recruited only from Chinese Han individuals and the sample size is relatively small. The results presented here need to be confirmed in other ethnic populations and using larger samples. Finally, although molecular biology study provides functional evidence for the rs3825427 polymorphism, this result is generally suggestive and does not exactly explain how the genetic variant translates into physiologic processes and then affects disease susceptibility. Therefore, the association results presented here should be investigated further using more functional experiments.UBAC2, rs3825427, to be associated with the increased risk for BD in Chinese. The functional study showed that this SNP may be involved in the development of BD through transcriptional modulation of UBAC2.In conclusion, our study replicated the association of UBAC2 with BD and identified a promoter SNP of BD: Behçet's disease; EAE: experimental autoimmune encephalomyelitis; EDTA: ethylenediaminetetraacetic acid; GWAS: genome-wide association study; HWE: Hardy-Weiberg equilibrium; LD: Linkage disequilibrium; OR: odds ratio; PBMCs: peripheral blood mononuclear cells; SD: standard deviation; SNP: single-nucleotide polymorphism; SOD: superoxide dismutase; UBAC2: ubiquitin-associated domain containing 2.None of the authors has a proprietary or financial interest in any of the products mentioned.HSP carried out the genotyping, Dual Luciferase Reporter Gene Assay and expression studies and drafted the manuscript. SQM and JZX carried out the genotyping study. CYY, LFZ and CFL participated in the samples collection. HSP and YPZ participated in the design of the study and performed the statistical analysis. HSP and YPZ conceived of the study, and participated in its design and coordination. AK and YPZ revised the manuscript. All authors read and approved the final manuscript.UBAC2 gene with Behcet's disease and linkage disequilibrium of UBAC2 geneThe association of . Table S1 presented the association analysis of three SNPs in UBAC2 gene with Behcet's disease in Han Chinese population. Figure S1 showed linkage disequilibrium plots of UBAC2 gene based on the HapMap Phase II dataset for the Han Chinese from the Beijing population by Haploview 4.2 software.Click here for file


The association between aldosterone synthase (CYP11B2) C-344T gene polymorphism and ischemic stroke remains controversial and ambiguous. To better explain the association between CYP11B2 polymorphism and ischemic stroke risk, a meta-analysis was performed.Based on comprehensive searches of Medline, Embase, Web of Science, CNKI and CBM databases, we identified and abstracted outcome data from all articles to evaluate the association between CYP11B2 polymorphism and ischemic stroke. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were performed in all genetic models. Fixed or random effects model was separately used depending on the heterogeneity between studies. Publication bias was tested by Begg's funnel plot and Egger's regression test.A total of 12 studies including 3,620 ischemic stroke cases and 4,090 controls were identified. There was no statistical evidence of association between CYP11B2 C-344T polymorphism and ischemic stroke in all genetic models . On subgroup analysis by ethnicity, similarly results were found in both Asians and non-Asians. For Asians, the combined ORs and 95% CIs were . For none-Asians, the combined ORs and 95% CIs were .The present meta-analysis suggested that CYP11B2 C-344T polymorphism was unlikely contribute to ischemic stroke susceptibility. The renin-angiotensin-aldosterone system (RAAS) is important for cerebrovascular research, because of its influence on blood pressure, vasoconstriction, thrombosis, and vessel wall damage This meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) criteria Two reviewers (Y.P. and K.C.) independently screened titles and abstracts of all studies for relevancy. Disagreements were resolved by a third opinion (J.L.). The inclusion criteria were: (1) case-control studies to evaluate the association between CYP11B2 C-344T polymorphism and risk of ischemic stroke, (2) useful data including genotype number or frequency given, (3) studies clearly describe ischemic stroke diagnoses and the sources of cases and controls, (4) studies written in English and Chinese with full-text, (5) genotype distribution of controls in Hardy-Weinberg equilibrium (HWE). The exclusion criteria were: (1) not case-control studies, (2) studies without available genotype number or frequency, (3) genotype distribution of controls not in HWE, (4) animal studies, reviews, case reports, and abstracts. For the studies with the same or overlapping data by the same authors, the most recent or largest population was selected.Data were extracted independently from each study based on the inclusion and exclusion criteria listed above. Agreement was reached after discussion for conflicting data. The following data were collected from each study: first author, publication year, original country, ethnicity, sample size, mean age, genotyping method, and genotype number in cases and controls. Different ethnicities were categorized as Asian, and non-Asian. Study design was stratified to population-based (PB) studies and hospital-based (HB) studies.The quality of included studies was assessed independently using the Newcastle-Ottawa Scale (NOS) 2 statistics. If the result of the Q test was PQ >0.1 and I2 <50%, the fixed-effects model was used to calculate the pooled ORs The strength of association between CYP11B2 C-344T polymorphism and ischemic stroke risk was estimated by odds ratios (ORs) and corresponding 95% confidence intervals (CIs). The pooled ORs were calculated respectively for allelic model (T vs. C), additive model (TT vs. CC), dominant model (TT+TC vs. CC), and recessive model (TT vs. TC+CC). Heterogeneity assumption was assessed by the Q test and IHWE was assessed using the Fisher's exact test, with the significance level was set at P<0.05. Sensitivity analysis was performed based on the high quality studies (according to the NOS score ≥7) and control source. Publication bias was tested by Begg's funnel plot and Egger's linear regression test  The initial search identified one hundred and one potentially relevant publications, eleven of which met the inclusion criteria in the final. A flowchart detailing the process for study identification and selection was shown in Random effects models were used to calculate the pooled ORs in all genetic models. Overall, the combined results showed no significant association between the CYP11B2 C-344T polymorphism and ischemic stroke for all genetic models . On subgroup analysis by ethnicity of study population, no evidence of association was also found in all genetic models. For Asians, the combined ORs and 95% CIs were . For none-Asians, the combined ORs and 95% CIs were . 