After 28 days, ALT levels were highest in the rats treated with ketoconazole followed by itraconazole, fluconazole, griseofulvin, and terbinafine, respectively. The AST levels were highest in the rats treated with ketoconazole followed by itraconazole, fluconazole, terbinafine, and griseofulvin, respectively. All drugs significantly elevated ALP levels after 14 days and 28 days of treatment (p < 0.0001). The liver enzyme levels suggested that ketoconazole had the highest risk in causing liver injury followed by itraconazole, fluconazole, terbinafine, and griseofulvin. However, histopathological changes revealed that fluconazole was the most hepatotoxic, followed by ketoconazole, itraconazole, terbinafine, and griseofulvin, respectively. Given the poor correlation between liver enzymes and the extent of liver injury, it is important to confirm liver injury through histological examination.Oral ketoconazole was recently the subject of regulatory safety warnings because of its association with increased risk of inducing hepatic injury. However, the relative hepatotoxicity of antifungal agents has not been clearly established. The aim of this study was to compare the hepatotoxicity induced by five commonly prescribed oral antifungal agents. Rats were treated with therapeutic oral doses of griseofulvin, fluconazole, itraconazole, ketoconazole, and terbinafine. After 14 days, only ketoconazole had significantly higher ALT levels ( In 2013, the United States Food and Drug Administration (US FDA) and the European Medicines Agency's Committee on Medical Products for Human Use (EMA-CHMP) concurrently issued safety warnings and limited the use of oral ketoconazole because of its association with increased risk of inducing hepatic injury, risk of drug interactions, and increased risk of adrenal insufficiency –6.The link between ketoconazole and hepatotoxicity is well established –10. HoweAlthough the hepatotoxicity of antifungal agents is well established , 13–19, Based on the currently available evidence, it is uncertain which antifungal agent poses the greatest risk of hepatotoxicity. The small number of cases in the two epidemiological studies that reported on the relative hepatotoxicity of antifungal agents limits the interpretation of their findings , 22. TheAll biochemical kits for alanine aminotransferase (ALT), aspartate amino transferases (AST), and alkaline phosphatase (ALP) were sourced from Beckman Coulter Inc. . Terbinafine , itraconazole , griseofulvin , fluconazole , and ketoconazole  were all sourced from a local pharmaceutical wholesaler. Standard diet pellets were obtained from National Foods Pvt. Ltd., Zimbabwe. Formaldehyde (37% solution), paraffin wax, haematoxylin, eosin, and other standard laboratory chemicals were sourced from Sigma-Aldrich (United Kingdom). Blood collection tubes (Vacuette® Z serum clot activator tubes) were sourced from Greiner Bio-One (United Kingdom). Microcentrifuge tubes  were sourced from Alpha Laboratories .Sixty-six 6-week-old male Sprague Dawley rats weighing 180–200 g were adapted to laboratory conditions for five days before experimentation. The rats were housed in plastic cages in groups of six with wood shavings as bedding under a 12-hour light/12-hour dark cycle. The rats were maintained in a conventional animal house with an ambient temperature of 25 ± 2°C and were given commercial standard diet rat pellets and tap water ad libitum. Ethical clearance to conduct the study was obtained from the Joint Parirenyatwa Hospital and College of Health Sciences Research Ethics Committee . The animals were handled and treated following the principles outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 Rev. 1985).The rats were divided into eleven groups (each group with six rats) including the control group. The rats in the control group were sacrificed one day before drug administration (day 0) in the active treatment groups. Five groups of rats received a daily single oral antifungal agent dose for 14 days. The other five groups of rats received a daily single oral antifungal agent dose for 28 days. The intragastric method  was used during drug administration. The treatment interventions were 20 mg/kg fluconazole, 50 mg/kg griseofulvin, 20 mg/kg ketoconazole, 20 mg/kg itraconazole, and 25 mg/kg terbinafine. Antifungal agents are frequently prescribed for two weeks or four weeks for most systemic and topical fungal infections. The equivalent doses in rats for common adult dose ranges for systemic and topical infections for the antifungal agents are as follows: fluconazole (10–20 mg/kg), griseofulvin (25–50 mg/kg), ketoconazole (10–20 mg/kg), itraconazole (10–40 mg/kg), and terbinafine (10–25 mg/kg). A suspension of each drug in distilled water was prepared two hours prior to administration. The doses were calculated using the formula provided by the US FDA  .Blood samples were collected on three different occasions on days 0, 15, and 29, using the cardiac puncture method. Day 0 was the day before initial drug administration. Blood samples collected from rats sacrificed on day 0 were for the determination of baseline liver enzyme levels in the clan of the rats. Day 15 was defined as the day after the rats had completed 14-day courses of drug treatment, that is, 24 hours after last dose. Day 29 was defined as the day after the rats had completed 28-day courses of treatment. Blood samples (4.0 ml) were collected using red top (black ring) vacutainers  and were left to clot for 30 minutes before being spun down. Blood samples were spun down at 1200 rpm for 10 minutes and the serum transferred to 1.5 ml plastic microcentrifuge tubes . The serum was then stored at −18°C up to the day of analysis (at most 7 days). The Beckman AU680® chemistry analyser was used to determine plasma levels of ALP, AST, and ALT. Aspartate transaminase (AST) and alanine transaminase (ALT) activity in serum were assayed using a procedure based on the method developed by Wróblewski and Ladue –29. Alkaµm thickness, mounted on clean glass slides coated with Mayer's egg albumin, and were stained with hematoxylin and eosin (H&E). Liver sections containing the central venule were used to make comparisons across treatment groups. Light microscopy (Motic BA210®) was used to generate photomicrographs during histological examinations. All histologic examinations were performed by the same histologist (DN).All the animals were sacrificed humanly under chloroform anaesthesia at the end of each treatment period. Livers were removed and fixed in 10% formalin for 24 hours. The whole liver tissue samples were then put in an automated tissue processer Leica TP10202 for 24 hours for dehydration using alcohol and clearing using xylol. The liver samples were then embedded in paraffin wax and cut into sections of 5 t-test. Kruskal-Wallis test and Mann–Whitney test were used as appropriate whenever the normality assumption was violated. The significance level was set at α = 0.05. Statistical analysis was carried out using Graph Pad® Prism Version 6.0 for Windows .Data are presented as the mean value ± standard error of the mean (SEM). Comparisons among multiple groups were done using one way ANOVA followed Tukey's post hoc test as appropriate. Two group comparisons were done using Student's p = 0.0017) and AST levels (p = 0.0008) than the control group. The ALT and AST levels were highest in rats treated with ketoconazole, followed by fluconazole, itraconazole, terbinafine, and griseofulvin. ALT levels in rats treated with ketoconazole were significantly higher than the levels in rats treated with griseofulvin (p = 0.0066) and terbinafine (p = 0.0074). Similarly, AST levels in rats treated with ketoconazole were significantly higher than the levels in rats treated with griseofulvin (p = 0.0076) and terbinafine (p = 0.0109). However, no significant differences in ALT and AST levels were observed between ketoconazole and fluconazole and between ketoconazole and itraconazole (p > 0.05). All drugs had significantly higher ALP levels than the control group after 14 days of treatment (p < 0.0001). Rats treated with terbinafine had the highest ALP levels, followed by those treated with itraconazole, fluconazole, griseofulvin, and ketoconazole, respectively. After 14 days of treatment, only ketoconazole had significantly higher ALT levels (p < 0.001). The AST levels were highest in the rats treated with ketoconazole followed by itraconazole, fluconazole, terbinafine, and griseofulvin, respectively. All drugs, except terbinafine, had significantly higher ALT levels compared to the control group after 28 days of treatment (p < 0.05). The ALT levels were highest in the rats treated with ketoconazole followed by itraconazole, fluconazole, griseofulvin, and terbinafine, respectively. All drugs had significantly higher ALP levels than the control group after 28 days of treatment (p < 0.001). Fluconazole caused the highest ALP levels, followed by itraconazole, terbinafine, ketoconazole, and griseofulvin, respectively. After 28 days of treatment, all the drugs had significantly higher AST levels compared to the control group (p = 0.0071). Similarly, treatment with itraconazole (p = 0.0226), terbinafine (p = 0.0434), and griseofulvin (p = 0.0190) for 28 days resulted in higher ALT levels than treatment for 14-day courses, respectively. Furthermore, there were significant duration-dependent elevations in ALP levels after treatment with fluconazole (p = 0.0009), ketoconazole (p = 0.0004), itraconazole (p = 0.0009), and terbinafine (p = 0.0300) for 28 days compared with treatments for 14 days. AST levels after 28 days of treatment with itraconazole were significantly higher than the levels after 14 days of treatment  while griseofulvin and terbinafine appear to have predominantly resulted in cholestatic injury. Azole antifungal agents have been reported to cause both hepatocellular and cholestatic injury . The incBased on liver enzyme levels observed in this study, ketoconazole had the highest risk in causing liver injury followed by itraconazole, fluconazole, terbinafine, and griseofulvin. The relative hepatotoxicity of antifungal agents based on liver enzymes is consistent with several studies. The higher ALT levels during treatment with ketoconazole than during treatment with fluconazole observed in this study is consistent with an in vitro study that reported that ketoconazole significantly increased the levels of ALT and lactate dehydrogenase (LDH) in cultured rat hepatocytes while fluconazole had minimal effects on both biomarkers . HepatotThe observation that fluconazole causes more hepatic damage than ketoconazole based on histological examinations is not consistent with an in vitro study which reported that ketoconazole caused more hepatotoxicity than fluconazole in cultured rat hepatocytes (Rodriguez and Acosta Jr. 1995) . AnotherThe observation during histological examination that fluconazole is more hepatotoxic than ketoconazole and itraconazole is consistent with the results from a large population-based study of 90,847 users of antifungal agents in Taiwan . In thisIn the present study, biochemical assays revealed that ketoconazole was the most hepatotoxic antifungal agent while histological examinations indicated that fluconazole was the most hepatotoxic. Similarly, a population-based study that used biochemical assays as the only diagnostic tool reported that ketoconazole was the most hepatotoxic while the study that used a combination of biochemical assays, biopsy, and tissue pathology reported that fluconazole was the most hepatotoxic , 22. TheLiver enzyme levels suggested that ketoconazole is likely to cause liver injury than fluconazole while histopathological examinations revealed that fluconazole is more hepatotoxic than ketoconazole. The diagnostic criteria used in the evaluation of hepatotoxicity of antifungal agents should be taken into consideration when reviewing the evidence on their relative hepatotoxicity. Given the poor correlation between liver enzymes and the extent of liver injury, it is important to confirm liver injury through histological examination before a diagnosis of hepatotoxicity can be made in clinical settings.


