MYO15A gene (7.2%), followed by GJB2 (6.9%), MYO7A (5.5%), SLC26A4 (4.6%), TMC1 (1.8%), ESPN (1.8%), POU3F4 (1.8%), MYH14 (1.8%), EYA1 (1.8%), and MR-RNR1 (1.1%). The unique spectrum of causative genes in the Vietnamese deaf population was similar to that in the southern Chinese deaf population. It is our hope that the mutation spectrum provided here could aid in establishing an efficient protocol for genetic analysis of severe-to-profound hearing loss and a customized screening kit for the Vietnamese population.The mutational spectrum of deafness in Indochina Peninsula, including Vietnam, remains mostly undetermined. This significantly hampers the progress toward establishing an effective genetic screening method and early customized rehabilitation modalities for hearing loss. In this study, we evaluated the genetic profile of severe-to-profound hearing loss in a Vietnamese pediatric population using a hierarchical genetic analysis protocol that screened 11 known deafness-causing variants, followed by massively parallel sequencing targeting 129 deafness-associated genes. Eighty-seven children with isolated severe-to-profound non-syndromic hearing loss without family history were included. The overall molecular diagnostic yield was estimated to be 31.7%. The mutational spectrum for severe-to-profound non-syndromic hearing loss in our Vietnamese population was unique: The most prevalent variants resided in the  It has been shown that genetic causes account for about half of all known causes of these congenital hearing loss cases3. Hence, understanding the genetic etiology of hearing loss could aid practitioners to get a better understanding of the progression and risk of hearing loss in children, ultimately leading to the development of an optimal intervention strategy6. Most children with hearing loss were born to parents with normal hearing7, and genetic counseling for auditory rehabilitation and future family planning might be needed.The prevalence of infants born with moderate to severe bilateral hearing impairment is one in 1000, and the prevalence of infants with profound hearing loss, which causes severe speech impairment requiring cochlear implantation, is 4 in 10,000http://hereditaryhearingloss.org as of Apr 2018). It has been reported that not only are there numerous genetic variants responsible for hearing loss, but also the prevalence of the genetic variants is variable according to ethnicity and country of the study population. GJB2 variants are the most common cause of NSHL, and its detection rate differs depending on the ethnicity and country of origin; 20% in Caucasians of European descent8, 43% in Israelis9, 21~27% in Japan and Chinese12, 17% in Tunisians13, 17% in Iranian14, 14% in Australians15, and 10% in Koreans16. Furthermore, the GJB2 mutations were rarely found in African patients (0~1.0%)19. As such, the prevalence of GJB2 variants in hearing loss varied widely across different regions even within a single country. According to several genetic studies performed in Iran20, the prevalence of GJB2 varied from 0% to 38.3%, depending on the region. This diversity reflects the ethnic footprint of the Iranian population from neighboring countries. SLC26A4 (4.8~18%), MYO15A (9.6%), and MYO7A (~5%) were other prevalent causative genes in the Iranian population with NSHL20. In East Asian countries, mutations of SLC26A4 (10~13%) and mitochondrial DNA (1.8~2.1%) were frequently found following the mutation of GJB221. The mutations of GJB2, USH2A, MYO15A, and STRC genes were frequently found in the European population with autosomal recessive hearing loss22.However, molecular genetic diagnosis of congenital hearing loss is not always feasible. Deafness is an extremely heterogeneous disorder; to date, more than 400 various types of syndromic hearing loss as well as more than 163 chromosomal loci and 115 genes of non-syndromic hearing loss (NSHL) have been identified . The first step was to screen for the 11 known deafness-causing variants using the U-TOPTM HL Genotyping Kit. The second step was to perform Sanger sequencing of all the coding regions of GJB2 in those who remained undiagnosed after the first step (N = 76). Finally, the third step involved massive parallel sequencing (MPS) in a subset of affected subjects (N = 55) that remained undiagnosed after the former two steps.To evaluate the genetic profile of our Vietnamese population for severe-to-profound NSHL, we utilized a three-step genetic analysis protocol Fig. . The fir. These eleven subjects were divided into two groups. The first group (N = 9) included those with causative variants detected by the screening genotyping kit—c.235delC of GJB2 homozygotes (N = 1), p.V37I of GJB2 homozygotes (N = 4), p.V37I/c.235delC of GJB compound heterozygote (N = 1), c.IVS7-2A > G of SLC26A4 homozygotes (N = 1), p.T410M/c.IVS7-2A > G of SLC26A4 compound heterozygote (N = 1), and c.1555 A > G of MR-RNR1 (N = 1) . The second group (N = 2) included those who underwent Sanger sequencing of SLC26A4 for further analysis—two compound heterozygotes of SLC26A4:p.H723R/c.1344_1355dup and c.IVS7-2A > G/c.1547dupC . To address the pathogenic potential of p.V37I in this population, the Vietnamese control group with normal hearing was also screened using U-TOPTM HL Genotyping Kit, with a special focus on p.V37I. A total of 19 single heterozygotes of p.V37I of GJB2 and two homozygotes of p.V37I of GJB2 were identified from the control group with normal hearing (N = 117) . Resultantly, the frequency of p.V37I homozygote in the HL group  was higher than that of the ethnicity-matched control group , although the difference was not statistically significant (p = 0.154).Among the 87 subjects segregating the severe-to-profound NSHL, complete molecular diagnosis was made on 11 subjects  through the first screening step using the U_TOP HL genotyping kit 17) Figs . ResultaGJB2 – to exclude other pathogenic variants, if any, elsewhere in GJB2. Single heterozygotes of p.V37I allele (N = 22) and single heterozygote of c.299delAT (N = 1) of GJB2, with no other pathogenic variants discovered elsewhere in GJB2 through GJB2 Sanger sequencing, were considered undiagnosed in the HL group . None of these subjects carried the known non-coding region variants of GJB2 or the known large deletions of GJB6.All the remaining 76 subjects underwent the second step – Sanger sequencing of JB2Fig. . Single elAT N =  of GJB2,. Among the 55 subjects, four (7.3%) subjects turned out to have ‘causative pathogenic variants’ involving two genes . . Meanwhile, eight subjects  with severe-to-profound NSHL were classified as the group with variants of uncertain significance. Five subjects with variants in MYO15A, MYO7A, TMC1, and ESPN genes, which were inherited in autosomal recessive mode, were identified. In addition, one boy (HL-78) with a single novel variant of uncertain significance in POU3F4 with X-linked inheritance pattern and two subjects with a single variant in MYH14 and EYA1 genes with autosomal dominant inheritance pattern were identified  without reaching a final molecular genetic diagnosis, and the other 24 subjects (43.6%) did not carry any potentially pathogenic variant as documented by MPS . In this study, the overall molecular diagnostic rate through our three-step protocol in our Vietnamese population with severe-to-profound NSHL was estimated to be 31.7% when the subjects with variants of uncertain significance were included and 19.0% when they were not.To further elucidate the causative variants in subjects who remained undiagnosed after the first two steps, the third step – MPS targeting the genes related to hearing loss – was performed Fig. . Among tgenes MYO5A, MYO7Agenes MYO5A, MYO7Agenes MYO5A, MYO7Agenes MYO5A, MYO7Aied Fig.  without genes MYO5A, MYO7AMYO15A variants were the most common cause of severe-to-profound NSHL in our Vietnamese population, and the prevalence of the MYO15A variants that were classified as (likely) pathogenic variants or uncertain significance was 7.2% (N = 4/55) (Table 2). Next, the autosomal recessive variants of GJB2 , MYO7A , and SLC26A4  were identified as the next tier of causes in this population (Table 2). The frequency of variants in ESPN , TMC1 , POU3F4 , MYH14 , and MR-RNR1  were less common in our Vietnamese population with severe or greater degree of HL.The MYO15A, GJB2, MYO7A, and SLC26A4 variants were shown to be the leading causes in our cohort, and the estimated prevalence was 7.2%, 6.9%, 5.5%, and 4.6%, respectively. The variants of TMC1, ESPN, POU3F4, MYH14, EYA1, and MR-RNR1 genes followed those from the top tier deafness genes in the order of frequency as a molecular etiology of severe-to-profound NSHL in Vietnamese.In this study, we elucidated the mutation spectrum of severe-to-profound NSHL in the Vietnamese pediatric population for the first time. GJB2 variants compared with other ethnic populations, including East Asians. The prevalence of the DFNB1 due to GJB2 variants in our Vietnamese pediatric population with severe-to-profound NSHL was calculated to be 6.9%, which could be further lowered to 2.3% if the p.V37I homozygotes were excluded. GJB2 variants are the leading genetic etiology of NSHL in many populations, even though the detection rate of GJB2 variants can significantly differ depending on the ethnicity and country. The prevalence of GJB2-associated hearing loss was quite variable, ranging from 0% in South Africa to 40~50% in Eastern Europe25. According to a nationwide genetic study about the GJB2 mutation spectrum in China, there was a significant difference in the detection rate of pathogenic GJB2 allele depending on the region within China, varying from 4% in Guangxi to 30.4% in Jiangsu, while the overall frequency of pathogenic GJB2 allele among moderate to severe hearing loss was 17.9%26. Interestingly, Guangxi, a city associated with a low prevalence of GJB2 variants, is in the southern part of China, bordering Vietnam. Vietnam is the easternmost country on the Indochina Peninsula, bordering China to the north, Laos to the northwest, Cambodia to the southwest, and Thailand across the Gulf of Thailand to the southwest. The population of Vietnam was approximately 94.6 million in 2016, and the ethnic group, “Kinh,” comprised 86% of the total population. Kinh has been known to originate from northern Vietnam. Recent phylogenetic studies revealed that the majority of Kinh originated from South China and a minority from Thailand and Indonesia27. Therefore, the low prevalence