Matrix metalloproteinases (MMPs) polymorphisms have been implicated in the pathogenesis of glaucoma risk. However, the results were controversial. We performed a meta-analysis to evaluate the precise associations between MMPs polymorphisms and glaucoma risk.Related studies were reviewed by searching electronic databases within four databases. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the association between the most common polymorphisms of MMPs and glaucoma risk. Heterogeneity, publication bias and sensitivity analysis were conducted to guarantee the statistical power.P = 0.02, I2 = 0%), MMP-9 rs3918249 C > T polymorphism  and glaucoma risk in the general population. Subgroup analysis also suggested that MMP-9 rs17576 G > A was related to glaucoma in the Caucasian population .Overall, 11 selected articles involving 2,388 cases and 2,319 controls were included in this meta-analysis. Significant associations were only found between MMP-9 rs17576 G > A polymorphism (GA vs. GG: OR = 0.80, 95%CI = 0.67-0.97, Our meta-analysis demonstrates that MMP-9 rs17576 G > A polymorphism might be a protective factor against the development of glaucoma in Caucasian population. Glaucoma is a heterogeneous disease of the eye characterized by the progressive degeneration of retinal ganglion cells and loss of vision associated with elevated intraocular pressure (IOP) . After cAs we know, ocular hypertension is the most important risk factor for glaucoma, but its etiology is still unclear. Many molecular epidemiological studies have suggested that glaucoma is a complex multifactorial disease. Various risk factors such as diabetes, hypertension, lifestyle habits , age, and genetics play interacting roles in the development of glaucoma. Recently, certain genetic factors, including matrix metalloproteinases (MMPs), were found to be associated with glaucoma. MMPs are a group of zinc and calcium-dependent endopeptidases that are involved in extracellular matrix (ECM) homeostasis and remodeling , 5. In gPrevious molecular research has demonstrated that genetic mutations, including single nucleotide polymorphisms (SNPs), can alter the level of gene expression or the function of gene products, thereby affecting the susceptibility of individuals to specific diseases , 9. In 2This meta-analysis was conducted according to the guidelines of Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) , 12. No Four online databases  were used to search for case control studies evaluating the association between MMPs polymorphisms and glaucoma risk published up to February 1, 2016, with the following search terms: “glaucoma,” “MMP,” “matrix metalloproteinases,” “polymorphism,” and “variant”. Manual searches of references from original studies and review articles on this topic were conducted to identify other relevant studies.The inclusion criteria for studies in our meta-analysis were as follows: (1) designed as a case control study, (2) reported an association between MMP polymorphism(s) and glaucoma risk,(3) sufficient genotype frequency to estimate odds ratios (ORs) and 95% confidence intervals (CIs), and (4) no deviation from Hardy-Weinberg equilibrium (HWE) in the genotype distribution of the control group. For results that were reported in multiple publications, only the largest or latest dataset was included. The exclusion criteria included: (1) review articles, (2) case reports, (3) results without sufficient genotype frequency data, and (4) animal model research.Two reviewers (MYW and YW) independently reviewed the full articles and collected the following characteristics: the first author’s name, publication year, study country/region, ethnicity of participants (such as Asian or Caucasian), disease subtype, genotyping method, sources of controls, and frequencies of genotypes in glaucoma cases and controls. Hardy-Weinberg equilibrium (HWE) was estimated based on the genotypes of the controls. Discrepancies were resolved by discussion between the 2 reviewers or by consulting with an expert in ophthalmology (ZY).2 method [2 was less than 50% [P-values and P < 0.05 was considered statistically significant.Crude ORs with 95% CIs were calculated to evaluate the strength of the association between each reported MMP polymorphism and glaucoma risk. For the MMP-9 rs17576G > A polymorphism, the pooled ORs were obtained for the allele contrast (A vs. G), co-dominant , dominant (GA + AA vs. GG), and recessive (AA vs. GG + GA) models. Subgroup analyses according to disease type, ethnicity, study design, and genotyping methods were also conducted. Heterogeneity was assessed using Cochran’s Q statistic and the I2 method . ORs estthan 50% ; otherwithan 50% , 16. CumIn total, 45 related articles were identified. Seventeen of these studies were excluded through title and duplicate screening. Subsequently, 1 study was excluded for not involving glaucoma research; 2 studies without related polymorphisms locus were excluded; 6 studies were excluded for fundamental molecular biology researches; and 8 studies were excluded because they were reviews. Finally, 11 articles involving 2,388 cases and 2,319 controls met the inclusion criteria , 17–26 , MMP-9 rs3918249 C > T polymorphism  and glaucoma risk. Furthermore, in the subsequent analyses based on disease type, ethnicity, control design and genotyping methods, a significant protective effect against glaucoma risk was observed for MMP-9 rs17576 G > A in Caucasian populations   involving 2,028 cases and 1,794 controls were included in our analyses of the 6 most commonly reported SNP loci. Overall, significant associations were only found between MMP-9 rs17576 G > A polymorphism , indicating that there was no detectable publication bias.Sensitivity analyses were conducted by deleting each included study step by step in MMP-9 rs17576 G > A polymorphism. No single study qualitatively changed the pooled ORs when removed, indicating that the results of this meta-analysis are stable  involving 1,065 cases and 836 controls and 2 articles  with 792 cases and 559 controls were included in our meta-analysis of the MMP-1 rs1799750 1G > 2G and MMP-2 rs243865 C > T polymorphisms and glaucoma risk. No significant association was found for all models for these 2 SNP loci , can degrade and remodel ECM molecules, thereby influencing cellular activities and maintaining the homeostasis of theepithelialbasementmembrane , 28. TheP = 0.03). Subsequently, more epidemiological studies were conducted, with inconsistent and even contradictory results.SNPs are the most common type of genetic mutation and have been associated with altered disease susceptibility through changes in gene transcription and expression as well as amino acid substitutions. In 2006, Wang et al. conducted the first case control study investigating the potential association between MMP-9 SNPs and primary angle-closure glaucoma. Significant differences in the frequencies of the MMP-9 rs17576 G > A SNP genotypes were found between the glaucoma and healthy control groups, which suggested an increased risk for glaucoma in the Chinese population according to a dominant model  conferred different significantly susceptibility to glaucoma. Stratified analysis by ethnicity, control design, and genotyping method were performed to estimate the association for each subgroup. The subgroup analysis according to ethnicity demonstrated a significant protective association between the MMP-9 rs17576 G > A polymorphism and glaucoma susceptibility in the Caucasian population. Thus, ethnic differences may be the most important factor underlying differences in glaucoma susceptibility between the Asian and Caucasian populations. MMP-9 rs17576 polymorphism is locate in exon 6 of MMP-9 gene with a nucleic acid substitution from G to A, and this mutation located fibronectin type II domains that presumably enhance substrate binding , 35. AltTo the best of our knowledge, this is the first comprehensive meta-analysis evaluating the potential association between MMP gene polymorphisms and glaucoma susceptibility. Some limitations of this meta-analysis were inevitable and should be addressed. First, the number of included studies and the amount of data available were limited, which constrained the statistical power. Second, heterogeneity was observed in the data for some SNP loci, which further hindered our ability to confidently identify any potential associations. Third, due to the deficiency of available data, it was not possible to study the interactions between MMP gene SNPs and glaucoma risk factors, such as haplotype, tobacco smoking, alcohol drinking, hypertension, and diabetes mellitus.Despite these limitations, the findings further enhance our understanding of the potential associations between MMP gene polymorphisms and glaucoma susceptibility. Positive aspects of the analysis were also identified. First, the distributions of genotypes in the control subjects for all of the investigated SNPs conformed to HWE. Second, evaluation using Egger’s test and Begg’s funnel plots did not show significant publication bias. Third, a large amount of heterogeneity was alleviated through subgroup analyses.In conclusion, our meta-analysis indicated that the MMP-9 rs17576 G > A polymorphism maybe an important protective factor against glaucoma, especially in the Caucasian population. Moreover, the present findings highlight the need for further investigations of the potential associations between MMP gene polymorphisms and glaucoma, which should ideally large sample sizes and multiple ethnic groups.


Staphylococcus aureus, a major cause of bovine mastitis, produces a wide range of immune-evasion molecules. The bi-component leukocidin LukMF’ is a potent killer of bovine neutrophils in vitro. Since the role of LukMF’ in development of bovine mastitis has not been studied in natural infections, we aimed to clarify whether presence of the lukM-lukF’ genes and production levels of LukMF’ are associated with clinical severity of the disease. Staphylococcus aureus was isolated from mastitis milk samples  from 33 different farms. The lukM-lukF’ genes were present in 96% of the isolates. Remarkably, 22% of the lukM-lukF’-positive S. aureus isolates displayed a 10-fold higher in vitro LukMF’ production than the average of the lower-producing ones. These high producing isolates were cultured significantly more frequently from clinical than subclinical mastitis cases. Also, the detection of LukM protein in milk samples was significantly associated with clinical mastitis and high production in vitro. The high producing LukMF’ strains all belonged to the same genetic lineage, spa-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (rot) gene which results in a non-functional start codon, preventing translation of rot. This mutation was only identified in high LukMF’ producing isolates and not in low LukMF’ producing isolates. Since rot suppresses the expression of various toxins including leukocidins, this mutation is a possible explanation for increased LukMF’ production. Identification of high LukMF’ producing strains is of clinical relevance and can potentially be used as a prognostic marker for severity of mastitis. Staphylococcus aureus is one of the major causes of mastitis in cows [Staphylococcus aureus possesses many virulence factors, some of which enable it to manipulate the innate and adaptive immune responses of the host [S. aureus lineages [S. aureus immune evasion molecules is that of the leukocidins: pore-forming, bi-component toxins that specifically target immune cells [S. aureus strains of ruminant origin [S. aureus strains resulted in more severe mastitis compared to challenge with intermediate producing S. aureus strains [Mastitis—inflammation of the mammary gland—is a major cause of economic losses in the dairy industry associated with costs of treatment, reduced milk yield, discarded milk and premature culling of animals ,2. In ad in cows . These i in cows . Staphylthe host . These vthe host  and inclthe host , proteasthe host , factorsthe host  and cytothe host . Some vilineages . An impone cells . So far,ne cells ,14, of tne cells ,13,14. Lt origin ,16,17 ant origin ,19. LukMt origin , it is e strains . Also, h strains . It is, S. aureus with increased LukMF’ production that is associated with clinical rather than subclinical mastitis. In this lineage, we found a nonsense mutation in the start codon of the global expression regulator Repressor of toxins (rot). This mutation is the likely cause of increased toxin production associated with severity of clinical signs of mastitis.In this study, we investigated the impact of LukMF’ on clinical severity of bovine mastitis under field conditions. We identified a genetic lineage of S. aureus isolates were collected at 33 Dutch dairy farms by bacteriological culture of milk samples from clinical (n = 37) and subclinical (n = 18) cases of bovine mastitis and the presence of the femA, lukM, and lukF’ genes was detected using PCR. All isolates were positive for the S. aureus-specific gene femA, confirming that the selected bacteria were S. aureus, and 53/55 (96%) of isolates were positive for lukM and lukF’. The proportion of lukM-lukF’ positive S. aureus was similar for clinical  and subclinical  mastitis cases.Fifty-five S. aureus isolates for eight hours under controlled conditions and measuring the LukM concentration in supernatant using ELISA. All lukM-lukF’ positive S. aureus isolates produced LukM in vitro, with levels ranging from 1.5 to 60 µg/mL. The production levels had a clear bimodal distribution (S. aureus (n = 41) were LukMF’ low producers . The remaining isolates (n = 12) were LukMF’ high producers  and were significantly more often isolated from cases of clinical mastitis . Although some of the 55 S. aureus isolates originated from the same farm, the 12 high producing isolates were cultured from