Dexmedetomidine has been shown to confer direct renoprotection by stabilizing the sympathetic system, exerting anti-inflammatory effects and attenuating ischemia/reperfusion (I/R) injury in preclinical studies. Results from clinical trials of dexmedetomidine on acute kidney injury (AKI) following adult cardiac surgery are controversial.We searched EMBASE, PubMed, and Cochrane CENTRAL databases for randomized controlled trials (RCTs) comparing the renal effect of dexmedetomidine versus placebo or other anesthetic drugs in adult patients undergoing cardiac surgery. The primary outcome was the incidence of AKI. The secondary outcomes were mechanical ventilation (MV) duration, intensive care unit (ICU) stay and hospital length of stay(LOS), and postoperative mortality .P = 0.02; I2 = 0.0%], and there was no difference between groups in postoperative mortality , MV duration , ICU stay , and hospital LOS .Ten trials with a total of 1575 study patients were selected. Compared with controls, dexmedetomidine significantly reduced the incidence of postoperative AKI [68/788 vs 97/787; odds ratio(OR), 0.65; 95% confidence interval (CI), 0.45–0.92; Perioperative administration of dexmedetomidine in adult patients undergoing cardiac surgery may reduce the incidence of postoperative AKI. Future trials are needed to determine the dose and timing of dexmedetomidine in improving outcomes, especially in patients with decreased baseline kidney function. Acute kidney injury (AKI) following cardiac surgery is a widely recognized complication in association with high mortality risk , 2. AKI Dexmedetomidine, a highly selective α2 adrenoreceptor agonist, induces sedation, analgesia, hemodynamic stabilization, anti-inflammation, as well as diuresis , and hasThis meta-analysis was performed according to the PRISMA  guidelines  and apprThe literature review and data extraction were independently completed by 2 investigators (BS and SZW). In case of duplicate records pertaining to a single study, we considered the PubMed database to take precedence. Disagreements were handled by discussion for consensus. Quality assessment was completed using the Cochrane risk of bias tool and Jadad scale. Data extraction included patient characteristics , as well as dexmedetomidine dosage.The primary end point was incidence of AKI . Secondary outcomes included all-cause mortality , mechanical ventilation(MV) duration, ICU length of stay, and hospital length of stay(LOS).2). Publication bias was assessed by Begg’s test and Egger’s test. P < 0.05 (2 sided) was considered to be statistically significant for hypothesis testing. All statistical analyses were performed in REVMAN  and Stata .For dichotomous outcomes (reported with incidence), we calculated the odds ratio (OR) with 95% confidence interval (CI). For continuous outcomes , we calculated mean differences for each study according to the statistical method of Hozo et al.  and usedFigure For postoperative outcomes, AKI incidence was reported in 9 trials , 19–23, Study design and patient characteristics were summarized in Tables P = 0.02; I2 = 0.0%; Fig. P = 0.22; Egger’s test, P = 0.32; Fig. The outcome of AKI was reported in 1575 study participants, and the overall incidence was 10.48% . The postoperative incidence of AKI was significantly reduced by dexmedetomidine ,and the other studies were consistent with the direction and size of the overall AKI- reducing effect of dexmedetomidine .Sensitivity analysis excluding each included study at a time revealed that the Cho 2015 study was inconsistent with the direction and size of the overall AKI- reducing effect of dexmedetomidine . There were no statistically significant reduction for mortality owing to perioperative dexmedetomidine  injury , 26. In P = 0.86 and P = 0.08).To date, the optimal dose of dexmedetomidine to improve kidney function after cardiac surgery is unclear. The optimal dose of dexmedetomidine on postoperative renal events can’t be drew because of the lack of detailed patient data. Future large and well-designed randomized trails should explore the more appropriate dose of dexmedetomidine to maximize its renal protective effect with less side effects affecting prognosis.In the included trials, dexmedetomidine was used with a loading dose (0.4μg/kg-1μg/kg) and continuous infusion (0.04–0.6μg/kg/h). Balkanay enrolled adult patients undergoing CABG found a significant difference between high dose group (8 μg/kg) and low dose group (4 μg/kg) for the 24th postoperative hour in the mean values of neutrophil gelatinase-associated lipocalin (NGAL) , indicatP = 0.21).Our findings do not provide a strong guidance on this question, and it merits further investigation. Future trials in this area would most likely be of greatest benefit.The timing of dexmedetomidine administration in relation to cardiac surgery is emerging as an important consideration. In 6 of 10 included trials , 22, 23,P = 0.33). The advantages of dexmedetomidine compared with other anesthetics still call for further research.Two recent expert consensus articles on postoperative AKI have been recently published, which discussed also new possible therapies/preventive measures , 31. OurOur analysis has several disadvantages. First, AKI in cardiac surgery is common and may have several different causes. It is difficult to establish a protective role for dexmedetomidine. We were unable to access individual patient data, so the influences of age, sex, and other confounding factors may be underestimated. Second, the definition of AKI was not uniform in the included trials. Third, sample size in each study is relatively low, so future large clinical studies were needed. Fourth, the exclusion of non-English studies may be inappropriate, however, the assessment of publication bias did not show statistical significance. Fifth, Bland  and KwonIn summary, available evidence from the present meta-analysis suggests that perioperative administration of dexmedetomidine in adult cardiac surgery might reduce the incidence of AKI. Future trials are needed to be much larger and ascertain the optimal dose and, more importantly, the time of the dose, especially in patients with decreased kidney function at baseline.


The smell sense reduction was considered to represent the potentially warning of early stage of neurodegenerative disorders. The Shanghai Aging Study provided us a unique opportunity to explore the association between olfactory identification (OI) and cognitive function among community-dwelling elderly in China.OI of each participant was measured by the 12-item identification tests from Sniffin’ Sticks Screening test (SSST-12). Participants with mild cognitive impairment (MCI) were diagnosed by Petersen criteria. We used the logistic regression analysis to explore the association between OI scores and cognitive function by adjusting potential confounders.P < 0.0001]. After adjusted for age, gender, education, lifestyles, medical history, Apolipoprotein E genotype, lower OI score was found to be an independent influence factor related to MCI .Among 1782 non-demented participants, 345 (19.4 %) participants were diagnosed as MCI. The mean OI score for participants with MCI [7.1 (SD 2.3)] was significantly lower than that for those with normal cognition [8.2 (SD 2.0), Our study suggests that poor OI may be associated with MCI in elderly population. Further prospective studies may confirm the OI as a reliable and early marker predicting the decline of cognitive function. Olfactory function is an important role in health and behavior because it may be a valid marker of the integrity of the aging brain. The prevalence and severity of olfactory dysfunction increase substantially with aging. Olfactory dysfunction represents an important clinical symptom suggestive of early stage of neurodegenerative disorders, including Parkinson’s disease (PD) .A series of community-based and hospital-based studies in western population have demonstrated that in older adults, impaired olfactory function was closely associated with the decline of cognitive functions, especially Alzheimer’s disease (AD) in the preclinical stage, although this dysfunction is more likely to be due to problems of olfactory identification (OI) than detection –6. As a There was no general standard test for olfactory because of the cultural differences in diverse regions. Modern tests for OI include the Connecticut Chemosensory Clinical Research Center Test (CCCRCT), University of Pennsylvania Smell Identification Test (UPSIT), Cross-Cultural Smell Identification Test (CC-SIT), Pocket Smell Test (PST), Odorant confusion matrix, Biolfa olfactory test, Sniffin’ Sticks (SS), Smell Disketts Test, Scandinavian Odor-identification test (SOIT), and San Diego Odor Identification Test (SDOIT) in western countries . As in CChina has one of the fastest ageing in the world. According to the 2014 census, the number of people aged 60 years and older was 212 million, occupying 15.5 % of the population . With a The Shanghai Aging Study was designed to establish a prospective community-based cohort with elderly in downtown Shanghai, China. Eligible participants were registered residents aged 60 or older in Jingansi community, able to communicate and accept physical and cognitive examinations, and they were not suffered from mental retardation or schizophrenia based on their medical records. Recruitment procedures were reported elsewhere . AccordiParticipants were interviewed face-to-face by trained research nurses to obtain information on their demographic characteristics, including age, gender, education, lifestyle factors . History of chronic diseases, such as physician-diagnosed hypertension, diabetes, and heart disease (including coronary artery disease and arrhythmia), were asked and confirmed from their medical records. Each participant was examined by neurologists for motor responses and reflexes. Neurologists were assigned to administer the Zung Self-Rating Anxiety Scale (ZSAS) and the Center for Epidemiologic Studies Depression Scale (CESD) for each participant to indicate his mood episode within the past week. Anxiety and depression were determined if ZSAS >44 and CESD ≥ 16 , 20. NeuCognitive function of each participant was tested by a neuropsychological test battery, which covers domains of global cognition, executive function, spatial construction function, memory, language, and attention. The battery contained: 1) Mini Mental State Examination (MMSE); 2) Conflicting Instructions Task (Go/No Go Task); 3) Stick Test; 4) Modified Common Objects Sorting Test; 5) Auditory Verbal Learning Test; 6) Modified Fuld Object Memory Evaluation; 7)Trail-making test A&B; 8) RMB (Chinese currency) test. Normative data and detail description of these tests were reported elsewhere , 24. TheAfter each clinical and neuropsychological assessment, study neurologists and neuropsychologists  reviewed the functional, medical, neurological, psychiatric, and neuropsychological data and reached a consensus for dementia, MCI and normal cognition. DSM-IV criteria  was used“Sniffin’ Sticks” is a test of nasal chemosensory function based on felt-tip pens that was devised by G. Kobal in Erlangen, Germany. In its most elaborate version, it comprises 3 tests of olfactory function , and takes approximately 20 to 30 min for application . The SSSThe administrator of OI test was blind for the cognitive diagnosis of