2 = 90%, PQ = 0.13), additive , dominant , and recessive  genetic models. To explore the sources of heterogeneity, the following meta-regression  was subsequently conducted in all genetic models. Under the allelic model, the heterogeneity could be explained by TT genotype number of ischemic stroke group sample size , and TT genotype number of control group sample size . Under the additive model, the heterogeneity could be explained by TT1 (P = 0.027) and TT0 (P = 0.005). Under the dominant genetic model, the heterogeneity could be explained by TT1 (P = 0.033) and TT0 (P = 0.011). Under the recessive model, the heterogeneity could be explained by TT0 (P = 0.005). . Robustness of our results with regard to different assumptions was examined by performing a sensitivity analysis. We carried out sensitivity analysis for CYP11B2 C-344T polymorphism by limiting the meta-analysis to the high NOS score (≥7) and basing on control source (PB studies). One study with relatively low NOS score (<7) and six studies without PB study design were excluded from the sensitivity analysis. The sensitivity analysis indicated that our data were stability and liability in this meta-analysis. The results were shown in The shapes of the funnel plots did not reveal any evidence of obvious asymmetry . Also thStroke is a multifactorial and polygenic disease with major clinical manifestations and multiple aetiologies, and a significant cause of disability and death in developed countries In the current meta-analysis, a total of twelve studies involving 7,710 subjects were included. No statistically significant evidence of an association between CYP11B2 C-344T polymorphism and ischemic stroke was found in the overall study population. The limited statistical findings may be arguably due to the differences of the ethnic population included in the meta-analysis, because different racial or ethnic populations have different frequencies of alleles, and different genetic backgrounds may affect ischemic stroke susceptibilities. So, subgroup analysis was performed based on ethnicity, and the results also demonstrated no significant differences.Considering the heterogeneity under all genetic models, a meta-regression was conducted to explore the sources of heterogeneity. In the heterogeneity analysis, TT1 and TT0 were found to be the possible sources of heterogeneity under the allelic, additive, and dominant genetic models, and TT0 was found to be the possible sources of heterogeneity under the recessive genetic model. Sensitivity analysis was performed based on the high NOS score and control source, and no statistically significant evidence of an association between CYP11B2 C-344T polymorphism and ischemic stroke was also found. The results of sensitivity analysis indicated the present meta-analysis was stable and reliable.In view of the complex effect of genetic polymorphisms on ischemic stroke progression, the lack of an association between CYP11B2 polymorphism and ischemic stroke susceptibility may attribute to other polymorphisms in RAAS , which could affect the expression of aldosterone and cerebrovascular disease susceptibility For better interpreting the results, some limitations of our meta-analysis should be noted. Firstly, the retrieved literature is potentially not comprehensive enough. We did not track the unpublished articles to obtain data for analysis. The potential effect of this publication bias is unknown. Secondly, the small number of studies and sample sizes limited the ability to draw more solid conclusions. So, a larger number of available studies with a large sample size and adequate representation of ethnicities will help us to determine a more confident estimate of effect of this polymorphism on ischemic stroke. Thirdly, ischemic stroke is a multifactorial disease, and potential interactions between gene-gene and gene-environment should be considered.In conclusion, the present meta-analysis provides evidence that CYP11B2 C-344T polymorphism was unlikely to be associated with genetic susceptibility of ischemic stroke based on the current published studies. Further studies with large sample size of different ethnic populations are required.Figure S1Flow diagram of the selection of eligible studies.(TIF)Click here for additional data file.Table S1quality assessment of included studies.(DOC)Click here for additional data file.Table S2Sensitivity analysis for CYP11B2 C-344T polymorphism and ischemic stroke.(DOC)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


Although an increased risk of preeclampsia in pregnancies conceived by in vitro fertilization (IVF) has been reported, it remains unknown whether IVF is associated with preeclampsia. In the present study, we sought to investigate whether IVF is associated with preeclampsia in pregnant women using propensity score matching analysis.This study included 3,084 pregnant women who visited the National Center for Child Health and Development before 20 weeks of gestation without hypertension or renal disease and delivered a singleton after 22 weeks of gestation between 2009 and 2011. Of the 3084 patients, 474 (15.4%) conceived by IVF (IVF group) and 2,610 (84.6%) conceived without IVF (non-IVF group). The propensity score for receiving IVF was estimated using multiple logistic regression with 27 maternal and paternal variables. This model yielded a c-statistic of 0.852, indicating a strong ability to differentiate between those conceiving with and without IVF. The association between IVF and onset of preeclampsia was assessed by the propensity matched sample (pair of N = 474).There were 46 preeclampsia cases (1.5%) in the total study population, with a higher proportion of cases in the IVF group  than the non-IVF group . Before propensity score matching, the IVF group was 2.72  times more likely to have preeclampsia when unadjusted, and 2.32 (95% CI: 1.08-4.99) times more likely to have preeclampsia when adjusted for maternal and paternal variables by logistic regression. After propensity score matching, the IVF group did not show a significantly greater association with preeclampsia compared to the non-IVF group , although point estimates showed a positive direction.Propensity score matching analysis revealed that the association between IVF and preeclampsia became weaker than when conventional adjustments are made in multivariate logistic regression analysis, suggesting