of detectable GJB2 variants could be viewed as the genetic similarity between the Kinh people and the people from southern parts of China. This suggests a close ethnic relationship between these groups. Low prevalence of detectable GJB2 variants does not necessarily indicate low prevalence of DFNB1 in these populations and might suggest the presence of unidentified variants, such as large genomic deletions residing somewhere in the DFNB1 locus, and thus affecting GJB2 expression and function. These variants would have evaded our current molecular diagnostic protocol. This hypothesis requires future confirmation. The genetic similarity with the southern parts of China can also be seen in the SLC26A4 mutation. Unlike the northern area of China, in which the prevalence of SLC26A4 mutation has been reported to range from 10 to 13%30, patients with severe-to-profound hearing from the Guangxi province showed limited prevalence of SLC26A4 variants (2.2%)31, similar to our Vietnamese cohort.It may be worth noting that the mutation spectrum of this Vietnamese deaf population is unique and distinct. First, there was a significantly lower genetic load of 33 and European populations (0.6%)25. This allele was also frequently detected in the population of Thailand, which is bordered by Vietnam (16.7%)34, questioning the causative role of this allele to severe to profound deafness in Vietnamese. Among the 23 subjects in the HL group with one p.V37I allele detected on the first screening step, four subjects had pathogenic or uncertain significance variants in other deafness genes unrelated to GJB2  (Table ). The other 19 subjects showed either one  or no  variant with pathogenic potential from other unrelated deafness genes (see Supplementary Table S1). Interpretation of causality between p.V37I homozygotes and severe-to-profound deafness warrants further caution because two homozygotes of p.V37I of GJB2 were also identified in the control group with normal hearing , and the prevalence of the homozygotes of p.V37I of GJB2 (1.7%) in the control group was not significantly different from that (4.6%) in the HL group (p = 0.154). However, the p.V37I variant has been previously reported to have a diverse clinical presentation, ranging from normal-to-profound hearing loss36, and the prevalence of p.V37I homozygote was, even if not statistically significant, higher in the HL group than in the control group (4.6% vs 1.7%) in our study. Given this, at least a subset of p.V37I homozygotes in the Vietnamese population can potentially cause severe-to-profound hearing loss. Both normal hearing from the p.V37I homozygotes in the control group and profound hearing loss from some p.V37I homozygotes in the HL group might be phenotypes of both extremes from the same genetic alteration, which is modulated by modifiers. To clarify the pathogenic role of p.V37I in severe-to-profound hearing loss, further study evaluating the influence of genetic modifiers on the phenotype of homozygous p.V37I variant or exploring the yet-to-be identified, pathogenic DFNB1 allele in trans with single heterozygous p.V37I, such as copy number variation (CNV)40, is warranted.Second, the detection frequency of p.V37I in our Vietnamese population with severe-to-profound NSHL 18.4%) was significantly higher than that in East Asian populations (1~6.7%)8.4% was MYO15A was shown to be the most frequent causative gene (7.2%) in our Vietnamese population, with a much higher prevalence than that in the European populations (3.1%)41. In fact, the MYO15A gene, which is a causative gene of DFNB3 (OMIM 600316) and has been known to be associated with moderate to severe hearing loss as well as profound hearing loss, is usually ranked as the third or fourth most common cause of DFNB deafness in many ethnicities45. The MYO7A variants also accounted for a significant proportion (5.5%) of severe-to-profound hearing loss in our Vietnamese population, which was higher than the estimates found in other studies44. MYO7A is located on 11p13.5, encoding the myosin 7 A protein. To date, there have been more than 280 different pathogenic or likely pathogenic mutations and 380 variants of uncertain significance reported from MYO7A (https://www.ncbi.nlm.nih.gov/clinvar as of Aug 2018), which have been associated with Usher syndrome 1B46, non-syndromic autosomal recessive hearing loss (DFNB2)47, and autosomal dominant hearing loss (DFNA11)48. Patients diagnosed with Usher syndrome type 1B having MYO7A variants typically show profound hearing loss at birth and progressive retinal degeneration49.Third, MYH14 was considered as a contributor to severe-to-profound hearing loss in our Vietnamese population. The MYH14 gene encodes myosin-14 protein and was reported to be an important causative gene for prelingual severe NSHL with autosomal dominant inheritance. This is unique considering the usual auditory phenotype of autosomal dominant deafness genes50. In our present study, we were unable to determine whether this MYH14 variant occurred de novo due to the lack of parental samples, even though auditory phenotypes of parents were allegedly normal. Therefore, we classified these as variants of uncertain significance. Nonetheless, the MYH14 variant was still considered the variant with pathogenic potential due to their extremely rare MAF in publicly available databases.Another interesting finding is that the potentially pathogenic autosomal dominant variant of TM HL genotyping kit and Sanger sequencing of GJB2, respectively – does not seem high, only reaching 12.6% including the homozygous p.V37I variants of GJB2. The U-TOPTM HL genotyping kit, initially designed to screen East Asian subjects with hearing loss, does not appear to be suitable for the Vietnamese population with hearing loss, where MYO15A and MYO7A were the most frequent causative genes. Accordingly, an efficient screening kit customized for the mutation spectrum of this population should be designed. Moreover, we were able to detect the causative pathogenic variants or variants of uncertain significance using MPS in about 21.8% (N = 12/55) of our subjects with NSHL. In total, the diagnostic yield of our three-step molecular diagnostic protocol, including MPS, was estimated to be 31.7% in our Vietnamese population, which was inferior to other studies that used massively parallel sequencing51.Currently, the diagnostic yield from the first two steps – U-TOPtrans-configuration of two compound heterozygous due to lack of parental samples in our cohort. Therefore, some genotypes obtained from the MPS analysis may not be conclusive, and the diagnostic yield of the three-step protocol employed in our study for molecular diagnosis of severe-to-profound hearing loss may be slightly overestimated. These issues could be addressed in a future study using segregation analysis or MPS with the inclusion of family members. Secondly, the variants of other deafness genes or CNVs, which were not included in the targeted deafness genes in this study or could not be detected with our diagnostic protocol, may have been missed. CNV has been identified as one of the frequent causes of ARNSHL. According to the literature, at least one CNV in a known deafness gene was found in 15.2% of patients with hearing loss, and causative CNVs were identified in 7.3% of these patients as homozygous, hemizygous in conjunction with a second pathogenic mutation, or biallelic CNVs37. Large genomic deletions, involving GJB2 or GJB6, have previously been reported as a causative mutation related to hearing loss40. Therefore, novel or unrevealed CNVs in the coding or noncoding region of GJB2 or GJB6, which would lead to null function, can explain severe-to-profound auditory phenotypes in our participants in a trans configuration with the single heterozygous p.V37I . As aforementioned, the prevalence of GJB2 variants in our Vietnamese population was estimated to be only 2.3% when the p.V37I homozygotes were excluded. However, upon discovery of such occult genomic deletion, some of the single heterozygotes of p.V37I can later be classified as DFNB1, leading to an increase in the prevalence of DFNB1 in the Vietnamese population. Hence, in the next study, the presence of occult genomic deletion, if any, in the DFNB1 locus (either DFNB1A or DFNB1B) among these putative p.V37I carriers in a Vietnamese population will be explored by multiple ligation probe amplification (MLPA) covering the entire DFNB1 locus. Lastly, MPS test was not performed in 25 patients and their genetic diagnosis remained inconclusive, which may have been another limitation of this study.There were several limitations in this study, with respect to the confirmation of pathogenicity of detected variants and diagnostic rate of our molecular diagnostic protocol. We were unable to perform additional analyses, including a segregation study necessary to confirm the co-segregation of the variants with deafness among the family members and the In conclusion, our study is the first attempt to reveal the characteristics of mutation spectrum of severe-to-profound NSHL in the Vietnamese population, providing the basis for the establishment of a comprehensive molecular genetic diagnostic protocol. Furthermore, our data provide supportive evidence suggesting an ethnic relationship between the peoples of Vietnam and those in the southern parts of China.Between September 2015 and May 2016, children who visited the Children Hospital 1 in Vietnam with delayed language development or decreased responses to sound stimuli were included in this study. A total of 87 children  were selected and included. All participants underwent an audiological evaluation. The hearing thresholds at 0.5, 1, 2, and 4 kHz were evaluated with pure-tone audiometry, and the average threshold was calculated. In the cases where pure-tone audiometry could not be performed due to the subjects’ young age, the hearing level was determined using the auditory brainstem response threshold test or visual reinforcement audiometry. The inclusion criteria were severe-to-profound hearing loss with over 70 dB of hearing thresholds. Children who had syndromic hearing loss or familial history of hearing loss were excluded; to avoid any bias from acquired hearing loss, subjects who were older than 12 years were also excluded. The mean age was 4.6 ± 2.2 (0–11) years at the time of their initial visitation. Moreover, a total of 117 participants  with normal hearing under 25 dB were included in the control group. The mean age of the control