each participant. Before the test, participants were reminded to stay away from chewing gum, sweets or cigarettes. Testing was performed in a quiet, air-conditioned room. A brief history including questions related to the participant’s olfactory experience, previous diseases, drug intake, occupation and smoking habits were recorded. The administrator was wearing cotton gloves when presenting the odors. The opened odor sticks were positioned approximately 2 cm in front of both nostrils of each participant. Participants were then asked to sniff for no longer than 3–4 s and to choose one of four answers from a list that described best the odor. An interval of 30 s was between the different sticks.DNA was extracted from blood or saliva, collected from the study participants. Apolipoprotein (APOE) genotyping was conducted by the TaqmanSNP method . The preP-values and 95 % CIs were estimated in a two-tailed tests. Differences were considered to be statistically significant at P < 0.050. The data were analyzed using SAS 9.3 .The categorical variables were expressed as frequencies (%), and the continuous variables were expressed as the mean and standard deviation (SD). The Pearson Chi-squared test was used to compare the categorical variables. The Student t-test, analysis of variance (ANOVA) and generalized linear model (GLM) were used to compare the continuous variables. We used two multivariate logistic regression models to detect the association between OI and cognitive function, by adjusting for variables of age, gender and education (model 1) and by adjusting additional confounding variables, such as lifestyles , medical history  and APOE-ε4 allele (model 2). Risk was presented as odds ratio (OR) and 95 % confidence interval (CI). All of the p < 0.0001].From April 2010 to March 2011, the Shanghai Aging Study consequently enrolled 1808 participants who had completed both of the cognitive assessment and OI test. Twenty-six were excluded due to the diagnosis of dementia. Table P = 0.254). The median and percentile scores of OI were also showed in Table Table P trend < 0.0001). The mean OI scores of MCI group decreased more dramatically by increasing age than that of group with cognitive normal (P < 0.0001). Figure vs. MCI, 91.9 % vs. 87.3 %%, P = 0.015), orange , pineapple , and cinnamon , coffee , fish , banana , rose , leather , and liquorice .Figure The multivariate logistic model 1 indicated that, lower OI score was an independent influence factor associated with MCI , after adjusted for age, gender and education. Additionally, after adjusting for variables of age, gender, education, living alone, cigarette smoking, alcohol drinking, anxiety, depression, heart disease, hypertension, diabetes, and APOE-ε4 allele, model 2 also indicated that lower OI score was a influence factor independently associated with MCI  Table .Table 3OTo our knowledge, this is the first epidemiological study that reports the normative data of OI among community-dwelling elderly in China by using the SSST-12. The main finding of this community-based study was that, poor OI was an independent factor associated with the cognitive decline in elderly, after adjusting possible confounders. The advantages of this study include community-based design with large sample size, reliable diagnosis of cognitive function based on comprehensive clinical and neuropsychological exams, and detailed influence factor profiles (including APOE genotype) for each participant.Some small-sampled case-control studies have reported the relation between poor olfaction and AD/MCI. AD patients were more impaired on OI and recognition tasks than on odor detection thresholds task. They were also more strongly impaired on higher-order olfactory tasks involving specific cognitive processes . A studyP = 0.680); 1.95 (1.25–3.03) for Q2 (P = 0.003); and 2.18 (1.36–3.51) for Q1 (P = 0.001)  after adjustment for gender and education, with same age. The B-SIT score also predicted progression from aMCI to AD, with a significant dose-response with worsening B-SIT quartiles [Some prospective studies, mostly in western countries, demonstrated that older persons with impaired OI were more apt to experience cognitive decline than those with relatively preserved OI. The Mayo Clinic Study of Aging followed the 1430 cognitively normal participants over a mean 3.5 years, and observed an association between decreasing olfactory identification with a decreasing number of correct responses in Brief Smell Identification Test (B-SIT) score, and a higher risk of amnestic MCI (aMCI). Compared with the upper B-SIT quartile , hazard ratios (HRs) (95 % CI) were 1.12 0.65–1.92) for Q3 (5–1.92 fod 2.18 1.–3.51 foruartiles . DevananP < 0.010). It suggested that the decreased OI in MCI may be an indicator for the early diagnosis of AD, and APOE genotype may be a portion of the basis of OI decline [APOE genotype was identified as an important factor interacting with olfaction. Murphy C et al. have indicated that APOE-ε4 allele is related to OI deficits (tested by SDOIT) in 27 non-demented older persons . Olfacto decline . Our stuThere were several mechanisms or explanations for the association between olfactory dysfunction and cognition impairment. OI is more strictly involved in cognitive functions since it is processed in the mesial structures of the temporal lobe. The olfactory threshold, however, is mostly influenced by peripheral deficits of the smell sense. It has been shown that typical lesions of AD  were already detectable in the preclinical stages in central olfactory pathways . With reOur study also explored an interesting finding that, some odors, such as coffee, fish, banana, rose, leather, and liquorice were identified more significantly different between individuals with MCI and cognitive normal, than odors of lemon and cloves. It suggests that some odors might be the sensitive indicators for the early stage of cognitive decline, while some might be not. The transformational mechanism of stimulation by different odors may need more attention.Olfactory function includes threshold, discrimination, identification and olfactory recognition . In the Our study suggests that poor OI may be associated with MCI in elderly population. Follow-up assessment of our Shanghai Aging Study cohort will be carried out to confirm olfactory dysfunction may be an important predictor for cognitive impairment, as well as its pathological mechanism.


P = 0.011 in an additive model, P = 0.033 after Bonferroni's correction). The further stratified analyses showed that the link between variant genotypes of rs7853346 and decreased GC risk was more obvious in older subjects (≥60 years), nonsmokers, nondrinkers, and subjects without family history of GC. We also found that relative PTENP1 mRNA expression levels were higher in rs7853346 CG/GG genotype carriers than those with common genotype in both GC and normal tissues (P < 0.05). Besides, bioinformatics analyses revealed that rs7853346 may change the local folding structure and alter the target microRNAs (miRNAs) of PTENP1. In conclusion, our results suggested that lncRNA PTENP1 polymorphism rs7853346 may predict GC susceptibility.Long noncoding RNA (lncRNA) phosphatase and tensin homolog pseudogene 1 (PTENP1) is significantly downregulated in gastric cancer (GC), playing critical roles in GC progression. However, the association between PTENP1 genetic variants and GC risk has not yet been reported. Using TaqMan technology, three lncRNA PTENP1 tag single nucleotide polymorphisms (tagSNPs)  were genotyped in 768 GC patients and 768 cancer-free controls in a Chinese population. We found that subjects with rs7853346 G allele had a remarkably decreased risk of GC, compared with those carrying C allele ( Gastric cancer (GC) is one of the most common cancers worldwide, whose incidence rate is higher in Eastern Asia generally  . The NatLong noncoding RNAs (lncRNAs) are defined as transcripts more than 200 nucleotides in length, which are associated with a variety of biological progress through interacting with DNA, RNA, and proteins as molecular scaffolds, sponges, or activators . LncRNA Considering the role of PTENP1 in GC suppression, based on the 1000 Genomes data, we selected three tagSNPs  in lncRNA PTENP1. The genotyping assay was carried out to evaluate the influence of three tagSNPs on the risk of GC in this hospital-based, case-control study in a Chinese population.The case-control study received approval from the ethics committee of the First Affiliated Hospital of Nanjing Medical University. Written informed consents were provided by all participants. All subjects consisting of 768 GC patients and 768 cancer-free controls were consecutively recruited from the First Affiliated Hospital of Nanjing Medical University during May 2010 to December 2016. Besides, there was a portion of nonresponse patients accounting for 10.2 percent of all GC patients. The participants, who lived in Jiangsu province or surrounding areas, were genetically unrelated ethnic Han Chinese, and none received blood transfusion from nonself in the preceding six months. All cases were primary and histopathologically confirmed GC by two pathologists, and patients who had any history of malignancies (including any metastatic cancer) and received radiotherapy or chemotherapy previously were eliminated from this study. All controls were randomly selected from vascular surgery or orthopedics department of the abovementioned hospital during the same period, none had self-reported history of malignancies or gastric polyp. We collected relevant information, such as age, gender, hypertension, diabetes, smoking history, drinking history, residence, family history of GC (FH), and clinical data from the medical history, or face-to-face interviews were conducted to supplement the above information. The definitions of hypertension and diabetes were described in the previous studies , 6. The GC tissues and corresponding adjacent tissues were obtained from a total of 70 patients, who underwent surgery at the First Affiliated Hospital of Nanjing Medical University during 2010 to 2014. The resected samples then were frozen with liquid nitrogen immediately and stored at −80°C for RNA extraction. The diagnosis of all patients was histopathologically confirmed by two pathologists.http://asia.ensembl.org/index.html). The pairwise option of Haploview version 4.2 software , setting r2 > 0.8 [http://gvs.gs.washington.edu/GVS144/).Based on Chinese Han in Beijing population data from the 1000 Genomes Project (Ensembl GRCh37 release 87—Dec 2016), we selected tagSNPs of PTENP1 in chromosome 9, position 33673502-33677497 . The 10 μl PCR reaction volume included 1 μl DNA template, 5 μl 2× HotTaq PCR Reaction Mix, 0.5 μl primer, 0.25 μl probe, and 2.5 μl double distilled water. The sequences of the probes and primers were showed in Supplementary Table S1 available online at https://doi.org/10.1155/2017/6807452 . Amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The call rates were more than 98% for all SNPs. Over 10% of samples were selected randomly for repeated assays, and the results were in line with the previous genotypes.Genomic DNA was extracted from the leukocytes of 2 ml peripheral blood of the subjects using RelaxGene Blood DNA System kit , following the manufacturer's directions. The NanoDrop Spectrophotometer (ND2000) was conducted to measure the