Apolipoprotein B (apoB) is emerging as the crucial lipoprotein trait for the role of lipoprotein lipids in the aetiology of coronary heart disease. In this study, we evaluated the effects of genetically predicted apoB on outcomes in first-degree relatives.Data on lipoprotein lipids and disease outcomes in first-degree relatives were obtained from the UK Biobank study. We did a univariable mendelian randomisation analysis using a weighted genetic instrument for apoB. For outcomes with which apoB was associated at a false discovery rate (FDR) of less than 5%, multivariable mendelian randomisation analyses were done, including genetic instruments for LDL cholesterol and triglycerides. Associations between apoB and self-reported outcomes in first-degree relatives were characterised for 12 diseases  and parental vital status together with age at death. Estimates were inferred causal effects per 1 SD elevated lipoprotein trait . Replication of estimates for lifespan and type 2 diabetes was done using conventional two-sample mendelian randomisation with summary estimates from genome-wide association study consortia.−10; mothers: 0·48 years of life lost per 1 SD higher apoB in offspring, 0·25–0·71, FDR-adjusted p=1·7 × 10–4). The effects were strengthened to around 2 years of life lost in multivariable mendelian randomisation and were replicated in conventional two-sample mendelian randomisation . Genetically elevated apoB caused higher risks of heart disease in all first-degree relatives and a higher risk of stroke in mothers. Findings in first-degree relatives were replicated in two-sample multivariable mendelian randomisation, which identified apoB to increase  and LDL cholesterol to decrease  the risk of type 2 diabetes.In univariable mendelian randomisation, genetically elevated apoB in participants was identified to lead to a shorter lifespan in parents (fathers: 0·89 years of life lost per 1 SD higher apoB in offspring, 95% CI 0·63–1·16, FDR-adjusted p=4·0 × 10Higher apoB shortens lifespan, increases risks of heart disease and stroke, and in multivariable analyses that account for LDL cholesterol, increases risk of diabetes.British Heart Foundation, UK Medical Research Council, and UK Research and Innovation. In this study, we explore a broad repertoire of outcomes, taking advantage of events reported in first-degree relatives in UK Biobank. In studies using outcomes in first-degree relatives, the genetic instrument is constructed in individuals who share around 50% of their DNA with those in whom outcomes occur, which should halve the effect size. Associations identified in this way can provide insights into likely underlying causal relationships, which, if consistent with more conventional mendelian randomisation approaches, bolster confidence that the associations are real.Although the crucial role of apoB in coronary heart disease is increasingly emerging,12 Finally, we sought replication of our findings for lifespan and type 2 diabetes using findings from large-scale genome-wide association study (GWAS) consortia, given that multivariable analyses using parental data showed evidence of an effect of apoB on these outcomes in the current study.In this study, we explored the association between an apoB polygenic instrument and self-reported outcomes in first-degree relatives in participants of the UK Biobank for 12 diseases  and parental vital status together with age at death. We assessed the rigour of these findings using multivariable mendelian randomisation by including genetic instruments for other major atherogenic lipoprotein lipids (LDL cholesterol and triglycerides), and comparing findings to previously published results.14 as have general characteristics of the cohort, such as geographical regions and recruitment processes.15 Details on genotyping quality control, phasing, imputation, and association testing in UK Biobank are reported elsewhere.16 Ethical approval for this study was obtained from the UK Biobank Ethics Advisory Committee  and informed consent was collected from all participants enrolled in UK Biobank.Data on lipoprotein lipids and disease outcomes in first-degree relatives were obtained from the UK Biobank study under application #15825. UK Biobank comprises approximately 500 000 participants who were recruited between 2006 and 2010 from 22 assessment centres across the UK. Details on sample handling and assays for lipoprotein lipids in UK Biobank have been described previously,17 Data for type 2 diabetes were derived from the most recent large-scale European type 2 diabetes GWAS that included data from 32 studies and excluded participants from UK Biobank.18We sought to replicate estimates from UK Biobank using conventional two-sample mendelian randomisation. Data for lifespan were derived from a GWAS meta-analysis of cohorts with participants dichotomised according to whether they survived to an age corresponding to the 90th centile of lifespan.20 Analyses were adjusted for age, sex, fasting status, and a binary variable denoting the genotyping chip used in individuals (the UK Biobank Axiom array or the UK BiLEVE array).Each lipoprotein lipid trait was normalised to have a mean of 0 and SD of 1 using inverse rank-normalisation. We did a GWAS on all UK Biobank participants with genotype and trait data after excluding individuals of non-European descent (based on K-means clustering of K=4) and standard exclusions . BOLT-LMM version 2.3.2 (linear mixed model) software was used for the GWAS because it is robust to potential confounding caused by relatedness or population structure.–8) were identified using a reference panel of 503 Europeans from phase 3 (version 5) of the 1000 Genomes Project.21 Clumping parameters of r2<0·001 and a distance of 1 megabase were used for instrument identification. Univariable sets of instruments for each lipoprotein trait were derived by applying linkage disequilibrium clumping on each trait’s GWAS results in turn. For multivariable instruments, we combined all GWAS results before applying linkage disequilibrium clumping to ensure independent instruments were identified among these correlated traits.Genetic instruments for each lipid-related trait were derived by linkage disequilibrium clumping of GWAS results  per 1 SD higher lipid trait. Effect estimates for the association of lipid traits with vital status and age at death of parents are also presented in this way because, by being obtained from UK Biobank individuals at study entry, data on parental vital status are effectively cross-sectional.25 weighted mode,26 and MR-Egger27 regression approaches. For multivariable analyses,13 we additionally did multivariable MR-Egger.28For effects in the univariable mendelian randomisation analysis that survived correction for false discovery rate (FDR) of less than 5% based on estimates from the IVW method, we applied the weighted median,18 The GWAS for lifespan used UK Biobank as a form of validation and the estimates we use here do not include UK Biobank. However, the latest release of data from DIAMANTE included type 2 diabetes cases from UK Biobank; since including these data might have led to overfitting of the mendelian randomisation estimate, we analysed type 2 diabetes GWAS estimates on individuals of European descent with UK Biobank data removed. We did conventional univariable IVW two-sample mendelian randomisation. We then fitted a multivariable mendelian randomisation model simultaneously including genetic instruments for apoB, LDL cholesterol, and triglycerides to explore their direct effects on lifespan and type 2 diabetes. We did a MVMR-Egger28 sensitivity analysis to evaluate whether the mendelian randomisation estimates were influenced by dose−response confounding of the genetic instruments.To replicate the findings identified for lifespan and type 2 diabetes, we did a conventional two-sample mendelian randomisation analysis in which SNPs for exposures  were derived from UK Biobank participants and SNPs for lifespan (grouping study participants based on whether they survived to the age corresponding to the 90th centile) and type 2 diabetes were derived from DIAMANTE, a previous large-scale GWAS.12 398 SNPs were used in the instrument for WHRadjBMI using a previous GWAS within UK Biobank.29 We also used the IVW, MR-Egger, and weighted median approaches in a univariable setting to investigate the potential bidirectional relationship between apoB and type 2 diabetes.Because the apoB effects on risk of type 2 diabetes might be confounded by adiposity and fat distribution, we additionally included body-mass index (BMI) and waist–hip ratio adjusted for BMI (WHRadjBMI) in the multivariable mendelian randomisation analyses for type 2 diabetes. 641 SNPs used in the instrument for BMI were derived from 461 377 UK Biobank participants using the same GWAS pipeline as described previously.Because some outcomes  occur only or predominantly in men or women, we re-ran the GWAS stratifying by sex and subsequently derived genetic instruments and did mendelian randomisation analyses using the aforementioned methods. To quantify the extent to which coronary heart disease and type 2 diabetes mediated the effects of apoB on lifespan , we constructed multivariable mendelian randomisation models that included apoB, type 2 diabetes, coronary heart disease, and lifespan.Because first-degree relatives share around 50% of DNA, and because outcomes were reported multiple times among first-degree relatives, the exposure-to-outcome analyses are not truly independent and use of conventional Bonferroni approaches to address multiple testing would be overly stringent. We therefore used Benjamini-Hochberg FDR of less than 5% to guide our interpretation of results from the initial univariable mendelian randomisation analysis of apoB for all 38 outcomes. This strategy was used to highlight which outcomes to evaluate in further detail in all subsequent analyses. FDR corrections were also applied to all additional univariable and multivariable analyses using UK Biobank participant data for completeness.19 was used to do GWAS analyses and identify genetic instruments. The TwoSampleMR R package was used for all mendelian randomisation analyses.30 The ggplot2 R package was used to generate forest plots.31 Conditional F-statistics were generated using the MVMR R package.BOLT-LMM softwareThe funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.A median of 400 304 UK Biobank participants (range 273 111–454 999) reported information on prevalent diseases in first-degree relatives. A median of 361816 participants (361 199–364 661) reported data for their siblings, 423 692 (422 464–426 391) reported data for their mothers, and 400 687 (399 089–407 557) reported data for their fathers . 