that the association between IVF and preeclampsia might be confounded by residual unmeasured factors. Preeclampsia is a major obstetric problem worldwide that causes significant maternal and perinatal morbidity and mortality. SeveralPropensity score matching analysis is a statistical tool which can minimize selection bias and confounding factors in observational studies. EstimatWe collected data of women who delivered between June 2009 and June 2011 at the National Center for Child Health and Development (NCCHD) in Tokyo, Japan. This study was approved by the NCCHD ethics committee. All pregnant women were Japanese. Women who delivered singleton neonates at ≥22 weeks of gestation without hypertension or renal disease were enrolled in the study. In total, we identified 3,084 women who met our study criteria (eligible study sample), including 474 women who received IVF (IVF group) and 2,610 women who did not (non-IVF group). All 3,084 study participants had only one pregnancy during this study period. Written informed consent was not obtained from participants because the study involved a secondary data analysis and such studies do not require consent according to the NCCHD ethics committee.th week of gestation, then every two weeks up to the 36th week of gestation, and then weekly up to delivery. At each routine prenatal checkup, BP was measured in the sitting position with the right arm held at heart level after a five-minute rest period using an automated sphygmomanometer . Urine protein levels were assessed at each prenatal checkup by a dipstick test of a single voided urine sample. When the pregnant woman had an abnormal BP (>140/90 mmHg) and urine test (1+ proteinuria on dipstick) at a routine prenatal checkup, she was hospitalized for further evaluation of BP and urine protein and for bed rest.Preeclampsia was diagnosed after 20 weeks of gestation if the pregnant woman had a blood pressure (BP) >140/90 mmHg with proteinuria (>300 mg/24 hours). Early-onset preeclampsia was defined as preeclampsia diagnosed at <32 weeks of gestation, and late-onset preeclampsia was defined as preeclampsia diagnosed at 32+ weeks of gestation. Severe preeclampsia was diagnosed as BP >160/110 mmHg with proteinuria (>2 g/24 hours). Women received routine prenatal checkups every four weeks up to the 28Maternal characteristics, including age, age at marriage, parity, number of abortions, working status , height, weight before pregnancy, smoking and drinking during pregnancy, family history of hypertension, mean systolic and diastolic BP before 20 weeks of gestation, and underlying diseases , were collected from the perinatal database and medical charts. Paternal factors, including age, age at marriage, height, weight, and smoking status, were also collected. Missing data for continuous variables  were substituted with the average value of the variable. Missing data for categorical variables were treated as dummy variables .Maternal and paternal characteristics were compared between the IVF and non-IVF groups using chi-squared or Fisher’s exact tests for categorical variables and t-tests for continuous variables.Because IVF procedures were not randomly assigned in this population, potential confounding and selection biases were accounted for by developing a propensity score for the IVF procedure. This propensity score was determined using a multivariate logistic regression model adjusting for maternal and paternal variables. These variables included those listed above as baseline characteristics, except for drinking during pregnancy and two types of underlying diseases , due to their low frequency <0.9%). Twenty-seven independent variables were thus used to determine the propensity score. This model yielded a c-statistic of 0.852, indicating a strong ability to differentiate between those conceiving with and without IVF. The 474 IVF cases were then matched one-to-n with 474 non-IVF cases with the closest propensity score (nearest neighbor matching)%. Twenty. The maxTo assess the association between IVF and preeclampsia, we performed a logistic regression analysis in the initial eligible study sample (n = 3084), unadjusted and adjusted for baseline characteristics, and performed a conditional logistic regression analysis for the propensity score matched sample (n = 948). All analyses were conducted using STATA MP software .A comparison of baseline characteristics and pregnancy outcomes between the IVF (n = 474) and non-IVF n = 2,610) groups is shown in Table 0 groups Baseline characteristics of the IVF and non-IVF groups after propensity score matching (each n = 474) were not significantly different. For example, maternal age for the IVF and non-IVF groups was 38.3 and 38.4 years (p = 0.97), respectively, and mean diastolic BP was 64.8 and 65.0 mmHg (p = 0.86) for the IVF and non-IVF groups, respectively.Table Table Table In the present study, we used propensity score matching analysis and adjusted for 27 known variables to simulate random assignment of the IVF procedure. Using this analysis, we found that women who conceived by IVF were not at a significantly higher risk of preeclampsia than women who conceived without IVF, although the point estimation of the risk of IVF for preeclampsia was positive (i.e. 2.50), suggesting that IVF may be weakly associated with preeclampsia.To the best of our knowledge, this is the first study to investigate the impact of IVF on preeclampsia using propensity score matching analysis. In a 2004 meta-analysis of eight studies, Jackson et al. concluded that IVF was associated with an increased risk of preeclampsia ,11. ShevSpecifically, we demonstrated that IVF was not significantly associated with preeclampsia, although the point estimate of OR was 2.50, which implies that IVF is a risk factor for preeclampsia. The lack of a significant association might be due to the small sample size (n = 948); however, attenuation of the point estimate of OR from the unadjusted model  to the propensity score analysis (2.50) was substantial, suggesting that the association observed between IVF and preeclampsia in previous studies might have been due to confounders.Further, the timing of onset of preeclampsia, severity of preeclampsia, and BP during mid- and late-pregnancy did not differ between the IVF and non-IVF groups. These results suggest that IVF may not elevate BP or induce preeclampsia because women with preeclampsia have higher BP levels throughout pregnancy. MacdonaA plausible biological mechanism to explain how there could be a causal relationship between IVF and preeclampsia is unclear. Abnormal placentation is considered the