group was 33.5 ± 9.6 (20–66) years. The Institutional Review Boards (IRBs) of Seoul National University Bundang Hospital (SNUBH) in Korea (IRB-B-1007-105-402) and Research Center of Children Hospital 1 in Vietnam (CS/1/15/13) approved this study. All procedures were performed in accordance with relevant guidelines and regulations. We obtained written informed consent from all participants in accordance with the Declaration of Helsinki; in the case of children, written informed consent was obtained from their patient or guardian. After obtaining written informed consent, ten milliliters of whole blood for genetic analysis was obtained from all participants. All data generated or analyzed during this study are included in this published article and its supplementary information file.. This new screening tool was designed to detect variants known to be associated with mild-to-moderate hearing loss, as well as to screen variants associated with severe-to-profound hearing loss52. The molecular diagnostic platform, based on ethnicity-specific mutation spectrums of sensorineural hearing loss, contain 11 variants of 5 genes  showing high prevalence with varying degrees in the Korean population. The phenotype of the p.V37I variant of GJB2 was reported as mild to profound hearing loss55. Therefore, the homozygosity of p.V37I allele of GJB2 was considered as causative pathogenic variant in this study. The compound heterozygote of V37I and other pathogenic variant of GJB2, such as p.R143W, were proposed as possible causative variant of moderate to severe hearing loss55, and were included as causative pathogenic variant in this study.A total of 87 subjects in the HL group and 117 normal participants in the control group of our Vietnamese population were screened using the U-TOP™ HL Genotyping Kit , which is a real-time PCR-based MeltingArray genetic diagnostic kit GJB2 or SLC26A4 was detected with the screening kit, Sanger sequencing for the entire coding region of the corresponding gene, beyond the coverage of the screening kit, was additionally performed to find the second variant. A rare novel variant of SLC26A4 in a trans configuration with a definitely pathogenic SLC26A4 variant was considered as ‘pathogenic’, as previously proposed56.When only one variant of either GJB2 gene was performed . Next, Sanger sequencing for previously reported, non-coding variants of GJB2 and evaluation of known genomic deletion of GJB6 was performed. In detail, Sanger sequencing for the four known pathogenic variant on the noncoding regions, c.-22-2A > C57, c.-23G > T58, c.−23 + 1 G > A59, and c.−259C > T60 was performed with two primer sets, as previously described61. Next, a multiplex breakpoint PCR assay was performed for two previously reported large genomic deletions (del[GJB6-D13S1830] and del[GJB6-D13S1854])40. To detect other structural variations on the 5-kb regions upstream of GJB2 within the DFNB1 locus, we also verified the raw data of MPS, if available, using the Integrative Genomic Viewer (http://www.broadinstitute.org/igv/home).For 76 subjects whose causative genetic variants were not identified with a screening test, Sanger sequencing for the coding region of the med Fig. . Next, S63. In total, 32–69 million short reads (100-bp paired-end reads) were obtained via MPS. More than 85% of the target exon regions were covered by at least five sequence reads. The reads were mapped onto the UCSC hg19 reference genome. The non-synonymous single nucleotide polymorphisms (SNPs) were filtered with a depth ≥ 15. Minor allele frequency (MAF) of the variants was evaluated from publicly available databases, including ExAC (http://exac.broadinstitute.org/), 1000 Genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genome), GO-ESP (http://evs.gs.washington.edu/EVS/), and GnomAD (http://gnomad.broadinstitute.org/). Variants with MAF ≥ 0.5% were excluded, unless they were pathogenic according to the literature, ClinVar, or Deafness Variation Database (http://deafnessvariationdatabase.org/) 64. We used a threshold of 0.5% for ARNSHL and 0.05% for ADNSHL65. We interpreted and classified the pathogenicity of our variants as one of the followings: Pathogenic, likely pathogenic, benign, likely benign, and uncertain significance according to the ACMG guidelines (Table 66. Patient cohort was divided into four groups: (1) patients with causative (likely) pathogenic variants; (2) patients with variants of uncertain significance; (3) patients with only one variant of the gene with autosomal recessive inheritance pattern; (4) patients without detected causative variants 22.A subset of subjects whose causal genetic variants were not identified with the U-TOPTM HL genotyping kit were evaluated with targeted exome sequencing  and whole exome sequencing . TES and WES were captured using the NimbleGen Sequence Catcher  and SureSelect 50 Mb Hybridization and Capture kit, respectively. Bioinformatics analyses were performed, as previously described, against the targeted genes related to hearing loss Fig. 62,63. InSupplementary information


Combined laparoscopic resection of liver metastases and colorectal cancer (LLCR) may hold benefits for selected patients but could increase complication rates. Previous studies have compared LLCR with liver resection alone. Propensity score-matched studies comparing LLCR with laparoscopic colorectal cancer resection (LCR) alone have not been performed.A multicenter, case-matched study was performed comparing LLCR  with LCR alone . Patients were matched based on propensity scores in a 1:1 ratio. Propensity scores were calculated with the following preoperative variables: age, sex, ASA grade, neoadjuvant radiotherapy, type of colorectal resection and T and N stage of the primary tumor. Outcomes were compared using paired tests.n = 44, 69%) of small size (≤ 3 cm) . LLCR was associated with a modest increase in operative time  and blood loss . The rate of Clavien–Dindo grade 3 or higher complications , anastomotic leakage , conversion rate  and 30-day mortality  did not differ between LLCR and LCR.Out of 1020 LCR and 64 LLCR procedures, 122 (2 × 61) patients could be matched. All 61 laparoscopic liver resections were minor hepatectomies, mostly because of a solitary liver metastasis (In selected patients requiring minor hepatectomy, LLCR can be safely performed without increasing the risk of postoperative morbidity compared to LCR alone. Approximately 15–25% of patients with colorectal cancer will have synchronous colorectal liver metastases at the time of diagnosis , left lateral sectionectomies [7 (11%)], and total segmentectomies [12 (19%)]. In 17 patients (25%), two or more resections were performed. For the primary tumor, low anterior/sigmoid resection  and 125 ml increase in blood loss . Other operative outcomes did not differ between the groups. All outcomes after matching are displayed in Tables LLCR was associated with a 9-min longer operative time [206 166–308) vs. 197  and blood loss (125 ml). Hospital stay was similar between LLCR and LCR alone, indicating a benefit of LLCR in these highly selected patients by omitting the need for a second hospital admission with its associated risks, costs and emotional burden for the patient. Based on these results, it seems worthwhile for experienced centers to screen and select patients with synchronous colorectal liver metastases who require minor hepatectomy for LLCR.Despite single center reports on the feasibility and safety of LLCR, the true impact of adding a laparoscopic liver resection to a LCR on postoperative morbidity has never been investigated –17. The Laparoscopy could have played a role in the relatively low rate of major morbidity after LLCR in this series. In most centers nowadays, laparoscopic surgery is considered standard of care for primary resectable colorectal cancer, and most recent consensus meetings on laparoscopic liver surgery have declared laparoscopy the standard for minor liver resections as well –25. In bThe current study had several limitations. First, the retrospective design clearly introduced a risk of selection bias. Selection criteria were, however, essentially similar in the four participating centers and are described in the methods section. Even though laparoscopic major liver resections were performed in all centers during the study period, these patients were not considered to be candidates for LLCR. Second, the size of the cohort did not allow for identification of subgroups, for instance comparing outcome after left- and right-sided colon cancer resection. A randomized controlled trial designed to answer the question whether LLCR is superior to a staged resection seems unlikely, so matching based on propensity scores is probably the next best methodology. Larger cohorts could help to further identify subgroups when it comes to the surgical treatment of synchronous colorectal liver metastases. In order to increase the potential of finding a matching LCR control patient, metastasized colorectal tumors were not excluded. This means that some of these patients might undergo further surgery for metastatic disease after colorectal resection. The aim of this study, however, was to investigate short-term outcomes and there is no current literature available suggesting that in situ metastatic disease influences the outcomes of colorectal or liver surgery. Long-term results of simultaneous resections, such as disease-free and overall survival, remain uncertain, especially considering the possible extravasation of tumor cells during colorectal resection that could settle down in areas of tissue damage and inflammation such as the liver surface after resection. Finally, the potential advantages of LLCR were not specifically addressed in this study, which can be seen as a limitation. The most accurate comparison would be between LLRC and the cumulative of both colorectal and liver resection in a delayed setting. These patients were difficult to identify and due to the selection bias for the extent of liver resection matching would not have been possible.Postoperative hospital stay has consistently been shown to be decreased with open combined resection when compared to the cumulative length of stay when performing sequential resections . The curIn conclusion, this study showed that LLCR is feasible and does not increase postoperative morbidity compared to LCR alone, in selected patients with synchronous colorectal liver metastases requiring a minor liver resection, operated in experienced centers.


