Intermediate Phenotypes Research Articles

Impact of CYP3A5 Status on the Clinical and Financial Outcomes Among African American Kidney Transplant Recipients.

Background.Pharmacogenetic profiling of transplant recipients demonstrates that the marked variation in the metabolism of immunosuppressive medications, particularly tacrolimus, is related to genetic variants. Patients of African ancestry are less likely to carry loss-of-function (LoF) variants in the CYP3A5 gene and therefore retain a rapid metabolism phenotype and higher clearance of tacrolimus. Patients with this rapid metabolism typically require higher dosing to achieve therapeutic trough concentrations. This study aims to further characterize the impact of CYP3A5 genotype on clinical outcomes and financial expenditure.Methods.The CYP3A5 phenotype status was identified in 438 adult kidney transplant (KTx) recipients (96% were African American) using 3 LoF alleles (CYP3A5*3, *6 or *7). Individuals were categorized as rapid metabolism phenotype without LoF alleles‚ intermediate phenotype for 1 LoF allele‚ and slow phenotype for 2 LoF alleles. KTx outcomes (patient/kidney survival and Medicare spending) were determined using linked transplant registry and claims data.Results.Among the cohort, 23% had a rapid, 47% intermediate, and 30% a slow metabolism phenotype based on genotype. At 3 y, the rate of death censored graft failure and all cause graft failure was highest in the rapid metabolism phenotype and lowest in the intermediate metabolism phenotype group. First-year Medicare reimbursement differed significantly by genotype (rapid: $79 535, intermediate: $72 796, slow: $79 346, P = 0.03). After adjustment for donor and recipient characteristics, care for patients with intermediate metabolism was $4790 less expensive (P = 0.003).Conclusions.Pharmacogenomic assessment of African American KTx recipients may be useful to guide therapy when as CYP3A5 functional variants appear to be associated with differential outcome and spending after transplant.

Septic Shock: Phenotypes and Outcomes.

Sepsis is a heterogeneous syndrome that results in life-threatening organ dysfunction. Our goal was to determine the relevant variables and patient phenotypes to use in predicting sepsis outcomes. We performed an ancillary study concerning 119 patients with septic shock at intensive care unit (ICU) admittance (T0). We defined clinical worsening as having an increased sequential organ failure assessment (SOFA) score of ≥ 1, 48h after admission (ΔSOFA ≥ 1). We performed univariate and multivariate analyses based on the 28-day mortality rate and ΔSOFA ≥ 1 and determined three patient phenotypes: safe, intermediate and unsafe. The persistence of the intermediate and unsafe phenotypes after T0 was defined as a poor outcome. At T0, the multivariate analysis showed two variables associated with 28-day mortality rate: norepinephrine dose and serum lactate concentration. Regarding ΔSOFA ≥ 1, we identified three variables at T0: norepinephrine dose, lactate concentration and venous-to-arterial carbon dioxide difference (P(v-a)CO2). At T0, the three phenotypes (safe, intermediate and unsafe) were found in 28 (24%), 70 (59%) and 21 (18%) patients, respectively. We thus suggested using an algorithm featuring norepinephrine dose, lactate concentration and P(v-a)CO2 to predict patient outcomes and obtained an area under the curve (AUC) of 74% (63-85%). Our findings highlight the fact that identifying relevant variables and phenotypes may help physicians predict patient outcomes.

Frequency of functional exonic single-nucleotide polymorphisms and haplotype distribution in the SLCO1B1 gene across genetic ancestry groups in the Qatari population

