ACE (I/D), AGTR1 (A +1166 C), BDKRB2 (+9/−9) and LEP (G–2548A) genomic variations in 175 Greek athletes who excelled at a national and/or international level and 169 healthy Greek adults to identify whether some particular combinations of these loci might serve as predictive markers for superior physical condition.The aim of this study was to determine the genotype distribution and allelic frequencies of ACE gene (p = 0.034) combined with the simultaneous existence of BDKRB2 (+9/−9) (p = 0.001) or LEP (G/A) (p = 0.021) genotypes was the most prevalent among female athletes compared to female controls. A statistical trend was also observed in BDKRB2 (+9/−9) and LEP (G–2548A) heterozygous genotypes among male and female Greek athletes, and in ACE (I/D) only in male athletes. Finally, both male and female athletes showed the highest rates in the AGTR1 (A/A) genotype.The D/D genotype of the ACE (D/D), BDKRB2 (+9/−9) or LEP (G/A) genotypes in female athletes might be correlated with a superior level of physical performance.Our results suggest that the co-existence of  Genetic polymorphisms that act as potential mediators of the human health and physical performance are targets for many research groups attempting to unravel their role to the genetic predisposition for a superior performance and endurance. There are up to 170 gene variant sequences, 17 mitochondrial DNA markers and 25 additional nuclear genetic markers in the human genetic map which are related to physical performance phenotypes as well as to good physical fitness [Alu repeats within intron 16 of the angiotensin-converting enzyme (ACE) gene [rs1799752]  has been strongly associated with the serum leptin levels in overweight individuals and obesity and an increased risk for obesity  . ACE plapressure . Additiopressure , where tpressure , reveali obesity -14. A st release , which sdiabetes ,16.AGTR1 (A +1166 C) allele in 3′ UTR of the AT II type-1 receptor gene [rs12721276], results in increased expression of the receptor gene [BDKRB2 (β2 receptor of bradykinin) gene [rs72348790] results in a higher transcriptional activity and, consequently, to a quicker receptor's response to bradykinin molecules [ACE D/D genotype responsible for elevated ACE enzyme activity might counterbalance this activity, preventing bradykinin's hydrolysation, by withdrawing it in a higher rate.The presence of other polymorphisms, like the tor gene , and theolecules ,19. AddiACE (I/D), AGTR1 (A +1166 C) and BDKRB2 (+9/−9) along the RAS biochemical pathway as well as the one in the promoter of the LEP gene (G–2548A). Leptin exhibits a cross-interaction with RAS components. The presence of all the above polymorphisms in specific combination showed to play a role not only in the blood pressure control, but also in other metabolic pathways that might affect fitness and physical performance in humans.In this study, we have investigated the presence of known polymorphisms named ACE (I/D), BDKRB2 (+9/−9) and LEP (G/A) genes and homozygotes (A/A) for AGTR1 gene polymorphism. In total, there were no statistically significant differences in genotypes between male athletes versus male controls (Table ACE (I/I) genotype was absent in the international male athlete group of 39 out of 102 male athletes.For all four polymorphisms studied, among male athletes versus male controls, the highest percentages of male athletes appeared as heterozygotes for p = 0.034) of the ACE (D/D) genotype, while the ACE (I/D) genotype exhibited a higher score in female controls (51.81%) versus the female athlete group (34.25%) (p = 0.027). All other genotypic distributions did not reach statistical significance (Table ACE (D/D) genotype, the BDKRB2 (+9/−9) or LEP (G/A) genotypes were more prevalent , compared to the female control group (Table BDKRB2 (+9/+9) genotype (27.40%) versus that of the BDKRB2 (−9/−9) genotype (10.96%) (p = 0.042). This trend was also observed in male athletes but did not reach statistical significance. Finally, the distribution of the LEP (A/A) and LEP (G/G) genotypes was similar among female athletes .In both female athletes and female controls, their genotypic distribution is shown in Table ontrols 5.81% versgroup 34.5%  versus female athletes (52.1%) (p = 0.034).Allele frequencies concerning both cohorts of male athletes versus male controls and female athletes versus female controls are shown in Table ACE (I/D), LEP (G/A), BDKRB2 (+9/−9) and AGTR1 (A/C); Table p = 0.017 and 0.004, respectively) among female athletes.The impact of the allele frequencies was further assessed by analysing the 16 possible allelic combinations coming from the four different studied polymorphisms .ACE and proteins participating in skeletal muscle activity such as a-actinins [ACE I/D [Physical performance seems to be controlled by many genetic factors that interact with the environment to affect complex interactions in human physical performance characteristics. All these render such investigations quite complex, and extended studies are needed to unravel possible interactions. To date, genetic studies, attempting to ascertain the role of genetic variants involved in the human superior physical performance, have focused on candidate genes mainly associated with cardiovascular functions, including actinins ,21. Much[ACE I/D .ACE with high athletic performance phenotypes is mediated through changes in kinin activity and is related to the existence of the BDKRB2(−9) allele as it provides a higher expression and abundance of the bradykinin receptor [ACE (I/D) genotype may be mediated through changes in AT II activity, which acts via the AGTR1 receptor. AGTR1 (A +1166 C) gene polymorphism appears functional, with the C allele acting as an enhancer of the receptor activity; however, it does not seem to be associated with differences in high-level human performance [Both I and D alleles have been so far successfully associated in sports and with superior athletic performance in South African triathletes , Britishreceptor . Besidesreceptor . Alternaformance .ACE (D/D) polymorphism among female athletes, which is in line with previous publications [ACE (D/D) genotype could influence sprint performance in Greek athletes. This might be due to both, i.e. the limited number of the participating athletes and the different kinds of sports they are considered elite.In our study, we observed a strong statistical trend towards ications ,25. The ications ; this stBDKRB2 (−9/−9) genotype was associated with the actual performance of 701 South African males who completed an Ironman Triathlon [LEP (G−2548A) in the human capacity for athletic performance indicated that the G allele provides an advantage to the reduction of body mass index as a response to physical training [BDKRB2 (+9/+9) genotype were statistically higher than those of the BDKRB2 (−9/−9) homozygotes, while a statistically significant increase was obtained for the co-existence of the ACE (D/D) genotype together with the heterozygosity of the LEP (G/A) or BDKRB2 (+9/−9) genotypes , LEP (G/A) and BDKRB2 (+9/−9) genotypes was also highlighted among male Greek athletes, which may require a larger sample size of athletes in order to be demonstrated. Finally, for the AT II type-1 receptor gene, both male and female athletes showed the highest rates in the AGTR1 (A/A) genotype, but with no statistical significant results compared to the corresponding control groups.An interesting trend towards the heterozygous state of New approaches should be identified to evaluate the impact of DNA polymorphisms in human fitness and high-level performance. One possible approach is conducting genome-wide association studies as the one performed by De Moor et al. , yet it The limitation of our study is the small number of elite athletes, both as a whole and also in each sport. As such, this study can be considered a pilot investigation that can be expanded with the inclusion of further athletes. For the same reason, the abovementioned genomic markers have to be considered with caution and under no circumstances can be exploited to predict ones athletic performance. A meta-analysis study is currently under way to confirm or to overrule the predictive value of these biomarkers to assess athletic performance.ACE (I/D), BDKRB2 (+9/−9) and LEP (G/A), out of the four gene polymorphic sites studied was shown, but not proved by the statistical analysis in male athletes. Among female athletes, the co-existence of ACE (D/D) with BDKRB2 (+9/−9) or LEP (G/A) genotypes and the reduction of I allele frequency and of both IG+9A and IG−9A allelic combinations were proved to be significant, compared to the female control group. Probably, a broader and more homogeneous sampling of athletes would demonstrate how strong the results highlighted in this study are and examine the effects of multiple genetic variants and allele combinations in superior physical performance.The tendency for the heterozygous state in three: One hundred and seventy-five Greek athletes and 169 Greek normal individuals who served as the control group were recruited. The athlete group consisted of 102 males and 73 females, whereas the control group consisted of 88 males and 83 females. Inclusion criteria for athletes were their participation and excellence at least once at international and/or national competitions, respectively. In particular, 39 out 102 men and 35 out 73 women had represented Greece at an international level in various sports: swimming , volleyball , handball  and athletics .ACE (I/D), AGTR1 (A +1166 C), BDKRB2 (+9/−9) and LEP (G−2548A) polymorphisms, respectively.DNA was collected with consent from 10 ml of peripheral blood or 10 ml of saline mouth rinse samples, and the DNA was isolated by QIAamp DNA Blood Kit . DNA amplification was performed with polymerase chain reaction, and subsequent restriction fragment length polymorphism analysis was carried out, according to the protocols described by Rigat et al. , Di Maurp values less than 0.05 were considered significant, and they were further assessed by Fisher's exact test.Statistical analysis was performed with SPSS statistical software package . The statistical differences between groups in genotype distribution, allele frequencies and allelic combination frequencies are presented in Tables We have statistically cross-tested genotypic distribution, allele frequencies and furthermore the frequencies of the 16 allelic combinations derived from the four genes examined  between all groups. The tests were initially applied to the whole athlete group in comparison to the non-athlete group, and then each of the above groups were subdivided into cohorts of males and females where the same tests were applied again.The authors declare that they have no competing interests.AS designed the study, collected the samples from  athletes and international teams, performed the experiments and drafted the manuscript. VF collected the data, carried out the statistical analysis and gave technical support and conceptual advice for the manuscript. VK participated in the design of the study, collected the control  samples and performed the initial experiments. GPP and AP developed the concept, designed the experiments and compiled the manuscript. AP also administered the experiments and supervised the statistical analysis. All authors read and approved the final manuscript.