Nevertheless, extensive investigations on the molecular aspects of TCS action have identified numerous adversaries associated with the disinfectant including oxidative injury and influence of physiological lifespan and longevity. This review presents a summary of the biochemical alterations pertaining to TCS exposure, with special emphasis on the diverse molecular pathways responsive to TCS that have been elucidated during the present decade.Triclosan (TCS) is a synthetic, chlorinated phenolic antimicrobial agent commonly used in commercial and healthcare products. Items made with TCS include soaps, deodorants, shampoos, cosmetics, textiles, plastics, surgical sutures, and prosthetics. A wealth of information obtained from  M. MCarasstadpoles  and indug larvae .2) and a heterogeneous mixture of organic compounds including TCS using the clam Ruditapes philippinarum. Modulations in EROD, SOD, CAT, GPx, GST, and GR enzyme activities were noted in the clam's digestive gland, in parallel with increased lipid peroxidation. TCS exposure caused alterations in Cat, Sod, Gpx1, Gpx2, Gsta, Hsp90bb, Hsp90ba, and Hsc70a genes in rainbow trout Oncorhynchus mykiss estradiol was successfully displaced from the ER by the antimicrobial [μM TCS attenuated E2-dependent ERE-CAT reporter gene induction, while 0.1 and 1 μM TCS inhibited TSN-stimulated LTR-CAT reporter gene in both T47D cells and S115 mouse mammary tumor cells [With regard to the androgenic properties of TCS, it was revealed that TCS interferes with testosterone- (TSN-) related transcription but promotes that dependent on androgen , 182. Ind et al.  reportedd et al. . These fd et al.  in whichicrobial . Furtheror cells . TCS wasor cells  showed tor cells .α in HepG2 cells and mouse hepatoma Hepa1c1c7 cells, distinct responses were observed by Wu et al. [α downstream target, acyl-coenzyme A oxidase, were decreased in HepG2 cells but were increased in Hepa1c1c7, which also showed higher DNA synthesis and blunted apoptosis through transforming growth factor (TGF-β). PPAR signaling was similarly identified as a target of TCS through genome-wide CRISPR-Cas9 screening in HepG2 cells [Gallus gallus chicken embryos [Cyp7a1, fatty acid-binding protein 1 (Fabp1), acyl-CoA synthetase long-chain family member 5 (Acsl5), acyl-CoA oxidase 2 (Acox2), and perilipin 1 (Plin1) were upregulated, whereas angiopoietin-like 4 (Angptl) was downregulated.Controversy surrounding the interaction between TCS and members of the peroxisome proliferator-activated receptors (PPARs) has gained considerable attention as of late. This has essentially stemmed from the apparent discrepancy between data obtained from humans and those from rodents. In comparing the differential modulation of TCS on PPARu et al. . ProteinG2 cells , zebrafiG2 cells , and Gal embryos . In the γ activator, rosiglitazone, solely reversed the decrease in Akt phosphorylation in adipose tissue and in muscle [γ is known to ameliorate mTOR suppression-induced glucose intolerance in rats [TCS administered to pregnant mice caused insulin resistance, hypothyroidism, diminished glucose transporter 4 (GLUT4) expression, and inhibition of Akt and mTOR phosphorylation , 192. Wh in rats , furtherα following TCS treatment. Importantly, the authors also identified constitutive androstane receptor (CAR) as a possible aggravator of TCS-induced tumorigenesis, given the halved tumor number in Car–/− mice compared to their Car+/− counterparts. TCS, as is the case with PPARs, is reported to exhibit varying affinities for CAR and pregnane X receptor (PXR) in humans and rodents. A weak agonist for human CAR, TCS was found to be a reverse agonist for rodent CAR, an agonist for human PXR, and had no effect on rodent PXR [Although TCS has been reported to promote hepatocyte proliferation in mice through PPAR , Yueh etdent PXR .2+, on the other hand, maintains the ion's content within a physiological range and sets forth diverse cellular activities related to gene expression, motility, secretion, and survival [2+ levels are modulated by a variety of stimuli, including xenobiotic exposure. Through the Ca2+ channel ryanodine (Ry) receptor type 1 (RyR1), TCS increased cytosolic Ca2+ dose-dependently in primary skeletal myotubes irrespective of extracellular Ca2+ [in vitro and in vivo [2+ transients in myotubes without depleting intracellular Ca2+ and notwithstanding RyR1 blockage. TCS also efficiently blocked excitation-coupled Ca2+ entry and interfered with the bidirectional signaling between RyR1 channels and Ca2+ ions. Likewise, TCS compromised ECC in larval fathead minnows Pimephales promelas, as evidenced by altered RyR and dihydropyridine receptor (DHPR) mRNA and protein levels and weakened ligand binding to both receptors in adult muscle homogenates [Calcium concentration within cells influences protein conformation and dynamics. Protein binding of Casurvival . Beside lar Ca2+ . Accordi in vivo . Resultsogenates .2+ levels and opened Ca2+-responsive K+ channels, eventually leading to membrane hyperpolarization [2+-induced mitochondrial swelling in rat liver [2+ homeostasis was conducted on rat basophilic leukemia (RBL) mast cells [2+ and depleted cytosolic Ca2+ levels following antigen stimulation. Accordingly, TCS-induced degranulation of mast cell may at least in part be attributed to Ca2+ mobilization.In rat thymocytes, TCS elevated intracellular Carization . Also, Tat liver . A more st cells . In thisC. reinhardtii exposed to 14 μM TCS, increased Ca2+ levels with oxidative stress, cell and mitochondrial membrane depolarization, compromised photosynthesis, and caspase activation were noted [2+ ions by BAPTA-AM protected the algae from TCS-induced Ca2+ dysregulation. These observations strongly implicate Ca2+ as a mediator of a wide array of toxic anomalies attributed to TCS.Calcium modulation by TCS has also been investigated in other organisms. In re noted . ImportaLiterature concerning the xenobiotic response to TCS has revealed important signaling pathways activated or suppressed by TCS . DistincEscherichia coli was noted following exposure to TCS [in vivo. Further, a mutated or overexpressed ACP, encoded by fabI, was shown to confer TCS resistance in the bacterium. These findings established ACP as a specific, subcellular TCS target. Efforts have thus far revealed the susceptibility of a host of other pathogens to inhibition of fatty acid synthesis by TCS. These include Staphylococcus aureus, M. tuberculosis, Helicobacter pylori, Haemophilus influenzae, Plasmodium falciparum, Toxoplasma gondii, Leishmania spp., and Trypanosoma spp. [E. coli FabI protein and FAS showed no homology, appreciable sequence similarities were nevertheless found with polyketide synthase and type I FAS of M. tuberculosis [The first specific action mechanism of TCS in prokaryotes was only demonstrated 20 years ago, when inhibition of fatty acid synthesis in e to TCS , 201. TComa spp. , 202–208oma spp. , 210. Alrculosis .in vivo has spawned several reports in support of fatty acid synthesis inhibition as an emerging target for chemotherapy [μM is cytotoxic and antiproliferative, induces morphological alterations, and inhibits FAS. These findings corroborate an earlier observation linking FAS inhibition with apoptotic death of breast cancer cells [μM [50 values as low as 4.5-7.8 μM [μM, values of IC50 ranging from 0.74 to 62 μM were nonetheless observed in nonmalignant prostate cells. This suggests two things; first, that prostate cells are relatively more sensitive to TCS toxicity than fibroblasts and presumably other nonmalignant cell types, and second, that malignant prostate cells exhibit higher chemosensitivity compared to their nonmalignant counterparts. This differential susceptibility could be due to overexpressed FAS in malignant cells. However, in contrast to these reports, at concentrations up to 345 μM, TCS was found to be preferentially cytotoxic to Y79 RB cells over mouse 3T3 fibroblasts and human MIO-M1 Müller glial cells as indicated by IC50 values, creating a large therapeutic index of 7.1 and 5.3, respectively [μM was also shown to be effective against MiaPaCa-2 and AsPC-1 pancreatic cancer cells suppressing proliferation and eliciting apoptotic death [The success of cerulenin, a mycotoxin with fatty acid inhibitory action, in suppressing tumor progression otherapy . The earotherapy . It was er cells , 214. TCer cells . In humaells [μM . TCS was5-7.8 μM . Whereasectively . FAS supectively . Recentlic death . Of noteic death . Given tin vivo results, cements the antimicrobial as a promising candidate for chemotherapy. As noted earlier, it must be stressed that variations in the final outcome of TCS treatment largely depend on experimental setup. Moreover, limited data from animal studies suggest that in the presence of a preexisting tumor, TCS administration seems to exacerbate the condition. This observation is concerning and indeed warrants further investigation before TCS can be invested in for clinical trials.The differential expression and activity of FAS in healthy and malignant tissues, where it is upregulated in the latter , 221, inin vitro and in vivo studies have provided evidence of adverse effects on diverse molecular pathways. Most alarmingly is TCS enhancement of malignant cell proliferation in vitro and tumor growth in vivo. On the other hand, TCS has also been shown to be protective against malignant cell growth and proliferation, possibly opening the door for its use in chemotherapy. Clearly, dose and time dependence is an important factor in determining the eventual denouement of the chemical. In spite of the numerous publications dissecting the signaling pathways responsive to TCS, it is evident that a severe paucity surrounding human-based in vivo and in vitro studies still remains today. Future studies, thus, should focus on identifying signaling molecules differentially regulated by TCS and characterize their roles in toxic or protective effects in different cell types. Insights gained from such revelations will be invaluable to possibly validate targets for drug development or devise possible TCS adjuvants or inhibitors.TCS is a synthetic antimicrobial with a long history of human use. At concentrations well below those present in commercial products, data from