primary stage of pathogenesis in preeclampsia. In a reThere are several limitations to our study. First, given our observational study design, we could not completely avoid selection biases and unknown confounding factors  that existed before IVF treatment. These factors may have affected pregnancy outcomes, including the incidence of preeclampsia, despite adjusting for 27 variables using a propensity score. Moreover, as we used secondary data, assessment of subfertility was not possible, although it was reported to be associated with an increased risk of preeclampsia,16. To oIn conclusion, we found that IVF was weakly associated with preeclampsia when using propensity score matching analysis  than when using simple adjustments in multivariate logistic regression analysis. This suggests that the influence of the IVF procedure itself was weaker than observed in previous studies. Future studies with a larger study population that can assess more confounding variables are needed to replicate our findings.Propensity score matching analysis revealed that the association between IVF and preeclampsia became weaker than with conventional adjustments in a multivariate logistic regression, suggesting that the association between IVF and preeclampsia might be confounded by residual unmeasured factors.IVF: In vitro fertilization; BP: Blood pressure; OR: Odds ratios; CI: Confidence interval.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.WN and FT conceived and designed the study. WN, ST, TK, YY, KK and SH contributed to clinical data collection. WN and FT contributed to statistical analysis and interpretation of data. WN drafted the manuscript and TF finalized it. All authors contributed to critical revision and final approval of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/14/69/prepub


COMT) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxidase (MAO) and COMT in the cells. We hypothesized that the COMT-158G > A polymorphism was associated with the risk of psoriasis vulgaris in Han Chinese people. In a hospital-based case-control study, 524 patients with psoriasis vulgaris and 549 psoriasis-free controls were studied. COMT-158 G > A polymorphism was genotyped using the PCR sequence-specific primer (PCR-SSP) technique. We found no statistically significant association between the COMT-158 allele A and the risk of psoriasis vulgaris . This suggests that the COMT-158 G > A polymorphism may not contribute to the etiology of psoriasis vulgaris in the Han Chinese population.Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase ( Psoriasis is a common skin disorder characterized by the focal formation of inflamed, raised plaques that constantly shed scales derived from excessive growth of epithelial cells. The disease is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia and infiltration of T lymphocytes, neutrophils, and other types of leukocytes in the affected skin . The gloet al. found that human T and B lymphocytes had catechol-O-methyltransferase (COMT) immunoreactivity. Later, Psoriasis is a T-cell mediated autoimmune process. Activated lymphocytes, other immune accessory cells and lymphokines have been detected in psoriatic plaques . SeveralAlthough the pathogenesis of psoriasis is still unclear, many studies suggest that the disease has an exceedingly complex genetic basis .COMT gene, resulting in an amino acid change  at codon 158 of MB-COMT and codon 108 of S-COMT, reduces the thermostability and the activity of the enzyme  codes for a substitution of valine  by methionine (Met), resulting in the reduced thermostability and activity of the enzyme (Catechol-O-methyltransferase (COMT) is an enzyme that catalyses the O-methylation of biologically active toxic catechols and plays an important role in the metabolism of drugs and neurotransmitters  patients and control subjects were included in the analysis, because genotype frequencies can vary among ethnic groups. The psoriasis-free control subjects were persons who came to the hospital for health examinations and did not have individual or family histories of psoriasis. All cases and controls were examined and diagnosed by dermatologists. We used a questionnaire to collect demographic and other information , and matched the controls to the cases by age (±10 years) and gender. The response rates of patients and controls approached for participation were both over 90%.Based on this concept, we genotyped the All subjects signed informed consent forms, and donated 5 mL venous blood, used for genomic DNA extraction. This research protocol was approved by the ethics review board of the Fourth Military Medical University.COMT-A, 5'-TggTggATTTCgCTggCA-3' (forward) and 5'-ACACC CATACAAgcaTTCATCAgTT-3' (reverse); for COMT-G, 5'-gCATgCACACCTTgTCCTTCAC-3' (forward) and 5'-TgAgCATAgAggCTAAgggACCAT-3' (reverse). The PCR conditions were as follows: preheating for 1 min at 96 °C to achieve a hot start, then a touch-down (TD) procedure consisting of denaturation at 96 °C for 20 s and annealing at 70 °C for 45 s during the first five cycles, followed by 21 cycles of 96 °C for 25 s, 65 °C for 50 s, and 72 °C for 30 s each, then 21 cycles at 96 °C for 30 s, 55 °C for 60 s, and 72 °C for 90 s, and a final extension at 20 °C for 2 min.Venous blood collected from each subject was added to tubes containing 50 mmol EDTA/L, and genomic DNA was isolated from 1 mL anti-coagulated peripheral blood leukocytes using a DNA extraction kit . After DNA extraction, reactions were carried out in a final volume of 20 μL containing 100 ng DNA, 2.5 mM MgCl2, 1.25 units of Taq DNA polymerase, 1 μL of each primer, 250 μL dNTPs mixture, and 1X PCR buffer. All PCR reaction reagents were purchased from Fermentas (Lithuania). Genotyping for the G/A polymorphism was carried out using polymerase chain reaction with sequence-specific primers (PCR-SSP). The following primers were used to amplify the target fragments: for COMT-158 G allele and 322 bp for the COMT-158 A allele. These products were analyzed by 2% agarose gel electrophoresis and visualized with 0.5 μg/mL ethidium bromide staining under an ultraviolet illuminator. Genotypes were scored by two independent individuals, and any ambiguous genotypes were repeated or omitted. PCR products were identified by sequencing.The size of the amplified PCR products was 455 bp for the COMT polymorphisms. Unconditional univariate and multivariate logistic regression analyses were performed to obtain the crude and adjusted odds ratios (ORs) for the risk of psoriasis and their 95% confidence intervals (CIs). The multivariate adjustment included age and gender variables. Two-tailed