180 472Across the diseases reported as occurring in first-degree relatives , the preF-statistic of 160. For multivariable mendelian randomisation, in which genetic instruments for apoB, LDL cholesterol, and triglycerides were analysed simultaneously, an additional 197 SNPs associated with LDL cholesterol (F-statistic of 167) and 411 SNPs associated with triglycerides (F-statistic of 121) were included in the analysis. In multivariable mendelian randomisation, the conditional F-statistics for apoB and LDL cholesterol were similar (37 and 34), and for triglycerides the F-statistic was 80. Thus, in both univariable and multivariable mendelian randomisation settings, our genetic instruments would not be considered weak.Our apoB GWAS in the UK Biobank explained 10·4% of the heritability in this trait. Linkage disequilibrium clumping identified 229 apoB-associated SNPs with an –10) earlier (corresponding to a mean of 10·7 months of life lost per 1 SD higher apoB in offspring) and mothers 0·48 years  earlier (corresponding to a mean of 5·8 months of life lost per 1 SD higher apoB in offspring). When taking into account the effects of LDL cholesterol and triglycerides in multivariable mendelian randomisation, these estimates for apoB increased to 1·94 years of life lost  in fathers and 2·02 years of life lost  in mothers, with the inverse relative odds for being alive also becoming more pronounced . Mothers of individuals with genetically elevated apoB had a higher risk of stroke , whereas the decrease in risk was weaker in siblings . For fathers, the increase in the odds of being alive was weaker  in univariable mendelian randomisation became weaker when accounting for apoB and triglycerides in the multivariable analysis, as did the inverse relationships with lung cancer, Parkinson’s disease, and prostate cancer .Estimates for triglycerides were generally robust to multivariable mendelian randomisation analyses . Higher Given the findings for vital status and risk of type 2 diabetes in first-degree relatives, we sought to further elucidate these relationships using a conventional two-sample mendelian randomisation framework with data from two GWAS consortia: a GWAS of lifespan with 11 262 individuals surviving to the age corresponding to the 90th survival centile and DIAMANTE, including 55 927 cases of type 2 diabetes. The analyses of lifespan replicated our findings from age of death and vital status of parents . ApoB waNo clear effect of genetically predicted apoB on type 2 diabetes was identified in univariable mendelian randomisation. In contrast, higher LDL cholesterol caused a lower risk of type 2 diabetes  and higher triglycerides caused a higher type 2 diabetes risk  in univariable mendelian randomisation. In multivariable mendelian randomisation, the estimate for triglycerides was largely unchanged . However, the protective effect of LDL cholesterol against type 2 diabetes became more pronounced  and a strong positive association between apoB and type 2 diabetes risk emerged  deficiency is associated with an increased incidence of diabetes.44 to induce adipose tissue dysfunction with an increased inflammatory response, resulting in reduced insulin sensitivity. Faraj44 has also suggested that cytotoxic injury to adipocytes due to increased uptake of LDL might be a mechanism by which statins and PCSK9 inhibitors, agents which increase activity of the LDL pathway, could be diabetogenic. Accordingly, we hypothesise that increased uptake of LDL particles might injure adipocytes or islet cells or both, which might explain the strong positive association between apoB and the risk of diabetes that was observed in this study.Alternatively, increased uptake of LDL particles by adipocytes has been posited by Faraj45 Our findings suggest that therapies that do not increase pancreatic uptake of LDL particles while increasing hepatic uptake of LDL particles or precursor VLDL particles might lead to a reduction in risk of both coronary heart disease and type 2 diabetes.The resilience of our findings to inclusion of adiposity measured by BMI and WHRadjBMI  in the multivariable mendelian randomisation suggests that the associations we identify with risk of type 2 diabetes are not confounded by upstream adiposity or fat distribution measures, which are recognised to causally affect blood lipid traits.46 and also has additional issues related to the particularities of its design. First, we cannot claim that apoB directly causes outcomes in the first-degree relatives of the UK Biobank participants because the genetically elevated apoB is only an approximation to unconfounded estimates within first-degree relatives. However, the replication of our analyses for lifespan and type 2 diabetes corroborates our findings and therefore strengthens these hypotheses. Second, because first-degree relatives share 50% of their DNA, the effect estimates generated by our genetic instruments have been attenuated through regression dilution. Thus, the magnitudes of effect estimates for coronary heart disease, type 2 diabetes, and lifespan associations using conventional two-sample mendelian randomisation approaches are several times greater than the equivalent estimates obtained using outcomes reported as occurring in first-degree relatives. Third, because outcomes are self-reported, measurement error is likely. For example, an individual who has heart disease might be more likely to report a first-degree relative as also having heart disease. Study participants might also have accidentally answered questions regarding non-blood relatives  or half-siblings, which would have weakened effect estimates. Fourth, outcomes related to siblings might represent multiple individuals collapsed into a single trait because individuals were asked about siblings only cumulatively rather than individually. This would dilute effects if more than one sibling has an outcome .This study has the usual caveats and limitations that apply to mendelian randomisation studies,12 A similar difference in the magnitudes of effect is evident between estimates of apoB with risk of type 2 diabetes in first-degree relatives (ORs of 1·17 to 1·33) versus data from DIAMANTE (OR >2). Of note, both estimates for heart disease (using CARDIoGRAMplusC4) and type 2 diabetes (using DIAMANTE) might be inflated by spectrum-bias effects arising from case-control studies that sample cases at the extremes of the phenotypic distribution. Future studies that apply alternative approaches when analysing highly correlated lipoprotein traits might therefore be valuable in corroborating our findings, such as the mendelian randomisation Bayesian model averaging approach.11The cumulative effect of these limitations probably explains why the effect estimates we report for heart disease (ORs of 1·14 to 1·27) are of considerably smaller magnitude than the approximation to within-individual effect estimates we previously reported using data from UK Biobank and CARDIoGRAMplusC4D (OR 1·7) scaled to the same difference in exposure (1 SD higher apoB in the UK Biobank participants).In conclusion, our evaluation of apoB using outcomes in first-degree relatives identified that higher apoB is detrimental to lifespan and increases the risk of coronary heart disease and type 2 diabetes. Lifestyle and pharmacological approaches to lowering apoB should have widespread beneficial effects, including preventing common diseases and prolonging life.Supplementary FiguresSupplementary Tables


MC1R) variants could inform prevention and screening recommendations for Hispanics, but limited evidence exists on the impact of MC1R variants in Hispanic populations. We studied Hispanic subjects, predominately of Puerto Rican heritage, from Tampa, Florida, US, and Ponce, PR. Blood or saliva samples were collected by prospective recruitment or retrieved from biobanks for genotyping of MC1R variants and ancestry informative markers. Participant demographic and self-reported phenotypic information was collected via biobank records or questionnaires. We determined associations of MC1R genetic risk categories and phenotypic variables and genetic ancestry. Over half of participants carried MC1R variants known to increase risk of skin cancer, and there was diversity in the observed variants across sample populations. Associations between MC1R genetic risk groups and some pigmentation characteristics were identified. Among Puerto Ricans, the proportion of participants carrying MC1R variants imparting elevated skin cancer risk was consistent across quartiles of European, African, and Native American genetic ancestry. These findings demonstrate that MC1R variants are important for pigmentation characteristics in Hispanics and that carriage of high risk MC1R alleles occurs even among Hispanics with stronger African or Native American genetic ancestry.Skin cancer risk information based on melanocortin-1 receptor ( For preventable cancers such as skin cancer, identification of common genetic variants can improve the precision of risk prediction models and inform risk-stratified prevention and early detection strategies to potentially reduce mortality, morbidity and healthcare costs5.As knowledge of associations between common genetic variants and elevated disease risk rapidly advances, expectations are high for the implementation of genetic testing in routine clinical practice and public health strategiesMC1R) gene, a primary regulator of skin pigmentation, is associated with increased risk of melanoma and non-melanoma (keratinocyte) skin cancers, including basal cell and squamous cell carcinomas8. Among individuals with ‘sun-resistant’ phenotypes , associations between MC1R variants and skin cancer are often stronger than those among individuals with ‘sun-sensitive’ phenotypes 9. It is likely that individuals with sun resistant phenotypes who carry MC1R risk genotypes may be unaware of their elevated skin cancer risk. However, most genetic epidemiology studies of skin cancer to date have focused on non-Hispanic white populations, which potentially limits the generalizability of skin cancer risk information based on MC1R genotypes to at-risk populations with more diverse genetic ancestry, such as Hispanics.Numerous studies have demonstrated that inherited genetic variation in the melanocortin-1 receptor  can affect change in skin cancer prevention behaviors, we first addressed some gaps in research evidence by conducting a pilot study to examine the prevalence of MC1R variants among Hispanics living in the Tampa Bay region of Florida, US and in Puerto Rico. These geographies were selected because of an ongoing federally-funded partnership initiative between Ponce Health Sciences University (PHSU) in Ponce, PR, and Moffitt Cancer Center (MCC) in Tampa, Florida, US, the overall goal of which is to improve cancer care outcomes for Hispanics in Puerto Rico and Florida. We further assessed associations between MC1R variation and traditional skin cancer risk factors and genetic ancestry in this population.As a prelude to conducting an intervention study among Hispanics to determine whether feedback of Three different sources were used to obtain samples and/or recruit study participants: the Puerto Rico Biobank (PRBB), located in Ponce, PR; a Community Participant Registry (CPR) covering Puerto Rico and Florida, US; and the Morsani Family Medicine clinics (MFMC) at the University of South Florida, in Tampa, Florida, US. All participant information, informed consent forms, and questionnaires were available in both Spanish and English. We also obtained genotype data from the 1000 Genomes project.19. Briefly, we obtained de-identified stored peripheral blood samples from 122 healthy controls and 78 randomly selected cancer patients diagnosed with cancers other than melanoma  Committee, and all research was performed in accordance with relevant regulations.The PRBB is a cancer tissue biobank housed at PHSU that was established as part of a PHSU-MCC partnership initiative. In order to contribute to the PRBB, participants were required to confirm their Puerto Rican heritage as indicated by having at least three Puerto Rican grandparents. Processes of informed consent, collection, processing, and storage of samples are published in detail elsewhere® DNA (DNA Genotek) kit. Mailed kits included detailed instructions to promote maximize yield and minimize contamination of collected saliva. Ethics approval was obtained from Chesapeake IRB with subsequent continuing renewal approval by Advarra, and all research was performed in accordance with relevant regulations.The CPR was a resource also developed as part of the PHSU-MCC partnership and comprised Hispanic residents in Tampa Bay and Puerto Rico who had provided consent to be contacted about cancer prevention and control research studies. Invitation packets that included an information statement, consent