The purpose of this study was to investigate physiological responses to various intermittent and continuous small-sided games (SSGs) – including 2-a-side, 3-a-side, and 4-a-side games – in young soccer players. Twenty soccer players  voluntarily participated in this study. The subjects underwent anthropometric measurements followed by the YoYo intermittent recovery test. Then, they played intermittent (SSGint) and continuous (SSGcon) 2-a-side, 3-a-side, and 4-a-side soccer-specific SSGs in random order at 2-day intervals. Paired t-tests were used to assess differences between the training regimens (intermittent and continuous) in terms of heart rate (HR), percentage of maximum HR (%HRmax), and blood lactate concentration (LA). The differences in LA, HR and %HRmax between the 2-a-side, 3-a-side and 4-a-side SSGint or the 2-a-side, 3-a-side and 4-a-side SSGcon were identified using a one-way analysis of variance with repeated measures. The results demonstrated that the 3-a-side SSGint and SSGcon measurements were significantly higher than the 2-a-side and 4-a-side games in terms of HR and %HRmax, whereas the 2-a-side SSGint and SSGcon resulted in higher LA responses compared to other SSG types. The study results also demonstrated that SSGint and SSGcon are similar in terms of physiological responses except for 2-a-side game LA responses. The results of this study suggest that both SSGint and SSGcon could be used for the physiological adaptations required for soccer specific aerobic endurance. IT is dntervals . On the  than IT .Traditionally, most coaches used running drills without the ball to develop soccer players’ aerobic endurance . HoweverIntermittent (SSGint) or continuous (SSGcon) type small-sided games are used by coaches in young and senior teams’ soccer training in order to improve aerobic endurance . SeveralThere have also been some studies examining physiological responses during SSGcon. Although previous studies have investigated physiological responses to either SSGint or SSGcon, only one study has compared the acute physiological responses associated with both SSGcon and SSGint. −1) participated in this study voluntarily. All the players had a minimum 5 years of training experience and were the members of the same youth team competing in an elite academy league. This study was approved by the Research Ethics Committee of Pamukkale University, and was consistent with the institutional ethical requirements for human experimentation in accordance with the Declaration of Helsinki. The subjects were fully informed about the procedures to be used and the experimental risk.Twenty young soccer players  for the subjects. At the conclusion of pre-season training, players completed the YIRT and were ranked according to the distance covered. The scores gained on the YIRT were as follows: players who covered the least distance were given a score of 1 and those who covered the most ground were awarded a score of 5. Also, the coach provided an overall subjective technical/tactical skill level of each player using a 5-point scale (from 1 “below average” to 5 “outstanding”). The total score for each payer was the sum of their technical/tactical skill and YIRT scores. In an attempt to avoid skill and fitness mismatches and a consequent imbalance in opposing SSG teams, each side was balanced in terms of the players’ skill and fitness rankings.The study was conducted over a 2-week period. On the first day, anthropometric measurements  were taken for each player. They were followed by the YIRT.Then, intermittent and continuous 2-a-side, 3-aside, and 4-a-side soccer-specific SSGs were organized in random order with 2-day intervals. Each SSG was played after a 20-minute warm-up, which consisted of low intensity running, striding, and stretching. The HRmax for each player was determined during the YIRT; LA concentrations were taken after the SSGs and HRs were measured during the SSGs. The YIRT and SSGs were carried out on a synthetic grass pitch between 4 and 6 pm.The YIRT consists of repeated 20-m runs back and forth between the starting, turning, and finishing lines, and at a progressively increased speed, which is controlled by audio beeps from a tape recorder. The test was performed on a synthetic grass field in groups of 6 players, as suggested by 2) for the SSGint and SSGcon. The soccer players were asked to perform at maximum effort during the games. No specific rules were utilized in the games to influence their intensity, but extra balls were placed in the goals and along the side lines surrounding the entire pitch to ensure that there were no breaks in play. Moreover, the coaches constantly encouraged the players verbally during the games. SSGcon and SSGint were played in random order at two day intervals without a goalkeeper.Heart rate was recorded at 5-s intervals during each small-sided game via short-range radio telemetry . The heart rate monitors were also worn during the YIRT to determine each player’s maximum heart rate (HRmax). The HR was stored by Polar S810 HR monitors throughout the games and transferred to the computer and filtered by Polar Precision Performance Software TM . The mean HR for each SSGint was calculated by taking the means of the 3 bouts played (HRgame). The mean HR for each SSGcon was calculated by taking the means of all games (HRgame). The %HRmax was then calculated by formula 1 for each SSGs.Blood lactate samples were taken within two minute of the end of each SSG. The twenty-five microliter samples, taken from the ear lobes, were kept in YSI Preservative Kits  during the games. Once the games were finished, lactate analyses were carried out using an YSI 1500 SPORT analyzer (YSI Incorporated Life Sciences). The calibration of the analyzer was carried out according to the manufacturer’s instructions.The data are reported as means and standard deviations. Before using parametric tests, the assumption of normality was verified using the Shapiro-Wilk test. A paired t-test was performed on each dependent variable, including HR, %HRmax and LA differences between the training methods (intermittent and continuous). A one-way repeated-measures analysis of variance was performed on each dependent variable, including HR, %HRmax and LA either between the 2-a-side, 3-a-side and 4-a-side SSGint or bewteen the 2-a-side, 3-a-side and 4-a-side SSGcon. The Bonferroni Post Hoc test was applied to make a pairwise comparison between the different levels of within subjects’ factors (games). The level of statistical significance was set at p < 0.05.No significant differences were found between SSGint and SSGcon for 2-a-side , 3-a-side , and 4-a-side  formats in terms of HR .There were no significant differences between SSGint and SSGcon including 2-a-side , 3-a-side , and 4-a-side  in terms of %HRmax .Finally, there were significant differences between SSGint and SSGcon for the 2-a-side format  in terms of LA concentration, whereas no significant differences were found between SSGint and SSGcon for 3-a-side  and 4-a-side  games .The aim of this study was to investigate physiological responses in young soccer players to various SSGint and SSGcon played with different bout durations. The study results demonstrate that the 3-a-side SSGint and SSGcon responses were significantly higher than the 2-a-side and 4-a-side games in terms of HR and %HRmax, whereas the 2-a-side SSGint and SSGcon resulted in higher LA responses compared to the other SSG formats. The present study findings also indicate that SSGint and SSGcon are similar in terms of HR, %HRmax and LA responses except for the 2-a-side game LA responses.High intensity aerobic intervals at 90–95% HRmax are an effective tool for increasing aerobic endurance in soccer players . AlthougThe present study has demonstrated that lactate responses in SSGcon are higher than in SSGint. Two minutes of recovery between each bout in SSGint may help to remove the lactate and reduce its production, whereas the lack of a rest period in SSGcon may result in the accumulation of lactate. This finding is similar to the results of the study by SSGs studies have generally examined interval formats. However, the present study has shown that both SSGint and SSGcon could be used for the physiological adaptations required for soccer specific aerobic endurance. Our findings indicate that 3-a-side and 4-a-side SSGint and 3-a-side SSGcon could be used to improve maximum oxygen uptake (> 90% of HRmax ), whereas 4-a-side SSGcon games might be used to develop the anaerobic threshold (85–90% of HRmax). In addition, 2-a-side SSGint and SSGcon games can be used to improve lactate tolerance. Unfortunately, we were not able to measure the distance covered at various running speeds and technical actions, and therefore future studies which investigate time-motion analysis and technical actions during the SSGcon and SSGint are needed.