cows on 12 different farms. To compensate for a possible farm effect, we randomly selected one isolate per farm (n = 33) and found the same association between the LukMF’ high producing isolates and clinical mastitis .The in vitro LukMF’ production potential was investigated by growing ribution . Based oS. aureus isolates. Eight milk samples  could not be tested for LukM because the milk was clotted and therefore not fit for use in the ELISA, or because insufficient milk was available. LukM was detected in 15/45 milk samples, with concentrations ranging from 0.31 ng/mL to 96 ng/mL, significantly more often in milk from clinical mastitis cases (13/29 = 45%) than from subclinical cases  and in samples that yielded LukMF’ high producing isolates (9/11 = 82%) compared to samples that yielded LukMF’ low producing isolates . After again selecting one isolate per farm, the association between high in vitro production and LukM in milk, ex vivo, remained , but the association between LukM in milk and clinical severity was no longer statistically significant . The average LukM concentration in milk of cows that hosted LukMF’ high producing isolates  was higher than of those carrying LukMF’ low producing isolates    .spa-typed to investigate whether differences in LukMF’ production associated with certain genetic lineages. spa-types identified in our isolate collection, and shows that all high producing S. aureus isolates belonged to spa-type t543. Whole genome sequencing was performed on a subset of seven isolates, selected based on differences in LukM production levels, spa-type and farm. Sequencing results showed that all three spa-type t543 strains belonged to multilocus sequence type (ST) 479, which belongs to clonal complex (CC) 479. The three spa-type t529 isolates all belonged to CC151, with two isolates identified as ST151 and one as ST504, a double-locus variant (DLV) of ST151. The single spa-type t1403 strain belonged to ST133 (CC133).All isolates were S. aureus isolates. First, the prophages containing lukM-lukF’ were identified using PHAST [lukM-lukF’ were indeed present in all sequenced isolates, and were identified by PHAST to be most similar to reference phage phiPV83 (Genbank accession NC_002486.1). Prophages from CC479 and CC133 isolates were very similar to each other in size and gene content, but both differed from the smaller CC151 prophage. Variations were observed in lukM (four synonymous and five non-synonymous single nucleotide polymorphisms (SNPs)) and lukF’ (three synonymous and six non-synonymous SNPs) among the different CCs (lukM-lukF’ operon (up to 200 bp upstream from the start codon) contained five SNPs, but none were exclusively present in high producing CC479 strains  and the exoprotein two-component system SaeRS (saeS and saeR) [saeR gene, whereas a single non-synonymous SNP (not associated with CC479) was found in the saeS gene (data not shown). Two non-synonymous SNPs (position 2 and 452) were found in the rot gene, exclusively present in the CC479 isolates. The SNP at position 2 renders the rot gene non-functional due to the loss of a start codon. To corroborate these findings, the region surrounding the start codon of rot was identified in five additional S. aureus isolates (three LukMF’ high producing isolates and two LukMF’ low producing isolates) using PCR and sequencing. In these additional isolates, the mutation in the rot start codon was also only present in the high producing S. aureus isolates  ,24. Thesisolates .lukM-lukF’ carriage (96%) among S. aureus field isolates cultured both from cases of clinical and subclinical mastitis. A subpopulation of S. aureus, spa-type t543-ST479, with a very high in vitro LukMF’ production was significantly associated with clinical rather than subclinical mastitis. A mutation in the rot gene, leading to loss of the primary start codon, found exclusively in spa-type t543-ST479 isolates, may be functionally linked to the increased LukMF’ production. We observed a high spa-types identified in this study have previously been associated with bovine mastitis [lukM-lukF’ carriage of S. aureus, and this reveals that it is strongly associated with specific ruminant-associated clonal complexes, namely CC151, CC133, CC705, and CC479 [lukM-lukF’ [lukM-lukF’ harboring (pro)phage [All sequence types and mastitis ,27,28,29nd CC479 ,32,33,34kM-lukF’ ,31. Thisro)phage  is lineaS. aureus [S. aureus, and hence pro-inflammatory responsiveness increasing the clinical severity of mastitis. In our study, LukMF’ high production in vitro by ST479 S. aureus is indeed associated with clinical rather than subclinical mastitis. High production was also strongly correlated with substantial levels of LukM present in milk in vivo. Still, also the presence of LukMF’ low producing S. aureus in milk may lead to detectable levels. Although differences in bacterial load of LukMF’ producing bacteria in the mammary gland could also explain differences in milk LukM levels, we assume that ST479 are also LukMF’ high producers in vivo during the course of infection. In a recent study, cattle were challenged intramammary with LukMF’ high and low producing S. aureus strains [LukMF’ is a potent killer of bovine neutrophils, which play a critical role in the innate immune defense against . aureus ,19. We h strains . Quarter strains .rot start codon that was strongly associated with high LukMF’ production. Rot is a global regulator of S. aureus virulence gene expression and can directly bind to the promotor region of various toxin genes, such as hla, hlgC-hlgB, lukE-lukD, and lukA-lukB [rot can both activate or repress gene expression, depending on the site of the mutation and the target gene [S. aureus increases when the rot gene is made inoperative [S. aureus (CA-MRSA) clone USA500 is believed to be a result of increased leukocidin  production compared to other CA-MRSA, induced by an insertion in the promotor region of rot that prevents expression of this gene [rot as the one identified in our work. This demonstrates that the absence of, or impaired or altered function of rot likely explains the increased LukMF’ production of ST479 strains observed in our study. To further substantiate this, ST479 could be complemented with a functional rot copy which should lead to decreased LukMF’ production compared to the WT ST479. Likewise, rot knockout strains of ST151 or ST133 are expected to produce higher amounts of LukMF’ compared to the WT. These strains could also be used to identify how this mutation in rot affects the regulation of other leukocidins. Because of the limited geographic range from which our samples originated, it is unclear how prevalent the mutation in rot is in CC479 isolates cultured from cattle in other countries. Still, the strain S1444 [Although, to our knowledge, the regulation of LukMF’ expression has not been studied, it is plausible to assume that the regulation system of LukMF’ expression is