form, and questionnaire on demographics and pigmentation characteristics  at the MFMC were identified via clinical records and invited to participate at scheduled appointments. At the time of providing written informed consent, participants completed a questionnaire eliciting information on demographic variables and pigmentation characteristics  participants available from the 1000 Genomes Project data portal20.Genotype data at the MC1R to identify all existing variants21. We also genotyped 106 ancestry informative markers (AIM) that discriminate between Native American, African, and European ancestry. To maximize genetic information, SNPs with a large difference in allele frequency among ancestral populations were chosen. As well, representation across all 22 autosomal chromosomes was considered when selecting SNP markers. This AIM panel has been described previously22. AIM genotyping used a multiplex PCR coupled with single base extension methodology with alleles called using a Sequenom analyzer. Genotyping quality control for AIM was assessed using standard sample-level and SNP-level metrics. MC1R variants and genotype calls for the same 106 AIM were abstracted from 1000 Genomes genotyping data using VCFTools23.For DNA samples retrieved from the PRBB and obtained from participants recruited through the CPR and from the MFMC, we performed direct Sanger sequencing of the one exon coding region of MC1R variant(s) carried using an algorithm similar to that described in Hernando et al.24 Participants in the low risk group did not carry any MC1R variant (consensus) or carried only variants that do not impact on receptor function, i.e. pseudoalleles, based on published functional analyses or as predicted from bioinformatical algorithms. Participants in the medium risk group carried only a single MC1R variant that results in partial loss of receptor function, i.e. “r” allele, based on published functional analyses or as predicted from bioinformatical algorithms. Participants in the high risk group carried either two “r” variants or carried at least one variant known to result in loss of receptor function based on published functional analyses or as predicted from bioinformatical algorithms.We categorized participants into three groups based on the number and type of 25. The parental reference populations were genotyped on the Affymetrix 100 K SNP chip, and included 42 Europeans (Coriell’s North American Caucasian panel), 37 West Africans , and 30 Native Americans (15 Mayans and 15 Nahuas) and have been described previously22.For each individual, global ancestry proportions were measured using the software Admixture v1.3 at a k = 3 and under a supervised modelMC1R minor allele frequencies were calculated. We compared MC1R risk categories by phenotypic skin cancer risk characteristics after dichotomizing phenotypic measures, and we used logistic regression models to determine odd ratios (OR) and corresponding 95% confidence interval (CI) with adjustment for gender. We compared MC1R risk categories by quartiles of European, African and Native American genetic ancestry and tested for differences in proportions using the Jonckheere-Terpstra test or the Cochran-Armitage trend tests. Quartiles of genetic ancestry were based on the overall participant sample (n = 315) that had successful ancestry genotyping. All analyses were conducted using SAS.MC1R  and demographic information was obtained for 167 (87%) participants . From the CPR, 32 (82%) of the responders completed the questionnaire on demographics and self-reported pigmentation characteristics and provided a saliva sample, and 30 (77%) samples were successfully genotyped for MC1R. Eighty-eight (84%) participants from the MFMC completed the questionnaire on demographics and self-reported pigmentation characteristics and provided a saliva sample; 79 (90%) samples were successfully genotyped for MC1R. Flow diagrams summarizing participant enrollment, biosample collection and genotyping, and availability for analyses are given in Supplemental Figure MC1R genotyping are summarized in Table From the PRBB, 193 (97%) samples were successfully genotyped for MC1R genotype. Of these individuals, 137 (34%) were in the medium risk group and 89 (22%) were in the high risk group  and high  MC1R risk categories were more likely to report skin that burned (including sunburn without blistering and severe burning with blistering) compared to those in the low MC1R risk category. A similar, but weaker, trend (p = 0.025) was noted with tendency to tan: Hispanic individuals in the medium  and high  MC1R risk categories were more likely to report skin that only mildly tanned at best compared to those in the low MC1R risk category. Only the trend for burning remained significant after Bonferroni correction for multiple comparisons. Although individuals in the medium  and high  MC1R risk categories were more likely to freckle  compared to individuals in the low risk category, associations were not statistically significant. We did not find an association between MC1R risk categories and eye or hair color, in part because of the limited proportion (6–7%) of individuals reporting light eye color  or red or blonde hair color.Among CPR and MFMC participants who completed a questionnaire capturing phenotypic information, most reported having darker phenotypic characteristics Table . AssociaMC1R risk categories according to quartiles of genetic ancestry among individuals of sole Puerto Rican heritage. Within European, African, and Native American genetic ancestries, the proportions of Hispanic participants carrying low, medium, or high MC1R risk variants were similar across genetic quartiles, and all statistical tests were not significant (p > 0.05).Genotyping of AIM was completed on 181 samples from the PRBB, 30 individuals recruited from the MFMC, and was available on 104 Puerto Rican individuals from the 1000 Genomes Project. Mean genetic ancestry proportions for these 315 individuals are listed in Table MC1R variants in a Hispanic population in Tampa and Puerto Rico found that 56% of participants carried a MC1R allele(s) that placed them at elevated risk for skin cancer, the vast majority of which have been shown to increase the odds of melanoma, SCC, or BCC by at least 80%8. Our analyses demonstrate that MC1R variants are associated with some pigmentation characteristics in this overwhelmingly Puerto Rican sample, consistent with observations seen in European populations26. Among individuals reporting only Puerto Rican heritage, the proportion of participants categorized at elevated MC1R risk was comparable across quartiles of European ancestry, across quartiles of African ancestry, and across quartiles Native American genetic ancestry, indicating that those with greater African or Native American genetic ancestry carried MC1R risk alleles. Although our findings are limited by the overall and sub-group sample sizes and self-reported pigmentation characteristics, these results contribute novel evidence on the MC1R gene in an underserved population, which will inform future research studies.This pilot study on the prevalence of MC1R variants and their association with skin cancer risk is based on studies predominately in non-Hispanic whites. However, some international studies in general population and melanoma family settings have demonstrated a range in the prevalence and variation of MC1R variants according to geographic location28. Studies conducted in Spanish populations have found approximately 50–70% of individuals carry at least one risk variant30, which is comparable to the combined prevalence of medium and high risk alleles in our study. One study compared the association between melanoma and MC1R variants in German and Spanish populations, and the authors found significant differences in the frequency of, and risk attributable to, MC1R variants in the two populations31.Currently, our understanding of 32. We observed minimal variation in MC1R risk according to genetic ancestry among Hispanics of Puerto Rican heritage. Our findings suggest that MC1R variants are relevant to a diverse population and that even among populations with less European and stronger African or Native American genetic ancestry there may be carriers of alleles conferring elevated risk. Furthermore, these data reinforce the need for future research on the varied functional impact of MC1R variants according to population subgroups that are genetically and geographically diverse31.In our study, the overall proportions of genetic ancestry were consistent with those from published data, which demonstrate that Hispanic and Latino populations have high admixture of predominately European, African, and Native American ancestryMC1R is associated with melanoma risk independent of traditional phenotypic characteristics (e.g. hair and skin color) and that MC1R may even confer higher risk among individuals with a darker phenotype than among those with a lighter phenotype28. However, to the best of our knowledge, studies of associations between MC1R and skin cancer risk in Hispanic populations are lacking. Some studies have reported on the prevalence of some MC1R risk variants in Hispanic and Latinx sub-groups such as Mexican-American, Uruguayan and Brazilian, but most have either focused on populations at elevated risk of melanoma due to personal/family disease history34 or they were conducted in the context of other diseases, such as depression36. One recent study set in New Mexico reported carriage of a medium or high risk MC1R variant in 63% of enrolled Hispanics37. Some studies have examined associations between MC1R variation and genetic ancestry39, but there appears to be limited research that also incorporates pigmentation characteristics in Hispanic populations. Our findings contribute novel evidence on the association between MC1R variation and pigmentation characteristics in Hispanics who were not selected to participate on the basis of their personal or family history of melanoma (or non-melanoma skin cancers).Several studies among non-Hispanics have shown that genetic variation at 16. As expected, the majority of participants in our study who completed the questionnaire reported darker phenotypic characteristics. Among those who reported moderate to strong tanning ability, 53% carried either medium or high MC1R risk variants, and this group particularly stands to benefit from receiving information on their MC1R genotype as it may change their perceived skin cancer risk (from lower to higher). High uptake and interest in MC1R testing among Hispanics has previously been demonstrated18, and the impact of receiving such genetics-based skin cancer risk information on behavioral, psycho-social and ethical outcomes is being investigated in ongoing trials in Hispanic and broader population contexts41. Additionally, our findings indicate a need for further research on the pathways and attributable risk of MC1R variants in individuals with sun resistant phenotypes.The belief that darker skin pigmentation is infallibly protective against skin cancer can serve as a barrier to Hispanics undertaking, and receiving education in, prevention and early detection behaviorsMC1R genetic variants may be relevant to a diverse, Hispanic population and could inform skin cancer risk assessment, prevention and early detection recommendations in this setting. Our findings further highlight the need to ensure that prevention and early detection recommendations are inclusive of populations with low risk phenotypic characteristics alongside general population strategies, and the need for research on the pathways and risk of MC1R variants in groups with diverse genetic ancestry and phenotypic characteristics.This pilot study demonstrated that risk information based on Supplementary information.


Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.Environmental and agricultural pollination services by honey bees,  Varroa destructor—the most important pathogen of the European honey bee (Apis mellifera). DWV has a near-global distribution  is arguably—in concert with its vector the ectoparasitic mite smission ) and, inthogenic . In contthogenic ,4,5,6, athogenic . Evidencthogenic ,9, selecvariants , and synvariants  have beevariants  and othevariants . AlthougIflaviridae family [Varroa and named Varroa Destructor Virus type 1 (VDV-1) [Varroa to naïve colonies with a diverse virus population was interpreted as an indication that this variant was more virulent [DVD (GenBank HM067437) and VDV-1VVD , both bearing the DWV A capsid proteins coding region and DWV B non-structural coding region [Varroa transmission toward the emergence of variants with enhanced virulence [DWV is a picorna-like virus from the e family ,14. The e family  exhibit  (VDV-1) . As a co (VDV-1)  and its  (VDV-1) ,18. A th (VDV-1) ,20,21,22virulent ,22. Convvirulent . In addivirulent ,25,26. Firulence . Therefode facto standard approach to address questions about virus replication, virulence and pathogenesis [Varroa destructor. Finally, by introducing a reporter encoding sequence to the virus genome we visualise the in vivo tissue distribution of DWV in infected honey bee brood. The results of this study provide new insights into understanding the nature of DWV infection and introduce new molecular tools for honey bee research.In virology, RG involves the manipulation of the genotype, the recovery of the virus, and the investigation of the phenotype. Over almost four decades, it has become the ogenesis ,28. To fogenesis . Moleculogenesis ,30. In tVarroa-infested honey bee colony from Warwick-HRI apiary (2014), and a cloned full-length cDNA was incorporated into a plasmid vector containing all required elements for the transcription of the viral RNA. VVD RG clone is identical to the source VDV-1-DWV-No-9, with the exception of two nucleotide substitutions , resulting in creation of an HpaI restriction site. In order to obtain VDD and VVV constructs bases 2727–4888 (capsid proteins encoding region) and 4885-9783  were replaced with corresponding DWV A and B fragments respectively (HpaI (5275-5280 nt in VVD variant), Kpn2I and AvrII (2751-2756 and 4884-4889 nt in VDD variant), and AvrII, PflFI and BglII .RG constructs used in this study were based on the cDNA clone of a recombinant DWV variant VDV-1-DWV-No-9 (GenBank HM067438.1). VDV-1-DWV-No-9 sequence was rescued using samples from a ectively . Insertsapiary 20, and a cE) were obtained by NEBuilder Hifi assembly  reaction applied to previously made RG constructs. EGFP sequence flanked by 32 additional nucleotides (ATGGATAACCCT from 5’ and GCAAAACCAGAG from 3’ end) resulting in duplication of the protease cleavage site (amino acid composition of the site is AKPEMDNP [in silico cloning simulation. Full cDNA sequences of DWV clones used in this study are available in EGFP-encoding RG constructs (DWVAKPEMDNP ) was insPme I (cutting at the end of the sequence encoding the poly-A tail) or Nru I  respectively. Linearized DNA was purified by phenol-chloroform extraction and ethanol precipitation and T7 transcription was performed with T7 RiboMAXTM Express Large Scale RNA Production System  according to the manufacturer’s protocol. In order to account for the stabilization effect of the poly-A tail truncated transcripts were subjected to an additional polyadenylation step with poly(A) Tailing Kit . RNA transcripts were purified with GeneJet RNA Purification Kit  using clean up protocol and eluted in RNAse free H2O. All transcripts were analysed for integrity by gel electrophoresis and stored at −80 °C.DWV RNA was synthesized using linearized plasmid templates. Full length and truncated templates were linearised with in vitro transcribed RNA. Homogenized tissue was diluted with sterile PBS in 1:1 (w:v) ratio and centrifuged at 13 000× g, 4 °C for 10 min. The supernatant was sterilized by passing through 0.22 μM PES filter  and treated with RNase A to destroy all non-encapsidated RNA. RNA was extracted from 100 μL of the virus stock using RNeasy kit  and analysed by RT-qPCR.DWV stocks were prepared from honey bee pupae injected with Apis mellifera) brood in this study was obtained from the research apiary of the University of St Andrews. Hives used for sampling were regularly treated for Varroa with an appropriate miticide and routinely screened for DWV. DWV level in bees obtained from these hives was found to range within 102–106 genome equivalents (GE) per 1 μg of total RNA. No phenotypic evidence for other honey bee viruses being present was found within the colonies throughout the course of the studies. Pupae and larvae of the required age were collected from the comb and transferred to the incubator set at 34.5 °C and 90% relative humidity. Pupae were kept on folded sheets of filter paper, and larvae were transferred into 96-well plates with round bottom wells containing feeding diet (6% (w/v) glucose, 6% (w/v) fructose, 1% (w/v) yeast extract, and 50% (w/v) royal jelly in H2O).All honey bee . On the next day, feeding packets containing artificial feeding diet and supplemented with DWV stock or equal amount of PBS were prepared. The feeding packets were prepared by wrapping a 175 μL drop of the diet containing RNase A treated DWV inoculate (prepared as described in Locusta migratoria) haemolymph was frozen-thawed, heat-treated, and centrifuged for 30 s at 7500× g.Mites were placed in groups of 5–10 mites per feeding packet with four replicates for each treatment and kept in the incubator for four days. At the end of incubation live mites were collected, snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. Total RNA was extracted from pooled mite samples using the standard TriReagent protocol .RNA and virus injections into pupae were performed with insulin syringes ). 5 μL injections were introduced by inserting the syringe needle between the second and third abdominal segments of a pupa. All pupae were maintained on a warm heating plate during the injections.10 and 8.7 × 1011 of DWV RNA copies) of in vitro transcribed RNA was injected individually into white-eyed honey bee pupae. Truncated VVD transcript injections were used as a negative control in RNA transcript injections. RNA-injected pupae were analysed at 72 h post-injection. 0.5 and 5 μg . Total RNA was extracted with GeneJet RNA Purification Kit  using a protocol adapted for vacuum manifold application. cDNA was prepared with qScript cDNA Synthesis Kit  from 1 μg of total RNA following the manufacturer’s protocol with both oligo dT and random hexamer primers included in the reaction mixture in the reaction volume of 20 μL.Total RNA was extracted individually from all bee samples, actin, used as an internal RNA quality control, was carried out by end-point PCR with Taq DNA polymerase  and 2 μL of cDNA. DWV_RTPCR primers were designed to detect all three DWV variants under study. To amplify the cDNA regions containing restriction site tags in VDD and VVV virus variants Kpn2I_F/R and PflFI_F/R primer sets were used respectively. PCR cycling conditions were 30 cycles of 95 °C (15 s), 55 °C (15 s), 68 °C (2 min) with an initial 95 °C step (30 s), and a final extension at 68 °C (5 min). PCR samples were analysed on a 1% agarose gel stained with ethidium bromide. When required, DWV PCR products were subjected to restriction digest prior to loading on the gel.All sequences of primers used in this study are shown in E RNA was assayed by a set of primers amplifying a junction region between EGFP and viral sequence (primers EGFP_qPCR_F and VDD_VP2qPCR_RP or VVV_VP2qPCR_RP depending on the virus variant used). DWV and DWVE titers were calculated by relating the resulting Ct value to the standard curve generated by performing qPCR from a serial dilution of the cDNA obtained from 1 μg of VVD or DWVE RNA transcript respectively.The quantification of DWV genome copies was performed by SYBR-Green Real-Time Quantitative PCR (qPCR). Reactions were carried out in a C1000 Thermal Cycler  using Luna Universal qPCR master mix , 0.25 μM forward and reverse DWV_qPCR primers, and 2 μL of cDNA with the following thermal profile: 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C with a final post-amplification melting curve analysis step. Accumulation of DWVTaq DNA polymerase  using a forward primer identical to the adapter sequence (primer 388) and DWV(-RNA)_RT R primer. PCR was run for 35 cycles in the same conditions as described above.Strand-specific detection of DWV RNA was performed as described earlier . BrieflyE was done using Leica TCS SP8 confocal microscope with 10× HC PL FLUOTAR objective . All imaging to detect EGFP signal in samples infected with DWVLive larvae were washed with increasing concentrations of aqueous ethanol solution and fixed in 4% paraformaldehyde in PBS for 2 h at 4 °C. Fixed larvae were allowed to sink in 30% sucrose in PBS, mounted in NEG-50 Frozen Section Medium  and subjected to microsectioning on CM1860 Cryostat . Sections of 50–80 µm thickness were placed on Superfrost Plus microscope slides , mounted in ProLong Gold antifade medium, and analysed by microscopy.in vitro transcribed viral RNA from relevant cDNAs. All RG templates were derived from an infectious cDNA of a recombinant DWV variant (GenBank HM067438.1) [To examine the biology and pathogenesis of different strains of DWV we prepared near-clonal virus stocks from honey bee pupae injected with 67438.1) . The 5′ 67438.1) . Each cDin vitro synthesised RNA enabled recovery of genetically tagged virus at high efficiency. Using qPCR analysis, the average viral load in RNA-injected pupae was shown to be 1.5 × 1010 DWV GE per 1 μg of total RNA , but there was no discernible difference in yield of the three variants when ≥10 GE were inoculated (p > 0.05). Incubation of pupae for a further 24 h resulted in virus yields of ~1010 GE/μg of RNA with no notable difference between the three DWV variants tested. This shows that RG-generated DWV is infectious and the kinetics of virus replication is very rapid, with the genome being amplified ~108–1013 times within 48 h of injection of pupae (minimum yield of RNA/pupa was 200 μg).We investigated the infectivity of RG-recovered VDD, VVV and VVD variants by inoculation of white-eyed honey bee pupae using serial dilutions of virus stocks and analysed all samples individually 24 h post-inoculation by RT-qPCR . Under tVarroa feed throughout the development of the pupa and possibly during the phoretic phase of the life cycle. We investigated whether pupal development phases were partially or totally refractory to DWV replication by inoculating morphologically distinct stages with 102 GE of VVD DWV . High DWV levels (up to 1010 GE/μg of RNA) were detected in 5 × 106 GE fed groups for VVV and VVD inoculates and for all three variants in 5 × 107 GE fed larvae, clearly indicating virus replication. It was notable that a proportion of larvae showed little or no amplification of DWV over the fed amount suggesting, in comparison to inoculation via injection, a significant barrier to the development of a productive infection may exist for virus acquired per os. Having demonstrated pupal susceptibility to directly inoculated virus we then examined virus transmission to developing larvae via the oral route. First instar larvae were individually fed a diet containing dilutions of RG-derived DWV, and the virus was detected and quantified after 48 h. Although RG-derived DWV was detectable when 5 × 10s tested c. UninocVarroa. A confounding issue in some studies is the pre-existing presence of DWV within the mite, and the potential presence of contaminating DWV genomic negative strands (a marker of replication) in either the mite or the inoculum. To address this, we investigated the replication of RG-derived genetically tagged VVD in Varroa using an in vitro feeding system [There are conflicting reports on the replication of DWV in g system  containiVarroa were maintained on artificial feed packets supplemented with 1.75 × 109 GE VVD (107 