PAX9, Msx1 and Axin2 genes. Limited numbers of studies are present to show etiological factors beyond this anomaly in Turkish community belonging to Caucasian racial family. The purpose of this study is to investigate the relationships between the two different single nucleotide polymorphisms that are G-1031A and T-912C with hypodontia in Caucasians.Hypodontia, the congenital absence of one or a few teeth is one of the most common alterations of the human dentition. Familial hypodontia is caused by mutations in 200 individuals having hypodontia and 114 normal individuals having all 32 teeth present were selected for the study. Blood samples were collected from each individual and DNA was extracted. To determine the polymorphisms, PCR-RFLP method was used.The outcomes suggest that the individuals having AC haplotype carry less risk in having hypodontia compared with the rest of the haplotype groups . The ratio of GT haplotype is less in the hypodontia group meaning that the GT carriers are in risk group in terms of hypodontia risk.PAX9 gene may have an influence on the transcriptional factors and activity of this gene and are associated with hypodontia in Caucasian individuals.These results indicate that polymorphisms in the promoter region of  Teeth are an important part of the digestive system. There are more than 200 genes in the control mechanism of tooth development . The majCongenital tooth agenesis can be found in many patients during dental practices, and the prevalence of tooth agenesis can be between 0.027%  and 11.3MSX1 allele of rs2073244 (adenine) A > G was 25% in a Brazilian population  but 48% agenesis , the purPAX9 -1031 and −912 promoter regions are shown in Table The permanent dentition profile of the individuals suffering from different types of tooth agenesis and their genotypes of the PAX9 gene promoter region was found to be 34.75% in the hypodontia group, 37.70% in the third molar agenesis group, 31.60% in the hypodontia group except the third molars, and 35.50% in the control group (Table Photographs of gel products obtained from restriction enzyme digestion with the restriction fragment length polymorphism (RFLP) technique for each polymorphic site are given in Figure up Table . There wup Table . There wPAX9 gene in the control and test groups. The haplotype distribution of both polymorphic sites was consistent with Hardy–Weinberg equilibrium (p > 0.09). When haplotypes as alleles were compared, statistically significant differences were found between the control and test groups (p = 0.05). When compared with other groups, the ratio of individuals having the AC haplotype was found to be less than control groups in congenitally tooth agenesis cases . It was noticed that in the test group, the individuals having the GT haplotype were less than any other group . No differences between control and test groups were found when haplotypes were organized as genotypes (p = 0.622). It was noticed that the AC/GC haplotype group was found three times more often in the test group than the control group, but this was not significant (p > 0.05).As shown in Table A comparison of the genotype variations of polymorphic sites with various congenitally missing teeth groups is shown in Table PAX9 gene, which were responsible for activating the protein synthesis process. The first mutation described in the human PAX9 gene was a guanine insertion to the 219th nucleotide in the second exon of chromosome 14 of an oligodontia family [PAX9 gene have been reported [We examined two mutagenic regions (polymorphic sites) of the a family . Since treported ,22,26. MIn our study, two polymorphic regions were examined. No differences were found between the control and hypodontia groups for the A or G alleles (p > 0.05) Table . Our finPAX9 mutations have a strong connection with third molar agenesis, indicating that PAX9 mutations have a lesser but distinct effect on incisors and premolars.Our study comprised third molar agenesis cases, and we consequently found no significant difference between the control and test groups on the basis of alleles or genotypes. In contrast, Peres and colleagues  found siIn our study, the haplotype frequencies of genotypes and their relationship with the patient and control groups were also examined. GT, GC, and AT alleles seen in cases of patients with congenital missing teeth were found to be a significant risk. In other words, the individuals having an AC allele were found to have a lower risk of facing tooth agenesis (p ≤ 0.05). We conclude that no relationship exists between the AC haplotype and tooth agenesis. In parallel with this finding, when comparing the AC allele with GT, GC, and AT alleles, the risk of missing teeth in individuals having an AC allele is 3.88 times lower than individuals having other alleles . The ratio of the GT haplotype in the control groups was significant. It is possible that subjects having this allele have a low risk of tooth agenesis . The findings of Peres et al. were consistent with the present study. After examining the two related polymorphic sites, compared to the GA and AA genotypes, the GG genotype was seen less in the third molar missing group but there was higher frequency in groups missing lower and upper canines, upper central incisors, and lower and upper second molars (p = 0.026). Thus, we conclude that individuals with a GG genotype are unlikely to show a lack of congenital third molars and are likely to show a lack of canines, first and second molars, and upper central incisors. However, it is not possible to infer this for T-912C (p > 0.05).PAX9 gene polymorphism and congenital tooth agenesis. The second reason is that the diversity of missing teeth phenotypes of patients may give rise to different outcomes. For example 70% of patients lack the third molar in the study by Peres (2005), whereas this percentage was 55% in our study. Pan (2008) had excluded the third molars. We suggest that this diversity may disappear if future studies are based on genotypes of different populations with the same missing teeth groups. Past studies have contributed a great deal to our knowledge about the process of congenital missing teeth. However, the molecular mechanism underlying this abnormality is still not fully described. The mismatch between high incidences of tooth agenesis with low-value findings of agenesis studies [The discrepancy of the results between the studies may be explained as follows. As demonstrated in our study, genetic polymorphisms often show ethnic variation. For example, in the study of Peres and colleagues on the G-1031A polymorphism, the G allele frequency was 25%, but in our study this frequency was 65.25%. According to the study of Pan et al. (2008), this frequency value was 48%. Thus, further studies of different ethnic populations are needed to illuminate the exact relationship between the  studies ,29,30 suPAX9 gene and the other candidate genes, by increasing the sample size and using the latest laboratory techniques that are proven to give more definitive results. In order to develop the curing techniques and connect these results with contemporary medical treatment alternatives in the future, investigators dealing with tooth agenesis may think of gathering samples from oral tissues and build the studies up to the tissue-like structures and engineering methods which might be one of the highest limitations of these kinds of studies in addition with gathering ethical approval for both animal and human studies.For the Turkish population, limited number of studies investigating the genetic etiology of congenital missing teeth anomaly was found. To understand the molecular basis of tooth agenesis, to investigate the variations of different populations, and to explain the relationship between genetic variants and variable phenotypes clearly, further studies should analyze both the PAX9 gene, no significant relationship between the hypodontia group and the control group was found in terms of both the A or G allele in the G-1031A polymorphic site; (2) the risk of missing teeth in individuals having the AC allele is 3.88 times lower than individuals with other alleles; (3) individuals having the GG genotype have a low risk of third molar agenesis; (4) the subjects having the GT allele can be assumed to have a low risk of tooth agenesis; (5) polymorphisms in the promoter region of the PAX9 gene may have an influence on hypodontia in humans.Our study revealed that (1) in the Inclusion criteria were as follows:1. All patients having hypodontia were between 12 and 32 years of age and every non-missing permanent tooth was apparent in panoramic X-rays, including third molars and/or their germs.2. Patients and families were off systemic diseases and hereditary abnormalities.3. Spaces in the arches of patients were only from deciduous teeth.4. Patients having doubts about the extraction history were excluded.Two hundred patients meeting these criteria were chosen for the study group. The control group was organized according to these criteria:1. Subjects must have proper tooth number and shape.2. Thirty-two teeth must be properly seen in panoramic radiographs.3. Subjects must be genetically and hereditarily healthy.EDTA (Ethylenediaminetetraacetic acid - an anticoagulant for blood samples-) tubes from these 314 subjects.One hundred and fourteen controls were included. All received detailed clinical, intra/extra oral, radiological examination. General characteristics including age, gender, ethnicity, health status, birth defects, and family history, were documented. Subjects declared that their ancestors were Turkish. Two milliliters of venous blood samples were collected in The study design was approved by Selcuk University Faculty of Dentistry Ethical Committee . Informed consent was prepared and signed by every patient participating in the study or in the case of children, by the parents or legal guardians.TM DNA BloodSpin Kit; Mobio Lab Inc., Carlsbad, CA, USA). A microcentrifuge  , water incubator (65°C), micropipette (20 ml and 500 ml volume), and a vortex were also used throughout the process.DNA was extracted using a special isolation kit , 50 μM KCl, 1 μM of each primer, 200 μM of each dATP, dCTP, dGTP, dTTP, 2.5 μM MgCl2, and 2.5 U Tag DNA polymerase .The incubation process of the thermo cycle of the PCR  was performed as follows: 5 min at 95°C, 10 cycles of the triple steps that are 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C; then another 10 cycles of the triple steps that are 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C; and finally 5 min at 72°C.2% agarose  was mixed with TAE  solution and boiled for 1 min to prepare agarose gel. 5 μl of the PCR product was mixed with the loading dye and inserted into the pits. The mixture was processed through gel electrophoresis  for 45 minutes under 120 V. 100-bp DNA ladder was used in the first pit of the gel. The 202-bp PCR product was observed under a ultra-violet (UV) illuminator  and recorded.2O, 2 μl of restriction enzyme buffer, and 0.5 μl restriction enzymes (HaeIII and MseI). This mixture was added into the PCR product and rested in a water bath at 37°C for 12–16 h.The rest of the 15-μl PCR product was used for restriction enzyme digestion. A 6-μl mixture was prepared consisting of 3.5 μl sterile dHA 3% agarose gel was prepared again, and the obtained digestion product was loaded into this gel. A 100-bp DNA ladder was added, and after 45 min at 120 V, the bands were observed under a UV illuminator .Acquired data were statistically analyzed using the program SPSS (version 15.0). The chi-square test was used, and the power of significance of the data was tested. Inter-group relations were evaluated using an odds ratio (OR), and the results were arranged and shown in tables. The significance level was given as 0.05.There are no financial or non-financial competing interests  to declare in relation to this manuscript.EI did the decision making and worked in clinical section. He examined the patients, collected bloods and stored them. SN took the bloods and participated in the genetical laboratory part. HA designed the primers and helped in connecting with companies. ZS helped Dr. EI to analyze the outcomes statistically and to write the manuscript. All authors read and approved the final manuscript.Dr. Eren Isman is the head of the department of Orthodontics, Gaziantep University, Gaziantep, Turkey. Dr. Suleyman Nergis is working as a post-doctor in Medical genetics department in Selcuk University, Konya, Turkey. Dr. Hasan Acar is the head of Medical Genetics Department in the same faculty as Dr. Suleyman. Dr. Zafer Sari is the dean and Orthodontic department head of Akdeniz University, Faculty of Dentistry, Antalya, Turkey.