tests of statistical significance were performed with SAS software .Chi-square tests were used to evaluate the differences in the frequency distributions of selected demographic variables between the cases and controls, including each allele and genotype of the We found no significant differences in age and gender between the psoriasis patients and the control group: mean age was 32.1 ± 13.6 years in the psoriasis group and 31.5 ± 13.9 years in the control group (p = 0.232), while the gender distributions (M:F) were 54.8%:45.2% in the study group and 50.5%:49.5% in the control group (p = 0.157) .COMT158 in the study and control groups are shown in COMT 158 polymorphism were in agreement with the Hardy-Weinberg equilibrium (p = 0.168). No significant differences were found between the psoriasis and control subjects regarding the frequencies of genotypes GG, GA, and AA (p = 0.759) (A (p = 0.624).The genotype and allele frequencies of = 0.759) . SimilarG > A) of the COMT gene, which leads to a valine-methionine substitution resulting in the difference in COMT activity, we analyzed the association between combined genotypes  and the risk of psoriasis vulgaris. We investigated whether the distributions of combined genotypes  were different among type 1 and type 2 psoriasis patients and control subjects. However, as shown in GA+AA) was not statistically different in the type 1 psoriasis (43.5%) and type 2 psoriasis (47.8%) cases compared to controls (43.0%).To consider the single nucleotide polymorphism in codon 158 (GA+AA) was 45.5% in level 1 and 41.5% in level 2 psoriasis patients. There was no statistical difference between the two levels of psoriasis and the controls.The Psoriasis Area and Severity Index (PASI) is a widely used method to characterize the severity of the disease (COMT158 (G > A) might be associated with the risk of psoriasis vulgaris. Our study, however, found no significant association between COMT158 (G > A) and psoriasis vulgaris, and also no significant difference between COMT158 (G > A) and different types of psoriasis vulgaris. Our study included only statistical research, not functional investigation of the association between the COMT polymorphism and psoriasis vulgaris, so this hypothesis needs to be tested by further functional studies.Based on previous research, we hypothesized that polymorphism COMT-158 (G > A) polymorphism between psoriasis vulgaris patients and control subjects in a Han Chinese population. The difference between these results and those of COMT 158 may not be associated with the risk of psoriasis vulgaris. Larger population-based studies, different clinical subgroups and studies among different ethnic groups are needed to confirm these findings.In conclusion, we found no significant difference regarding the


XPD Asp312Asn polymorphism and head and neck cancer risk through this meta-analysis.To investigate the association between XPD Asp312Asn polymorphism and head and neck cancer risk.We performed a meta-analysis of 9 published case-control studies including 2,670 patients with head and neck cancer and 4,452 controls. An odds ratio (OR) with a 95% confidence interval (CI) was applied to assess the association between XPD Asp312Asn polymorphism and head and neck cancer risk was found in this meta-analysis . In the subgroup analysis by HWE, ethnicity, and study design, there was still no significant association detected in all genetic models.Overall, no significant association between XPD Asp312Asn polymorphism may not be a risk factor for developing head and neck cancer.This meta-analysis demonstrates that  Head and neck cancers (HNC) constitute about 5% of all cancers recorded in the US, and the incidence is increasing in most developed and developing countries. These cancers have been estimated to be about six times more common among smokers than non-smokers and are most common in males over 50 years old XPD, ERCC1, and XPCXPD polymorphisms may be associated with reduced DNA repair because of a possible reduction in helicase activity XPD polymorphisms in the coding regions is Asp312Asn in exon 10. The functional significance is not yet completely clear, although the amino acid mutations in exon 10 give rise to a loss of an acidic residue and a complete change in the electronic configuration of the amino acid Many environmental factors, such as radiation, diet, smoking, and endogenous or exogenous estrogens, are associated with DNA damage. Unrepaired or misrepaired DNA results in gene mutations, chromosomal alterations, and genomic instability. Several studies have suggested that genes involved in DNA repair play a crucial role in protecting against mutations. Patients with certain cancers have reduced capacities for DNA repair. Similarly, the enzymes of the nucleotide excision repair (NER) pathway have been implicated in cancer. Associations between polymorphisms in several DNA repair genes and the risks of several types of cancer have been extensively examined. Many epidemiologic cancer studies have focused on single nucleotide polymorphisms (SNPs) in genes in the NER pathway such as XPD Asp312Asn polymorphism was conducted by Sturgis et al. XPD Asp312Asn polymorphism and HNC. Since then, a lot of studies have confirmed or refuted this finding XPD Asp312Asn polymorphism. Worthy of note, that meta-analysis included five studies were conducted in Caucasian populations and one in an Asian population XPD Asp312Asn polymorphism and HNC risk have been published. A comprehensive meta-analysis is needed to provide an updated approach on the overall relationship. Subgroup analyses were also performed on Caucasian and Asian populations to investigate ethnicity-specific effects.The first study on the relationship between HNC and XPD polymorphism and HNC risk from 2000, when the first study of the association between XPD Asp312Asn polymorphism and HNC risk was reported, to October 2011. We also used the “Related Articles" option in PubMed to identify additional studies on the same topic. Reference lists in retrieved articles were also screened for. All selected studies complied with the following three criteria: (a) case–control study on the XPD Asp312Asn polymorphism and HNC risk; (b) sufficient published data for estimating the odds ratio (OR) with 95% confidence interval (CI); (c) For multiple publications reporting on the same data or overlapping data, the largest or most recent publication was selected The PubMed database was searched with terms “head and neck cancer", “oral cancer", “oropharyngeal cancer", “laryngeal cancer", “pharyngeal cancer", “XPD", “excision repair cross-complementing