GE/μl). VVD RG-derived virus stock was amplified in honey bee pupae, extracted and treated with ribonuclease A to remove any non-encapsidated viral or cellular RNA. Varroa, and consequently high DWV, levels. Upon digestion with the relevant restriction enzyme (HpaI), this product was partially cleaved in the VVD groups only, indicating that at least some of it originated from the negative-strand replication intermediate of VVD  results in predominantly asymptomatic  infection, with no apparent phenotypic deformities. We tested the morbidity in honey bees infected orally with clonal DWV by feeding larvae with a virus-supplemented diet, allowing pupation and eclosion and scoring viability and the presence of overt symptoms. Although lower levels of virus (≤105 GE) in the diet again failed to establish robust replication in the experimental groups, the feeding of 2 × 107 GE of RG-derived DWV to honey bee larvae  resulted in high morbidity rates (12 GE/μg of RNA (In the field non-ty rates . In larvg of RNA . Notablyg of RNA . It is kE), analogous to that previously reported for DWV A [pro proteolytic cleavage site [The tropism and pathogenesis of DWV remains poorly understood. Relatively little is known about the sites of virus replication and whether differences in tissue tropism after oral or mite-mediated transmission account for the appearance of symptomatic and asymptomatic forms of disease. To facilitate virus localization in experimentally inoculated bees, we developed an EGFP-expressing chimeric DWV , but was largely absent from the Malphigian tubules and thoracic muscles c. Since s before , all hadlication .7 GE of DWVE purified from previously inoculated pupae and analysed larval fluorescence 4 and 6 days later. Samples infected with DWVE exhibited extensive fluorescence; external observation localized this to the larval head, two thoracic segments containing wing rudiments, the caudal part of the body and to cells surrounding the spiracle openings  and a recombinant that has previously been reported to predominate in colonies ,26. We ain vitro gene synthesis, we constructed infectious cDNAs for type A (protein coding sequence), type B and a recombinant variant of DWV, recovered molecularly tagged virus after RNA inoculation and investigated the kinetics of virus replication in honey bee pupae. Replication was rapid, amplifying to ~108 GE/μg of RNA within 24 h, and plateauing at ~1010 GE per μg of total cellular RNA within 48 h. There were no major differences between the replication rates of the three DWV variants under study, and all stages of pupal development tested appeared equally susceptible to virus infection , any virus replication in the mite would likely guarantee the transmitted dose would significantly exceed the ID50. The viral load in mites has been reported to be high [per se. Previous analysis of virus replication in Varroa has produced conflicting results. Mass spectroscopy studies failed to detect viral non-structural proteins implying that DWV may not replicate in the mite [in vitro on a diet containing no honey bee-derived components. The only DWV virus  present would therefore be derived from the last pupa the mites had fed on, together with any subsequent replication in the mite. We supplemented the diet with the RG-derived VVD variant of DWV carrying a unique genetic tag allowing its unambiguous identification. The presence of genetically tagged negative strand RNA of DWV in the pooled mites samples fed on this diet demonstrates that VVD does replicate in Varroa and that newly acquired virus can replicate in the presence of a pre-existing virus population in the mite  is probably critical. Analysis of whether infection of all stages of pupal development are as likely to result in wing deformities may be informative in this regard, though Varroa-mediated virus transmission will initially occur when the foundress mite feeds on the just-capped pre-pupa. One of the factors which can potentially influence the development of overt disease are the sites of virus replication in the developing honey bee. This may result in direct cytopathicity and tissue damage or indirectly by dysregulation leading to damage at remote locations. To address the tissue tropism of DWV, we constructed a modified virus genome co-translationally expressing the green fluorescent protein. Inoculation of pupae, or ingestion by larvae, resulted in distinct fluorescence in a range of organs and tissues. In fed larvae, the EGFP signal accumulated in thoracic segments where the developing wing buds are located [It remains unclear what determines whether virus exposure results in overt disease or asymptomatic infection  although located , and inf located , would pVarroa as a vector the oral route no longer plays the decisive role in DWV spread, but the observed tropism of DWV infection indicates the initial evolutionary trait used by the virus. Robust virus replication in the primary tissues that enable subsequent transmission often results in spillover to secondary sites that are permissive for virus infection, but that offer no further route to a new host, e.g., the neurotropism of the faecal-orally transmitted poliovirus [in vivo time course studies of virus dissemination and facilitate host-vector transmission studies. More generally, the ability to introduce a ‘payload’ to the virus genome may also be exploited by using genetically modified DWV as a virus-based gene delivery system [Varroa or Bombus [Our observations indicate that DWV selectively targets organs most favourable for its oral and vertical transmission. Sites of DWV replication colocalize with exocrine glands, which are responsible for the secretion of larval diet components, and digestive tract tissues, from which it can presumably be transmitted in faeces  and, folliovirus . It is ty system . Our resr Bombus  or, morein vitro. With no system for propagating DWV in vitro these studies have had to be conducted in vivo. We show that the availability of a reverse genetic system allows the kinetics of virus replication in larvae and pupae to be examined. Comparative studies demonstrate that the two predominant variants of DWV essentially replicate equivalently, and that the symptomatic outcome of DWV infection is not linked to a particular genotype of the virus nor to the route of transmission. Unique genetic tags introduced to the genome enabled discrimination of input from the endogenous virus population and provide unequivocal evidence for virus replication in Varroa following detection of de novo synthesised negative-sense viral RNA. A viable GFP-tagged DWV genome supplies the basis for a better understanding of virus tropism and pathogenesis in infected brood and will provide a useful tool for elucidating the mechanisms behind symptomatic DWV infection in developing honey bees.In many single stranded positive sense RNA viruses, the development of reverse genetic systems was facilitated by a good understanding of virus replication


SCN1A are identified in up to 80% of individuals with clinical Dravet syndrome, and hundreds of individuals are diagnosed every year. SCN1A encodes a voltage-gated sodium channel subunit that is critical for neuronal function, specifically the function of GABAergic interneurons. While Dravet syndrome represent the main clinical phenotype for SCN1A-related disorders, milder clinical presentations are known that are typically seen in families [In 1978, the causes of most severe childhood epilepsies were entirely unknown, and any ability to systematically apply massive parallel sequencing to understand the underlying etiologies was still in the far future, while targeted therapy for a genetically defined epilepsy was at best a fantasy. During this period, Charlotte Dravet and collaborators first described a distinct epilepsy syndrome characterized by fever-induced seizures, generalized features on electroencephalography, and developmental plateauing starting in the second year of life, a condition initially called Severe Myoclonic Epilepsy of Infancy . Diseasefamilies . This grfamilies ,5.SCN1A-related disorders. First, noncoding de novo variants in highly conserved intronic regions of SCN1A were found in individuals with Dravet syndrome [SCN1A gene expression identified physiological nonproductive splicing events that could subsequently be reduced through specifically designed antisense oligonucleotides (ASO) [SCN1A expression in a Dravet mouse model with a reduction of seizures and Sudden Unexpected Death in Epilepsy (SUDEP) [SYNGAP1, SCN2A, or SCN8A [Haploinsufficiency is the generally accepted disease mechanism in Dravet syndrome, and until 2 years ago, no strategies were available to affect this primary disease mechanism. Two findings have revolutionized the current understanding of syndrome . This coes (ASO) . This me (SUDEP) . This noor SCN8A . This raSCN1A through poison exons. The SCN1A gene contains the naturally occurring poison exon 20N, and inclusion of this exon results in nonproductive splicing and nonsense-mediated RNA decay, effectively reducing the amount of Nav1.1-containing sodium channels made available to neurons. The authors characterize a Dravet syndrome mouse model with a poison exon mutation resulting in aberrant Scn1a regulation. The specific disease-causing poison exon variant was previously identified in a patient with Dravet syndrome reported by Carvill and colleagues [Scn1a transcripts carry poison exon 20N in wild type, this ratio is increased 5-fold in animals with the poison-exon mutation. This increase in poison exon inclusion alone resulted in a decrease of Scn1a mRNA to 50%, resulting in a reduced gene expression known to occur in Dravet syndrome. The presence of poison exons hints at complex underlying regulatory mechanisms that remain unknown to date and that may represent a promising avenue of future research.In the study by Voskobiynyk and colleagues , the autlleagues . VoskobiSCN1A transcripts included exon 20N during embryonal development, which decreased over time to 10% postnatally. This suggests that poison exon inclusion is not just an epiphenomenon, but an active regulatory process to suppress SCN1A expression during early development. Poison exon inclusion during normal development then decreases successively postnatally (SCN1A expression to be released from the poison exon-mediated shutdown. Therefore, mutations resulting in increased poison exon inclusion do not create an entirely novel transcriptional scenario. Poison exon mutations maintain the embryonal pattern of SCN1A regulation, a disease mechanism that could best be referred to as “persistent poison exon inclusion.” In fact, the authors also find a similar pattern of poison exon inclusion for SCN8A, a gene also strongly associated with DEE. This indicated that poison exon regulation may be a common feature regulating genes for neurodevelopmental disorders where the precise timing of gene expression is important.Voskobiynyk and colleagues  then protnatally . This alWhere does the study by Voskobiynyk and colleagues leave us? In the past, understanding the basis of genetic causes for epilepsies and neurodevelopmental disorder was considered a question of genomics. Massive parallel sequencing approaches allow up to 30% of DEE to be explained through genetic causes, and the number of known disease genes is increasing steadily. However, as in cancer genomics, it is reasonable to expect a paradigm shift from genes to transcripts. A large number of genes are recognized to contain appreciable amounts of nonproductive alternative splicing through poison exons. Therefore, conceptualizing disease-causing variation as “transcript issues” rather than “gene issues” will enable us to explore the utility of gene regulation approaches in more detail. Many disease-associated genes possess inherent regulatory mechanisms that may provide new targets to restore physiological levels of gene expression. Understanding how these mechanisms change in age-dependent patterns will help identify novel interventional strategies—and the required timing for such interventions—for conditions that are currently still largely treated symptomatically.