similar to that of other leukocidins, which are controlled by global gene regulators Agr, Rot and SaeRS . We obseukA-lukB ,35. Mutaperative ,36. The his gene  also conin S1444 , a high S. aureus isolates were submitted by farmers, sometimes in consultation with their veterinarian, hence not randomly collected and not likely to be representative for the population in the field. Due to that approach, under- or overestimation of the actual proportions of LukMF’ high producing isolates in the population as well as of isolates belonging to the various spa-types cannot be excluded. The association between high LukMF’ production and clinical versus subclinical mastitisis, however, not likely to be affected by this sampling bias, but the clinical importance of this association in terms of the population attributable fraction depends on the prevalence of LukMF’ high producing isolates in the field and cannot be calculated from our data.Mastitis milk samples used in our study as source of rot gene is part of the core genome of S. aureus, transfer of the ability of ST479 to produce high levels of LukMF’ seems unlikely. It is unclear if increased production of LukMF’ by ST479 also increases the transmission of this lineage in the population, as a severe clinical infection is expected to result in a quicker death or culling from the herd of the host or quicker treatment with antibiotics, reducing the chances to further spread in the population. In previous studies, ST479 made up 26% of Dutch [S. aureus mastitis isolates [Since the of Dutch  and 17% isolates , suggestS. aureus is strongly associated to the type of mastitis, sequence typing of mastitis isolates would enable farmers to implement specific interventions in case of infection with the high LukMF’ producing lineages.Screening tools, like loop-mediated isothermal amplification (LAMP) , currentn = 33) belonged to the same veterinary practice, the farms were geographically clustered around the Utrecht region in the Netherlands. Samples were collected between April 2014 and December 2015. Clinical or subclinical mastitis was diagnosed by the farmer, with clinical mastitis defined as visibly abnormal appearance of the udder, the milk or both. Subclinical mastitis was characterized by absence of clinical signs and generally were animals with a high somatic cell count. A total of 55 S. aureus positive milk samples from cases of bovine mastitis  were used for this study and stored at −18 °C before further use. After thawing of the milk samples, 200 µL aliquots of the S. aureus positive milk samples were plated on sheep blood agar plates and cultured overnight at 37 °C to isolate fresh bacterial colonies. Of milk samples showing signs of a clinical mastitis , a smaller volume of 50 µL was used to prevent bacterial overgrowth of the plate. Single colonies were picked and added to 2 mL T1438 Todd Hewitt Broth (THB)  and incubated overnight at 37 °C with agitation. Bacterial glycerol stocks (25% glycerol) were made by adding 0.5 mL of bacterial broth to 0.5 mL 50% glycerol solution in distilled water. Stocks were stored at −80 °C before use in further experiments.Quarter milk samples from cows with subclinical or clinical mastitis were aseptically collected by farmers belonging to the University Farm Animal Practice  and sent in for bacteriological culture and species identification, which were performed according to National Mastitis Council protocols . As partg for 1 min), bacteria were resuspended in 200 µL distilled water and heated at 100 °C for 10 min, centrifuged at 17,000× g for 1 min, and diluted 1:10 in distilled water and stored at −20 °C.Bacterial isolates were plated from glycerol stocks on blood agar plates and cultured overnight at 37 °C. Single colonies were picked for DNA extraction and washed in 1 mL distilled water. After centrifugation  in separate, single PCR. The reaction was performed in a total volume of 25 µL containing 10 µL 1:10 diluted boiled DNA sample, 5 µL GoTaQ Green buffer 5× , 1.5 mM MgCl2 (Promega), 0.2 mM dNTPs , 0.4 µM of the lukF’ primer pair and 0.6 µM of the other primer pairs  and 0.625 U of GoTaQ DNA polymerase . After an initial denaturation step at 95 °C for 2 min, 35 cycles  and supernatant was collected. The supernatant was sterilized using a microfilter  and stored at −20 °C before use in further experiments. Bacterial supernatants were produced in triplicate using separate, single colonies from the same cultured plate.Bacteria were cultured from glycerol stock on blood agar plates overnight at 37 °C. Single colonies were picked and added to 1.5 mL THB. Bacteria were incubated with agitation for 30 min at 37 °C. Optical density (OD) at 600 nm was measured and samples were diluted to an OD of 0.01 in 2.5 mL of THB. Next, bacteria were incubated with agitation for 8 h at 37 °C. After incubation, samples were centrifuged  gene of all S. aureus isolates was amplified according to the Ridom StaphType standard protocol (www.ridom.org). PCR amplicons were purified using ExoSAP-IT PCR Cleanup Reagent  according to manufacturer’s instructions and sequenced using Sanger sequencing . BioNumerics v7.5 software  was used to analyze sequence data and to assign spa-types.The polymorphic X-region of the Staphylococcal Protein A . MiSeq sequencing  was performed at the Utrecht Sequencing Facility , using 300 bp paired end reads. Reads were assembled into a scaffold genome using SPAdes v3.1.1. [https://cge.cbs.dtu.dk/services/MLST/) [LukM-lukF’ genes were identified using PHAST (accessible on http://phast.wishartlab.com/index.html) [lukM-lukF’ encoding gene sequences and putative promotor region (200 bp upstream of start LukM gene) were identified by BLASTN using reference sequences for lukM (GenBank accession: 1262967) and lukF’ (GenBank accession: 1262954). Sequences for candidate LukMF’ regulator genes were extracted from available genomes using reference gene sequences of rot (Genbank accession: AF189239.2) and saeRS locus (Genbank accession: AF129010.1). Nucleic acid sequences were translated to their corresponding protein sequences using EMBOSS Transeg software (accessible on http://www.ebi.ac.uk/Tools/st/emboss_transeq/). Gene and protein sequences were aligned using MegALIGN Pro software .Whole genome sequencing was performed on seven isolates, selected from LukMF’ high and low producers of different  v3.1.1. . This Whs/MLST/) . Prophagex.html) . The lukStatistical analysis was performed using GraphPad Prism 7 software . In vitro differences in LukM levels between clinical/subclinical isolates and in vivo LukM levels between LukMF’ high/low producers were compared using the Mann–Whitney U test. The associations between in vitro LukMF’ production levels, presence of detectable LukM in milk and mastitis type were tested using Fisher’s exact test. A subset of the dataset with a single sample per farm was assembled using the random number generator function in Microsoft Excel .