concentration and purity of all DNA specimens, which were diluted to a concentration of 10 ng/β-Actin  was selected as the endogenous control to standardize data. The 10 μl amplification reaction volume contained 5 μl SYBR Green Master Mix , 0.2 μl primer, and 100 ng cDNA. RT-PCR was carried out with the condition: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.Total RNA was extracted from 70 paired GC and adjacent normal tissues using TRIzol reagent , which then was reverse transcribed into cDNA with Primescript RT Reagent . Real-time reverse transcription PCR (RT-PCR) was conducted to evaluate relative PTENP1 expression with ABI StepOnePlus real-time PCR system . All procedures were performed in triplicate. The PTENP1 primers were as follows: forward 5′-TCAGAACATGGCATACACCAA-3′; reverse 5′-TGATGACGTCCGATTTTTCA-3′. P values were two-sided, and the data were considered to be statistically significant when P < 0.05. The goodness-of-χ2 test was conducted to assess the Hardy-Weinberg equilibrium among cancer-free subjects. Differences in demographic data between cases and controls were calculated by the χ2 tests  or Student's t-tests (for continuous variables). The risk caused by genetic variants was assessed with odds ratios (OR) and 95% confidence intervals (CI). Crude ORs were computed by the unconditional univariate analysis and adjusted OR, was evaluated by unconditional logistic regression method with age, gender, smoking condition, drinking condition, hypertension, diabetes, residence, and FH adjusted. The results were corrected with the Bonferroni method conservatively for multiple comparisons in association analyses. The stratification analysis was performed under the dominant model, due to the small sample size of GG genotype group for rs7853346. We carried out the stratified analysis to exclude impacts of the potential confounders on GC risk in the logistic regression model. Moreover, we can concretely assess the magnitude of the interested association in every two strata for all subgroups and calculate the heterogeneity to know whether rs7853346 exists interaction with these stratified factors. The χ2-based Q-test and I2 by Stata 14.0 software were used to detect the heterogeneity from corresponding subgroups [−ΔCT method normalized to the levels of β-actin. The one-way ANOVA test was adopted to evaluate the associations between the expression levels of PTENP1 mRNA and PTENP1 polymorphisms. The Power and Sample Size Calculation software  was used to calculate the statistical power.The SPSS 22.0 software  was used for statistical analyses. All ubgroups , 21. TheP > 0.05). However, the percentage of smokers was apparently higher among GC group compared with controls , so was that of drinkers . The Supplementary Table S2 summarized the characteristics of 87 nonresponse GC patients. Compared with included 768 GC cases, the proportion of rural population in nonresponse GC patients was larger .The characteristics of 768 GC patients and 768 cancer-free controls were detailed in P = 0.900 for rs7853346, P = 0.098 for rs865005, and P = 0.342 for rs10971638). For rs7853346, the frequency of G allele of GC cases was significantly lower than that of the controls . In the additive model, logistic regression analysis indicated that rs7853346 variant genotypes significantly decreased GC risk . In codominant and dominant models, the variant CG and CG+GG genotypes were significantly associated with the decreased risk of GC. After Bonferroni's multiple adjustment applied to our results, we found that the variant genotypes of rs7853346 were still obviously correlated to decreased GC risk in additive and dominant models . With the increasing number of G alleles, the proportion of controls increased . Based on the fact that the tumor differentiation of 78% GC patients was poor, we took the well + moderate differentiation group as a sensitivity analysis to evaluate the association between rs7853346 and GC risk. As shown in Supplementary Table S4, no impacts of rs7853346 variant genotypes on the susceptibility to GC risk were observed. Using the PS software, given PTENP1 rs7853346 C>G mutant alleles in the control group, OR, GC samples, and control sample sizes, the power of our analysis (α = 0.017) was 0.708 in 768 GC cases and 768 controls with unadjusted OR = 0.69.The genotype and allele frequencies of PTENP1 tagSNPs in cases and controls as well as their associations with GC risk were calculated by the following five methods  (P > 0.05).However, no significant associations were detected between the genotypes of the other two PTENP1 tagSNPs  and GC risk , nonsmokers , nondrinkers , and non-FH . However, no significant association was identified between rs7853346 and GC risk in terms of sex or residence. The differences in every two strata of all stratified factors were not statistically significant , which showed that these stratified factors  had no multiplicative interaction with rs7853346.To eliminate influences of the potential confounders on GC risk, the stratified analysis was conducted for rs7853346 according to age (60 years), gender, smoking status, drinking status, residence, and FH. All results were obtained under the dominant model and summarized in P = 0.048, adjusted OR = 0.70, 95% CI = 0.49–1.00). Nonetheless, no distinct association was found between rs7853346 and other clinicopathological characteristics, such as tumor infiltration depth, lymph node metastasis, and localization.Next, the subgroup analysis was conducted to evaluate the correlations between the variant genotypes of rs7853346 and clinicopathological features in GC patients . We founThe RNAfold algorithm was performed to analyze the impact of rs7853346 on the local secondary structure of PTENP1. As shown in Supplementary Figure S1, the folding structure of PTENP1 was notably changed with rs7853346 C/G alleles.Given the fact that rs7853346 C>G is located in the 3′ untranslated region (UTR) of PTENP1 gene, we applied the RegRNA2.0 program to predict that rs7853346 C>G may create hsa-miR-1245b-3p, hsa-miR-4679 binding sites on PTENP1.P = 0.019, 0.007, and 0.002, resp.; adjacent normal tissues: P = 0.032, 0.025, and 0.007, resp.) (P = 0.021 and 0.006). In adjacent normal tissues, only the PTENP1 expression levels of those with CG+GG genotypes were higher than CC genotype (corrected P = 0.021).We then evaluated the effects of the rs7853346 genotypes on PTENP1 levels by qRT-PCR in 70 paired GC and adjacent normal samples including 43 CC, 20 CG, and 7 GG genotypes. The results showed that the relative PTENP1 mRNA expression levels (mean ± standard error) were significantly higher for the CG, GG, or CG+GG genotypes than for the CC genotype in both GC and adjacent normal tissues  . After BIn the case-control study, the associations between lncRNA PTENP1 polymorphisms  and the risk of GC were firstly evaluated in Chinese Han population. Our results showed that variant genotypes (CG/GG) of rs7853346 were associated with a prominent reduced risk of GC. Moreover, PTENP1 expression levels in individuals carrying rs7853346 CG/GG genotypes were higher than CC genotype in both GC and adjacent normal tissues. The statistical power calculated by using the PS Software was 0.708 and was relatively small.LncRNAs are a class of noncoding RNAs, and increasing evidences have demonstrated that aberrant expression of lncRNAs could result in the occurrence and development of various malignancies , 23. ForSubsequently, our stratified analysis by age showed that the decreased GC risk related to rs7853346 CG/GG genotypes was more pronounced in older subjects (age ≥ 60 years), indicating that protective effects of PTENP1 variants may be regulated by specific epidemiological features . Furtherhttp://rna.tbi.univie.ac.at/) [http://regrna2.mbc.nctu.edu.tw/detection.html) [How the polymorphism rs7853346 in PTENP1 could influence its function was yet unclear. Considering that a single base conversion may change the RNA local secondary structure, we used the RNAfold (.ac.at/)  algorith.ac.at/) , 30. How.ac.at/) , 32. BasSeveral limitations should be considered. Firstly, the sample size is relatively small, especially for stratified analyses , limiting the statistical power. We will expand the quantity of sample to make statistical power satisfy stricter criterion in future studies. And when evaluating the influences rs7853346 on PTENP1 expression, the sample size of GG group was relatively small, which could not guarantee enough statistical power. Secondly, some controls recruited from a single hospital during the same period suffered from various nonmalignant diseases, which may cause selection bias. However, the distribution of genotype among controls met the Hardy-Weinberg equilibrium. Also, in the case group, GC patients with poor differentiation accounted for 78%. After taking the well + moderate differentiation group as a sensitivity analysis, we did not find the relationship of rs7853346 with GC risk. The data suggested that the marginally significant results in this study might attribute to this selection bias or residual confounding . Thirdly, given the ethical issues, we did not carry out a Helicobacter pylori test for each subject, particularly in controls. Fourthly, we failed to perform the further analyses because of missing relevant clinical data, such as a high-salt diet, chronic gastric ulcer, and BMI, which are also risk factors in gastric carcinogenesis. Finally, our study was conducted in a Chinese population, so the data should be extrapolated to other ethnic groups with caution. Our study also had its own advantages. Firstly, the selection of tagSNPs in PTENP1 was based on the same analysis results of 1000 Genomes Project and Genome Variation Server 144. Secondarily, about analytic strategy, stratified analyses were used to eliminate influences of the potential confounders on GC risk, and Bonferroni's multiple adjustment was conducted to decrease the type I error. Thirdly, the bioinformatics analyses in the present study were therefore more reliable and convictive in the aspect of understanding the potential function of rs7853346 in PTENP1.Taken together, the study firstly identified that a potentially functional lncRNA PTENP1 tagSNP (rs7853346 C>G) reduced the susceptibility to GC in a Chinese population. However, further prospective studies with larger sample size are needed to validate the initial findings. In addition, in view of the fact that the difference of PTENP1 expression levels caused by different genotypes was much smaller than that between GC tissues and adjacent normal tissues, so more functional researches should be carried out to illuminate whether rs7853346 decreases gastric cancer risk by altering the expression levels of lncRNA PTENP1 or other specific mechanism.Supplementary Figture S1. Prediction of the effects of the rs7853346 C>G change on PTENP1 folding structure. The folding structure alterations  were demonstrated by RNAfold (http://rna.tbi.univie.ac.at/). Supplementary Table S1: The detailed sequences of primers and probes for tag SNPs. Supplementary Table S2: Demographic information. Supplementary Table S3: Association between PTENP1 gene polymorphisms and risk of gastric cancer (rs865005 and rs10971638). Supplementary Table S4: Association between PTENP1 gene polymorphisms and risk of gastric cancer based on the well+moderate differentiation group (rs7853346).