group 2", “polymorphism", and the combined phrases for all genetic studies on the relationship between Two investigators (Hu and Yuan) independently extracted the following data from each included publication: the first author's name, publication data, sources of controls, racial descent of the study population (categorized as either Asian or Caucasian), genotyping method, number of cases, cases and controls with different genotypes, and Hardy-Weinberg equilibrium(HWE).XPD Asp312Asn polymorphism and HNC risk. The pooled ORs were performed for codominant model , dominant model (Asn/Asn+Asp/Asn vs. Asp/Asp), and recessive model (Asn/Asn vs. Asp/Asp+Asp/Asn), respectively. In the subgroup analysis, statistical analysis was conducted on Asians and Caucasians. Heterogeneity assumption was assessed by the chi-square based Q-test P>0.10. Otherwise, the random-effects model (the DerSimonian and Laird method) was used P<0.05 considered statistically significant.Crude ORs with 95% CIs were computed to assess the strength of the correlation between the XPD Asp312Asn polymorphism and HNC risk were included in this meta-analysis This meta-analysis is guided by the PRISMA statement . A totalXPD Asp312Asn polymorphism and HNC risk in the total populations . Similarly, in the succeeding analysis of HWE studies, no significant association was found between XPD Asp312Asn polymorphism and HNC risk. Finally, in the stratified analysis of ethnicity and study design, we also did not find any significant association between XPD Asp312Asn polymorphism and HNC.The main results of this meta-analysis and the heterogeneity test are shown in A single study involved in the meta-analysis was deleted each time to reflect the influence of the individual dataset to the pooled ORs. The analysis results demonstrate a borderline increased risk after excluding the studies that in Asp/Asn vs. Asp/Asp model P = 0.093 for Asn/Asn vs. Asp/Asp; P = 0.370 for Asp/Asn vs. Asp/Asp; P = 0.173 for Asn/Asn+Asp/Asn vs. Asp/Asp; P = 0.215 for Asn/Asn vs. Asp/Asp+Asp/Asn).Funnel plot and modified Egger's test were performed to estimate the publication bias of literature. The shapes of the funnel plots in all genetic models did not reveal any evidence of obvious asymmetry. XPD gene has been mapped to chromosome 19q13.3 and it is composed of 23 exons. Germline mutations in the XPD gene can result in xeroderma pigmentosum and other diseases. The XPD protein is involved in transcription-coupled NER and is an integral member of the basal transcription factor BTF2/TFIIH complex.Today, genetic susceptibility to cancer has attracted growing attention to the study of gene polymorphisms involved in tumorigenesis. The XPD 312 variant allele may be associated with the reduced repair of aromatic DNA adducts XPDThe Asp to Asn change at position 312 of XPD changes the electronic configuration of amino acid and alters the interaction between XPD protein and its helicase activator XPD Asp312Asn polymorphism has been shown to increase the risk of bladder cancer and lung cancer, but it is not associated with breast cancer Correlations between the polymorphisms and some cancer risks have been studied, but the results remain controversial. The XPD Asp312Asn polymorphism and HNC risk in codominant model  XPD Asp312Asn polymorphism and HNC risk. Majumder et al. XPD is associated with increased risk of cancer among rapid and intermediate acetylators . However, other studies showed that HNC risk is not significantly related to XPD Asp312Asn polymorphism. Ji et al. The first study, published in 2002, revealed a borderline correlation between XPD Asp312Asn heterozygous and combined variants and HNC in their study. The considerably larger sample size of our study may account for this difference relative to the previous study.The present meta-analysis of nine eligible studies, including 2670 cases and 4452 controls focused on XPD Asp312Asn polymorphism and HNC risk, was performed to derive a more precise estimate of the association, but no significant association was found in the total population when all the studies were pooled. Similarly, no significant association was detected in all genetic models during the satisfied analysis based on the HWE, ethnicity and study design. Our finding is not in accordance with that previously published by Flores-Obando et al XPD Asp312Asn polymorphism and HNC risk, some limitations should be addressed. First, these results are based on unadjusted estimates that lack the original data from the eligible studies, which limits the evaluation of the effects of the gene-gene and gene-environment interactions during HNC development. Second, the sample size is still relatively small. Thus, we could not have enough statistical data to find the true relationship between XPD Asp312Asn polymorphism and HNC risk. Finally, each gene is known to have a moderate effect on HNC development. The combinations of certain genotypes may be more discriminating as risk factors than a single locus genotype. In our meta-analysis, linkage disequilibrium (LD) and haplotype analysis were not performed. In spite of these limitations, no publication bias was observed, and a large number of subjects still significantly guarantee the statistical power of the analysis.Despite the considerable efforts to test for possible association between XPD Asp312Asn polymorphism may not be associated with HNC development. In the future, large-scale case-control and population-based association studies are necessary to validate the risks identified in the present meta-analysis and to investigate the potential gene-gene and gene-environment interactions between XPD Asp312Asn polymorphism and HNC cancer.In conclusion, despite these limitations,our meta-analysis suggests that Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