The outcome of bladder cancer after radical cystectomy is heterogeneous. We aim to evaluate the prognostic value of HALP  and explore novel prognostic indexes for patients with bladder cancer after radical cystectomy. In this retrospective study, 516 patients with bladder cancer after radical cystectomy were included. The median follow-up was 37 months (2 to 99 mo). Risk factors of decreased overall survival were older age, high TNM stage, high American Society of Anesthesiologists (ASA) grade and low HALP score. The predictive accuracy was better with HALP-based nomogram than TNM stage (C- index 0.76 ± 0.039 vs. 0.708 ± 0.041). By combining ASA grade and HALP, we created a novel index—HALPA score and found it an independent risk factor for decreased survival .The present study identified the prognostic value of HALP and provided a novel index HALPA score for bladder cancer after radical cystectomy. In 2017, there were 79030 new cases estimated in United States; the cancer ranks 6th in all cancers and 4th in males2. Radical cystectomy is the typical treatment for bladder cancer including muscle-invasive bladder cancer (MIBC) and high risk non-MIBC (NMIBC)4. Despite the advancements in surgical techniques and chemotherapy, the outcome of bladder cancer is poor, especially for patients with advanced and metastatic disease5.Bladder cancer is the most common cancer in the urogenital systemin situ disease, 70.2% with invasive tumors and 3.0% and 5.4% with regional and distant-stage disease6. Therefore, it is crucial to stratify the risk of mortality and establish the optimal therapy and follow-up strategies.The outcome of bladder cancer is heterogeneous, with 77.9% survival at 5 years for patients at all stages, 96.4% with 11. The combination of those indices have better predictive ability, for example, neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR)14. Recently, a novel index, HALP  was developed and demonstrated as a significant prognostic factor for patients with colorectal cancer and gastric carcinoma16.Besides the traditional TNM system, numerous prognostic factors may predict outcome with bladder cancer. Haematological parameters including leukocyte count , platelet count and levels of hemoglobin, albumin, C-reactive protein (CRP), and fibrinogen are all easily assessed and reliable indicators of postoperative prognosisHere we evaluated the prognostic value of HALP and explored the development of a novel prognostic index for patients with bladder cancer after radical cystectomy.A total of 516 patients were enrolled in this study  ; HALPA = 1 (HALP < 22.2 or ASA grade = 3&4); and HALPA = 2 (HALP < 22.2 and ASA grade = 3&4). Therefore, a higher HALPA score indicated higher risk of decreased survival. Univariate and multivariable analyses were performed again with HALPA score. On log-rank test, HALPA score was a significant indicator of poor survival for all patients and NMIBC or MIBC patients Fig. . Notably17. In the present study, we evaluated the prognostic value of a recently reported index, HALP, which combined hemoglobin and albumin levels and lymphocyte and platelet count, and found it as a good prognostic index for overall survival of patients with bladder cancer after radical cystectomy. By combining ASA grade and HALP score, we also created HALPA score and found it as an independent risk factor of decreased overall survival.Besides traditional TNM stage and histologic classification, several haematological-based indices such as hemoglobin, albumin, and CRP levels, NLR, PLR and LMR were found associated with outcome with bladder canceret al.7 reported that 81 of 200 (40.5%) bladder cancer patients undergoing radical cystectomy had preoperative anemia and that anemia was significantly associated with disease recurrence and cancer-specific mortality and for MIBC patients, 53.3% patients were anemic, and older age, neoadjuvant chemotherapy, female sex, and low BMI were independent predictors of preoperative anemia. In this study, anemia was present in 27.9% patients , which was lower than in previous reports. This difference may be due to use of different population. On univariate analysis, anemia was significantly associated with decreased overall survival (P < 0.001).Cancer-related anemia is a common complication for cancer patients because of chromic blood loss, iron deficiency, vitamin deficiency and inflammation imbalance in terms of increased expression of interleukin and tumor necrosis factor. Jung et al.11 reported that for bladder cancer patients after radical cystectomy, 16.5% had low-albumin level (<3.5 g/dL), and low albumin level was significantly associated with increased risk of mortality . Another study found that 197 of 1471 (13.4%) bladder cancer patients had a low serum albumin level, and low albumin level was independently associated with reduced recurrence-free survival  and overall survival . Moreover, the authors also found low serum albumin associated with higher 90-day complication rate  and 90-day mortality 17. These findings document the adverse effects of nutritional deficiency on survival of bladder cancer after radical cystectomy and demonstrate the importance of nutritional interventions.Hypoalbuminemia is another nutrition deficiency index. Lambert 18. For example, neutrophils and platelets could promote carcinogenesis, angiogenesis, invasion, or metastasis by secreting proinflammatory cytokines20. However, lymphocytes could inhibit tumor proliferation and migration via cytotoxicity21. Therefore, high PLR level, which reflects high platelet count and low lymphocyte count was found associated with overall survival in patients with bladder cancer undergoing radical cystectomy14.Tumor-promoting inflammation is one of the hallmarks of cancer17. In this study, a high ASA score was also significant independent risk factor for decreased overall survival and the nomogram including ASA grade and HALP score had better predictive accuracy than TNM stage. By combining ASA grade and HALP score, we created a novel index, HALPA score, and found it to have a specific distribution in patients at different stages and a significant independent risk factor with higher risk ratio than HALP