The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) and aspirin both have proof of concept for colorectal cancer chemoprevention, aligned with an excellent safety profile. Therefore, we aimed to test the efficacy of EPA and aspirin, alone and in combination and compared with a placebo, in individuals with sporadic colorectal neoplasia detected at colonoscopy.In a multicentre, randomised, double-blind, placebo-controlled, 2 × 2 factorial trial, patients aged 55–73 years who were identified during colonoscopy as being at high risk in the English Bowel Cancer Screening Programme  were recruited from 53 BCSP endoscopy units in England, UK. Patients were randomly allocated (1:1:1:1) using a secure web-based server to receive 2 g EPA-free fatty acid (FFA) per day (either as the FFA or triglyceride), 300 mg aspirin per day, both treatments in combination, or placebo for 12 months using random permuted blocks of randomly varying size, and stratified by BCSP site. Research staff and participants were masked to group assignment. The primary endpoint was the adenoma detection rate  at 1 year surveillance colonoscopy analysed in all participants with observable follow-up data using a so-called at-the-margins approach, adjusted for BCSP site and repeat endoscopy at baseline. The safety population included all participants who received at least one dose of study drug. The trial is registered with the International Standard Randomised Controlled Trials Number registry, number ISRCTN05926847.Between Nov 11, 2011, and June 10, 2016, 709 participants were randomly assigned to four treatment groups . Adenoma outcome data were available for 163 (93%) patients in the placebo group, 153 (85%) in the EPA group, 163 (92%) in the aspirin group, and 161 (91%) in the EPA plus aspirin group. The ADR was 61% (100 of 163) in the placebo group, 63% (97 of 153) in the EPA group, 61% (100 of 163) in the aspirin group, and 61% (98 of 161) in the EPA plus aspirin group, with no evidence of any effect for EPA  or aspirin . EPA and aspirin were well tolerated , although the number of gastrointestinal adverse events was increased in the EPA alone group at 146 events . Six upper-gastrointestinal bleeding events were reported across the treatment groups .Neither EPA nor aspirin treatment were associated with a reduction in the proportion of patients with at least one colorectal adenoma. Further research is needed regarding the effect on colorectal adenoma number according to adenoma type and location. Optimal use of EPA and aspirin might need a precision medicine approach to adenoma recurrence.Efficacy and Mechanism Evaluation Programme, a UK Medical Research Council and National Institute for Health Research partnership. Despite substantial advances in diagnosis and treatment, colorectal cancer remains the second most common cause of cancer death in the UK, causing 16 000 deaths in 2014, with an estimated 1·4 million cases diagnosed worldwide in 2012.One strategy to reduce colorectal cancer incidence and mortality is prevention. Prevention measures include population screening  and endoscopic surveillance of high-risk groups.Evidence before this studyWe searched PubMed for articles published in English between Jan 1, 1990, and Jan 1, 2011, using the terms “colorectal cancer”, “colorectal adenoma”, “chemoprevention”, “omega-3 polyunsaturated fatty acid”, “aspirin”, and “clinical trial”. We found one small randomised trial of eicosapentaenoic acid (EPA) for colorectal cancer chemoprevention in familial adenomatous polyposis and four randomised sporadic polyp prevention trials of aspirin, which used varying doses in different risk populations.Added value of this studyTo our knowledge, this is the first randomised trial of EPA for sporadic colorectal cancer chemoprevention and the first evaluation of aspirin in a high-risk population within a high quality-assured national bowel cancer screening programme.EPA and aspirin did not reduce the proportion of individuals with any colorectal adenoma (adenoma detection rate [ADR]), but they both decreased the recurrence of some subtypes of adenoma, measured by adenoma number, 1 year after clearance screening colonoscopy. There was evidence of selectivity for adenoma type and location.Implications of all the available evidenceAlthough our trial found no effect on the primary outcome of the proportion of patients with at least one adenoma, both EPA and aspirin show some chemoprevention efficacy for colorectal cancer. The larger effect size of aspirin adds to the weight of evidence for its use in combination with endoscopic screening and surveillance, which provides suboptimal protection against right-sided colorectal cancer.Our findings suggest that a precision medicine approach (for adenoma type and location) to colorectal cancer chemoprevention will be necessary, which mirrors best practice in colorectal cancer treatment based on molecular stratification.The trial raises the crucial question of whether ADR or adenoma number is the best measure of chemoprevention efficacy in polyp prevention trials that are based on quality-assured colonoscopy in individuals at high risk of colorectal cancer, in which the ADR is used as a performance indicator, and the adenoma recurrence rate is high.Colorectal cancer develops over a period of years via a benign lesion termed the colorectal adenoma ,n3 eicosapentaenoic acid (EPA) is licensed for treatment of severe hypertriglyceridaemia that is unresponsive to other therapies and has clinical proof-of-concept as a colorectal cancer chemoprevention agent from a randomised controlled trial (RCT) in patients with familial adenomatous polyposis, in which 2 g EPA free fatty acid (FFA) per day for 6 months was associated with a significant reduction in rectal adenoma number and size.The naturally occurring omega-3 polyunsaturated fatty acid (PUFA) C20:5A robust body of evidence exists from polyp prevention RCTs in individuals undergoing colonoscopic surveillance after colorectal adenoma clearance,The National Health Service (NHS) Bowel Cancer Screening Programme (BCSP) in England provides population-level colorectal cancer screening using guaiac faecal occult blood testing, high quality-assured screening colonoscopy, and surveillance on the basis of colorectal adenoma number and characteristics,Colorectal adenoma risk reduction has previously been shown at 1 year in polyp prevention RCTs of aspirin and metformin.The Systematic Evaluation of Aspirin and Fish Oil (seAFOod) Polyp Prevention Trial was a multicentre, randomised, double-blind, placebo-controlled, 2 × 2 factorial trial. The trial protocol has been published elsewhere.Individuals aged 55–73 years who were identified as high risk at a complete BCSP screening colonoscopy  were screened for trial eligibility. For participants who had a bowel scope flexible sigmoidoscopy in the BCSP, eligibility was established on the basis of combined colorectal adenoma findings from the flexible sigmoidoscopy and subsequent colonoscopy.protocol summary on the International Standard Randomised Controlled Trials Number (ISRCTN) registry.All individuals at high risk were provided with written trial information after colonoscopy and then attended a routine BCSP outpatient visit 7–14 days later, during which written informed consent was obtained if they were eligible. Exclusion criteria included requirement for more than one repeat colonoscopy or flexible sigmoidoscopy within the 3 month BCSP screening window, regular (>3 doses per week) prescribed or over-the-counter (OTC) aspirin or non-aspirin non-steroidal anti-inflammatory drug (NSAID) use, and concomitant warfarin therapy or use of any other anticoagulant or antiplatelet agent.Participants were randomly allocated in a 1:1:1:1 ratio to either EPA plus aspirin; EPA plus placebo; aspirin plus placebo; or placebo plus placebo. Randomisation occurred within 4 weeks from the screening colonoscopy by research staff at each site using a secure web-based system with treatment assignment established by a pseudorandom code, using random permuted blocks of randomly varying size. Stratification was done by BCSP site.The sequence of treatment allocations was concealed until recruitment, data collection, and database lock were completed. Investigational medicinal product (IMP) allocation was not divulged to any research staff or participant.Participants who were randomly assigned to receive EPA received either 2 g 99% EPA-FFA per day , as used in the previous RCT in patients with familial adenomatous polyposis,Participants could withdraw from the trial at any time. When a participant withdrew from the intervention only, follow-up occurred per protocol. If a participant withdrew from the trial, data collected up to the point of withdrawal were included in the analyses.Participants completed a European Prospective Investigation into Cancer and Nutrition short food frequency questionnaire at study entry and after surveillance colonoscopy at 12 months, for categorisation of total and oily fish intake . A prescFatty acids were extracted from red blood cell membranes and rectal mucosa and then measured by liquid chromatography tandem mass spectrometry as previously described.Colorectal adenoma outcomes at the 12 month surveillance colonoscopy were collected as per routine BCSP practice,The primary outcome was the proportion of participants with one or more colorectal adenomas detected at surveillance colonoscopy—ie, the adenoma detection rate . The secondary outcomes were as follows: the proportion of participants with advanced  colorectal adenomas and with conventional , serrated, left-sided, and right-sided colorectal adenoma subtypes (serrated adenoma was added as a secondary endpoint to the SAP before database lock); the mean number of colorectal adenomas per participant for all colorectal adenomas, and for advanced, conventional, serrated, left-sided, and right-sided colorectal adenoma subtypes; the number of participants at high risk who were reclassified as being at intermediate risk ; development of colorectal cancer; dietary fish and other seafood intake at baseline and at 12 months; red blood cell and rectal mucosal PUFA concentrations; and adverse events, including clinically significant bleeding episodes .A sample size of 768 evaluable individuals defined as high risk by BCSP allowed detection of a minimum 18% relative (10% absolute) reduction in ADR from 60% for both agents within a 2 × 2 factorial design, assuming independent intervention effects, with 80% power and a 5% two-sided significance level, as described in the protocol.Primary and secondary outcomes were analysed according to allocation, regardless of compliance with treatment, for all participants who were randomly assigned and who had observed follow-up data. Per-protocol analysis of the primary outcome was based on all participants who were randomly assigned without a major protocol violation  before treatment codes were revealed. Adverse events are reported in the safety population of all participants who were randomly assigned and who received at least one dose of IMP.The primary outcome was analysed with a so-called at-the-margins approach,Treatment-emergent adverse events and adverse drug reactions were summarised by MedDRA system organ class. Gastrointestinal adverse events were also summarised by MedDRA preferred term and EPA formulation (FFA or triglyceride). The worst case  was assumed if severity or causality data were missing. Clinically significant bleeding episodes  were identified by the chief investigator (MAH) using a manual search of all adverse events. Common