The purpose of this study was to evaluate the distribution of the polymorphisms of the SCN1A gene in a series of children and adolescents with primary headache and idiopathic or cryptogenic epilepsy compared to controls. Five non-synonymous exonic polymorphisms  of the SCN1A gene were selected and their genotyping was performed, by high resolution melting (HRM), in 49 cases and 100 controls. We found that among the five polymorphisms, only 3199A > G was a true polymorphism. We did not find a statistically significant difference between distribution of 3199A > G genotypes between cases and controls. We excluded the role of the SCN1A gene in the pathogenesis of comorbidity between headache  and epilepsy. The SCN1A gene is a major gene in different epilepsies and epilepsy syndromes; the HRM could be the new methodology, more rapid and efficacious, for molecular analysis of the SCN1A gene. CACN1A, ATP1A2) in the common forms of migraine [Migraine and epilepsy are both common neurological disorders and can occur as comorbid conditions –7. From migraine , 11–13.Considering the multifactorial etiology of migraine, genetic studies on phenotypically homogenous populations are required . The comSCN1A gene encodes the α-subunit of the neuronal voltage-gated sodium channel Nav1.1 (MIM# 182389). Mutations in SCN1A are associated with a spectrum of epilepsy syndromes [SCN1A gene has never been investigated in subjects with common forms of migraine.The yndromes ; a few myndromes . So far,SCN1A gene in the pathogenesis of comorbidity between primary headache and idiopathic or cryptogenic epilepsy in a large series of children and adolescents.The purpose of this study was to evaluate the role of the SCN1A gene between patients and healthy controls.This was a case–control study comparing the genotype distribution and allele frequency of single nucleotide polymorphisms (SNPs) of Fifty-six subjects out of 1,795 headache sufferers under the age of 18 consecutively diagnosed at the juvenile Headache Center of the Department of Pediatrics of Padua Hospital between 1995 and 2008 were selected according to the following criteria: (1) primary headache and (2) idiopathic or cryptogenic epilepsy or unprovoked seizures. Patients with secondary headache or symptomatic epilepsy were excluded from the study. The methodology of patients’ recruitment and phenotype selection has been previously reported [Forty-nine patients and 100 healthy controls participated in the genetic study. All the healthy controls were interviewed by a board certified child neurologist regarding, in particular, the personal history of headache; those affected by migraine or probable migraine, according to the criteria of the International Classification of Headache Disorders (ICHD-II)  were excThe molecular analysis was conducted at the Genetic Laboratory of the Department of Pediatrics (University of Padua). Parental informed consent for genetic testing was obtained for each subject included in the study. Genomic DNA was extracted from peripheral blood using the High Pure PCR Template Preparation Kit .http://frodo.wi.mit.edu/primer3). Primer sequences and amplification conditions for SCN1A gene are available upon request. Genotypes were determined by high resolution melt (HRM) analysis [The five SNPs examined are described in Table analysis –19.TableThe polymerase chain reaction (PCR) cycling and HRM analysis were carried out sequentially on a Rotor Gene 6000  using Takara Ex Taq R-PCR custom . The HRM analysis was performed with the temperature ramping  and fluorescence acquisition setting recommended by the manufacturer. The melting curves were normalized through calculation of the “line of best fit” of two normalization regions before and after the major fluorescence drop, corresponding to the melting of the PCR product using the software provided with the Rotor Gene 6000 (Corbett Research). Melting profiles and HRM running conditions were determined experimentally in 10 reference controls that were sequenced.We obtained three different melting profiles only for the 3199A > G polymorphism. We sequenced two samples for each melting profile of each polymorphism, confirming that different profiles corresponded to different sequences. Sequence analysis revealed that heterozygotes and homozygotes have characteristic melting profiles that give rise to differently shaped melting curves. Therefore, in the following HRM analyses we used the curves of homozygotes and heterozygotes that were confirmed by sequencing as the reference for the genotype analysis of the unknown samples.® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) on ABI 310 automated DNA sequencer . The HRM profiles of cases with comorbidity were compared to HRM profiles of healthy reference controls that were sequenced.Direct sequencing of PCR products with abnormal HRM profiles was performed with the use of the BigDyeThe allele frequencies and genotypes in patients and controls were calculated. Genotype distributions in patients and controls were tested for Hardy–Weinberg equilibrium, and differences in genotype frequencies between patients and controls were tested using a Chi-square test for independence, with a level of significance set at 0.05.Among 1,795 headache sufferers under the age of 18 consecutively diagnosed at the juvenile Headache Center of the Department of Pediatrics of Padua Hospital between 1995 and 2008, fifty-six subjects having comorbidity between primary headache and idiopathic or cryptogenic epilepsy or unprovoked seizures were selected. The clinical characteristics and the statistical analysis of these two population study have been previously reported [SCN1A gene molecular analysis, which was undertaken in 49 cases and 100 healthy controls. Among the 49 cases, 45 subjects (92%) had migraine (M)  and 4 patients suffered from episodic tension type (ETTH) headache. Considering the type of epilepsy, there were 30 subjects with focal cryptogenic epilepsy , 11 with focal idiopathic epilepsy , and 8  with idiopathic generalized epilepsy.The present study focuses on the results of the The HRM profiles consist of three different graphs  that are visualized all together during the analysis of results. As an example, we report the three graphs concerning the HRM analysis of exon 16 Fig. .Fig. 1RoComparing the HRM profiles of the cases and healthy control samples with those of the sequenced reference controls, we could identify the genotypes of the samples examined.The polymorphism 3199A > G, having a significant heterozygosity (0.33), exhibited three HRM profiles consistent with the three genotypes , as confirmed by direct sequencing. The other four SNPs having a low or not known heterozygosity disclosed a unique HRM profile, consistent with the wild type genotype.During HRM analysis of exon 11, we found in one patient a different profile. The PCR product was analyzed by direct sequencing that disclosed a missense variation sequence 1811G > A that causes at protein level a replacement of arginine with histidine (R604H). The patient is a 15-year-old boy with a history of childhood absence epilepsy (since 3–6) suffering from episodic tension headache, since the age of 11. He never experienced febrile seizures or periodic syndromes. The analysis was carried out in both the parents; the mother was negative while the father, who was asymptomatic, carried the missense variation sequence 1811G > A.During the analysis of healthy