score or ASA alone.The association of ASA score and long-term survival outcomes of bladder cancer patients after radical cystectomy is rarely reported. One study found that 740 of 1964 (64.8%) patients who underwent radical cystectomy had a high ASA score (3 or 4), and a high ASA grade remained independently associated with decreased overall survival There are several limitations to this study. First, the retrospective nature has inherent limitations. Second, this was a single-center study and could not avoid the bias of population choice. Third, the use of cutoffs for continuous variables might weaken the reliability of the risk system. Finally, ASA is a subjective measure and this may limit the generalizability and overall utility. The prognostic ability and accuracy of HALP and HALPA score for bladder cancer patients warrants prospective multi-center validation.HALP may be a good prognostic index for overall survival for patients with bladder cancer after radical cystectomy. Combining ASA grade and HALP score in the HALPA score could distinguish tumor stage and was an independent risk factor for decreased overall survival. These indices could be used for risk stratification of individual bladder cancer patients and for choosing therapeutic strategy.This study was approved by the Institutional Review Board of Peking University First Hospital. Between 2006 and 2012, 516 bladder cancer consecutive patients underwent radical cystectomy in the Department of Urology, Peking University First Hospital. This research was carried out in accordance with the approved guidelines and informed consent was obtained from all patients. Hematological features including hemoglobin and albumin levels and blood cell counts were preoperative collected within 3 days before surgery. Other clinicopathological data included gender, age, smoking history, hypertension history, histology subtype, grade, TNM stage and American Society of Anesthesiologists (ASA) grade.The follow-up of bladder cancer patients after radical cystectomy was according to the routine procedure in our institution. The period from the operation date until the time of death resulting from any cause was calculated as overall survival time. HALP was calculated as hemoglobin (g/L) × albumin (g/L) levels × lymphocyte count (/L)/platelet count (/L) and PLR as platelet count/lymphocyte count. Anemia was defined according to preoperative hemoglobin level  and hypoalbuminemia according to preoperative albumin level (<35 g/L).22. SPSS v22.0  was used for all statistical analyses. Chi-square test was used to evaluate the association between clinicopathological data and HALP. Overall survival was estimated by the Kaplan-Meier method and the log-rank test was used for univariate analysis. Variables with significant differences on univariate analysis (P < 0.05) were then included in the multivariable survival analyses with a Cox proportional-hazards regression model, estimating hazard ratios (HRs) and 95% confidence intervals (CIs). Nomogram and production of calibration curves involved use of R v3.3.2  with the rms package (Regression Modeling Strategies). A two-tail P < 0.05 was considered statistically significant.X-tile software v3.6.1  was used to determine the cut-off values of PLR and HALP


CHM gene, which lead to the absence of the Rab escort protein 1 (REP-1), resulting in prenylation deficiency. Typical fundus appearances for choroideremia were found in 3 probands from three unrelated Chinese families in our study. We firstly used the targeted exome sequencing (TES) technology to detect mutations in CHM gene. Based on an established filtering strategy of data analyses, along with confirmation by co-segregation, a previously reported mutation  was identified in one family, while two novel mutations  were identified in the other two families. These findings not only expands the currently limited spectrum of Chinese disease-causing variants in CHM gene, but also increases our understanding of the phenotypic and genotypic correlations of choroideremia, and may potentially lead to improved genetic counseling and specific treatment for families with choroideremia as well.Choroideremia is a bilateral and progressive X-linked inherited disease characterized by widespread chorioretinal atrophy with relative sparing of the macular region. It is caused by mutations in the ubiquitously expressed  Choroideremia CHM; MIM 303100) is an X-linked recessive eye disorder, with the incidence ranging from 1:50,000 to 1:100,000103100 is CHM gene, which is mapped to chromosome Xq21.23. The CHM gene spans over 180 Kb on the X chromosome, and is expressed in many tissue types including retinal photoreceptor, choroid, RPE, and lymphocyte tissues. The mRNA consists of 15 exons that are conserved in transcription. All of the conserved exons, except exon 15 , are fewer than 400 bp longCHM, which results in prenylation deficiency, has been verified as the cause of retinal degeneration in choroideremia7In patients with choroideremia, diagnoses are confirmed with the identification of mutations in the xon 15 3,42 bp, arAlmost all reported cases of choroideremia so far have been attributed to functionally null mutations combined with the slow rate of degeneration and small size of the CHM protein coding sequence, which make gene therapy with adeno-associated viral (AAV) vectors an appealing treatment strategy. The subsequent preclinical data for the effects of the vector (AAV2.REP1) in restoring prenylation activity to human choroideremia fibroblasts and initial findings from a phase 1/2 clinical trial have been described10CHM gene. More than 100 different pathogenic CHM mutations are currently documented in the RetinoGeneticsCHM gene, the investigations of Chinese patients with choroideremia are rather limited. We herein describe the phenotype and genotype of members in three Chinese families with choroideremia, and disclose the detection of novel mutations of the CHM gene firstly with the targeted exome sequencing (TES) technology.In published literatures, a wealth of disease-causing mutations have been reported, including nonsense mutations, missense mutations, frameshifts, splice site defects, gross