Rhododendron oldhamii, test the difference in the average GC content of coding sites and of third codon positions with that of surrounding non-coding regions, and test exon variants associated with environmental variables. Spatial expansion was suggested by R2 index of the aligned intron sequences of all genes of the regional samples and sum of squared deviations statistic of the aligned intron sequences of all genes individually and of all genes of the regional and pooled samples. The level of genetic differentiation was significantly different between regional samples. Significantly lower and higher average GC contents across 94 sequences of the 12 genes at third codon positions of coding sequences than that of surrounding non-coding regions were found. We found seven exon variants associated strongly with environmental variables. Our results demonstrated spatial expansion of R. oldhamii in the late Pleistocene and the optimal third codon position could end in A or T rather than G or C as frequent alleles and could have been important for adaptive divergence in R. oldhamii.Demographic events are important in shaping the population genetic structure and exon variation can play roles in adaptive divergence. Twelve nuclear genes were used to investigate the species-level phylogeography of  Molecular techniques, such as amplified fragment length polymorphisms (AFLPs), expressed sequence tag simple sequence repeats (EST-SSRs), and methylation-sensitive amplification polymorphisms (MSAPs) have been commonly employed in investigation testing for environmentally dependent local adaptation6. Nonetheless, nuclear gene sequences can be amplified and sequenced spanning coding and non-coding regions. The variation in non-coding and coding sequences in nuclear genes can be used, respectively, in investigation of phylogeny and phylogeography and in testing for correlation with environmental variables contributing to adaptive evolution8. Within the coding region of a gene, the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site is commonly used for the inference of adaptive evolution of genes driven by natural selection11, and synonymous substitutions are thought to be inconsequential because of the conservative nature of amino acids. However, synonymous substitutions can have significant effects on gene expression, protein folding, and protein cellular function13, and hence synonymous substitutions may not be "silent"14.Spatial and temporal patterns underlie population demographic processes of plant species and the phylogenetic relationship between and within species can be revealed by using chloroplast and nuclear DNA sequence data17. Theory suggests that the strength of natural selection on synonymous sites may be weak and effective population size is thought to be the determining factor for natural selection to be effective on codon usage pattern19, and synonymous substitution is subjected to strong purifying selection21. Nonetheless, selection acting on codon bias has been found in prokaryotes with large effective population sizes23 and in eukaryotic species with low effective population sizes26.The difference in the relative frequency of synonymous codons for individual amino acids in protein coding sequences is coined as codon usage bias. Codon bias can vary among species and/or among genes of a genome and may be derived via mutational bias processes, GC biased conversion, or driven by selection co-adapting with tRNAs in optimizing the efficiency and accuracy of translation28. The level of GC content at third codon positions is considered as an indicator reflecting codon usage pattern, and the level of gene expression has been found to be positively correlated with the level of GC content at third codon positions30. Studies have shown that overall codon bias is more phenomenal in monocots than in dicots and, respectively, tended to use C/G and A/T at third codon positions32. However, optimal codons tend to end in G or C in dicots, resulting in higher average GC content at coding sites and at third codon positions compared with surrounding non-coding regions33.In genes with high codon bias, the "preferred codons" often end in either C or G according to major codon preference modelRhododendron oldhamii Maximowicz belongs to the subgenus Tsutsusi of Ericaceae is an endemic dicot species widely, but fragmented distributed in the lowlands and up to 2,500 m in the humid understory of broadleaf forests in Taiwan. Heterogeneity in flowering times of R. oldhamii populations located in different geographic areas, across the species’ distribution range, have been shown either via examination of herbarium specimen records34 or by field studies35. Differential flowering times of species distributed in different geographic areas can result in reproductive isolation or reproductive incompatibility36, and in consequence a limited gene flow between populations37. Hsieh et al.4 showed that gene dispersal was limited within geographic regions of R. oldhamii and inferred that the discontinuities of population distribution resulted from recent population bottlenecks during the Holocene based on EST-SSR data. Although organisms in isolated, small populations may be restricted in developing local adaptation in response to changing environments38, study based on EST-SSR found population divergence at regional level in association with environmental variables underlying local adaptation in R. oldhamii4. Moreover, genetic and epigenetic variations based on AFLP and MSAP also found environmentally dependent adaptive divergence in populations of R. oldhamii5. R. oldhamii discontinuously distributed in a wide geographic range makes it an excellent exemplar endemic subtropical forest tree species occurring in Taiwan for investigating not only the population divergence associated with environmental differences, but also the phylogeographic history related to the current genetic structure of this species.R. oldhamii based on EST-SSRs4, it is worthwhile to study further from the evolutionary perspective of natural selection on codon usage bias using DNA coding sequences. Additionally, the far-distant past demographic history shaping the genetic structure and distribution of R. oldhamii can be inferred using nuclear non-coding sequences in contrast to using frequency data of EST-SSRs reflecting more recent demography in the previous study4. Strong correlations of synonymous and nonsynonymous substitutions with environmental variables using regression approaches40 may provide as evidence suggesting that they would have experienced the effects of selection. We hypothesized that both synonymous and nonsynonymous substitutions of nuclear coding sequences, particularly synonymous substitutions at third codon positions, in natural populations of R. oldhamii  test the significant differences in the average GC content of coding sites and of third codon positions of nuclear coding sequences with that of surrounding non-coding regions, and (3) test the associations of synonymous and nonsynonymous variants in coding sequences with environmental variables.Since it has been shown that adaptive divergence driven by natural selection at local scales in mii Fig.  might hamii Fig. , we aime4) (Table GAPC1) to 908 bp (PCFS4) (Supplementary Table GRP7) to 372 bp (LACS8) and from 280 bp (LHCA1) to 859 bp (GRP7). The minimum number of recombination events (Rm) within gene ranged from 0 (CPD) to 10 (GAPC1) (Table π ranged from 0.00163 (population CJ) to 0.00785 (population EGS) and from 0.00586 (southeast) to 0.00659 , respectively, for the 18 populations and the four geographic regions. Waterson’s nucleotide diversity measure, θw, based on segregating sites ranged from 0.00163 (population CJ) to 0.00785 (population EGS) and from 0.00552 (south) to 0.00695  for the 18 populations and the four geographic regions, respectively. The nucleotide polymorphisms (π and θw) for individual gene in each population were reported in Supplementary Tables π nor in θw . However, significant differences of π and θw among populations were found . Moreover, significant differences of π and θw were found in pairwise population comparisons after correction for multiple comparisons at false discovery rate (FDR) of 5%, but no significant difference was found in between region comparisons (Supplementary Table FIS), indicative of departure from Hardy–Weinberg equilibrium, representing homozygote excess, was found for all populations that were estimable (sample size > 1) except the population LLK  estimation revealed that the spatial expansion model could not be rejected based on the aligned intron sequences of the pooled samples of the 12 loci individually. Spatial expansion was also suggested by SSD when analyzed using the total aligned intron sequences of the regional and the pooled samples. Comparable sample sizes among regions and approximately equal migration rates among populations within regions were found based on the goodness-of-fit test for mismatch distribution under spatial expansion model , but not significant when compared among populations . Significant pairwise FST was also found for between region comparisons  higher than the average GC content of surrounding non-coding regions  and seven  of the 12 genes, respectively, showed significantly lower and higher average GC content across 94 sequences at third codon positions than that of surrounding non-coding regions.The average GC content at coding sites, at third codon positions, and in surrounding non-coding regions across 94 sequences of the 12 genes were 0.453, 0.419, and 0.356, respectively (Table P = 1). However, significant environmental heterogeneity was found when compared among regions . Significant environmental heterogeneity was also found in all pairwise regional comparisons except between the central-south regional group comparison (P = 0.334)  , albeit large amount of exon variation was unaccountable (fraction [d]: 87.87%) . Nevertheless, exon variation was significantly explained by pure environment , albeit a larger portion of explainable exon variation was attributed to geographically-structured environmental differences (fraction [b]: adjusted R2 = 0.0769).In the total aligned exon sequences of the pooled samples, 31 exon variable sites were found in nine out of the 12 nuclear genes examined. Using a variation partitioning, the total amount of explainable exon variation (12.13%) was significantly explained by a combinatorial effect of environment and geography (%) Table . HoweverGCT → TCT in LACS8_4, CGA → CAA in PCFS4_1, CAC → CGC in SPA1_1, and AAT → AGT in SPA1_6, were found to be strongly associated either positively or negatively with various environmental variables  or generalized linear mixed effect model (GLMM), or by both GLM and GLMM Table . The frees Table . Three sons Fig. . For tho42. Climatic oscillations during the Pleistocene have been widely recognized as the main historical factor shaping current population genetic structure and distributions of species or populations42. Limited gene flow between geographic regions resulted from bottleneck events in the Holocene approximately 9168–13,092 years ago was revealed based on EST-SSRs in R. oldhamii4. This study inferred that R. oldhamii has experienced a process of transition from historical connectivity toward contemporary regional isolation. In the present study, we found no consistent evidence of spatial expansion based on Tajima’s D and Fu and Li’s D* and F* using DNA sequences of 12 nuclear gene loci , due to the retention of ancestral