Organic anion transporting polypeptides (OATP), which are encoded by SLCO genes, participate in the hepatic elimination of drugs and xenobiotics. SLCO1B1 is an important pharmacogenomic gene (encoding OATP1B1) associated with response to the uptake of endogenous compounds, such as statin and bilirubin. Ethnicity of the patient modulates the response to these drugs; the frequency and haplotype data for SLCO1B1 genetic variants in the Arab population is lacking. Therefore, we determined the frequencies of two well-characterized SLCO1B1 single nucleotide polymorphisms (SNP) and haplotypes that affect the OATP1B1 drugs transportation activity in Qatari population. Genotyping data for two SLCO1B1 SNPs (c.388A > G, c.521 T > C) were extracted from whole exome data of 1050 Qatari individuals, who were divided into three ancestry groups, namely Bedouins, Persians/South Asians, and Africans. By way of using Fisher's exact and Chi-square tests, we evaluated the differences in minor allele frequency (MAF) of the two functional SNPs and haplotype frequencies (HF) among the three ancestry groups. The OATP1B1 phenotypes were assigned according to their function by following the guidelines from the Clinical Pharmacogenetics Implementation Consortium for SLCO1B1 and Simvastatin-Induced Myopathy.The MAF of SLCO1B1:c.388A > G was higher compared to that of SLCO1B1:c.521 T > C in the study cohort. It was significantly high in the African ancestry group compared with the other two groups, whereas SLCO1B1:c.521 T > C was significantly low in the African ancestry group compared with the other two groups. The SLCO1B1 *15 haplotype had the highest HF, followed by *1b, *1a, and *5. Only the SLCO1B1 *5 haplotype showed no significant difference in frequency across the three ancestry groups. Furthermore, we observed that the OATP1B1 normal function phenotype accounted for 58% of the Qatari individuals, the intermediate function phenotype accounted for 35% with significant differences across the ancestry groups, and the low function phenotype accounted for 6% of the total Qatari individuals with a higher trend observed in the Bedouin group.The results indicate that the phenotype frequencies of the OATP1B1 intermediate and low function in the Qatari population appear at the higher end of the frequency range seen worldwide. Thus, a pharmacogenetic screening program for SLCO1B1 variants may be necessary for the Qatari population.

Rac-deficient cerebellar granule neurons die before they migrate to the internal granule layer

Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1flox/flox; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1flox/flox; Rac3+/−; Cre mice showed intermediate phenotype between Rac1flox/flox; Cre and Rac1flox/flox; Rac3−/−; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1+/flox; Rac3−/−; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons.

Brain-wide versus genome-wide vulnerability biomarkers for severe mental illnesses.

Severe mental illnesses (SMI), including major depressive (MDD), bipolar (BD), and schizophrenia spectrum (SSD) disorders have multifactorial risk factors and capturing their complex etiopathophysiology in an individual remains challenging. Regional vulnerability index (RVI) was used to measure individual's brain‐wide similarity to the expected SMI patterns derived from meta‐analytical studies. It is analogous to polygenic risk scores (PRS) that measure individual's similarity to genome‐wide patterns in SMI. We hypothesized that RVI is an intermediary phenotype between genome and symptoms and is sensitive to both genetic and environmental risks for SMI. UK Biobank sample of N = 17,053/19,265 M/F (age = 64.8 ± 7.4 years) and an independent sample of SSD patients and controls (N = 115/111 M/F, age = 35.2 ± 13.4) were used to test this hypothesis. UKBB participants with MDD had significantly higher RVI‐MDD (Cohen's d = 0.20, p = 1 × 10−23) and PRS‐MDD (d = 0.17, p = 1 × 10−15) than nonpsychiatric controls. UKBB participants with BD and SSD showed significant elevation in the respective RVIs (d = 0.65 and 0.60; p = 3 × 10−5 and .009, respectively) and PRS (d = 0.57 and 1.34; p = .002 and .002, respectively). Elevated RVI‐SSD were replicated in an independent sample (d = 0.53, p = 5 × 10−5). RVI‐MDD and RVI‐SSD but not RVI‐BD were associated with childhood adversity (p < .01). In nonpsychiatric controls, elevation in RVI and PRS were associated with lower cognitive performance (p < 10−5) in six out of seven domains and showed specificity with disorder‐associated deficits. In summary, the RVI is a novel brain index for SMI and shows similar or better specificity for SMI than PRS, and together they may complement each other in the efforts to characterize the genomic to brain level risks for SMI.

Investigating Causal Relations between Genetic-Related Intermediate Endophenotype and Risk of Chronic Prostatitis: Mendelian Randomization Study.