gastrointestinal adverse events were presented separately as clinically meaningful symptom categories, as defined by the chief investigator.The statistical analysis plan was finalised before database lock on Aug 31, 2017. The plan included comparison of the EPA formulations , analysis of serrated adenoma outcomes, and definition of the analysis population to include only participants with observed follow-up data, compared with the analysis described in the protocol.All statistical analyses were done using Stata version 15.0. The trial is registered with the ISRCTN registry, number ISRCTN05926847.The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Patients were recruited between Nov 11, 2011, and June 10, 2016 . Therefore, the ADR was analysed according to factorial margins  between red blood cell and rectal mucosal EPA concentrations was observed at 12 months  EPA incorporation and red blood cell EPA levels commonly used as a biomarker of tissue EPA exposure.2 months . We also2 months . Both EP2 months .The seAFOod Polyp Prevention trial found no evidence of an effect of either EPA or aspirin on the primary outcome of the proportion of individuals with one or more colorectal adenomas at 12 month surveillance colonoscopy (the ADR) in patients defined as high risk by the English BCSP. However, secondary analyses of the effects of EPA and aspirin on colorectal adenoma number provided evidence of the chemopreventive activity of both agents. Aspirin was effective at reducing the total number of colorectal adenomas per participant, but the effect of EPA on total number of adenomas per participant was insufficiently precise to draw a firm conclusion about efficacy. Other secondary analyses suggested that EPA and aspirin have colorectal adenoma subtype-selective and site-selective effects. Participants randomly assigned to receive EPA had a reduced total number of and ADR for conventional adenomas in the left colorectum than did those in the placebo group. As well as a reduction in total colorectal adenoma number, participants randomly assigned to receive aspirin had a reduced number of adenomas in the right colon, particularly for serrated adenomas, but also a reduced risk of conventional colorectal adenomas.The ADR was chosen as the primary outcome because previous polyp prevention trials have used this binary measure of colorectal adenoma occurrence as the primary endpoint.By contrast, previous RCTs in patients with familial adenomatous polyposis have reported efficacy on the basis of colorectal adenoma number.More recently, adenoma number has gained credence as a primary outcome measure in polyp prevention RCTs,Chemopreventive activity of EPA against conventional colorectal adenomas (but not serrated lesions), is consistent with the known efficacy of the same dose of EPA–FFA in patients with familial adenomatous polyposis and rectal adenomas,Total and left-sided colorectal adenoma multiplicity was lower in the group receiving combined treatment with EPA and aspirin, consistent with efficacy of both agents on left-sided, conventional colorectal adenomas. A factorial trial design that assumes independent intervention effects can only detect a large interaction between EPA and aspirin. A key objective of future work will be to apply precision medicine principles to establish which individuals might gain most from chemoprevention with one or both agents, based on baseline colorectal adenoma characteristics alone or together with other mucosal biomarkers.The seAFOod trial became a unique opportunity to compare the tissue bioavailability and tolerability of two EPA formulations of the same FFA dose in an RCT. There appeared to be no meaningful difference in compliance, tissue EPA incorporation, or tolerability between the FFA and triglyceride formulations, contrasting with many previous short-term studies of different omega-3 PUFA formulations.Omega-3 PUFA concentrations in red blood cells were similar to data from studies in other countries with relatively low dietary fish intake,No evidence of substantial conversion of EPA to docosahexaenoic acid either in red blood cell membranes or rectal mucosa was seen. Therefore, the trial data do not support EPA as a universal omega-3 PUFA donor in the colorectum and no conclusions can be drawn from the trial about the efficacy of mixed EPA and docosahexaenoic acid formulations, which represent the majority of prescription and nutritional supplement omega-3 PUFA formulations.Strengths of the trial include excellent, quality-assured colonoscopy performance and histological reporting, and uniform, protocol-driven care in the BCSP.In conclusion, the seAFOod Polyp Prevention trial has shown that the omega-3 PUFA EPA (2 g FFA per day) and aspirin (300 mg per day) did not reduce colorectal adenoma risk (as measured by the proportion of participants with at least one adenoma) at 1 year surveillance colonoscopy in individuals at high risk with colorectal neoplasia in the English BCSP. However, both agents had some chemopreventive efficacy on colorectal adenoma burden, as measured by a reduction in the mean number of adenomas per participant. The colorectal adenoma subtype-dependent and location-dependent specificity of EPA and aspirin are consistent with previous observations. Existing data on colorectal cancer risk reduction by aspirin suggest that the decrease in colorectal adenoma recurrence that we report for both agents is likely to translate into a clinically meaningful decrease in long-term colorectal cancer risk.


Vitis vinifera cultivars  seeds from veraison to harvest. The results showed: (1) polymers were the main form of flavan-3-ols in grape seeds and monomers accounted for only a small proportion; (2) the contents of flavan-3-ol monomers in the seeds of three grape cultivars all exhibited a gradually decreasing trend with a little fluctuation, whereas the patterns of the change of contents of oligomers and polymers were extremely different among grape cultivars; the contents of flavan-3-ol oligomers were enhanced in the seeds of ‘Cabernet Sauvignon’, but were reduced in the other two cultivars; (3) with regard to the proportion of flavan-3-ols with a certain DP to total flavan-3-ols, both flavan-3-ol monomers and flavan-3-ols with low DP fell in proportion, while the flavan-3-ols with high DP increased correspondingly. These findings indicate that flavan-3-ol polymerization in developing seeds is variety-dependent and may be genetically regulated.Flavan-3-ols consist of flavan-3-ol monomers and polymers with different degrees of polymerization (DP). In this study, flavan-3-ol extracts from grape seeds were well separated into three fractions including monomers, oligomers (2 < DP < 10) and polymers (DP > 10), by means of normal-phase HPLC-MS. The different patterns of these three fractions were analyzed in three  Flavan-3-ols are widely distributed in higher plants like grape. Abundant flavan-3-ols are present in grape skins and seeds ,2,3, andetc.) taste more acid than astringent [et al. [et al. [et al. [The exact or mean degree of polymerization of flavan-3-ols is a major factor influencing the sensory properties of bitterness and astringency ,10,11,12tringent . Oligome [et al. , the ast18 Sep-Pak cartridges has been used [With the development of chromatographic and spectroscopic methods, quantization of individual small molecular size phenolic compounds in grapes has become possible , and someen used ,16,17. Ieen used . Howevereen used , and theeen used . Usuallyeen used ,23,24. BConsiderable research has been done to describe the flavan-3-ol changes during grape berry ripening ,27,28,29Although the biosynthesis of flavan-3-ol units is well known, the mechanism of flavan-3-ol polymerization remains unclear. Grape seeds are rich in flavan-3-ols, and thus can be used as an ideal material to study this polymerization during berry maturation. In this study, we fractioned flavan-3-ol extracts from grape seeds into three fractions according to their DP by normal phase HPLC-MS, and quantified each fraction. On this basis, the changes of flavan-3-ols with different DP were investigated in ripening seeds of three winegrape cultivars  after veraison. This work will provide some valuable information for ultimately uncovering mechanism of flavan-3-ol polymerization. Flavan-3-ol monomers to decamers were well separated and the polymers larger than decamers were eluted as a single peak corresponding to a retention time of 50 min . The monThree flavan-3-ol monomers, C, EC, and ECG, were detected, but was ECG present in trace amounts, representing less than 1% of the total concentration of flavan-3-ol monomers. In the seeds of ‘Shiraz’ grape, EC was the major flavan-3-ol monomer during whole experiment, except for the first sampling date  A-C , whiRegarding the relative proportion of the contents of individual monomer to total content of all flavan-3-ol monomers at the same stage, EC increased significantly in all three of these cultivars with seed maturation, from 47.8% to 60.7% for ‘Shiraz’, from 20.4% to 37.3% for ‘Cabernet Sauvignon’ and from 24.2% to 35.9% for ‘Marselan’ , respectFigure 4). At harvest, the seeds of ‘Cabernet Sauvignon’ contained the highest level of total flavan-3-ol oligomers (about 130 mg/g.DW). Total content of oligomers (2 < DP < 10) behaved differently in these three grape cultivars. It decreased with fluctuations in the seeds of both ‘Shiraz’ and ‘Marselan’, but an increase in total content of oligomers was observed in ‘Cabernet Sauvignon’  in ‘Marselan’ seeds declined at harvest in comparison with that at veraison. However, in ‘Shiraz’ and ‘Cabernet Sauvignon’ seeds the polymers had no significant changes in content between these two sampling dates. It was noteworthy that flavan-3-ol polymers in the seeds of ‘Cabernet Sauvignon’ rapidly declined during the first two weeks after veraison, which was concomitant with the progressive increase of oligomer levels. Following that, the total polymer level showed little significant change, which was similar to the report of Kennedy et al.  who had To better understand flavan-3-ol polymerization in maturing seeds, we assessed the relative proportion of all monomers, oligomers and polymers, respectively, to all the detected flavan-3-ols. Similar change patterns were observed in these three cultivars. The polymers accounted for the highest proportion in each cultivar, being more than 60% of total flavan-3-ols, whereas that of monomers was the lowest, about 10% . et al. [The proportion of flavan-3-ol monomers in each cultivar declined gradually during seed maturation. But those of oligomers and polymers behaved in different ways among these three grape varieties. In ‘Shiraz’ seeds, the proportion of polymers gradually increased, while that of oligomers was essentially unchanged from grape veraison to harvest. The polymers in ‘Marselan’ seeds also showed an increasing tendency in proportion during