polymorphisms, based on nuclear DNA intron sequence data in the present study. Hence, no clear genetic structuring was also observed based on DAPC  was observed which is consistent with the regional population divergence inferred based on EST-SSR and AFLP5.Since nuclear DNA is the fastest evolving among the three genomes plants harboredπ = 0.00659) was found based on the total aligned intron sequences of the 12 nuclear genes examined 3 and Quercus glauca 51. Moreover, the level of nucleotide diversity of R. oldhamii was found to be higher than the level of nucleotide diversity (π = 0.00134) of a mainland Rhododendron species, R. delavayi, based on sequences of a major RNA Polymerase II subunit52 and the nucleotide diversity (π = 0.0039) based on eight nuclear loci of a Rhododendron species, R. weyrichii, distributed in Japan and South Korea53. Nonetheless, the comparisons may not be appropriate because DNA sequences used in the calculation of nucleotide diversity were derived from different genes. Although there are no nucleotide diversity estimates based on DNA sequences available for comparison, the level of genetic diversity can be compared between congeneric species occurring in Taiwan based on EST-SSRs genotyped using the same set of amplification primer pairs. Comparable levels of R. oldhamii EST-SSR genetic diversity across populations (average HE = 0.284)4 were found when compared with other Rhododendron species belonging to the same subgenus Tsutsusi occurring in Taiwan (average HE = 0.293)54.Higher or comparable level of nucleotide diversity 59. The level of EST-SSR genetic diversity may not only reflect the outcome of a long evolutionary history, but also influenced by recent demographic events60. The lower level of EST-SSR genetic diversity in R. oldhamii compared with species of the R. pseudochrysanthum complex might have been resulted from inbreeding . In addition, background compositional bias may also play an important role in the synonymous codon selection in a gene78. Although, intron length may have a positive relationship with the level of intron GC content79, we found no significant correlation between length of intron and its GC content of the 12 nuclear genes examined . Additionally, the average GC content of coding sites and of third codon positions were significantly different from the average GC content of surrounding non-coding regions . Nonetheless, selection might have played an important role in the levels of GC content at both synonymous and nonsynonymous substitution sites14.Selection was demonstrated to play an important role in driving codon usage patternLACS8 and SPA1 were the two genes that had lesser amounts of average GC content at third codon positions compared to that of surrounding non-coding regions  and A/C (SPA1_5), possessed lesser amounts of GC content compared to surrounding non-coding regions . These samples were used for DNA extraction82 and in direct sequencing of polymerase chain reaction (PCR)-amplified DNA products of 12 nuclear loci  based on EST sequences of R. catawbiense83. PCR amplifications were performed in a PTC-100 DNA programmable thermal cycler  and done by initial denaturation , 40 cycles of denaturation , annealing  . The amplification products were electrophoresed on a 1% agarose gel and the corresponding bands of the 12 genes under study were purified with Viogene Gel Extraction Kit  and directly sequenced using an ABI 3730 DNA sequencer . Heterozygous site resolution, haplotype phasing, and functional annotation 87, the average nucleotide diversity of segregating sites (θw)88, and haplotype diversity (Hd) were computed using DNASP v.689. Neutrality test statistics including Tajima’s D90, Fu and Li’s D* and F*91, and R247 were also estimated using DNASP and tested for deviation from neutral expectation using 10,000 coalescent simulations. Summary statistics were estimated based on the total aligned intron sequences of the population, regional and pooled samples, and computed for the aligned intron sequences of the pooled samples for each gene separately. Neutrality test statistics were computed based on the total aligned intron sequences of the regional samples and the pooled samples and the aligned intron sequences of the pooled samples for each locus individually. Significant negative values of D, D*, and F* and significant small positive values of R2 represent an excess of low frequency mutations, indicating unimodal mismatch distributions, representative of sudden expansion relative to a null model of demographic stability with multimodal mismatch distributions. The minimum number of Rm following the four-gamete test92 and the number of segregating sites were estimated for each gene based on the aligned intron sequences of the pooled samples using DNASP. A goodness-of-fit test based on the SSD statistic was calculated using ARLEQUIN v.3.593 for the total aligned intron sequences of the regional and the pooled samples and the aligned intron sequences of the pooled samples of each gene separately considering population subdivision. A significant SSD value represents departure from the estimated demographic model of spatial expansion. In the goodness-of-fit test, though the error estimate is generally high, the time of spatial expansion was calculated using the formula t = τ/2μk, where t is the time since the expansion, τ is the estimated number of generations since the expansion, μ is the mutation rate per site per generation, and k is the sequence length. We adopted a generation time of 15 years95 and the mutation rate of 1.581 × 10–9 per site per year53 used in the study of population demographic history of R. weyrichii53, which is also belongs to the subgenus Tsutsusi, for calculation of the expansion time. Friedman test was used to assess the overall difference of nucleotide diversity (π and θw) at the population and regional levels using the friedman function of R agricolae package96. In Freidman test, nuclear locus was used as a blocking effect. Pairwise population and regional comparisons were performed and P values adjusted using Fisher’s least significant difference.Sequence alignment was performed using the https://codonw.sourceforge.net//culong.html). Differences in the mean GC content of coding sites and of third codon positions compared with the average GC content of surrounding non-coding regions of each gene were assessed using paired Wilcoxon test (the R wilcox.test function of R).The GC content of 94 sequences derived from 47 individuals at coding sites, at third codon positions, and in surrounding non-coding regions of the 12 genes were calculated using CodonW  of Environmental variables with variance inflation factor (VIF) > 5 and highly correlated with other variables (|r|> 0.8) were removed  and absence ("0") of the rare alleles and implemented in a GLM and a GLMM as response variables to assess the correlations of exon variant alleles with environmental variables, with binomially distributed residuals, and significance assessed with 95%, 99%, and 99.5% CIs. GLMs were performed using the R varpart and anova.cca functions of R package vegan were used, respectively, for variation partitioning and testing for significance with 999 permutations. Exon variation was partitioned into four fractions explained by pure environmental variables (fraction [a]), geographically-structured environmental variables (fraction [b]), pure geographic variables (fraction [c]), and residual effects (fraction [d])106, based on adjusted R2 values107. Sample site coordinates were used as geographic variables in variation partitioning.The frequencies of the frequent alleles of exon variants were used in a variation partitioning analysis to disentangle the effects of environment and geography explaining exon variation. The Supplementary information.


Physical exercise plays an important role in both the prevention and treatment of cardiometabolic diseases. Peptides secreted or released from skeletal muscle, so-called myokines, contribute to the beneficial anti-inflammatory and insulin-sensitizing effects of increased muscle activity and may, therefore, counteract pathomechanisms of obesity and type 2 diabetes (T2D) . HoweverWe included cross-sectional data from 400 Korean participants of the ongoing Korean Sarcopenic Obesity Study (KSOS) described in detail elsewhere . Blood aWe included a total of 400 participants  with an established diagnosis of T2D which have been consecutively recruited at the Department of Medicine of the University Hospital in Leipzig (Germany). Patients were excluded from the analyses according to the criteria defined for the Korean subcohort. The study has been approved by the ethics committee of the University of Leipzig , and all subjects gave written informed consent before taking part in the study. All blood samples were collected between 8 and 10 a.m. after 12 h overnight fast. T2D complications were defined as described in the Korean cohort.Serum samples were analyzed centrally at Woongbee Meditech Inc., Korea. Irisin  and IL-15  concentrations were measured using specific enzyme-linked immunosorbent assays (ELISA). U test or independent t test. Fisher’s exact test or Pearson’s Chi-square was utilized to evaluate the differences in the categorical variable distribution. To investigate correlations between myokines and cardiometabolic variables, Spearman partial correlation analysis was used after adjusting for age and sex. Data were analyzed using SAS 9.2 . A p value < 0.05 indicates statistical significance.Korean and German study populations were compared using the Mann–Whitneyp = 0.002). Independent of ethnicity, irisin and IL-15 serum concentrations were not correlated with most cardiometabolic risk factors  in Korean T2D patients with albuminuria.In analyses of the entire study population and in the German and Korean subgroups, irisin and IL-15 serum concentrations were not significantly different between T2D patients with or without evidence for diabetic retinopathy (data not shown). Comparisons of participants with or without albuminuria only revealed significantly lower irisin (but not IL-15) serum concentrations (The key result of our study is that despite significant differences in almost all anthropometric parameters and cardiometabolic risk factors between German and Korean T2D patients, both irisin and IL-15 serum concentrations were indistinguishable. Noteworthily, Korean women displayed slightly higher irisin levels than German women. In addition, we could reproducibly show for the German and Korean cohort that irisin and IL-15 serum concentrations are not related to cardiometabolic risk factors or the presence of diabetes complications. There was only one exception that Korean T2D patients with albuminuria showed lower irisin levels compared to those without albuminuria, generating the hypothesis that there are ethnic differences in the interplay between irisin secretion or clearance and renal function. An important limitation of our study is that we only analyzed the circulating myokines at one time point. We can, therefore, not exclude that these myokines may be relevant biomarkers for the prognosis of cardiometabolic diseases and/or for the evaluation of treatment response. Another limitation is that our study combined two separate data collections into one evaluation, which may have created a potential bias.In conclusion, our data suggest that neither irisin nor IL-15 serum concentrations reflect cardiometabolic risk factors or T2D complications in two independent cohorts from Korea and Germany.