insertions, small deletions, and deletion of the entire The study protocol was in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Committee of The Eye Hospital of Wenzhou Medical University. Informed consent was obtained from each participant. Three probands and two of their family members were enrolled in this study. Each of them underwent a comprehensive ophthalmologic examination comprising best-corrected visual acuity (BCVA), slit-lamp biomicroscopy, and color vision screening, which was supplemented with a series of imaging studies including fundus photography and optical coherence tomography (OCT). A panel of ophthalmologists and optometrists reviewed all ocular findings, and confirmed the clinical diagnosis of choroideremia in affected individuals. We contacted all of the genetically related family members, and obtained a detailed family history from each of that were recruited. Moreover, the five participants donated their blood for molecular studies. Physical examinations were performed to exclude systemic diseases, and no other remarkable diseases were found in the subjects’ medical histories.Genomic DNA was extracted from peripheral blood using standard organic extraction procedures. The DNA samples were quantified using a Nanodrop 2000 , and stored below −20 °C until experimental use. Libraries were prepared in the light of Illumina standard protocol. Briefly, a minimum of 3 μg of the DNA was sheared into 350- to 400-bp fragments. The fragmented DNA was end-repaired, a single adenine base was added to 3′ end, Illumina adapters were then ligated to the fragments, and the final products were PCR amplified. The probes, aiming to be hybridized in solution with the target library for TES experiments, were designed to potentially tile along 164 known causative inherited retinal dystrophy (IRD) genes, including one choroideremia-related gene. Afterwards, Illumina sequencing was performed in according to a previous studyhttp://bio-bwa.sourceforge.net/), and afterwards, base quality recalibration and local realignment were conducted using the GATK program (http://www.broadinstitute.org/gsa/wiki/index.php/Home_Page)14http://122.228.158.106/exomeassistant) was then used for annotation of all identified variants. MagicViewer was applied to view the short read alignment and ascertain the candidate SNPs and InDels. Missense variants were assessed with PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/www/SIFT_enst_submit.html), while the splice-site effect was predicted using the BDGP (http://www.fruitfly.org/seq_tools/splice.html) and ASSP programs (http://wangcomputing.com/assp/index.html).Sequencing reads were aligned to human hg19 reference using BWA (CHM gene were amplified by PCR. The reagents used for a 50 μl reaction were as follows: 25 μl of 2 × Power Taq PCR MasterMix (BIOTEKE CORPORATION), 2.5 μl each of 10 μM forward and reverse primer, 100 ng of genomic DNA template, and sterile H2O. Amplification was performed in a thermal cycler (Applied Biosystems Veriti 96-Well) with the following PCR protocol: initial DNA denaturation occurred at 95 °C for 5 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60s, and a final extension phase at 72 °C for 7 min. Afterward, the temperature was held at 4 °C. PCR products were subjected to agarose gel electrophoresis, excised from the gel, and subsequently sequenced. The sequencing results were analyzed using FinchTV (version 1.4.0), and compared with the NCBI database reference sequences. Sanger sequencing and co-segregation analyses in the family member samples confirmed the candidate variants.The DNA samples from the 5 participants in this study, as well as 200 unrelated Chinese control individuals, were analyzed by DNA sequencing. All 15 coding exons of the The inheritance patterns of the three Chinese families were all X-linked, as indicated by the familial pedigrees . The proWe designed a capture panel to enrich the target DNA by selecting 164 known IRD-related genes from RetNet and OMIM. The enriched DNA was then sent for TES. More than 96.9% of the targeted disease gene regions were sequenced, with an average sequencing depth of 104.38. A total number of 650 variants were identified in the samples. Among them, there were 386 non-synonymous variants, including missense, nonsense, and splicing variants. This number was narrowed down to 46 variants by excluding variants reported in dbSNP137, Exome Variant Server, and ExAC that had minor allele frequencies of (MAF) > 0.01. For missense variants, computational prediction by PolyPhen and SIFT as well as imposing restriction such as the consistency of genetic transmission mode were applied to further reduce the number of candidate mutations. The remaining variants were likely to cause the disease in patients and were further subjected to validation by Sanger sequencing.CHM gene were detected, none of which occurred in the 200 controls  and degradation of the inappropriately folded protein or truncated mRNA27CHM gene, producing a truncated REP-1 protein of 81 aa, 534 aa, and 236 aa, respectively. The truncated REP-1 protein was assumed to be degraded enzymatically in vivo in the affected members of these CHM families.In this work, candidate variants were only considered pathogenic when they satisfied following criteria: (1) missense mutations were positioned in the amino-acid conserved region across species, (2) splice-site variations fulfilled the GT-AT rules, (3) stop/frameshift variants were present, and (4) the mutations were predicted to be damaging or disease-causing by no less than two of the bioinformatic programs In summary, our study uncovered the clinical and genetic characteristics of three Chinese families with choroideremia. The primary methods used to identify disease-causative genes are currently positional cloning and linkage analysis. Nevertheless, there are tremendous limitations within the conventional strategies. Recently, targeted exome sequencing (TES) technology has become an efficient for exploring disease-related genesHow to cite this article: Cai, X.-B. et al. Novel CHM mutations identified in Chinese families with Choroideremia. Sci. Rep.6, 35360; doi: 10.1038/srep35360 (2016).