maturation, while that of oligomers presented a decreasing tendency till a week before the last sampling date, but on the last sampling date, oligomer proportion increased dramatically to its initial level. In ‘Cabernet Sauvignon’ seeds, polymers greatly declined in proportion in the first two weeks after veraison and then increased up to the initial level within the following three weeks, and after that time, this proportion remained almost constant till harvest; the relative proportion of oligomers first increased in the first two weeks after veraison and then remained around this level before harvest . Althouget al.  have alsSeveral potential mechanisms of flavan-3-ol polymerization have been proposed . Some stThe standards of C, EC, ECG and procyanidin B1 were all purchased from Sigma Chemical Co. . Methanol and acetic acid (HPLC grade) were obtained from Fisher Company , and deionized water (<18 MΩ resistibility) from a Milli-Q Element water purification system .Vitis vinifera ‘Shiraz’, ‘Cabernet Sauvignon’ and ‘Marselan’ were collected from a single vineyard in Huailai County , Hebei province, northwest of Beijing (China) during the 2006 growing season. The vineyard is located on the southern bank of the Guan-ting reservoir. The average annual temperature is 10.5 °C, the coldest month being January with an average temperature of −8.3 °C and the hottest month being July at an average temperature of 25 °C. There is big temperature difference between day and night. The average annual rainfall is nearly 413 mm, most of which comes in July and August. Soils in the vineyard are uniform, typified by coarse sandy loam. Row and vine spacing are 2.5 m and 1 m respectively, with the orientated in a south-north direction. The berries began coloring on Aug. 2nd, 3rd, and 6th for ‘Cabernet Sauvignon’, ‘Shiraz’, and ‘Marselan’ respectively, and completed about a week later. The berries were sampled randomly at weekly intervals from the end of veraison (100% colored) to commercial harvest (24 Brix). To obtain samples representing a vineyard population, we sampled according to the method described by He et al. [Grape berries of e et al. . Three 1et al. [g for 10 min. The precipitate was extracted three times as discussed previously and the supernatants were combined. Acetone was removed by a rotary evaporation at 30 °C and the residue was dissolved in approximately 6 mL of 30% (v/v) aqueous methanol before loading onto a Sephadex LH-20 column (6 × 1.5 cm). After loading the sample, the column was washed with 30% (v/v) methanol/water (40 mL) to remove the sugars and other phenols with low molecular weight. Flavan-3-ols were obtained from the column by eluting with 70% (v/v) aqueous acetone (70 mL). The effluents were evaporated under partial vacuum at 30 ºC and the residues were freeze-dried. Then the dried residues were dissolved in methanol (HPLC grade) with a total volume of 5 mL. The methanol extract containing flavan-3-ols was filtered through 0.45 µm filters  prior to analysis by NP-HPLC-MS. For each sample, at least three independent extractions were carried out.The extraction and purification procedures were performed as described by Gu et al.  with somThe analysis of flavan-3-ols was carried out using an Agilent 1100 series LC and LC/MSD Trap VL mass spectrometer  equipped with electrospray ionization (ESI) interface, using Agilent Zorbax RX-SIL  column protected with a Zorbax RX-SIL  guard column. The mobile phase consisted of (A) methylene chloride, (B) methanol, and (C) acetic acid and water (1:1 v/v). The sample of 2 μL was injected and run at 30 °C with a flow rate of 0.2 mL/min. The gradient was 0-20 min, 14.0–25.0% B linear; 20–40 min, 25.0–33.2% B linear; 40–45 min, 33.2–86.0% B linear; 45–55 min 86.0% B isocratic; 55–60 min, 86.0–14.0% B linear followed by 10 min of re-equilibration of the column before the next run. A constant 4.0% C was kept throughout the gradient. The LC/MS conditions were at negative mode as follows: capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature 150 °C; desolvation gas temperature, 300 °C. For each extraction, duplicate analyses were carried out.EC, C, and ECG in the seeds were quantified using the respective EC, C and ECG standards as references, and oligomeric and polymeric flavan-3-ols using the standard procyanidin B1 as reference.Statistical analyses were performed with the aid of SPSS 11.5 software . One-way analysis of variance (ANOVA) was applied to maturing seeds to verify significant changes (significant at 5% level) according to flavan-3-ols with different DP. In this study, the developmental changes in the content and the relative proportion of flavan-3-ol monomers, oligomers and polymers in seeds of ‘Shiraz’, ‘Cabernet Sauvignon’, and ‘Marselan’ grape were monitored during grape maturation. From the result we can conclude that flavan-3-ols with different molecular sizes in grape seeds behave differently during fruit ripening, and the change patterns of these compounds are cultivar-dependent, which may be genetically regulated. The genetic regulation mechanism needs to be determined in future studies.


The NGS tests included analysis of sequence alterations in 129 genes. The variants were interpreted according to the ACMG/AMP guidelines for genetic hearing loss combined with NGS data from 616 ethnically matched normal hearing adult controls. We identified a positive molecular diagnosis in 226 patients with sporadic SNHL (52.19%) and in patients from 17 deafness pedigrees (56.67%). Ethnically matched MAF filtering reduced the variants of unknown significance by 8.7%, from 6216 to 5675. Some complexities that may restrict causative variant identification are discussed. This report highlight the clinical utility of NGS panels identifying disease-causing variants for the diagnosis of hearing loss and underlines the importance of a broad data of control and ACMG/AMP standards for accurate clinical delineation of VUS variants.Hereditary hearing loss is a monogenic disease with high genetic heterogeneity. Variants in more than 100 deafness genes underlie the basis of its pathogenesis. The aim of this study was to assess the ratio of SNVs in known deafness genes contributing to the etiology of both sporadic and familial sensorineural hearing loss patients from China. DNA samples from 1127 individuals, including normal hearing controls ( Hearing impairment is one of the most common human disabilities. According to the World Health Organization, 5% of the world’s population (~360 million people) suffer from disabling hearing. More people are affected by severe hearing loss than by epilepsy, multiple sclerosis, spinal injury, stroke, Huntington’s disease, and Parkinson’s disease combined . SensoriGJB2, SLC26A4, and mitochondrial 12S rRNA, which appear to account for 30–50% of congenital hearing loss cases [Molecular epidemiological studies have found several common deafness genes, such as ss cases –12. Howess cases –15. Howen = 616), sporadic patients clinically diagnosed SNHL (n = 433), and individuals from 30 deaf pedigrees including familial cases (n = 78). Ninety-nine family members with normal hearing from the 30 deaf pedigrees were enrolled for segregation analysis.This study was performed according to the protocol approved by the Ethics Committee of the Chinese People’s Liberation Army General Hospital. We collected DNA samples from 1127 individuals, including normal hearing controls , GJB3 c.538C>T (NM_001005752.1), SLC26A4 c.919-2A>G (NG_008489.1), c.2168A>G (NM_000441.1), mtDNA 12S rRNA, and were further referred to the otology clinic and diagnosed as hearing impairment by objective hearing tests. Chinese individuals with normal hearing (controls) were verified by pure-tone audiometry. Only those with a PTA (pure-tone average) threshold of 0–20 dB were enrolled. The average age of this population was 31.18 ± 10.18 years; no control individuals claimed hearing problems or family history, and all passed physical examinations. All participants were informed about the scope and requirements of the study, and all signed the consent forms approved by the Ethics Committees of the participating institutions.The sporadic cases were recruited from outpatients who visited the hospital during 2011–2013; only unique probands were included (each nuclear and/or extended family was represented only by the proband). The patients comprised those who claimed hearing loss and were verified by either subjective and/or objective hearing tests, those who failed the newborn hearing screening and were further diagnosed as deafness by objective hearing tests, and those who failed the newborn deafness gene screening carrying at least one variant of the nine including The clinical history was obtained by investigators, with special emphasis on the onset age of hearing loss; family history of deafness; pregnancy and labor history; general health condition; potential environmental causes of hearing loss such as infections and trauma; and information on exposure to known or possible ototoxic drugs. Cases involving trauma and otitis media were excluded from this study. Conventional clinical examinations included pure-tone audiometry, acoustic immittance, and auditory brainstem responses (ABR). Distortion product otoacoustic emissions, ABR, and auditory steady state response tests were carried out in babies who failed the hearing screening and in children who were uncooperative during the subjective audiometry examination. The level of hearing loss was described in terms of PTA  as follows: normal hearing, <20 dB; mild hearing impairment, 21–40 dB; moderate hearing impairment, 41–70 dB; severe hearing impairment, 71–90 dB; and profound hearing impairment, >91 dB. High-resolution computed tomography (CT) of the temporal bone was performed in the sporadic and pedigree SNHL cases to exclude middle ear pathological changes and to diagnose inner ear malformation. Magnetic resonance imaging was performed when the CT scan failed to reveal membranous labyrinth problems in a patient. Syndromic hearing loss was diagnosed based on a range of evidence, including self-reported symptoms, family history, and physical examination, with special attention to the external ears and neck, skin, hair, eyes, and digits. Data Management System for Clinical Otology software (Computer Software Copyright: 2008SR06229) was used to store and track the information.12S rRNA, the coding areas of GJB2 and SLC26A4 previously.The DNA was isolated from blood extracted from a peripheral vein by a registered nurse in the genetic testing center of the Chinese PLA General Hospital. Genomic DNA (gDNA) was extracted using gDNA blood extraction kits  within 1 week of sample collection. The quality of the gDNA was examined by assessing the optical density ratio (260/280 ratio) and by gel electrophoresis imaging for the presence of a high-molecular-weight gDNA band. All of the 433 sporadic cases were sequenced The DA3 