Grading of essential hypertension according to blood pressure (BP) level may not adequately reflect clinical heterogeneity of hypertensive patients. This study was carried out to explore clinical phenotypes in essential hypertensive patients using cluster analysis. This study recruited 513 hypertensive patients and evaluated BP variations with ambulatory blood pressure monitoring. Four distinct hypertension groups were identified using cluster analysis: (1) younger male smokers with relatively high BP had the most severe carotid plaque thickness but no coronary artery disease (CAD); (2) older women with relatively low diastolic BP had more diabetes; (3) non-smokers with a low systolic BP level had neither diabetes nor CAD; (4) hypertensive patients with BP reverse dipping were most likely to have CAD but had least severe carotid plaque thickness. In binary logistic analysis, reverse dipping was significantly associated with prevalence of CAD. Cluster analysis was shown to be a feasible approach for investigating the heterogeneity of essential hypertension in clinical studies. BP reverse dipping might be valuable for prediction of CAD in hypertensive patients when compared with carotid plaque thickness. However, large-scale prospective trials with more information of plaque morphology are necessary to further compare the predicative power between BP dipping pattern and carotid plaque. Hypertension is the most prevalent treatable risk factor, accounting for about 50% of cardiovascular events4In addition to BP variability and other clinical risk factors for CAD, carotid plaque load has also been widely used to evaluate the risk and prognosis of cardiovascular disease. Both carotid plaque and intima-media thickness are different measures to assess preclinical arterial pathology, but total plaque area appeared to be a stronger predictor than intima-media thickness for first-ever ischemic stroke6Several reports have suggested that cluster analysis could lead to improved characterization of a disease phenotype11A total of 513 patients, including 278 men and 235 women, were enrolled in this study . The aveAll study participants met common diagnostic criteria for essential hypertension. Nonetheless, a phenotype heat map was created with cluster analysis and demonstrated substantial heterogeneity among all subjects . Then weCluster analysis identified four groups that were significantly different from each other. Clinical and laboratory characteristics were stratified according to phenogroup . Key chaCluster 1 (n = 172) was the largest, with more than a quarter of all subjects in our study. The patients were smokers and tended to be younger men (average age 59 years) (77.90%). These patients had the lowest level of high density lipoprotein , while the 24-h systolic BP (SBP) (137.31 mmHg), 24-h diastolic BP (DBP) (81.62 mmHg), daytime SBP (139.05 mmHg), daytime DBP (83.33 mmHg), night-time SBP (131.28 mmHg), and night-time DBP (76.69 mmHg) were the highest among the four clusters. These patients were the most likely to have a non-dipper pattern.Cluster 2 (n = 70) had the lowest number of patients and were primarily older women (average age 64 years) (70.00%). All patients in this cluster were likely to have diabetes mellitus; the levels of fasting glucose (6.72 mmol/L) and total cholesterol (5.03 mmol/L) were the highest. Furthermore, cluster 2 patients had the lowest 24-h DBP (75.34 mmHg), daytime DBP (76.60 mmHg) and night-time DBP (70.47 mmHg). No patients in cluster 1 or 2 had CAD.Cluster 3 (n = 144) had no smokers or diabetics, and the level of fasting glucose (4.77 mmol/L) was the lowest. Compared with other three clusters, more patients tended to have normal thickness of carotid artery walls (48.61%). Moreover, in this cluster, 24-h SBP (131.84 mmHg), daytime SBP (134.02 mmHg) and night-time SBP (123.80 mmHg) were the lowest. Compared with cluster 1, cluster 3 had the most patients with a dipper pattern (40.97%).Cluster 4 (n = 127) was the least likely to have severe carotid narrowing (3.15%). Importantly, all of the hypertensive patients in this cluster also had CAD, while patients in the other three clusters had no CAD. The level of total cholesterol (4.47 mmol/L) was the lowest and the level of HDL was the highest (1.32 mmol/L). In addition, cluster 4 had the most patients with a reverse dipper pattern (28.34%) and the rates of decline for both SBP (3.75%) and DBP (6.39%) were the lowest, compared with the other three clusters.To further investigate the risk of CAD in the current patient population, binary logistic regression was carried out after cluster analysis. Multivariate models included variables of age, carotid plaque thickness, sex, smoking, diabetes, CAD, total cholesterol, HDL and BP pattern. For all subjects, carotid plaque , BP pattern , male , smoking , and HDL  were associated with CAD .Carotid plaque thickness  and total cholesterol  were associated with the risk of CAD in cluster 4 and cluster 1. For cluster 4 and cluster 2, male  and total cholesterol  were significantly correlated with CAD. BP pattern  was the only common risk factor significantly correlated with CAD when comparing cluster 4 with cluster 3 .Machine learning approaches are typically subdivided into two categories: supervised and unsupervised learning. Supervised learning seeks to classify or predict specified outputs or outcomes. In contrast, unsupervised learning analyzes the intrinsic structure within data, such as groupingOnce the four clusters were identified, the differences between them were striking. Study participants within the four clusters, despite having shared diagnostic features of hypertension, differed markedly on almost every characteristic. In cluster 1, most were younger male smokers, and had the most severe carotid narrowing and relatively high BP; however, none had CAD. Surprisingly, all patients in cluster 4 had CAD, but the least severe carotid narrowing and the most prominent reverse BP dipping pattern. To further investigate the potential significance of reverse dipping pattern, we studied the risk factors for CAD in hypertensive patients, and identified BP pattern as a risk factor. In particular, when comparing cluster 4 with cluster 3, the latter of which was a relatively healthy group, a reverse BP pattern was the only prominent risk factor for CAD. Moreover, carotid narrowing seemed not contribute to the risk of CAD in the results of binary logistic regression. This may be because only carotid plaque thickness were considered, while other parameters of carotid plaque, such as carotid plaque area or three-dimensional plaque characteristicsBased on the differences in BP levels between day and night, circadian BP variations were used to divide patients into dippers  and non-dippers Assessment of carotid artery plaque with ultrasound examination is a straightforward, inexpensive, and noninvasive method for evaluation of cardiovascular risk7In conclusion, our findings demonstrated the heterogeneity of hypertension with cluster analysis and clearly showed that every subgroup had its own characteristics. Additionally, reverse dipper pattern hypertension was found to contribute to the risk of CAD. However, our study was carried out in a Northern Chinese population with essential hypertension in a single center. Due to the cross-sectional design, prospective clinical observation may also facilitate better understanding of the underlying mechanism.Patients with essential hypertension were recruited continuously from January 2012 to June 2014. Data were extracted from our entire in-patient ABPM service database. Hypertension was diagnosed as systolic BP (SBP) ≥140 mm Hg and/or diastolic BP (DBP) ≥90 mm Hg in casual office recording, or daytime (or awake) SBP ≥ 135 mmHg and/or DBP ≥ 85 mm Hg, or night-time (or asleep) SBP ≥ 120 mm Hg and/or DBP ≥ 70 mm Hg in ABPMAll hypertensive patients were evaluated with 24-hour ABPM using an oscillometric device . The monitor was installed on the non-dominant arm between 7:00 and 9:00AM and removed 24 hours later. Frequency of recordings was made at every 15 minutes from 7:00AM to 11:00PM  and every 30 minutes from 11:00PM to 7:00AM . Strenuous physical activity was discouraged for all patients during the monitoring period. Recordings were accepted only if more than 70% of the raw data was valid. All ABPM recordings were manually edited by 2 individual physicians unaware of the objective and risk factors. Values of SBP < 70 or >250 mm Hg, DBP < 40 or >150 mm Hg, and heart rate < 40 or >150 beats per minute were excluded from the recording. The percent change in nocturnal BP decline was calculated as follows: /mean diurnal BP × 100%. According to the BP pattern, patients were divided into three groups: dippers (average SBP decreased 10% to 20% of daytime level during sleep), non-dippers  and reverse dippers We used the WEKA machine learning toolkit  for cluster analysis. K-means, one of the widely adopted clustering algorithms, was carried out to find structure in data and divide patients into different groups. Principal component analysis, mapping high-dimension data into low-dimension space, was used to reduce the primal data into two dimensions. As is necessary for clustering analysis, the missing values were globally replaced with mean values. The main steps in the K-means algorithm are as follows: (1) Select initial cluster centres with the number of K. Then, repeat steps 2 and 3 until cluster membership stabilizes. (2) Assign each point to its closest cluster centre. (3) Compute new cluster centres. In step 1, K points are selected randomly as initial cluster centres. In step 2, when we assign each point to its closest cluster centre, we need to compute the distance, typically the Euclidean distance, between points and centres. In step 3, the new cluster centres are computed as the means of all points belonging to each cluster.P < 0.05 was considered to be statistically significant.All statistical analyses were performed using the SPSS software package version 18.0 . Adequacy of all parameters to normal distribution, was tested by using Kolmogorov–Smirnov Test. Parametric tests were applied to data with normal distribution, while non-parametric tests were used to data without normal distribution. Descriptive statistics are presented as percentages for discrete variables and mean ± SD for continuous normally distributed variables. Chi-squared test and analysis of variance (ANOVA) were performed to check the significance of binary and continuous variables respectively between different clusters. Then, binary logistic regression was carried out to investigate the association between CAD and other risk factors. A calculated difference of All of the examinations were carried out for diagnosis of hypertension and assessment of cardiovascular risk in adults in according to guidelines from the European Society of CardiologyHow to cite this article: Guo, Q. et al. Cluster analysis: a new approach for identification of underlying risk factors for coronary artery disease in essential hypertensive patients. Sci. Rep.7, 43965; doi: 10.1038/srep43965 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To evaluate the joint effect of hypertension (HTN) and diabetes (DM) on coronary heart disease (CHD), and stroke event, all-cause, and cardiovascular disease (CVD) mortality in Middle Eastern older adults, 2747 people (1436 women) aged ≥ 50 years, free of CVD at baseline, were categorized into four groups . Multivariate Cox proportional hazard models were run for different outcomes. To compare the impact of HTN versus DM, HTN+/DM− was considered as reference. In a median of 13.9 years, incidence rate of CHD, and stroke event, all-cause and CVD mortality in total population were 19.0, 4.7, 13.5, and 6.4 per 1000 person-years, respectively. The multivariate sex-adjusted hazard ratios (HRs) of HTN−/DM+ for CHD, stroke, all-cause mortality and CVD mortality were 1.19 : 0.9–1.57), 1.07 (CI: 0.63–1.82), 1.62 (CI: 1.2–2.18), and 1.28 (CI: 0.83–1.97); the corresponding HRs for HTN+/DM+ were 1.96 (CI: 1.57–2.46), 1.66 (CI: 1.1–2.52), 2.32 (CI: 1.8–2.98), and 2.6 (CI: 1.85–3.65) respectively. The associations between HTN/DM status with stroke incidence and all-cause mortality were stronger among men than in women (P for interaction <0.05). Compared to HTN+/DM−, HTN−/DM+ increases all-cause mortality by 62%, however, they are not considerably different regarding CHD, stroke