P < 0.01). Fibrinogen (FIB), PLT, and D-D increased in DN patients compared with those without complications (P < 0.001). Both aPTT and PLT were the independent risk factors for T2DM , and FIB and PLT were the independent risk factors for DN . The area under ROC curve (AUC) of aPTT and PLT was 0.592 and 0.647, respectively, with low sensitivity in predicting T2DM. AUC of FIB was 0.874 with high sensitivity (85%) and specificity (76%) for DN, and that of PLT was 0.564, with sensitivity (60%) and specificity (89%) based on the cutoff values of 3.15 g/L and 245 × 109/L, respectively. This study suggests that hemostatic parameters have a low predicting value for T2DM, whereas fibrinogen is a powerful predictor for DN.It would be important to predict type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN). This study was aimed at evaluating the predicting significance of hemostatic parameters for T2DM and DN. Plasma coagulation and hematologic parameters before treatment were measured in 297 T2DM patients. The risk factors and their predicting power were evaluated. T2DM patients without complications exhibited significantly different activated partial thromboplastin time (aPTT), platelet (PLT), and D-dimer (D-D) levels compared with controls ( The development of T2DM is a chronic process with approximately a decade-long latent period before the clinical onset of the disease . As the Many biological markers or biomarkers, such as C-reactive protein, gamma-glutamyl transpeptidase, or adiponectin, are associated with the risk of developing T2DM . Moreove1, etc.) in two weeks before sample collection. T2DM patients were divided into two groups including T2DM without complications group (202 patients) and T2DM with nephropathy group (95 patients). For comparison, 141 healthy controls matched for age, gender, and race were included in the study. The controls came from the healthy management center of the three hospitals and also did not take any abovementioned antithrombotic and hemostatic agents in two weeks before sample collection. Informed consent was obtained from the controls and patients or their relatives, and the study was approved by the institutional review board of the three hospitals.A total of 297 patients with T2DM, including 189 males and 108 females aged 37–85 (mean: 57 ± 21) years from the Departments of Endocrinology, Sanmen Hospital of Traditional Chinese Medicine, Sanmen People's Hospital of Taizhou City, Zhejiang Province, and Zhejiang Provincial People's Hospital, China, between October 2015 and December 2016, were finally enrolled in this retrospective study. All patients were Han Chinese and diagnosed according to the criteria in diabetes guideline 2013 of the China Diabetes Association [g at room temperature for 15 minutes. Subsequently, the measurements of PT, aPTT, TT, FIB, and D-D were performed with a coagulation analyzer  and the commercial reagents  within two hours after sample collection, respectively; INR and FDR were also calculated. For PLT analysis, two milliliters of blood sample was collected into vacutainer tubes  containing 3.6 mg EDTA-K2 and mixed completely. Platelet count was performed with a blood cell analyzer  in one hour after sample collection. Routine biochemical parameters  were measured. Estimated glomerular filtration rate (eGFR) was calculated according to the formula eGFR = 133 × (CysC/0.8)−0.449(−1.328 if CysC > 0.8mg/L) × 0.996age ×  [Samples were collected and prepared as described previously . In brie female) . And thet-test and Mann–Whitney U test were used, respectively. And the chi-square test was used for categorical variables. Univariate and multivariate logistic regression analyses were performed to calculate the odds ratio and 95% confidence interval of all hemostatic parameters to screen the risk factors for T2DM and DN. Receiver operating characteristic curve was constructed, and the area under curves was calculated to demonstrate the predicting power of the independent risk factors. Statistical analyses were performed using statistical package SPSS 20.0 . P value of less than 0.05 was considered statistically significant.One-way analysis of variance (ANOVA) was used to analyze the difference of the data among control, T2DM, and DN groups. For samples of normal and nonnormal distribution data (PLT and D-D), Student's P < 0.05). RDW, HbA1c, and CHOL levels in DN patients were higher than those in controls (P < 0.01), and there was no statistical difference between the two T2DM groups (P > 0.05).Basic clinical characteristics of the controls and patients are listed in P < 0.01). DN patients demonstrated increased fibrinogen (FIB), PLT, and D-D levels compared with patients without complications . Other parameters did not exhibit significant difference between the groups. The detailed data are presented in Patients without complications exhibited significantly shortened activated partial thromboplastin time (aPTT), decreased platelet (PLT) count, and increased D-dimer (D-D) level compared with controls . However, multivariate analysis revealed that both aPTT and PLT were independently correlated with T2DM without complications  and PLT , P < 0.01, resp.). Data are presented in P < 0.01, resp.). Detailed results are presented in The association of each parameter with the risk for T2DM and diabetic nephropathy was analyzed separately. Univariate analysis showed that INR, aPTT, D-D, DFR, and PLT among the laboratory variables of hemostatic assays were the risk factors for T2DM without complications . Based on the cutoff values, combinations of aPTT and PLT could increase the predicting power . Further study exhibited the high predicting power , high sensitivity (85%), and high specificity (76%) of FIB for nephropathy based on the cutoff value (3.15 g/L). However, PLT showed low predicting power  and sensitivity (60%) but high specificity (89%) for nephropathy according to the cutoff value (205 × 109/L). When combining FIB and PLT, the predicting power increased . Data are presented in ROC curve was used to evaluate the predicting power of the independent risk factors for T2DM and diabetic nephropathy. For T2DM without complications, the area under ROC curve (AUC) of aPTT and PLT demonstrated that there was low total predicting power , and the sensitivity was also low , whereas there was high specificity  according to the cutoff values (24.3 seconds and 176 × 10Patients with T2DM or DN may exhibit the abnormalities of biochemical parameters as what the results demonstrated in the present study, which were sure to be associated with the metabolic disorders and consistent with those of the studies reported by other authors . It has Various studies have demonstrated that T2DM and diabetic microvascular disease patients, especially those with diabetic nephropathy, would exhibit a hypercoagulable state and hemostatic abnormalities , 26, 27.Clinical practice has proved that combinations of several diagnostic parameters could increase the diagnostic and predicting power. As the present study revealed, the predicting significance of aPTT and PLT was low for T2DM, whereas FIB had high predicting significance for DN. Therefore, it would be important to investigate whether the combination of FIB and PLT had higher predictive power for DN in T2DM patients than their single use. As expected, the results demonstrated a higher AUC of 0.883, a sensitivity of 90%, and a specificity of 72% for predicting DN when using their cutoff values, respectively, than those of FIB or PLT single use. From the results, the study further indicated that combining FIB and PLT measurements may serve as a more useful tool in the prediction for nephropathy in T2DM patients.There are at least two limitations concerning the present study. Firstly, there probably were some nephropathy patients with other complications and T2DM patients with complications which were not diagnosed before treatment. Therefore, the laboratory data from those patients would cause some uncertain results and might potentially increase the predicting power of the hemostatic parameters. Secondly, the percentage of patients with serious nephropathy also might potentially increase the predicting power, and patients were not stratified based on disease condition of nephropathy in this study. Therefore, it was unclear whether there was significant difference for levels of hemostatic parameters between slight and serious DN. Although there were limitations, the present study also revealed the valuable predicting significance of fibrinogen and the combination of fibrinogen and platelet count in the prediction for nephropathy in T2DM patients.This study suggests that hemostatic parameters have a lower predicting value for type 2 diabetes mellitus, and fibrinogen is the independent risk factor with high predicting power for nephropathy in patients with type 2 diabetes mellitus, and a combination of fibrinogen and platelet count can increase the predicting power. However, further multicentric and controlled prospective studies on large groups of patients may give more definite results.