panel assay  includes 119 nuclear deafness genes and 10 mitochondria genes Table . LibraryAfter sequencing the target region, quality control was performed to ensure data accuracy. Low-quality data were filtered out to obtain clean sequencing data. Burrows–Wheeler alignment was used to align the clean sequence to the human reference genome hg19 nuclear genes or GRCh37 for mitochondria genes. GATK was used to detect SNP and Indel variants. VEP and dbNSFP databases were used to obtain the variants information, including minor allele frequency (MAF), variant consequence, altered protein function, gene information, and related disease information.p-values using Bonferroni multiple testing correction. Variants with p-values less than 3.521E−04 (0.05/142) were considered to be significantly associated with disease.After raw variants were annotated, we performed further filtering to identify candidate variants. Variants meeting all the following requirements were included: (1) MAF of KG and Exome Aggregation Consortium (ExAC) database < 0.01; (2) 0.005 < MAF < 0.05; (3) variants affecting protein function, including loss-of-function and missense variants. After that, we obtained 142 variants from 433 hearing loss cases and 616 normal controls. We performed a case-control association study using Fisher’s exact test and adjusted GJB2 and SLC26A4. By combining the clinical phenotype, inherited model, and related previous studies, we obtained a list of candidate variants. Sanger analysis was performed to verify the variant identified by NGS. The pipeline of bioinformatic analysis was shown in Fig. The variants interpreted by the ACMG/AMP guidelines for genetic hearing loss were revalued using allele frequencies from the ethnically matched controls. The variants were categorized as BA1 if the MAF thresholds of autosomal-recessive variants were >0.005 and autosomal-dominant variants were >0.001 in the controls , 18. TheCNSA). The pathogenic and likely pathogenic variants were submitted to ClinVar (ClinVarAccessions: SCV000902290~SCV000902403).The FASTQ data and variant call format were permanently reserved on  and intron regions  were called after the preliminary bioinformatic analysis. We identified 73  pathogenic and 49  likely pathogenic variants Table . In all,GJB2 c.235delC, c.299_300delAT, SLC26A4 c.919-2A>G, c.2168A>G, c.1174A>T, USH2A c.6998T>C) achieved a conservative significance threshold. Among the 541 variants, the 1000 Genomes, ESP, ExAC databases reported no MAF thresholds for autosomal-recessive variants >0.005 (excluding specific variants in GJB2 and SLC26A4) and autosomal-dominant variants >0.001; however, MAF > 0.005 was found in our control group in the recessive inheritance pattern and >0.001 in the dominant inheritance pattern and accordingly we categorized them to BA1. This result illustrated the power of this step and stressed the need for deeper and richer databases of multiple ethnicities to identify low-frequency variants specific to ancestry groups that are often unique to specific populations [We chose 1000 Genomes, Exome Sequencing Project , ExAC, Hulations .GJB2 c.109G>A  was 3.8%  and 3%  in the case and control groups, respectively; the difference between the two groups was not statistically significant. The homozygous p.V37I variant in GJB2 is prevalent in East and Southeast Asians and may lead to a broad spectrum of hearing phenotypes from mild-to-moderate hearing loss with reduced penetrance to profound hearing loss. The homozygous p.V37I knockin mice developed progressive, mild-to-moderate hearing loss over the first 4–9 months which modeled the hearing phenotype of the human patients, and confocal immunostaining and electron microscopic scanning revealed minor loss of the outer hair cells [The allele frequency for ir cells . Thus, wGJB2, SLC26A4, 12S rRNA, and MYO15A. The allele and carrier frequencies for variants in common deafness gene are shown in Table In sporadic SNHL cases, variants in 24 genes were verified for genetic etiology in 52.19% (226/433) by Sanger sequencing and cosegregation analysis in their families  , higher than the 41% identified among cases with multiethnic background  and ~40%GJB2 and SLC26A4. Our study showed that disease-causing variants in GJB2 and SLC26A4 were found in exceptionally high numbers, followed by variants in USH2A, MYO15A, MYO1F, and MYO7A. Variants affecting protein function in GJB2 and SLC26A4 were the most common cause of autosomal-recessive NSHL, accounting for 80.42% (184/226) of the genetic basis in all hereditary hearing loss patients. The genetic spectrum for autosomal-dominant NSHL seems to be wide and not focused on one or a few genes; this result differs from the findings of a study involving Japanese subjects, which reported KCNQ4 as the most frequent cause for dominant NSHL [GJB2 (22%), STRC (16%), SLC26A4 (7%), and TECTA (5%), which is different from those of our study except GJB2. The pathogenesis of 433 Chinese sporadic hearing loss patients was shown in Fig. GJB2, SLC26A4, and 12S rRNA) in this case cohort with that in a larger sample  from our deafness genetic testing center revealed that our sample selection was representative of the Chinese deafness population  and c.538C>T (BS1 + BS4) as benign. Our study supports the findings that querified variants in GJB3 as a cause of deafness [GJB3 variants cause fully penetrant deafness.The reported u et al. . Three pdeafness . FurtherSLC26A4. Neither KCNJ10 nor FOXI I variants contributed to the molecular etiology of EVA among patients in this study. The relevance ratio of bi-allelic SLC26A4 variants is 36% in Caucasians, 66% in Japanese, and 81% in Koreans [POU3F4. Other inner ear malformations included cochlea aplasia (one case), cochlea hypoplasia (one case), isolated Mondini deformity (three cases), common cavity deformity (two cases), and narrow internal auditory canal (one case). No genetic pathogenesis was identified in the types of inner ear deformity other than EVA and IP-III, indicating that the etiology of inner ear malformation may be multifactorial.The etiologies of inner ear malformations have not been completely clarified. Genotype and phenotype correlations were determined in EVA and IP-III. In this study, we observed at least seven types of inner ear malformations in 100 patients; the rate of inner ear malformations in SNHL was 23.09% (100/433). The most common type was EVA (89 cases), which is an autosomal-recessive disease. In our study, 94.3% (84/89) of EVA cases carried bi-allelic causative variants, 3.37% (3/89) had monoallelic, and 2.25% (2/89) had no variant in  Koreans –50. In tPAX3 and MITF were identified, respectively, whereas the other case showed no causative variant in WS-related genes . No Pendred syndrome was diagnosed in our patient cohort, which may be due to the fact that the average age of our patients with EVA was 9.36 years. The phenotype of goiter is not present at birth, developing in early puberty (40%) or adulthood (60%). In addition, it is possible that our sample size was not large enough. We identified ten cases with bi-allelic variants in the Usher syndrome-related genes, USH2A, MYO7A, CDH23, and ADGRV1. Usher syndrome is a clinically and genetically heterogeneous autosomal-recessive disorder characterized by sensorineural hearing deficiencies and later development of progressive retinitis pigmentosa. It is the most frequent cause of combined deafness and blindness in adults and affects 3–6% of children born with hearing impairment. None of the ten cases had vestibular dysfunction or hypoplastic, and vestibular and ophthalmologic examinations were not performed at the beginning of this study. The average age of these ten patients was 12.77 years. Hearing loss occurred postlingually in five cases, prelingually in four, and one with ambiguous onset. In consideration of the variable extent of vestibular involvement and the usual onset of retinitis pigmentosa symptoms in the second decade of life, vestibular evaluation and ophthalmologic follow-up will be performed in the future. In addition, there is the possibility that variants in MYO7A and CDH23 caused DFNB2 and DFNB12.The clinical diagnosis of syndromic SNHL helps direct the test panel choice, thereby saving time and cost. Three patients with Waardenburg syndrome (WS) were diagnosed clinically by the phenotype of hearing loss with dermal pigmentation and iris heterochrosis. In two of the WS cases, genetic variants in We demonstrated that the DA3 panel assay covering 129 genes had a positive diagnostic rate of 52.19% on sequence alterations for sporadic SNHL patients and 56.67% for deafness pedigrees. WES or WGS should be reserved only for negative cases as an opportunity to discover novel candidate genes. Ethnically matched MAF filtering changed the categorization of 8.75% of our list of variants from VUS to benign. This report highlight the clinical utility of NGS panels identifying disease-causing variants for the clinical diagnosis of deafness and underlines the importance of a broad data of control and the ACMG/AMP guidelines for accurate clinical delineation of VUS variants. The results provide more detailed information for genetic consulting to predict the risk of recurrence.Tables S1–S4Table S5


Studies suggest that depression is closely linked to hearing impairment, which is highly prevalent among older adults in the United States. There is evidence that social engagement may be impacted by hearing impairment in older adults. However, there is relatively little research on these associations among Chinese older adults. This study examines the relationships between hearing impairment, social activities, and depressive symptoms among older adults in China. Using nationally representative data from the China Health and Retirement Longitudinal Study 2011, we conducted cross-sectional analysis on adults age 60 years and older . Depressive symptoms were assessed by the 10-item Center for Epidemiologic Studies Depression scale and we considered self-reported hearing status (if participants wear a hearing aid and how they would rate their hearing), and social activities . Models were controlled for age, gender, education, and other covariates. Descriptive analysis showed that 9% of older adults experienced hearing impairment. Multiple linear regression analyses revealed that hearing impairment was positively associated with depressive symptoms among older Chinese adults . Social activities were found to partially mediate the relationship between hearing status and depressive symptoms. Respondents with hearing impairment were less likely to engage in social activities  and those who did not participate in social activities reported more depressive symptoms . These findings suggest that Chinese older adults experiencing hearing loss are at greater risk of depression and that social activities play an important role in the relationship between hearing status and depression.