incidence and CVD mortality. The number of elderlies by 2050 is projected to more than double its number in 2015, getting to around 2.1 billion. Likewise, Iran’s elderly population has increased from 7.22% in 2006 to 8.20% in 2011. Having 70.9% of the population in the 15–64 years age group, Iran is awaiting a tremendous rise in its elderly population in the coming decades4, which are two well-known risk factors of cardiovascular disease (CVD)5, the leading cause of death worldwide6. On one hand, it has been estimated that in 2015, 7.8 million deaths and 143 million disability adjusted life years were related to systolic blood pressures (SBP) ≥ 140 mmHg7. On the other hand, DM is considered a coronary heart disease equivalent;9 and even among Iranian population the population attributed hazard fraction of all-cause mortality for diabetes was greater than the one for prevalent CVD10.Aging population is at increased risk of developing hypertension (HTN) and type 2 diabetes mellitus (DM)14. Regarding all-cause mortality, data is scarce14. Moreover, to the best of our knowledge, there is no evidence regarding the comparative effect of these risk factors on CVD morbidity and mortality in the older adults among the Middle East population which has the highest burden of CVD risk factors and its mortality15. To top it off, none of the available studies directly compared hypertensive non-diabetic individuals with diabetic patients who are free of hypertension regarding CVD morbidity and mortality as well as all-cause mortality. Understanding the extent of the association of different combinations of hypertension and diabetes status with CVD events as well as all-cause mortality is crucial especially in low middle income countries where the burden of both the risk factors and outcomes is high, yet, the resources are constrained.Studies have investigated the joint prognostic effect of hypertension and diabetes on CVD risk among American, European and Asian populationsThe aim of the current study is to first evaluate the total and sex-specified joint effect of hypertension and diabetes on coronary heart disease (CHD), and stroke event, all-cause, and CVD mortality in older adults in a long-term population based cohort study. Second, to compare the impact of hypertension versus diabetes on the abovementioned outcomes in this population.16. For the current study, 3890 participants, aged ≥ 50 years [3326 people from the baseline examination (1999–2001) and 546 new participants recruited from the second phase (2001–2005)], were selected. Subjects with prevalent CVD at their baseline examination (N = 501), missing data of baseline variables (N = 354) or without any follow-up data after baseline recruitment (N = 288) were excluded; leaving 2747 participants who were followed until 20 March 2014. All experiments were performed in accordance with relevant guidelines and regulations. All subjects filled a written consent after being informed about the general aspects of the work and the study was approved by the Ethical Committee of Research Institute for Endocrine Sciences.The Tehran Lipid and Glucose Study (TLGS), an ongoing prospective population-based study being performed on a representative sample of the Tehran population, aims to determine the prevalence and incidence of non-communicable diseases and their risk factors. Detailed descriptions of the TLGS have been reported elsewhere16.Using a pretested questionnaire, a trained interviewer collected information, which included demographic data, smoking and drug history. Physical activity level was assessed with the Lipid Research Clinic (LRC) questionnaire in the first phase of the TLGS. Due to the inexactness of LRC, it was substituted by the Modifiable Activity Questionnaire (MAQ) from the 2nd phase. This questionnaire measures all three forms of activities including leisure time, job, and household activities in the past yearWeight was measured, with subjects minimally clothed without shoes, using digital scales (Seca 707: range 0–150 kg) and recorded to the nearest 1 kg. Height was measured in a standing position without shoes, using a tape meter, while shoulders were in normal alignment. Waist circumference (WC) was measured at the umbilical level and that of the hip (HC) at the widest girth of the hip over light clothing, using a tape meter, without any pressure to body surface. Measurements were recorded to the nearest 1 cm. BMI was calculated as weight (Kg) divided by height squared (m2). Waist to hip (WHR) was calculated as WC (cm) divided by HC (cm).After a 15-minute rest in the sitting position, two measurements of systolic and diastolic blood pressure (SBP and DBP) were taken, on the right arm, using a standardized mercury sphygmomanometer ; the mean of the two measurements was considered as the participant’s blood pressure.A blood sample was taken between 7:00 and 9:00 AM from all study participants, after 12 to 14 hours overnight fasting. All blood analyses were carried out at the TLGS research laboratory on the day of blood collection. For all non- pharmacologically treated diabetic participants aged 20 years and above, an oral glucose tolerance test with 82.5 g glucose monohydrate solution  was performed and a second blood sample was obtained 2 hours after glucose ingestion. Fasting plasma glucose (FPG) and 2hour-post-challenge plasma glucose (2h-PCPG) were measured using an enzymatic colorimetric method with glucose oxidase; inter- and intra-assay coefficients of variation at baseline and follow-up phases were both less than 2.3%.Serum total cholesterol (TC) was measured using enzymatic calorimetric tests with cholesterol esterase and cholesterol oxidase. High density lipoprotein cholesterol (HDL-C) was measured after precipitation of the apolipoprotein B containing lipoproteins with phosphotungistic acid. Analyses were performed using Pars Azmon kits  and a Selectra 2 auto-analyzer . All samples were analyzed when internal quality control met the acceptable criteria.17. “Daily smoker” was defined as someone who smoked any kind of tobacco at least once a day and “occasional smoker” was one who smoked but not every day; both of them were considered as current smoker. Hypercholesterolemia was defined as total cholesterol ≥ 5.18 mmol/L or taking lipid lowering medications. In accordance with the definition provided by the American Diabetes Association, (29) participants were considered to have T2D if they met at least one of these criteria: FPG ≥ 7.0 mmol/L, or 2h-PCPG ≥ 11.1 mmol/L or taking anti-diabetic medication. According to JNC 7 guideline18, hypertension was defined as SBP ≥ 140 or DBP ≥ 90 mmHg or using antihypertensive medication. Participants were categorized into four distinct groups according to their baseline hypertension and diabetes status as followed: i) those who were free of both hypertension and diabetes (HTN−/DM−), ii) non-diabetic hypertensive individuals (HTN+/DM−), iii) diabetic individuals without hypertension (HTN−/DM+), iv) diabetic hypertensive individuals (HTN+/DM+).Education level was classified in 3 categories: i) those who had studied less than 6 years, ii) those who had studied for 6–12 years, and iii) those with more than 12 years of education. Sports or heavy physical activity less than three days per week was defined as low physical activity in the first exam of the TLGS using Lipid Research Clinic (LRC) questionnaire. In the second exam, low physical activity was defined as less than 600 MET  per week, using Modifiable Activity Questionnaire (MAQ)Mean (SD) values for continuous and frequencies (%) for categorical variables of the baseline characteristics were described. Baseline characteristics were compared between four categories of hypertension and diabetes  using ANOVA or Chi-square tests as appropriate among total population and in each gender.Multivariate Cox proportional hazard model adjusted for sex , age, body mass index, waist to hip ratio, education level, low physical activity, current smoking, and hypercholesterolemia was used to evaluate the associations of different combinations of hypertension and diabetes with incident CHD, and stroke events, all-cause and CVD mortality; given HTN−/DM− as the reference. To compare the impact of HTN versus DM, analysis was repeated considering the HTN+/DM− group as reference.The study survival time was the time from entrance to the study to the date of the first related event. The censoring time of an individual was the time from date of entry into the study until lost to follow-up, death or the study end (20 March 2014), whichever happened earlier.The proportionality of the multivariable Cox model was evaluated using Schoenfeld’s global test of residuals and all proportionality hypotheses were appropriate. The interactions between gender and different categories of blood pressure and diabetes were checked by log–likelihood ratio test, in multivariate analysis for each event separately. Since there was statistically significant interaction for all-cause mortality  and stroke  analysis was done separately for men and women. However, in order to report comparable results with other studies in this field, we also performed the analysis on a pooled sample.Statistical analyses were performed using Stata version 12.0 .The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.2. The prevalence of hypertension and diabetes at baseline examination were 43.68% and 23.3%, respectively. Among total population, the prevalence of HTN+/DM−, HTN−/DM+, and HTN+/DM+ were 30.3%, 9.9%, and 13.4%, respectively. All the baseline characteristics except low physical activity in total population were significantly different in four categories. Comparing baseline characteristics between the four categories in men and women revealed a significant difference except for low physical activity in both sexes, education level in men and smoking in women  older adults with mean (SD) age of 59.9 (7.4) years , and mean (SD) BMI 27.8 (4.5) kg/m436 olderIn a median (inter-quartile range) of 13.9 (9.8–14.5) years, incidence rate of CHD, and stroke event, all-cause and CVD mortality in total population were 19.0, 4.7, 13.5, and 6.4 per 1000 person years, respectively. Out of 1311 men at baseline, 331 and 90 cases developed CHD, stroke, while 268 and 135 died from all-cause and CVD. Out of 1436 women at baseline, 252 and 64 cases developed CHD and stroke, while 180 and 78 died from all-cause and CVD  for CVD mortality Fig.  in HTN+/For all-cause mortality Fig.  the HR  in total population and 72% (1.2–2.45) in men. Those with HTN+/DM+ had increased risk for CHD, stroke, all-cause and CVD mortality compared to HTN+/DM− individuals Table .Table 3MConducted among the Iranian adults aged 50 years or above during a more than a decade follow-up, the current study showed that compared to those who were HTN−/DM− at baseline, both hypertension and diabetes either alone or accompanied together increased the risk of CHD event, all-cause and CVD mortality both in men and women. Stroke incidence was positively associated with hypertension and diabetes  in men as well as sex-adjusted multivariate models in the total population. Having HTN+/DM− individuals as the reference group, the all-cause mortality in HTN−/DM+ group was increased for 72% in men and 62% in total population.25. As shown in many studies using HTN−/DM− as reference, we illustrated that HTN+/DM+ individuals had the highest risk of developing CHD, and stroke as well as death from all-cause and CVD28. A large cohort study conducted among Finnish population12, showed that those with both hypertension and diabetes at baseline examination were at highest risk for incident CHD, stroke and their mortalities; furthermore, Kokubu et al. showed the same results regarding CVD events. However, these findings were not obtained among the American population13. We extend the previous studies by showing individuals with both risk factors had higher risk for different outcomes compared with hypertensive non-diabetic individuals in multivariate sex adjusted analysis. This is important especially due to the fact that incidence of hypertension in diabetic patients and diabetes in hypertensive individuals increases with aging29.The independent impact of hypertension and diabetes on CHD, stroke, all-cause and CVD mortality in older adults has been shown in many studies30 a number that reached 25% in national surveys31. Regarding diabetic individuals, in the current study, 