To systematically review the effectiveness of yoga in patients with breast cancer.MEDLINE, PsychInfo, EMBASE, CAMBASE, and the Cochrane Library were screened through September 2011. Randomized controlled trials (RCTs) comparing yoga to controls were analyzed. Risk of bias was assessed using the Cochrane risk of bias tool. For each outcome, standardized mean differences (SMD) and 95% confidence intervals (CI) were calculated, if it at least 2 studies assessing this outcome were available. As a measure of heterogeneity, I2 was calculated.11 RCTs and 655 subjects were included. 7 RCTs compared yoga to wait-list control groups, 3 RCTs compared yoga to supportive therapy and 1 RCT compared a combination of physiotherapy and yoga to physiotherapy alone.2=79%), as well as in functional , social , and spiritual HRQoL . Greater improvements were also found in anxiety , depression , perceived stress , psychological distress , and fatigue . No significant group differences were found in physical, emotional and mental HRQoL, breast cancer specific concerns and sleep disturbances.Yoga compared to control showed significantly greater improvements in global health-related quality of life (HRQoL) (SMD=0.62,  p=0.04, IThere is encouraging evidence that yoga has beneficial effects on HRQoL, psychological health and fatigue in breast cancer patients. Due to methodological and statistical heterogeneity, larger studies with rigorous trial design and reporting are necessary to underpin these results.


The results obtained by applying the proposed two-staged procedure to analyze co-morbidities of migraine revealed that the proposed procedure could effectively identify a number of clusters of samples with distinctive characteristics. The results further revealed that the distinctive characteristics of the clusters extracted by the proposed procedure were in conformity with the observations reported in recently published articles. Accordingly, it is conceivable that the proposed analysis procedure can be exploited to provide valuable clues of pathogenesis and facilitate development of proper treatment strategies.In this study, we will propose a density estimation based data analysis procedure to investigate the co-morbid associations between migraine and the suspected diseases. The primary objective of this study has aimed to develop a novel analysis procedure that can discover insightful knowledge from large medical databases. The entire analysis procedure consists of two stages. During the first stage, a kernel density estimation algorithm named relaxed variable kernel density estimation (RVKDE) is invoked to identify the samples of interest. Then, in the second stage, a density estimation algorithm based on generalized Gaussian components and named GThe online version of this article (doi:10.1007/s13721-013-0028-8) contains supplementary material, which is available to authorized users. In recent years, data analysis based on large medical and clinical databases has gained attention among biomedical researchers  algorithm  algorithm  is the maximum distance between si and its k nearest training instances; Γ(·) is the gamma function  program in Taiwan was launched in 1995 and as in December 2010 covered about 23,074,000 insurants, which accounted for over 99 % of the entire population in Taiwan. In addition, almost all medical hospitals and clinics in Taiwan have joined the program. As in December 2010, there were 25,031 medical institutes enrolled in the program. Since 2000, the Bureau of the program began to release the National Health Insurance Research Database (NHIRD) to facilitate medical research. The updated version used in this study contains the ambulatory and hospitalization claims records of 1,000,000 randomly selected insurants over the period from 1996 to 2010 without significant difference in age, sex, and insurance cost relative to the whole population.http://icd9cm.chrisendres.com/) used for screening include 346.0×, 346.1×, 346.8×, and 346.9×, which correspond to patients with migraine with or without aura. In our study, for each migraine case, five controls without any migraine record during 1996–2010 and with matched gender and age were randomly selected from the NHIRD. As a result, the cohort contained 19,356 migraine cases and 96,780 controls. For a case, the date of the first migraine diagnosis was defined to be the index date and the same index date was assigned to the matched controls.The cases in this study include those patients who were diagnosed with migraine in outpatient and/or inpatient records during 2004–2008. The ICD-9 CM codes  for validation. The exposure to each category of medications was counted separately. Accordingly, the feature vector is composed of ten elements. In our analysis, we further normalized the feature values corresponding to the same element in the feature vector by applying the standard min–max normalization.In our analysis, each cohort subject was associated with a feature vector that recorded the exposure of the subject to the commonly used medications for migraine treatment during the study period, including amitriptyline, flunarizine, propranolol, topiramate, and valproic acid. The exposure was measured by the number of days and the dosage in milligrams. The dosage was also calculated in defined daily dose (DDD) by World Health Organization ; anxiety state ; bipolar disorder ; depression ; drug abuse ; psychoses Otolaryngology: allergic rhinitis (ICD-9 CM codes: 477.x); chronic pulmonary disease ; Meniere’s disease (codes: 386.0x)Musculoskeletal illnesses: low back pain (ICD-9 CM codes: 724.xx); neck pain (code: 723.1); neck sprain (code: 847.0); pain syndrome (codes: 719.4x and 729.1); rheumatoid arthritis ; spinal disk herniation ;Metabolism and endocrinology: diabetes mellitus ; fluid electrolyte disorder (codes: 253.6 and 276.x); hyperlipidemia (codes: 272.x); hypothyroidism ; obesity (codes: 278.0x);Cardiovascular and neurological diseases: cardiac arrhythmias ; cerebrovascular disease (codes: 430–438.xx); coronary artery disease (codes: 410.xx–414.xx); heart failure (codes: 428.x); hypertension (codes: 401.x); peripheral vascular disease ; epilepsy (codes: 345.xx)Gastroenterology and hepatology: kidney stone (ICD-9 CM codes: 592.0); liver disease ; peptic ulcer disease (codes: 531.xx–534.xx); renal disease .Our study focused on those diseases that had been reported to be the co-morbidities of migraine  with 95 % confidence interval to quantify the risk of a co-morbidity of migraine in different groups of patients. All tests were two-tailed, and p values of <0.05 were considered significant.For each subject, outpatient and/or inpatient diagnoses of these disorders during the study period would be analyzed. Demographics and clinical variables were compared between migraine cases and controls using the Chi-square test or student’s i-th case in the dataset. If The analysis procedure consists of two stages. During the first stage, the RVKDE algorithm was invoked to construct one approximate probability density function for the cases, denoted by 2DE algorithm was invoked to cluster the cases of interest and provided summarized descriptions of the clusters. However, as mentioned earlier, the number of features, which correspond to the dimension of the vector space and thus the dimension of the covariance matrix output by the G2DE algorithm, should be limited to a small integer for us to easily obtain an abstract image of the underlying distribution. Accordingly, we incorporated a feature selection process before invoking the G2DE algorithm. The feature selection process proceeded as follows. First, the correlation matrix of the original ten features is derived based on the cases of interest identified in the first stage of analysis. Then, those eigenvectors with the corresponding eigenvalue larger than 1 are selected to form the factor space. Finally, the factor space is rotated orthogonally and the component features of the rotated factors with a loading larger than 0.4 are selected to form a subspace into which the original dataset is projected.During the second stage, the GTable Figure The red bars in Fig. p < 0.001). Moreover, the mean age of the cases of interest is older than the mean age of the remaining migraine cases, 45.3 versus 41.8 with p value <0.001. For both preventive medicines and relief treatment of migraine, cases of interest have significant higher utilization proportions than the remaining migraine patients. However, for topiramate and valproic acid, the cases of interest have lower exposure dosages and durations. Figure According to the demographics shown in Table 2DE algorithm was invoked to identify the main clusters among the 7,146 cases of interest. As mentioned earlier, before invoking the G2DE algorithm, factor analysis was carried out to identify the most informative features. In this respect, it must be noted that the set of cases of interest passed the two criteria commonly adopted to measure the adequacy of applying factor analysis. In fact, applying the Kaiser–Meyer–Olkin (KMO) test on the set of cases of interest yielded a value of 0.502, which is higher than the commonly adopted threshold of 0.5, and applying the Bartlett’s test yielded a value smaller than 0.001, which is significant for variance homogeneity. The end result of the factor analysis is that exposure dosages (in unit of milligram) for the five preventive medicines of migraine: amitriptyline, flunarizine, propranolol, topiramate, and valproic acid, were selected respectively.Since Figs. 2DE algorithm identified two clusters with distinctive characteristics shown in Table p value <0.001) but they have almost the same gender distribution. Furthermore, for both preventive medicines and relief treatment of migraine attacks, the case samples in cluster 1 had significant larger exposure dosages and longer durations. According to the results shown in Fig. The GAccording to the results shown in Fig. The correlation between mental disorder and migraine has been studied extensively in recent years and our results match the previous observations. The American Migraine Prevalence and Prevention (AMPP) study demonstrated that both depression (OR = 2.0) and anxiety OR = 1.8) were included in the co-morbidity profiles of chronic migraine and episodic migraine patients  more prevalent among those with asthma than those without  higher among individuals with musculoskeletal symptoms than among those without  , and stroke (OR = 1.5–5.46)  . So the proposed density estimation-based analysis procedure conceivably provides valuable insights which might be overlooked by conventional methods.Although conventional algorithms of regression analysis are applicable for data mining in medical and/or clinical information, they borrow the idea from multi-dimensional contingency table to determine certain associations between the dependent variable and the risk factors. Rather than fitting a more saturated model, it might be more inclined to reflect an interaction structure between the dependent variables and corresponding risk factors. However, in this research, we would like to refer the concept of discriminate analysis: classifying an object that comes from one of two populations having associated densities A major strength of our study was utilization of a large population-based medical claims database, but there were some limitations. First, administrative claims reported by hospitals or clinics may be less accurate than clinical diagnoses and observer-rating scales. Second, prescriptions of medications for migraine do not guarantee drug adherence. Third, the administrative claims data of NHIRD did not include detailed personal information like body mass index, living habits, or results of laboratory tests, which might be important confounding factors. Finally, more confounding factors of the outcome diseases, e.g., age, sex, medication drugs, treatment procedures, or associated symptoms, should be taken into account.In recent years, data analysis based on large medical and clinical databases has gained attention among biomedical researchers. Furthermore, scientists have turned to exploit advanced machine learning and/or data mining approaches to extract valuable clues hidden in large medical and clinical databases. In this paper, we have proposed a density estimation-based data analysis procedure to investigate the co-morbid associations between migraine and the suspected diseases by characteristics of medication exposure. The primary objective of this study is to develop a novel analysis procedure that can discover insightful knowledge from large medical databases. The results obtained by applying the proposed two-staged procedure to analyze co-morbidities of migraine reveal that the proposed procedure can effectively identify a number of clusters of cases with distinctive characteristics. Furthermore, it has been observed that the distinctive characteristics of the clusters are in conformity with the recently discovered knowledge in biomedical research. Accordingly, it is conceivable that the proposed analysis procedure will be exploited to provide valuable clues of pathogenesis and facilitate development of proper treatment strategies.Three further courses are undertaken. Firstly, since effectiveness of the proposed analysis procedure has been verified, this method will be exploited to investigate characteristics of more epidemics, such as osteoporosis or herpes zoster. Secondly, appropriate statistical tests will be issued on the mined facts to strengthen persuasiveness of this approach. Finally, application of various advanced machine learning/data mining algorithms on medical and/or clinical databases will also be studied.Supplementary material 1 (PDF 238 kb)Below is the link to the electronic supplementary material.