controls, we found in one case a missense variation sequence 5782C > G that causes at protein level a replacement of arginine with glycine (R1928G).The allele and genotype frequencies, respectively, absolute and percentage, of the five SNPs observed in 49 patients and 100 healthy nonmigrainous controls, tested by HRM, are presented in Table p = 0.64) and controls (p = 0.44) . For this polymorphism, the comparison of the distribution of allele  and genotype  frequencies in cases and controls by Chi-square test was not statistically significant .The analysis of the frequency distribution showed that, only for the polymorphism 3199A > G, the two alleles  are represented both in cases and controls; while for the other four SNPs the allele frequency of the wild type was 100% both in cases and controls, as assessed on a total of 298 alleles, therefore these SNPs are not true polymorphisms in the tested population. In the case of polymorphism 3199A > G, the genotype distributions for the two alleles (A and G) were in equilibrium under the Hardy–Weinberg law both in cases , may have seizures [Previous studies have shown that cases affected by FHM, carrying mutations of genes encoding ion channels  examined and they are not to be further investigated.We did not find a statistical significant difference of 3199 A > G genotypes’ distribution between cases and controls; therefore our results confirm that the polymorphism 3199A > G is not associated to pathological phenotypes in headache sufferers and in migraineurs, similar to that found in other studies conducted on patients with epilepsy.From a statistical point of view, the group sample sizes achieve a power lower than 20% to detect, as statistically significant, the observed differences between the two groups for both allele and genotype analyses for the polymorphism 3199A > G. Actually, those differences could be considered negligible from a genetic point of view when bearing in mind the epidemiological and clinical characteristics of the disease. Furthermore, specifically in reference to genotypes, the observed prevalence would suggest that the A/A genotype is less prevalent among wild type subjects, and this would be inconsistent with expected. For these reasons, in our opinion, the results suggest that the observed differences are due to chance.SCN1A gene in cases with comorbidity between headache and epilepsy.To our knowledge, this is the first study that has assessed the genotype of polymorphisms in the SCN1A gene has so far been well studied in large series of patients with severe myoclonic epilepsy [The epilepsy –26 or inepilepsy , 28 and epilepsy .SCN1A gene, and documented genotypic and allelic frequencies comparable between patients and controls. The polymorphism 3199A > G was analyzed specifically in patients with febrile convulsions [These studies also contain data on the frequency of polymorphism 3199A > G of vulsions  or with SCN1A gene was conducted in Asiatic patients affected by epilepsy [In two recent studies, the analysis of polymorphism 3199A > G of the epilepsy , 33; in . [SCN1A gene is not associated with pathological phenotypes; our results confirm that this also applies to headache and migraine sufferers.Only in the study of Lakhan et al.  a statis. [We found a missense mutation 1811G > A (R604H) in exon 11 in a patient with episodic tension headache and childhood epilepsy with absences; in the literature this mutation is already known –36. Esca. . The pos. , 36. In We also confirm that the missense variant 5782C > G (R1928G), found in a single healthy nonmigrainous control among the 100 examined, is a rare missense polymorphism, as previously reported , 36.SCN1A gene in the pathogenesis of migraine and in particular in cases with comorbidity between migraine and epilepsy. The major limitation of the present study is that the genetic analysis has been focused on a part of the gene, since a complete genetic analysis seemed too expensive, given the large number of exons  of the SCN1A gene and based on the negative results in previous studies [Our study seems to exclude a role of the  studies , 38.SCN1A gene is not implicated in the pathogenesis of common forms of migraine, even when the phenotype is restricted by the presence of comorbidity with epilepsy.Our data support the hypothesis , that thSCN1A gene has a causative role in some epilepsy syndromes (severe myoclonic epilepsy and GEFS+) and has recently been associated with new and different clinical phenotypes, such as focal and generalized cryptogenic epilepsy [The epilepsy  and atypepilepsy .SCN1A gene. The HRM offers significant advantages over traditional methods , as it does not require manipulation of genetic material after amplification, it is inexpensive, it has excellent sensitivity and specificity and allows the concomitant analysis of a large number of samples, increasing the reproducibility of analysis and significantly reducing the work time.Another important aspect of this study was the use of an innovative method, the HRM, for the molecular analysis of the The sensitivity and specificity of variation detection of HRM are estimated, respectively, to be 100 and 95% .SCN1A gene, which is currently usually performed with screening techniques (DHPLC) and direct sequencing.These characteristics are of particular relevance, especially in the genetic analysis of multifactorial diseases, such as headache and epilepsy, which requires a large number of subjects and very often involves the analysis of several polymorphisms in the same patients. The HRM could then form the new method of molecular analysis of the SCN1A gene and comorbidity between primary headache  and epilepsy.To the best of our knowledge, this is the first study that evaluated the association between polymorphisms of the Our results confirm that the polymorphism 3199A > G is not associated with pathological phenotypes in patients with headaches and migraine, while the other four polymorphisms examined were homozygous in the tested populations and should not be further investigated.SCN1A gene in the pathogenesis of comorbidity between headache  and epilepsy.We exclude the role of the SCN1A gene is a major gene in different epilepsies and epilepsy syndromes, and in this field it has to be further investigated. The HRM could be a new methodology, more rapid and efficacious, for molecular analysis of the SCN1A gene.The SCN1A gene and of other genes encoding for ion channels in different epilepsies and epilepsy syndromes.The development of protocols for more efficient and reliable molecular diagnostics would allow a wider appreciation of the role of the