Oreochromis niloticus is the third most commonly farmed finfish species in the world, accounting for nearly 5% of global aquaculture production. In the past few decades much of the success of this species has been attributed to the development and distribution of Genetically Improved Farmed Tilapia (GIFT). Despite the increasing availability of GIFT, the productivity of small-scale farming remains highly variable, particularly in developing nations. Commercial fish-feed pellets can increase fish farm productivity; however, many small-scale farmers rely on other means of feeding fish due to the high cost and limited availability of commercial fish feed pellets. Therefore, understanding how locally-sourced feeds affect the production of GIFT is an important step towards improving feeding practices, particularly for farmers with low financial capital. This study used stable isotope analysis (SIA) and 16S rRNA gene sequencing to compare the effects of a locally-sourced vegetable-based diet and commercial pellet-based diets on the relative condition, nutrient assimilation patterns and gastrointestinal microbiota of GIFT. GIFT fed a locally-sourced diet were smaller, and in a significantly poorer condition than those fed with commercial fish feeds. SIA showed no differences in dietary carbon between the two diets; however, δ13C, poor fish condition and the abundance of specific bacterial taxa (of such as Fusobacteria) were correlated. SIA revealed that GIFT fed locally-sourced diets that predominantly consisted of vegetables were significantly enriched in δ15N despite a perceived lack of dietary protein. This enrichment suggests that GIFT fed a locally-sourced diet may be supplementing their diet via cannibalism, a behaviour representative of poor farming practice. Overall this study highlights the need to increase the availability of suitable GIFT feeds in developing nations. The development a low-cost feed alternative could improve the success of small-scale GIFT farmers in PNG, increasing both food and income security within the region.Nile tilapia,  The gloa (GIFT) , 6.Inland aquaculture in Papua New Guinea (PNG) is mostly small-scale subsistence farming with growth limited by infrastructure, the high cost and limited availability of commercial feed, the poor economic status of people and a lack of fish husbandry skills in the farming communities –9. The fThe productivity of small-scale fish farms in PNG and other developing countries could be increased by better understanding the dietary preferences and nutrient assimilation patterns of the fish. Previous studies have relied on gut content analysis to classify the diet of fish ; howeverKnowledge of the composition and microbial diversity within the gastrointestinal tract is vital because of the influence these symbionts have on the host’s growth and survival –23. For All experimental procedures involving live fish were approved by the University of New South Wales Animal Care & Ethics Committee (UNSW ACEC) under permit number 18/26B specifically for this study.Oreochromis niloticus (GIFT Strain) individuals were collected from six different fish farms within the Aiyura valley , located in the Eastern Highlands of Papua New Guinea. The stocked GIFT were from the same family line acquired from the Highlands Aquaculture Development Centre (HAQDEC) breeding program. The sampled farms all stock GIFT in earthen ponds, and represent one of two different feeding practices; a locally-sourced raw vegetable-based diet  (n = 3) (hereafter referred to as a ‘vegetable’ diet) and a mixed diet consisting of both the occasional supplementation of raw vegetables  and regular commercial feed pellets (n = 3) (hereafter referred to as a ‘pellet’ diet). The commercial fish feed pellets were all from a single imported source from Vietnam and included 30% crude protein, 5% crude fat, 16% ash, 6% crude fibre and 11% moisture with raw ingredients including fishmeal, wheat flour, soybean meal, fish oil, rice bran and vitamins and minerals. Parameters such as feeding frequency and pond size were recorded on site during sample collection. The age of the GIFT and stocking density of the sampled ponds was estimated as there were no records kept of fish being removed from the ponds  or restocking events. Water quality measurements of temperature (n = 3), pH (n = 3) and dissolved oxygen (DO) (n = 3) were recorded at each farm using a TPS WP91 water quality meters with an Ionode J44 intermediate junction pH probe and YSI DO probe. Probes were calibrated daily according to the manufacturer’s guidelines and standards.Individual fish (n = 10) of similar size were collected from each farm using handheld nets and immediately euthanised in an AQUI-S solution . All fish were devoid of any gross or clinical signs of disease. Standard measurements of length and weight were recorded prior to dissection. Dissections were undertaken at HAQDEC within 2 hours of collection. White dorsal tissue samples were aseptically removed, scaled and skinned before being rinsed with distilled water and stored at -20°C. In addition, the gastrointestinal tract was aseptically removed with a combined hindgut content and hindgut wall sample collected and frozen  and stored at -80°C prior to microbial analysis.13C and δ15N. The C: N molar ratio relates to the lipid content of a sample and affects δ13C values. When the C:N ratio was greater than 3.5, the result was mathematically corrected to account for the lipid content using the formula specified by Post, Layman [White dorsal tissue samples were prepared and analysed as per the methods previously described by Kinney, Hussey  and Gopi, Layman  as folloCCT ACG GGN GGC WGC AG) and 785R (GAC TAC HVG GGT ATC TAA TCC) [Bacterial DNA was extracted from the hindgut samples (8 mg) using the DNeasy® Blood &Tissue Kit  following the manufacturer’s protocol. The diversity and structure of the microbial community associated with each sample was assessed by amplicon sequencing of the V3-V4 variable region of the 16S rRNA gene, using the barcoded universal bacterial primers: 341F  [Firstly, sequencing reads of low quality  were re 132 NR) . zOTU se 132 NR) . Rare zOn) was used to assess the condition of the sampled fish and was calculated as described in Jisr, Younes [13C and δ15N) of GIFT between the two feeding practices. Differences isotopic values were visualised using the package ggplot2 version 2.2.1 [All data analyses were conducted in R version 3.4.3 . Relativ, Younes  and Le C, Younes . Two fison 2.2.1 .Alpha diversity (Shannon Weaver Index) was calculated in the ‘vegan’ package version 2.5–4 with statistical differences analysed using a one-way ANOVA . DiffereTo identify specific zOTUs that significantly correlated with relative fish condition and nutrient assimilation patterns, we assessed those with a relative abundance of over 1% that were present in at least 90% of the samples. Correlation coefficients were calculated using Pearson’s correlation with a significance threshold of P < 0.05.To identify specific zOTUs associated with each of the farming practices, multipattern analyses were conducted using the ‘multipatt’ function within the ‘indicspecies’ package version 1.7.6 . IndicatGIFT fed a diet incorporating commercial pellets were in a significantly better condition than those only fed vegetables . Pellet-13C values for both feeding practices showing some overlap  indicated that all were sequenced nearly to saturation and hence their bacterial communities were well covered .P = 0.683, F = 7.0596, P = 0.001) and dispersion . There was more significantly variability in the clustering pattern of microbial communities associated with the vegetable diets than the more tightly clustered pellet associated communities . Differences in species composition within each diet group (i.e vegetable and pellet) were also seen .Alpha diversity, as measured by the Shannon-Weaver Index, did not vary significantly between the two feeding practices  and were negatively correlated with fish condition and dietary carbon. Similarly, significant negative correlations were also identified for zOTU 2 .Pearsons correlation revealed that three zOTUs  were significantly correlated with fish condition and nutrient assimilation . zOTUs 1P <0.05) based either on presence and absence data, species abundances or both (Rhodoblastus acidophilus (zOTU 192) and Rickettsiella sp. GSU (zOTU 246) and Candidatus Udaeobacter (zOTU 73 and 510).Indicator species analysis revealed that 24 zOTUs were significantly associated with pellet-based diets  was the most common taxon found to be significantly associated with GIFT fed a vegetable-only diet  of the fish in our study is of interest as it implies that these taxa may have been acquired from the hatchery prior to distribution. This finding is significant because the microbial composition of larval and juvenile fish has a significant influence of the microbiome of adults . Three o13C . These r of GIFT . The proFusobacteria are commonly identified as a major constituent of freshwater fish microbiomes , 78–80. The results from this study contribute to a growing body of work on the influence of diet on the microbiota, trophic status and condition of freshwater fishes. We show that trophic level is not always indicative of a good diet and can represent poor farming practices. Specifically, our results demonstrate how poor feeding practices can negatively impact the success of GIFT farms. We found that fish fed an insufficient vegetable based diet were in a relatively poor condition and while yet to be confirmed, possibly supplementing their diet through filial cannibalism. These results further highlight the extent of challenges faced by low income, small-scale subsistence farmers in developing nations. Small-scale fish farms account for the majority of inland freshwater finfish aquaculture and play a fundamental role in enhancing not only food and income security but also quality of life . For GIFS1 FigOreochromis niloticus), a) after sequence quality filtering and without rarefication of sequencing depth, b) equalized sampling depths  and c) after the removal of rare species.Rarefaction curves of microbial communities sampled from the gastrointestinal tract of Genetically Improved Farmed Tilapia (GIFT) ((TIF)Click here for additional data file.S2 FigShannon Weaver diversity index of the bacterial communities within the gastrointestinal tract of GIFT fed two different diets. ‘Pellet’ is representative of GIFT fed commercial fish feed pellet based diet, and ‘Vegetable’ is representative of GIFT fed a locally-sourced vegetable-based diet.(TIF)Click here for additional data file.S1 TableTilapia samples taken in this study for isotopic and microbial analysis, including information about the number of fish taken from each sampling location and the number of samples remaining after quality filtering.(DOCX)Click here for additional data file.