TF, P589S), hemochromatosis (HFE) C282Y and (H63D)], iron biochemical variables  and apolipoprotein E (APOE) gene variants in 139 Alzheimer's disease (AD), 27 Mild Cognitive Impairment (MCI), 78 Parkinson's disease (PD) patients and 139 healthy controls to investigate mechanisms of iron regulation or toxicity. No difference in genetic variant distributions between patients and controls was found in our Italian sample, but the stratification for the APOEε4 allele revealed that among the APOEε4 carriers was higher the frequency of those carriers of at least a mutated TF P589S allele. Decreased Tf in both AD and MCI and increased Cp:Tf ratio in AD vs. controls were detected. A multinomial logistic regression model revealed that increased iron and Cp:Tf ratio and being man instead of woman increased the risk of having PD, that increased values of Cp:Tf ratio corresponded to a 4-fold increase of the relative risk of having MCI, while higher Cp levels were protective for PD and MCI. Our study has some limitations: the small size of the samples, one ethnic group considered, the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest the hypothesis that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk.Deregulation of iron metabolism has been observed in patients with neurodegenerative diseases. We have carried out a molecular analysis investigating the interaction between iron specific gene variants [transferrin ( Specifically, an excessive iron accumulation has been reported in neuritic plaques, neurofibrillary tangles and in specific brain areas of patients with AD  proteins seem to play a key role. These proteins, encoded by the TF and HFE genes . The C1 variant is the most prevalent (95%) and its encoding polymorphic gene has two allelic variants that produce transferrin C2 . Both TF C2 (rs1049296) and the 2 HFE, C282Y (rs1800562) and H63D (rs1799945) gene variants, have been investigated as potential risk factors for neurodegenerative disorders. Studies analyzing the association between HFE mutations and AD came to different conclusions regarding the hypothesis that H63D mutation may anticipate the disease onset in sporadic AD   gene risk factor is reported herein.The brain iron content increases with advancing age % 68, age mean ± standard deviation 63 ± 12 years), 139 AD , 27 MCI  and 78 PD patients . All individuals included in this study are Caucasian. The study was approved by the local IRB and all the subjects involved or legal guardians signed their informed consent.APOE genotyping was performed according to establish methods . Genotyping of SNPs rs1800562, rs1799945, rs1049296 were achieved by the TaqMan allelic discrimination assays from Applied Biosystems Inc. (Foster City CA). The predesigned SNPs genotyping assays ID are C_1085595_10, C_1085600_10, C_7505275_10 (Applied Biosystems). The total reaction volume per well was 20 μL, including 5 ng genomic DNA, 1 μL TaqMan SNP genotyping assay (containing two PCR primers and two dye (VIC or FAM) labeled TaqMan MGB probes) and 10 μL TaqMan Universal PCR Master Mix (Applied Biosystems), according to the manufacturer's manual. PCR was performed at 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. The samples were amplified, read, and analyzed using the ABI Prism 7900HT Sequence Detection Systemand ABI Prism SDS 2.4 software. Two blank controls in each 96-well-plate were used for the assay quality control. *1.25) and multiplying by 100. The ferritin was analyzed by immunoturbidimetry assay enhanced with latex ]. All biochemical measures were automated on a Cobas Mira Plus  and performed in duplicate.Patients' fasting blood samples were collected in the morning and serum was rapidly stored at −80°C. Ceruloplasmin (Cp) was analyzed by immunoturbidimetry assay  Wolf, . Iron wat-test and the chi-square (χ2) test, or a non parametric test when appropriate, were used to compare separately each diagnosis  group vs. healthy controls. The effects of both age and sex were considered in all statistical analyses. Diagnosis groups were compared using ANCOVA models adjusting for genetic variable as well as age and sex. A Multinomial Multiple Logistic Regression model was applied, considering the control group as the reference category, to evaluate the effects of the biological and genetic variables on the probability of being AD or PD or MCI. All tests were interpreted at the 0.05 level of significance. SPSS 16.0 software was used for all statistical analyses.Demographic and clinical characteristics in our patient and control samples were described either in terms of mean ± SD if quantitative, or in terms of proportions. Student's HFE gene and P589S (rs1049296) TF gene mutations in order to test their association with AD, MCI and PD. Gene frequencies were in Hardy-Weinberg equilibrium in each group.We screened participants for C282Y (rs1800562), H63D (rs1799945) HFE gene variants among AD, MCI and PD subjects vs. healthy controls, though a C282Y mutation higher frequency approached significance in the MCI group  = 4.055]; in particular only the estimated parameter for AD diagnosis resulted significative , a significant simple effect of age was evident too . The simple effect of diagnosis was significant on Cp:Tf ratio  but it was due to difference between MCI and controls . Also the simple effect of age  were significant on Cp:Tf ratio.We measured iron, transferrin, ceruloplasmin, and calculated the Cp:Tf ratio and %Tf saturation in the whole study population. Some of these variables were not normally distributed and so we applied a Mann-Whitney non parametric test and these data are presented as median (min-max) in Table HFE on iron concentrations  and, always on iron concentrations, a significant simple effect of sex , age [p = 0.028 F = 4.876]. The analysis reconfirmed the previous results on Cp:Tf ratio while the effect of HFE was not significant (p > 0.20). The effect of age was significant  on the Tf values and also the effect of diagnosis factor . Finally, we observed the only significant effect of sex factor on Cp concentrations . The probability of having AD, PD or MCI was estimated with a multinomial logistic regression model considering the diagnosis as the dependent variable and the biochemical and genetic markers, as well as sex and age, as the independent variables and the control group was set as the reference category. These statistical analyses confirmed age as the main risk factor: the increase of age (1 year) increased of 14% the risk of being AD . Concerning PD, increased of 1 μg/dL of iron , of Cp:Tf ratio  as well as being man instead of woman  resulted in a higher risk of having PD, while the increase of Cp concentration (1 mg/dL) was a protection factor . In MCI, we observed that increasing values of Cp:Tf corresponded to a 4-fold increase of the relative risk of having MCI , and conversely that higher Cp levels resulted as a protection factor .Then, we again performed the ANCOVA analysis taking into account factors like diagnosis, age, sex and genetic variants and their possible interaction on concentration of biochemical variables under study. The analysis revealed a significant influence of the interaction between sex and HFE and TF genes on the risk of having neurodegenerative disorders, in particular AD, PD and MCI even though separately. The results reported are often controversial and this inconsistency suggests that, beside the complex genetic etiology, additional interactions can contribute to the disease, as for example, environmental factors, age, sex, and metal ion involvement or oxidative stress. Considering the current literature, we examined the contribution of HFE and TF mutations in PD, AD and MCI testing if single gene variants have an impact on iron metabolism. Even though our results don't support the hypothesis that these mutations are a genetic risk factor for Italian cohort they have been reported to have a strong effect on the disease risk in several populations  to Fe (III) allows iron binding to Tf and its mobilization from iron tissue storage. More specifically, the result that increased Cp concentrations are a protecting factor for PD is in line with the evidence that CP mutations have been associated with increased deposits of iron in SN  results in an increased absorption of iron in men. A “disease effect” seems essential to explain this relationship, since increased iron levels cannot be ascribed to the HFE change alone, but rather it is caused by the synergy between being man and HFE change on iron levels, sustaining previous results (Fargion et al., A result worthy of interest is the effect of interaction between sex and Our study has some limitations that need to be taken into account: the small size of the sample, and specifically the small number of MCI subjects included; one ethnic group considered; the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genotype imputation is often used in the meta-analysis of genome-wide association studies (GWAS), for combining data from different studies and/or genotyping platforms, in order to improve the ability for detecting disease variants with small to moderate effects. However, how genotype imputation affects the performance of the meta-analysis of GWAS is largely unknown. In this study, we investigated the effects of genotype imputation on the performance of meta-analysis through simulations based on empirical data from the Framingham Heart Study. We found that when fix-effects models were used, considerable between-study heterogeneity was detected when causal variants were typed in only some but not all individual studies, resulting in up to ∼25% reduction of detection power. For certain situations, the power of the meta-analysis can be even less than that of individual studies. Additional analyses showed that the detection power was slightly improved when between-study heterogeneity was partially controlled through the random-effects model, relative to that of the fixed-effects model. Our study may aid in the planning, data analysis, and interpretation of GWAS meta-analysis results when genotype imputation is necessary. Genome-wide association studies (GWA studies or GWAS) using high-throughput genotyping data are a powerful tool and are of a great help in identifying susceptibility loci for human complex traits and common diseases A partial solution to this issue is meta-analysis, which combines data from multiple studies of relatively small sample sizes, with the expectation to detect genes underlying susceptibility loci with greater power and produce more precise estimation of genetic effects, and hence to provide more convincing conclusions than the original individual studies do A useful tool in GWAS is imputation, which can provide the same set of SNPs across individual studies by inferring millions of untyped/missing SNPs from typed SNPs and based on the known knowledge such as haplotype structure from HapMap To better assess the usefulness and limitation of the meta-analyses of GWAS using genotype imputation, several critical questions need to be answered:Does genotype imputation affect (or create) between-study heterogeneity? If yes, how does this heterogeneity caused by imputation affect the performance of meta-analysis of GWAS?Does imputation-based meta-analysis of GWAS with a much larger sample