40% consumed anti-diabetic medications. Similarly, previous studies in TLGS population showed that only half of diagnosed diabetic patients used anti-diabetic treatment32. Moreover, national surveys estimate that about half of the diabetes cases are undiagnosed33. Hence, it might be hypothesized that, the low frequency of antihypertensive and anti-diabetic drugs consumption in our population might be attributable to low awareness of the disorders in the population, inertia for initiating treatment and low adherence to treatment regimen36. This issue may contribute to the higher risk of CVD morbidity and mortality among hypertensive and diabetic individuals37.Importantly, among hypertensive individuals in our study, only 38% consumed antihypertensive drugs at baseline examination. Results of a cohort study conducted in northern region of Iran reported that merely17% of hypertensive subjects were using antihypertensive medications;14, however, all of them considered HTN−/DM− participants as the reference group. Moreover, only one study assessed this issue regarding all-cause mortality14. We found out that there is no statistically significant difference between HTN−/DM+ and HTN+/DM− individuals in developing CHD, stroke and CVD mortality, however, the former group showed more than 60% higher risk for all-cause death compared to the latter in the total population as well as men. In a recent study, Oh et al. argued that in American older adults, compared to diabetes, hypertension has a stronger association with all-cause and CVD mortality. Nevertheless, their conclusion might not be appropriate due to the overlapping confidence intervals of HTN−/DM+ and HTN+/DM− groups14. In line with our results, in the study published by Hu et al. the HR for CHD incidence in Finnish diabetic men and women who were free of hypertension was 2.39 (1.61–3.55) and 5.63 (3.20–9.88), respectively; the corresponding values for non-diabetic stage1 hypertensive individuals were 1.25 (1.13–1.39) in men and 1.52 (1.28–1.81) in women; all of which had CIs far enough from the former group. Even the risk for diabetes was higher than among non-diabetic stage 2 hypertensive women12. Regarding stroke, results were generally the same for stage 1 hypertension11. Several reasons could explain our finding; first, diseases such as cancer and autoimmune disorders may induce insulin resistance38. Thus, those with such pre-existing conditions may increase the all-cause mortality under the name of diabetes. For this issue, when we excluded the event mortality during the first 3 years of follow-up, results did not change (data not shown). Hence, the reverse causality was not responsible for the current results. Second, diabetes complications may have been considered independent causes of death. A recent meta-analysis proved that apart from increasing the risk of vascular diseases, diabetes is associated with substantial premature mortality from several cancers, infectious diseases, external causes, and intentional self-harm, and degenerative disorders, independent of several major risk factors including systolic blood pressure39.To the best of our knowledge, our study is the first to conduct joint effect analysis comparing HTN−/DM+ individuals with HTN+/DM− persons. There are few studies conducted among European, American and Asian examining different impact of hypertension and diabetes on CVD incidence and mortalityConsidering limitations, first, as inherent to any prospective study, the level of risk factors at the baseline examination might change during the follow up. Second, the power of the study to detect the HR for stroke incidence in women was low. Third, unfortunately, measurements of HbA1C were not performed for participant of TLGS; hence, we could not use HbA1c as a criterion for defining diabetic patients. Finally, the present study was carried out among Persian ethnicities, so the present results cannot be directly extrapolated to other populations.As for the strengths, our study is the first studying the joint effect of hypertension and diabetes in a large representative sample of Iranian older adults in a population with reliable follow-up data. We had reasonable number of events in the total population which allowed us to evaluate the long term effects of hypertension and diabetes on CHD, stroke incidence and all-cause and CVD mortality. Also, we used both FPG and 2h-PCPG as indicators of diabetes status allowing us to have an accurate estimation of incident T2D.Last but not least, considering HTN+/DM− participants as the reference group; we evaluated the impact of diabetes versus hypertension on a variety of outcomes.To the best of our knowledge, our study is the first investigating the joint effect of two worldwide common risk factors of CVD i.e. hypertension and diabetes considering HTN+/DM− people as the reference group, hence, comparing HTN−/DM+ individuals directly with the former group. We showed that the all-cause mortality in HTN−/DM+ people in the total population increased for 62%; a number that rose to 72% in men. Further studies are needed to figure out the impact of hypertension versus diabetes on CVD events as well as all-cause mortality in other ethnicities.


Recent genome-wide association studies have identified genetic loci that jointly make a considerable contribution to risk of developing Alzheimer’s disease (AD). Because neuropathological features of AD can be present several decades before disease onset, we investigated whether effects of polygenic risk are detectable by neuroimaging in young adults. We hypothesized that higher polygenic risk scores (PRSs) for AD would be associated with reduced volume of the hippocampus and other limbic and paralimbic areas. We further hypothesized that AD PRSs would affect the microstructure of fiber tracts connecting the hippocampus with other brain areas.n = 272) and diffusion-weighted scans (n = 197).We analyzed the association between AD PRSs and brain imaging parameters using T1-weighted structural (p = .001). This effect remained when the APOE gene was excluded (p = .031), suggesting that the relationship between hippocampal volume and AD is the result of multiple genetic factors and not exclusively variability in the APOE gene. The diffusion tensor imaging analysis revealed that fractional anisotropy of the right cingulum was inversely correlated with AD PRSs (p = .009). We thus show that polygenic effects of AD risk variants on brain structure can already be detected in young adults.We found a significant association between AD PRSs and left hippocampal volume, with higher risk associated with lower left hippocampal volume (This finding paves the way for further investigation of the effects of AD risk variants and may become useful for efforts to combine genotypic and phenotypic data for risk prediction and to enrich future prevention trials of AD. APOE) ε4 allele, each copy of which increases AD risk by a factor of about 3 (Alzheimer’s disease (AD) is the most common neurodegenerative disease, affecting about 5% to 7% of the population over 60 years of age . Althoug about 3 . PRSs ba about 3 .APOE ε4  with an average age at time of inclusion of 24.8 years (SD 6.9). Tractography data were available for a subset of 197 participants  with an average age at time of inclusion of 23.9 years (SD 5.1). For a subgroup of 87 participants , data on the Hopkins Verbal Learning Task were available. This task measures declarative verbal learning capacity  and form2 test for Hardy-Weinberg equilibrium had a p value less than 1 × 10−4. Individuals’ genotypes were imputed using the prephasing/imputation stepwise approach implemented in IMPUTE2/SHAPEIT . Genotyping was performed using custom Illumina HumanCoreExome-24 BeadChip genotyping arrays, which contain 570,038 genetic variants . Quality control was implemented in PLINK . Individ/SHAPEIT , 36 and /SHAPEIT  as the rhttp://www.pasteur-lille.fr/en/recherche/u744/igap/igap_download.php. SNPs were removed from all analyses if they had a low minor allele frequency (< .01). Subsequently, the data were pruned for linkage disequilibrium using the clumping function (--clump) in PLINK (2 > .25 (--clump-r2) with a more significantly associated SNP. We used the --score command in PLINK to calculate polygenic scores . Lower PT indicates that SNPs are more significantly associated with AD case status in the training dataset (AD case-control study) , to assess if the association was purely due to variance in APOE.These polygenic risk scores include the 3 to 256 × 256 × 256 mm3 (anterior–posterior/left–right/superior–inferior), with acquisition time ranging from approximately 6 minutes to 10 minutes.Magnetic resonance imaging was carried out in Cardiff University Brain Research Imaging Centre on a GE Signa HDx 3T scanner . T1-weighted structural data were acquired using an axial three-dimensional fast, spoiled gradient recalled sequence with the following parameters: repetition time/echo time/inversion time = 8/3/450 ms; flip angle = 20°; 1 mm resolution; field of view ranging from 256 × 192 × 160 mmDTI data were acquired using a cardiac-gated sequence with the following parameters; b-values 0 and 1200, repetition time ~20 seconds (dependent on heart rate); echo time = 90 ms; 60 2.4-mm slices aligned with the anterior commissure-posterior commissure, zero slice gap; acquisition matrix 96 × 96; field of view = 230 mm; 2.4-mm isotropic resolution. Data were either acquired from 30 unique diffusion directions plus 3 b0 images or from a subsample of 30 optimal directions from an acquired set of 60 directions with the first 3 b0 images.surfer.nmr.mgh.harvard.edu), which has been validated as a suitable method for hippocampal segmentation in large samples (http://enigma.ini.usc.edu/) (n = 270), entorhinal cortex left (n = 257) and right (n = 268), posterior cingulate gyrus left (n = 272) and right (n = 271), and parahippocampal gyrus left (n = 259) and right (n = 271).Hippocampal volume, ERC, PHG, PCG thickness, and intracranial volume (ICV) were determined through analysis with FreeSurfer ( samples . The ressc.edu/) . Wheneve3 resolution in the resulting output. A single diffusion tensor model was fitted to the DTI data , covarying for age, gender, and ICV. The hippocampal volumes were adjusted for ICV of each participant with the formula: hippocampal volume corrected = hippocampal volume − [beta * (ICV − mean ICV across the group studied)] . SubsequThe DTI data were also analyzed using hierarchical linear multiple regression, including FA as the dependent variable and controlling for independent variables age, gender, and scan type (30 or downsampled 60 directions).p values were then corrected for multiple comparisons using false discovery rate in statistics package R  as demonstrated in PTs were nominally associated with decreased left hippocampal volume, and four of these survived correction for multiple testing , particularly with the more inclusive pTs , after correction for multiple comparisons. This effect persisted after removal of the APOE locus from the PRSs , with a negative association between PRS and FA at five thresholds. This effect was conserved for one of these thresholds (p ≤ .05) when correcting for multiple comparisons over all five regions of interest. Subsequent analysis of cingulum FA, excluding the APOE locus from the AD PRSs, only showed a significant association at one threshold .No effect of AD PRSs on FA was found in the fornix or PHC . HoweverWe found no correlation between scores on the Hopkins Verbal Learning Task and polygenic risk for AD .APOE risk alleles on hippocampal volume in young participants, while others found a significant difference between young ε4 and ε2 carriers (APOE ε4 risk allele carriers in comparison with ε3 carriers in a middle-aged population (As predicted, PRS for AD was associated with lower hippocampal volume. While hippocampal volume reductions in patients are a robust finding in AD , 13 and carriers . Previoupulation , whereaspulation , which wAPOE locus, cumulatively they explain a large amount of variance (We may see significant effects where others have obtained mixed results with single locus studies because of the benefits of using polygenic risk scores. Although the SNPs contributing to the PRSs have much smaller individual effect sizes than the variance .Although previous reports have implicated the ERC in AD pathology , 21, we APOE ε4 was linked to alterations in FA in the left medial temporal lobe and in the corpus callosum in healthy individuals (APOE risk variants (It has been proposed that AD, like many other neuropsychiatric disorders, is a disconnection syndrome . Connectividuals . One stuvariants , while avariants . A populvariants .APOE locus, is in line with models that assume early white matter changes in the course of the development of AD pathology. The fact that this correlation between cingulum FA and AD PRSs was not driven by the PHC, which connects the medial temporal lobe with areas in the parietal and occipital lobes such as the posterior cingulate cortex (Our finding of reduced FA in the right cingulum, which was not exclusively driven by the e cortex , was conR2 values are highly affected by the sample size of the training dataset (We used the results from the largest AD GWASs undertaken to date as our training data . Therefo dataset .Hippocampal volume and cortical thickness are highly heritable, which makes them appropriate parameters for genetic imaging analysis , 73. A lAPOE, as the associations persisted after removal of the APOE locus. Overall, this work suggests that genetic risk for AD is mediated, in part, through brain morphological differences, mainly in the hippocampus, confirming hippocampal volume changes as an important early biomarker of risk for AD.In conclusion, AD polygenic risk was associated with smaller volume of the left hippocampus, increased volume of left PCG, and lower FA in the right cingulum bundle in healthy young adults. Thus, AD genetic risk can be linked to structural differences in brain areas that have been implicated in the early stages of AD pathology many decades before potential illness onset. This effect was not driven exclusively by contributions from