size always have greater power than that of individual component studies with smaller sample sizes?What to do in the presence of potential negative impacts of genotype imputation on the meta-analysis of GWAS?In order to address these questions, we performed comprehensive simulation studies based on the empirical GWAS dataset from the Framingham Heart Study (FHS). We found that genotype imputation may cause between-study heterogeneity and reduce the power of meta-analysis of GWAS. Strategies were proposed to alleviate this negative impact of genotype imputation on meta-analysis of GWAS.Data structure such as sample size and simulation parameters were indicated in The estimated type-I error rates and the power of meta-analysis with both fixed-effects model and random-effects model are shown in The power of the meta-analysis was different for various scenarios . In geneThe estimation accuracy of the genetic effects, measured by Power comparisons between meta-analysis and individual association analyses were conducted and were partially shown in In this study, we investigated the performance of imputation-based meta-analysis of GWAS through an empirical GWAS genotype data. Considerable between-study heterogeneity was detected and reduction in detection power was observed when causal variants were typed in some individual studies and imputed in the others. Specifically, for meta-analysis using fixed-effects model, the power loss was up to ∼25% for situations that causal genes were genotyped in some but not all individual component studies, comparing to relatively homogenous situations of typed causal variants across all individual studies. Notably, for situations of partially directly-typed causal variants, the power of meta-analysis may be lower than that of analyses performed in some individual studies.An important issue in meta-analysis is the potential existence of between-study heterogeneity, which can affect the power of the meta-analysis. In the context of genome-wide association studies, the between-study heterogeneity can be caused by various factors, such as the variation of genetic effects across different populations, incomparable measures of phenotype used in different studies, and/or the deviation from Hardy–Weinberg equilibrium for SNPs and population stratification. With the above-mentioned heterogeneity-causing factors removed by constructing our sub-samples from a single population and using the same distribution for genetic effects across sub-samples, the observed between-study heterogeneity in our simulated sub-samples will be most likely to be only due to imputation, indicating another possible source of between-study heterogeneity for the meta-analysis of GWAS.in silico replications are performed for candidate or genome-wide association analyses. In this case, statistically significant SNPs are genotyped in the discovery sample, and may not be genotyped in all the replication samples and thus need to be imputed, such as in our previous study Although our study is based on simulations for an empirical data set, the observations from our study are closely relevant to practical analyses. An interesting and useful observation is that a meta-analysis with imputation is not necessarily more powerful than that of analyses performed in individual samples, as shown in var, an increasing RAF results in less between-study heterogeneity; and for a specific range of RAF, an increasing value of var results in more between-study heterogeneity. These trends may be partially explained by effect sizes of the causal SNPs. On one hand, greater effect sizes may imply greater between-population variation for the causal SNPs, and thus greater chances for observing between-study heterogeneity. On the other hand, it is known from population genetics theory that the variation explained by the causal SNPs is proportional to RAF*(1-RAF)*a2, where a is the effect size for the causal SNP. Thus for a fixed value of var, an increasing RAF yields a smaller value for a; and for a specific range of RAF, an increasing var yields a greater value for a. For either case, the magnitudes of effect sizes are positively correlated with those of between-study heterogeneity.Both risk increasing allele frequency and the magnitude of variation explained by the causal SNPs can affect between-study heterogeneity, as shown in To deal with the negative impacts of genotype imputation on meta-analysis of GWAS, we provide the following suggestions based on our study. Firstly, focus on SNPs presented in all individual studies, as meta-analysis with direct genotypes in all individual studies help to avoid generating spurious between-study heterogeneity by genotype imputation. Secondly, random-effects model should be used when significant between-study heterogeneity is detected. Although the random-effects model may not guarantee higher power than that of the fixed-effects model, it may help obtain more accurate effect size estimation. Thirdly, when sample sizes of individual studies vary largely, the results from the largest individual sample should be carefully evaluated, as it may provide better power than that of mate-analysis with imputation. Lastly, improving imputation accuracy may be useful in reducing between-study heterogeneity introduced by genotype imputation.A number of issues in our studies may need further investigations. For example, only three GWAS sub-samples were used in our current study. Although a similar number of individual populations were used in various published meta-analysis of GWAS e.g., , increasIn this section, we will first summarize how the sub-populations are constructed; we then describe the model and procedure for phenotype simulation; and at the end, we describe several topics related to our analyses, including imputation method, analytical models and test statistic for meta-analysis, and heterogeneity detection.The individual study samples used for the meta-analysis were constructed from an empirical GWAS dataset, the genome-wide genotyping data obtained from FHS SNP Health Association Resource (SHARe) project. The application for using the data has been approved by Tulane University Institutional Review Board and the access to the data has been granted by NHLBI Data Access Committee. The dataset contains more than 9,300 subjects from three generations of over 900 families and was genotyped for ∼550,000 SNPs . Detailed information about the FHS and its genotyping dataset can be found at the dbGaP website. For simplicity, we only used the SNPs in the 500 K array for subsequent analyses.st generation (two at most in each pedigree), and married-ins in the 2nd and 3rd generations. Sample 2 and Sample 3 were constructed by randomly selecting one subject from the rest members of the 2nd and 3rd generations in each pedigree, respectively. This selection strategy helps to ensure unrelatedness among individuals within each sample. After data quality controls, including removing individuals with genome-wide genotype missing rates >10%, SNPs with genotyping call rates <90% or minor allele frequencies <0.01, and Hardy-Weinberg equilibrium test p-values< = 0.0001, the numbers of individuals for Samples 1–3 are, respectively, 2,023 , 1,055 , and 806 , and the number of common SNPs across all three populations is 392,261. Notice that throughout the simulations, genotypes were fixed and obtained from the FHS data set directly, and phenotypes were simulated as described in the next session.To imitate meta-analysis, three sub-samples (Samples 1–3) were constructed, with related information summarized in Genotypes of three independent populations were also generated through simulations using the software HAPGEN2. The sample sizes for the three populations were 2000, 1000, and 1000, respectively. The genotypes were generated based on the 1000 Genomes data provided by the software, and SNPs matching those in the Affymetrix 500 K mapping array were then selected to be the genotyped SNPs for the simulated populations. This simulation produced fully independent samples.p and q ( = 1−p), respectively. With an additive genetic effect of ith individual is modeled by: ith individual, var or For a di-allelic quantitative trait locus (QTL), the risk allele and the alternative allele are denoted by 1 and 0, respectively. The frequencies for the two alleles are assumed to be To cover various biologically plausible conditions, our analyses were performed with a range of parameter values. Briefly, the variance explained by an assumed QTL was set as 0–2.0% with a 0.5% increment. Risk increasing allele frequency (RAF) was binned into four intervals: 0.01<RAF≤0.05, 0.05<RAF≤0.10, 0.10<RAF≤0.20 and 0.20<RAF≤0.50, which were represented by RAF1-4, respectively. To simulate meta-analysis with or without genotype imputation in individual studies, four scenarios  F-test and accounts for the uncertainty in the imputed genotypes.SNP association tests were carried out by using the program SNPTEST (Version 1.5.1) Fixed-effects model considers the genetic effects to be the same across all individual studies and assumes that any difference is due to chance. In genetic association studies, however, genetic effects could be different across populations due to various reasons such as allele frequency differences, different biases and estimation errors across studies. Thus to take these differences into consideration, random-effects model may be a better choice, as random-effects model assumes and can accommodate the potential differential effects across studies.ith study, respectively. The test statistic In this study, we adopted the inverse variance method to construct the test statistic for meta-analysis, which was recently reviewed in the context of genetic association for quantitative traits by de Bakker and colleagues I2 index k is the number of studies and Q represents Cochran's Q statistic ith study and the combined study, respectively.To test between-study heterogeneity, we used the The


Indian subcontinent has the highest child mortality rates along with a very high frequency of low birthweight (LBW). Folate and vitamin B12 (Vit-B12) are necessary during foetal development and their deficiency prevalence in Indians is very high. The objective of the present paper is to assess whether foetal homocysteine (Hcy)/folate metabolic pathway genes, their cofactors and homocysteine level independently (or collectively) predispose children to Low birth weight.Cord blood was collected for the study. Frequency of 5 SNPs in 4-Hcy-pathway genes, and levels of Hcy, Vit-B12 and folate were evaluated.Of the 421 newborns recruited for the study, 38% showed low birth weight (<2.5kg) and 16% were preterm babies. 101 neonates developed neonatal hyperbilirubinemia (NNH). High prevalence of Vit-B12 (65%) and folate (27%) deficiency was observed in newborns along with hyperhomocystinemia (hypHcy-25%). Preterm delivery, micronutrient deficiency, hypHcy and MTHFR 677T SNP are associated as risk factor while G allele of TCN2 C776G is protective against LBW. MTHFR 677T allele and folate deficiency are also independent risk factors for NNH.We record the highest incidence of Vit-B12, folate deficiency and elevated Hcy levels, of all the studies so far reported on neonates. These together with MTHFR 677T are potential risk factors for LBW. Association of impaired folate/Hcy metabolism with NNH is reported for the first time and the possible way of interaction is discussed. It appears that proper nutritional management during pregnancy would reduce the risk of complex clinical outcomes. World Bank report 2011 shows that globally the infant 5.7%) and child mortality 6.7%) rate in India . Neonata.7% and c.7% rate MTHFR, CBS, MTRR, and SLC19A1) or deficiency of the micronutrient cofactors , acts as a risk factor for a variety of developmental disorders [Several studies have shown that accumulation of the thiol amino acid, Homocysteine , either due to mutations in the metabolism pathway genes , occurring in ~25% of the children, as independent risk factors. The results are presented in this paper.The dismal state of the micronutrient (Vit-B12 and folate) levels related to Hcy-metabolic pathway in the general Indian population and their association with multiple disorders, raise concerns for the community at large, more specifically neonatal and child health. The premise of the present study is that if similar condition prevails in child at birth, it could be a risk factor for the high incidence of low birth weight and related complications in child health. We explore the status of these parameters in a group of just born children from the eastern region of India, and its influence, if any, on LBW and other health parameters. We report that the evaluated micronutrients in the children are even poorer than the adult population of the same region. More importantly, elevated Hcy, low vit-B12 and folates and Cord blood samples (n=421) were collected from Division of Neonatology, Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University. In general, the babies belonged to lower middle economy group and were from rural area of eastern Uttar Pradesh, and western Bihar. The study was approved by Institutional ethical committee of Banaras Hindu University. Clinical information such as birth weight, foetal distress, sepsis, ABO incompatibility, G6PD deficiency, hypothyroidism, total serum bilirubin (TSB), hematological parameters, APGAR score etc were obtained from hospital data sheet. Children with less than 2.5kg birth weight were placed in the category of LBW. Hyperbilirubinemia in newborns was defined as peak TSB  levels >95th percentile while control had physiological range of jaundice with peak TSB levels <75th percentile as per-hour-specific nomogram of the American Academy of Pediatrics  during tAs majority of the mothers came only during the period of delivery to the hospital and left after a few days, the follow up of subjects was not done, and since they were not regular visitors to the same hospital during pregnancy no systematic record of their metabolic status during pregnancy was maintained. The time of pregnancy was calculated mostly by LMP and rarely by clinical observation of the newborn by standard Ballard procedure. We did question them about intake of folate and Vit-B12 during pregnancy but most of them were unaware of the nature of “medicines” prescribed during pregnancy, and those 154 women who admitted to taking iron and folates were not confident of the duration and the period of intake during pregnancy. Vit-B12, it appears was not administered to any one, at least not on a sustained basis. As our objective was to investigate the relationship of newborn nutritional status, with potential clinical outcome, and not of the mother on which comprehensive data exist, we believe lack of maternal details in the studied cohort have minimal effect on the set objective of the present study.° C until biochemical analysis of Hcy, B12 and folate was done. DNA was isolated from rest of the blood through salting out method and dissolved in T.E buffer (pH 8.0) and stored at 4oC.The cord blood was collected in heparinised syringe. Plasma was separated immediately after sampling from 1ml of blood and stored at -80Homocysteine was measured by reverse phase HPLC for 421 plasma samples  as previously described . Vit-B12SLC19A1 (RFC1) (G80A), MTHFR (C677T & A1298C), and TCN2 (C776G), as earlier described [CBS (844ins68 bp) was analysed by PCR with gene specific primers [Five gene polymorphisms in four genes of Folate/Hcy metabolic pathway were studied using gene-specific primers. Genomic DNA was subjected to PCR-RFLP for escribed ,15 while primers  and the Reproducibility of biochemical measurements was ensured by performing intra assays where certain samples were measured in duplicates and also a few at two different time intervals. The measurement difference of <5% between the duplicates was accepted range for Hcy, Vit-B12 and folate values. The genotyping was repeated twice randomly for 20% of analysed samples, which yielded 100% identical results with the previous results.For biochemical parameters like Hcy, Folate and Vit-B12, median and interquartile range was calculated. Comparison between two groups was done by non parametric test Mann–Whitney U-test, while for three groups One way ANOVA with Bonferroni correction was used. The allele frequencies of various studied SNP’s were tested for Hardy-Weinberg equilibrium and to assess the genotype and allele distributions among groups chi-square test was applied with Yates correction and p value, <0.05 was taken to be statistically significant. Binary Logistic regression analysis was done to find out potential risk factors for the dependent variables, LBW and NNH, by using SPSS version 16 statistical package . Odds ratios (OR) were calculated, adjusted and given with 95% confidence interval. Cox and Snell R-square and Nagelkerke R-square were calculated to know the percentage of the clinical outcome (both in LBW and NNH) that could be explained with the total risk factors taken in to accountability for the study.Cord blood samples of 421 neonates were examined. Of these, 16% were delivered prematurely (<37 weeks of gestation), 38% had low birth weight (<2.5kg) and 2 to 5% had other clinical features . A subgrC677T and A1298C in MTHFR, C776G for TCN2, G80A for RFC1) and 1 insertion (844ins68bp for CBS) were in Hardy-Weinberg equilibrium , 12.6 µmol/L (GA) VS 9.9 µmol/L (AA), CBS there were no homozygotes for 844ins68bp and heterozygotes also were only 30 out of 421 but their Hcy level was distinctly higher than in the homozygotes for the major allele. In contrast, SLC19A180AA homozygote was apparently a protective genotype against elevated Hcy levels because the median Hcy level in 80 AA was significantly lower than in 80GG and 80GA, even in presence of T allele of MTHFR C677T the SLC19A180AA (14.5 µmol/L) showed low levels of Hcy as compared with 80GA (16.3), 80GG (15.7) genotypes. MTHFR 1298C allele, though not statistically significant, showed some association with elevated levels of Hcy. The TCN2 C776G bore no significant impact on Hcy levels. MTHFR 677T allele and MTHFR 1298CC genotype were associated with low folate levels while other polymorphisms did not show any association with Vit-B12 and Folate levels. In the individuals genetically predisposed to elevated levels of Hcy, deficiency of Vit-B12 and/or folate led to further rise in Hcy levels, implying that the genotypic effect on Hcy levels was modulated by micronutrient levels. The study further showed a close association of micronutrient levels, Hcy levels and certain genotype with respect to clinical outcome of a new born.Genotypes of 4 SNPs  category. The proportion of LBW children with elevated levels of Hcy, deficiency of Vit-B12 and folate was greater than those with normal weight (NW- ≥2500gm). When subjected to Mann-Whitney test, the difference was statistically significant for all the three parameters  However,nificant . The significant . The CoxNNH is a clinical condition common in early neonatal life. In accordance with the hour-specific nomogram of the American Academy of Paediatrics, 101 children within the studied cohort fell under NNH (>95 percentile TSB level), 304 in the normal (<75 percentile TSB levels) categories, and 16 were excluded from either category as they fell between the <75 and >95 percentiles. When subjected to diverse clinical parameters such as prematurity, gender , excessive weight loss, foetal distress, sepsis and ABO incompatibility, all of them showed association with NNH to variable degrees.MTHFR C677T, and folate deficiency all independently associated with NNH while Vit-B12 and hypHcy did not qualify as independent risk factors  was twice that in controls , while other SNPs did not vary among case and control groups thereby showing no significant association with clinical outcome. Similarly, proportion of individuals with deficiency of Vit-B12 as well as folate and elevated levels of Hcy in the cases was greater than in controls, and the difference was statistically significant  but up o factors . The CoxMTHFR 677T and SLC19A1 80G is higher as risk factors for elevated Hcy levels, a trend similar to that of the adult population, only more severe. An earlier study [The present study originates from the fact that there is very high incidence of low birth weight cases in India, and that there is deficiency of Vit-B12 and folates and high level of Hcy in a large proportion of the adult population in the eastern region of India . The rater study  on a coher study ,18. In vThe focus of our study, however, is to explore possible association of the Hcy-pathway variables with LBW. About 38% 160/421) of the presently studied just-borns have LBW which is in accordance with the projected frequency of LBW (40%) in India 21 of the. In factMTHFR 677T is a risk factor while TCN 776G, which is the major allele in this population, could be protective against LBW.Therefore while maternal environment per se, is the major environmental contributor to the foetal health including the weight, if the nutrient levels dip too low in the child, it may add to the risk of lowering of the birth weight which may have health consequences in later part of life. In addition, the genotype MTHFR 677T, elevated homocysteine and Vit-B12 and folate deficiency. However, following Bonferrroni correction and the binary logistic regression analysis, it turns out that only MTHFR 677T and folate deficiency are independent risk factors. This is the first study to show an association of MTHFR677T/folate deficiency with the development of NNH. Interaction of T allele of MTHFR C677T with low folate levels has been reported in studies on Norwegian [MTHFR 677T and folate deficiency, instead of being two independent factors, are causally related with the mutation contributing to the low level of folates. The same could be the situation in present study.It is intriguing that neonatal jaundice, which is transient in nature, also shows statistically significant association with orwegian  and Czecorwegian , which sFolate and Vit-B12 (stored in liver) are crucial for maturation and proliferation of RBC. Their deficiency causes failure of RBC maturation leading to rapid lysis . This reIn summary, this work reemphasises the need of antenatal care which will not only improve maternal health but also lessen neonatal disease burden. It is important that those responsible for overseeing community health in countries like India should consider fortification with necessary micronutrients for mothers-to-be and neonates.