DLG5 R30Q (rs1248696) and P1371Q (rs2289310), but the results remain controversial. We conducted a meta-analysis including a total of 22 studies with 10,878 IBD patients and 7917 healthy controls for R30Q and 5277 IBD cases and 4367 controls for P1371Q in order to systematically assess their association with the disease. The results indicated that R30Q was significantly associated with reduced susceptibility to IBD in Europeans by allelic and dominant comparisons, but not in overall population. No significant association was found between R30Q and Crohn’s disease (CD) or ulcerative colitis (UC). P1371Q was associated with increased risk of IBD in Europeans and Americans. On the contrary, a decreased risk of IBD was observed in Asian population for P1371Q. In disease subgroup analysis, we found that P1371Q was also significantly associated with CD, but this relationship was not present for UC. In conclusion, our results strongly suggest that the both polymorphisms of DLG5 are correlated with IBD susceptibility in an ethnic-specific manner. Additional well-designed studies with large and diverse cohorts are needed to further strengthen our findings.Growing evidence from recent studies has demonstrated an association between inflammatory bowel disease (IBD) susceptibility and two polymorphisms of  NOD2)5ATG16L1)(STAT3)8IRGM)IL23R)11Inflammatory bowel disease (IBD) is a relapsing and chronic inflammatory disorder mainly affecting the small intestine and colon23DLG5) gene, a member of the membrane associated guanylate kinase (MAGUK) family, encodes a cell scaffolding protein, which is wildly expressed in human tissues including liver, heart, pancreas, small intestine and colon131315DLG5 relating to IBD in a German cohort1718et al. reported that the both variants of DLG5 were associated with increased risk for IBD, additional studies replicated the association in different populations162022232425262728Discs large homolog 5 , heterozygote model  and dominant model . Therefore, random effects model was applied in these analyses. No significant association was identified between R30Q and IBD in overall population . Nevertheless, a trend of reduced risk could be seen  and dominant model  by fixed-effect model because no significant statistical heterogeneity was identified . However, no association was observed for the other three genetic models. As for the American and Australasian populations, no significant correlation was found in either cohort (data not shown).Intriguingly, subgroup analysis based on ethnicity disclosed a significant correlation between R30Q and IBD susceptibility in Europeans in allelic model  were analyzed. However, no modifying effect of R30Q on the risk of UC or CD was discovered in any genetic model .P = 0.01), and in Europeans in homozygote comparison  and recessive comparison  using fixed-effect model. However, we observed an opposite relationship between P1371Q and IBD in Asian cohort   . ConsideP = 0.02; dominant model AA + CA vs. CC: OR = 0.80, 95%CI: 0.68–0.95, P = 0.01) by fixed-effect model . In addition, a significant statistical association between P1371Q and CD in Asian was identified by heterozygote comparison . However, no significant association was found in Europeans or Americans . Nevertheless, R30Q was not significantly related to CD susceptibility in men (P = 0.058). In summary, they provided an evidence of a trend toward reduced CD risk in both males and females. This finding is consistent with our subgroup analysis of CD in European cohort, mainly from Caucasian. Compared to the above two publications, our meta-analysis summarized 22 eligible case-control studies with a larger sample size and offered convincing evidence by systematically analyzing the association of IBD susceptibility with two DLG5 polymorphisms, including not only the most studied variant, R30Q, but also another significant variant, P1371Q.A previous meta-analysis including 13 studies was performed to analyze the association between R30Q polymorphism and IBD risk in CaucasiansDLG51142DLG5 encodes a cell scaffolding proteins that has been found to play an important role in the determination and maintenance of intestinal epithelial cells polarityDLG5 participates in epithelial cell proliferation, migration and adhesionin silico analysis, both the R30Q and P1371Q variants probably influence or change the structure and function of DLG5DLG5 mutations might interfere with the epithelial barrier function of the intestine, and therefore be implicated in IBD pathogenesis.As a chronic and relapsing inflammatory disease, a growing number of studies have shown that IBD is associated with both genetic and environmental factors40et al.DLG5 polymorphisms were associated with IBD in a European cohort, and among them, R30Q was strongly related to an increased risk for IBD. Subsequently, several studies observed similar results2127222324262833et al. summarized R30Q data from published studies and attempted to resolve this discrepancy but failed201826283037DLG5 and IBD susceptibility.In 2004, Stoll There were several strengths of our meta-analysis. First of all, we performed the present study based on large sample sizes including a total number of 10878 IBD patients with 7917 controls on R30Q polymorphism and 5277 IBD patients with 4367 controls on P1371Q polymorphism, which helped to add convincing evidence. Compared with previous meta-analyses, more studies were included. Additionally, subgroup analysis, sensitivity analysis and genotype comparison were also carried out. Furthermore, we systematically investigated the relationship between P1371Q and IBD risk. To the best of our knowledge, this is the first meta-analysis regarding P1371Q and IBD. Moreover, we set and implemented strict inclusion and exclusion criteria to guarantee the quality of the included studies and the reliability of the results. Finally, selection bias was well-controlled via literature search, and no publication bias was identified by Egger’s test.Nevertheless, the present meta-analysis has several limitations that should be considered. Firstly, potential heterogeneity observed among the studies of both R30Q and P1371Q, which could not be effectively reduced via subgroup and sensitivity analysis, might partially influence the results. Hence, some other details are needed to analyze the sources of the heterogeneity. For instance, sample size, age of patients and genotyping methods might contribute to the heterogeneity. Secondly, there were two studies with genotypes deprived from HWE, of which one was for R30Q and the other for P1371Q. Although the pooled ORs were not significantly influenced by this study for R30Q, the significant association between P1371Q and CD was diminished by omitting the study for P1371Q. Thirdly, although we found that individuals with P1371Q variant have lower risk of suffering IBD in Asian population, the number of eligible studies and patients was limited. Therefore, many more high quality case-control studies are needed to further explore the association between P1371Q and IBD susceptibility in Asian cohorts.DLG5 R30Q polymorphism is associated with a reduced risk of IBD in Europeans, while P1371Q is associated with an increased risk in Europeans and Americans, but could serve as a protective factor in Asian population. Particularly, P1371Q may decrease the risk of CD in overall population. However, caution should be taken for P1371Q because of insufficient data and population heterogeneity, which might have impact on the convincingness of the results. Well-designed case-control studies with larger sample sizes and multiple ethnicities are required to strengthen the results of the current study. More importantly, studies focusing on the biological functions and mechanisms involving the IBD pathogenesis of DLG5 are also urgently needed.In summary, despite the limitations above, our results suggest that the DLG5 polymorphisms (R30Q and P1371Q) and IBD risk in the Pubmed and Web of Science up to March 14, 2016. The following search terms were used: (inflammatory bowel disease OR IBD OR ulcerative colitis OR UC OR Crohn’s disease OR CD) and (genetic polymorphism OR polymorphism OR variant) and (DLG5 OR discs large homolog 5), without any limitation. Manual search was also performed to obtain additional publications through the reference lists of retrieved studies and reviews.Two independent investigators conducted a literature search for relevant studies evaluating the relationship between DLG5 R30Q or P1371Q polymorphism and IBD risk; (3) provided detail genotype or allelic distribution; (4) contained sufficient data to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Exclusion criteria: (1) reviews, meeting articles, laboratory studies or duplication of previous publications; (2) studies without detail genotype data; (3) researches did not focus on DLG5 R30Q or P1371Q polymorphism; (4) family-based studies. When there were several articles about a study or the same cohort included in two or more publications were identified, only the latest or complete study was selected.Studies meeting the following criteria were included: (1) cases-control study; (2) evaluation the association between The following information was independently extracted by two authors from eligible studies: name of the first author, year of publication, country and ethnicity of the study population, genotyping method, number of cases and controls, allele and genotype frequency in cases and controls. Any disagreement was resolved by discussing with a third author to reach a consensus.DLG5 R30Q and P1371Q variants and IBD risk. Meta-analyses were performed by allelic model , heterozygote model , homozygote model , dominant model , recessive model . Cochran’s Q statistic and I2 test were performed to evaluate the heterogeneity among the eligible studies. Statistical heterogeneity was defined as P < 0.1 or I2 > 50%. Random effects models or fixed-effect model was used to pool the effect size depending on the existence of statistical heterogeneity or not, respectively. Chi-square test was applied to evaluate the Hardy-Weinberg equilibrium (HWE) in control group. Publication bias was detected by Egger’s test. All the P value were two-sided and P < 0.05 was considered statistically significant. All statistical analyses were performed by Review Manager software version 5.2 and Stata software version 12.0 .The present meta-analysis was conducted according to the PRISMA checklists and following the guidelineHow to cite this article: Li, Y. et al. Meta-analysis of associations between DLG5 R30Q and P1371Q polymorphisms and susceptibility to inflammatory bowel disease. Sci. Rep.6, 33550; doi: 10.1038/srep33550 (2016).