Pectus excavatum (PE) is a common chest wall malformation, with surgery being the only method known to correct the defect. Although the Nuss and Ravitch procedures are commonly used, there is no consensus as to whether surgical repair improves pulmonary function. We therefore investigated whether pulmonary function recovers after surgical repair, and if recovery is dependent on the type of procedure or time after surgery.Literature searches were performed using PubMed, EMBASE, Health Periodicals Database, and CNKI  from January 1990 to December 2007. The following keywords were used: pectus excavatum, chest wall deformity, funnel chest, pulmonary function, respiratory, lung function, and pectus severity index. The primary outcome of interest was possible changes in pulmonary function following surgical repair.p < 0.05), changes in pulmonary functional indices, including forced expiratory volume over 1 s (FEV1), forced vital capacity (FVC), vital capacity (VC), and total lung capacity (TLC), were similar 1 year after the Ravitch and Nuss procedures. Several years after surgery and bar removal, however, the changes in pulmonary functional indices significantly favored the Nuss procedure.Meta-analysis of 23 studies showed that, although there was evidence of statistically significant heterogeneity among studies (Chi-square, 17.11, Pulmonary function tends to improve after the surgical correction of pectus excavatum. Although the Nuss procedure was not significantly better 1 year after surgery, long-term postoperative pulmonary function improvement was significantly better after bar removal. Pectus excavatum (PE) is a common congenital deformity of the anterior chest wall in children and adolescents. AlthougThe more important aspect of the pathophysiology of PE is that the depth and extent of the sternal depression determine the degree of pulmonary compression, which in turn determines the degree of incapacitation, often causing impairment of pulmonary function,8. AlthoThe standard surgical treatment of PE since 1949 has been the Ravitch technique. This method, however, was modified to meet requirements related to cosmetics, orthopedics, and effectiveness in relieving symptoms. The modEligible studies were identified by searching the PubMed, EMBASE, Health Periodicals Database, and CNKI  databases for relevant reports published from January 1990 to December 2007. The following keywords were searched, either alone or in various combinations: pectus excavatum, chest wall deformity, funnel chest, pulmonary function, respiratory, lung function, age group, follow up, costal cartilage, and pectus severity index. The titles and abstracts of studies obtained were examined to exclude any articles that were clearly irrelevant. The full texts of the remaining articles were retrieved, and each paper was read to determine whether it contained information on the topic of interest.Inclusion criteria for this meta-analysis included the following: (1) reports that included quantitative measurements of preoperative and postoperative pulmonary function; (2) reports published in the English or Chinese language; (3) reports indexed between January 1990 and December 2007. Abstracts from conference proceedings, doctoral dissertations, and masters' theses were not included because those sources are unlikely to report substantive research findings that are not published elsewhere. Studies meeting the inclusion criteria were examined to ensure that the same subjects were not included in more than one study.The following information was extracted from each study: study characteristics, physical characteristics of subjects, type of surgical repair performed, pulmonary outcomes, major presenting symptoms, and postoperative complications. Because not all studies reported the same index of pulmonary function, we elected to place the indices reported in each study into pulmonary function global categories. When specific results were not reported, we used available tabular data to calculate them. The indices included in this meta-analysis Table represenp < 0.05. In addition, 95% confidence intervals were reported.The primary outcome of interest was change in pulmonary function following surgical repair. Statistical analysis was performed preferentially using the Cochrane Review Manager Software (version 4.2.10). The Statistical Package for the Social Sciences software  and Microsoft Office Excel 2007 were also used. Results were considered statistically significant if The initial computerized searches identified 171 potentially relevant articles using the search terms ‘pectus excavatum’ and ‘pulmonary’. We also reviewed reference lists from original and review articles to identify any studies not previously identified from the computerized searches. Overall, we identified 23 studies for our analysis .The meta-analysis included a total of 2476 patients with PE, including 921 who underwent open surgical repair (Ravitch + Daniel) and 1555 who underwent the minimally invasive Nuss technique. Average patient age was 11.6 years, with a 3–4.5:1 male:female ratio. Patients who underwent Ravitch correction tended to be younger than patients who underwent the Nuss procedure  for the Nuss, but not for the Ravitch, procedure. At 1 year after treatment, FEV1 recovery was better in patients who underwent the Ravitch than the Nuss procedure. However, 3 years after surgical treatment and bar removal, FEV1 changes favored the Nuss procedure , with no significant heterogeneity among studies. For the Ravitch procedure, however, there was evidence of statistically significant heterogeneity among studies , but not for the Ravitch, procedure. Although FVC decreased within 1 year after surgical correction of PE using both minimal and open techniques, greater FVC improvement occurred 3 years after the Nuss  than after the Ravitch  procedure and bar removal, and there was no evidence of statistically significant heterogeneity for either technique , but not for the Ravitch, procedure. Three years after surgery and bar removal, however, VC increased significantly, with postoperative results better for the Nuss  than for the Ravitch procedure  procedure, and no evidence of statistically significant heterogeneity among studies for either technique . Only one study reported no variation in TLC within 1 year after the Ravitch procedure. Three years after surgery, TLC improved after the Nuss procedure, showed better postoperative results  than the Ravitch procedure , with no evidence of statistically significant heterogeneity among these studies (FigureAs shown in FigurePectus excavatum is a common chest wall deformity, diagnosed shortly after birth and progressing through adolescence. The defect is often regarded as cosmetic over a long period, primarily because very young patients are often asymptomatic, with symptoms not observed until teenage and later ages. SymptomBy contrast, pulmonary function improvement after surgical treatment of PE has not yet gained widespread acceptance. Few studies have compared surgically treated and untreated patients with PE, and many studies have used patients as their own controls. We therefore analyzed whether pulmonary functional recovery occurred after the surgical correction of PE, and whether this recovery differed by type of procedure performed or by time after surgery.Although PE patients with mild sternal depression seem unlikely to benefit greatly from surgical correction, partly because the intrathoracic volume might not be significantly reduced, it is uWe also assessed whether pulmonary functional recovery was dependent on type of surgical procedure. We found that forced expiratory volume, vital capacity, and total lung capacity did not increase greatly after either the Nuss or Ravitch procedure but before bar removal, whereas significant improvements in lung function were observed after bar removal. However, better long-term ventilatory outcomes were observed after Nuss correction. Specially, we observed slight differences in pre- and post-operative pulmonary function in patients with mild-to-moderate PE. In contWe observed recurrence of sternal depression after surgical correction during the pubertal growth spurt, suggesting that costal cartilage resection may be unnecessary during surgical repair of prepubertal patients. Unfortunately, we were unable to find studies with similar findings for a meta-analysis. Whereas the extraction force in adolescents is about 175 N, the average force in adults was greater than 200 N, making it impossible to elevate the sternum to the desired level in adults without surgical mobilization. Only about 50% of this tension can be eliminated by costal chondrotomy. This may contribute to the recurrence of PE after correction, suggesting a need for costal cartilage resection,41.Patient age at the time of surgery is important for both speed of recovery and long-term results. Before One limitation of our data was our inclusion of only surgical candidates, who are by definition more likely to have at least moderately restricted chest wall function. Thus, our results may not be generalizable to all patients with PE. Our findings were also limited by the absence of uniformity in recording pulmonary function tests, suggesting the need for objective pulmonary function tests that would be less affected by patient subjectivity.Although the Ravitch and Nuss procedures for the correction of PE tended to show the same improvements in pulmonary function within 1 year, the Nuss procedure was associated with greater improvements in pulmonary function after bar removal. There is a need for further large-scale prospective trials and meta-analyses, based on the homogeneity of assessment parameters, to determine the principles of PE regression or recurrence over time, and better assess the extent of the beneficial effects of PE correction on pulmonary function.CI: Confidence interval; FEV1: Forced expiratory volume over 1 second; FVC: Forced vital capacity; PE: Pectus excavatum; NA: Nonapplicable; NR: Non reported; Postop: Postoperative; Preop: Preoperative; PSI: Pectus severity index; VC: Vital capacity; TLC: Total lung capacity; WMD: Weighted mean difference.Drs. Zhenguang Chen, Honghe Luo, Amos Ela Bella, Chunhua Su, Beilong Zhong, Jianyong Zou, and Yiyan Lei have no competing interest or financial ties to disclose.ZC and EBA conceived the study, performed papers collection and statistical analysis of the paper. HL participated in the manuscript preparation and coordinating of the study. CS and BZ participated in the statistical analysis of all data. JZ and YL participated in the drafting of the manuscript. All authors have read and approved the final version of the manuscript.