Objective Prostatitis is a common disease of the male genitourinary system, which seriously disturbs the physical and mental health of male patients. It is related to many factors such as living habits, age, and race, but the etiology has not been fully elucidated. This study investigated whether there is a causal relationship between clinical biochemical indicators (i.e., intermediate phenotype) and prostatitis through Mendelian randomization. The subjects of the study were prostatitis patients and related SNPs in the Guangxi Fangchenggang health examination cohort. Methods According to the requirements of Mendelian randomization (MR), the single nucleotide polymorphisms (SNPs) related to prostatitis patients and 29 common SNPs related to clinical biochemical indicators were analyzed by linkage disequilibrium, and the calculated SNPs were selected. Finally, the related SNPs were analyzed by Mendelian randomization method. Results 15 biochemical indicators such as complement C4, FOL, CRP, HCY, and estradiol have shared chronic prostatitis SNP sites, and five qualified SNPs were finally screened for complement C4. Finally, complement C4 was obtained by Mendelian randomization method (P = 0.039), which was statistically significant. The other 28 clinical endophenotypes were all negative. Conclusion The results show that there was a causal relationship between complement C4 and prostatitis, and the more consistent SNP is rs2075799.

Impact of cytochrome P450 2D6 polymorphisms on decision-making and clinical outcomes in adjuvant hormonal therapy for breast cancer

BACKGROUNDThere are concerns that tamoxifen is less effective in Asian women because of the high prevalence of impaired function cytochrome P450 2D6 (CYP2D6) polymor-phisms.AIMTo evaluate how knowledge of CYP2D6 genotype impacted the choice of hormonal agent and how CYP2D6 genotype and agent were associated with clinical outcomes.METHODSEighty-two women were recruited. Seventy-eight completed CYP2D6 genotyping and were categorized into poor, intermediate (IM) and extensive or ultra metabolizer phenotypes. Women with poor metabolizer and IM phenotypes were recommended aromatase inhibitors as the preferred agent.RESULTSMore than 70% of the women had an IM phenotype, 32% an extensive or ultra metabolizer phenotype, and 0% had a poor metabolizer phenotype. Regardless of genotype, more women opted for aromatase inhibitors. Overall, 80% of women completed 5 years of hormonal therapy. Five women developed recurrence, 3 contralateral breast cancer, 5 died, and 1 was diagnosed with a second primary cancer. Five-year recurrence-free and overall survival were slightly better in women with the extensive or ultra metabolizer phenotype compared to those with the IM phenotype, though not statistically significant [P = 0.743, hazard ratio (HR): 1.441, 95% confidence interval (CI): 0.191 to 10.17 and P = 0.798, HR: 1.327, 95%CI: 0.172 to 9.915, respectively]. Women receiving aromatase inhibitors also appeared to have a better, but also nonsignificant, 5-year recurrence-free and overall survival (P = 0.253, HR: 0.368, 95%CI: 0.031 to 0.258 and P = 0.292, HR: 0.252, 95%CI: 0.005 to 4.951, respectively).CONCLUSIONThe IM phenotype was highly prevalent but was not associated with clinical outcome.

Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants’ abilities to potentially infect different animals.IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.

Consistencies and Differences in Intermediate Physiological Phenotypes of Vascular Ageing between Ischaemic Stroke Aetiologies

Objective: Arterial stiffness, cerebral pulsatility, and beat-to-beat blood pressure variability partly mediate the relationship between hypertension and stroke, but it is unknown if these intermediate phenotypes of vascular ageing differ between stroke aetiologies. We therefore aimed to characterize differences in these intermediate cardiovascular phenotypes between patients presenting with strokes of different aetiologies. Methods: In consecutive patients on best medical management 1 month after TIA or nondisabling stroke (Oxford Vascular Study), arterial stiffness (PWV) was measured by applanation tonometry (Sphygmocor), middle cerebral blood flow velocity, and pulsatility index (MCA-PI) were measured by transcranial ultrasound (TCD, DWL Doppler Box), and beat-to-beat BP variability was measured with a Finometer. Differences between patients with large artery (LAS), small vessel (SVD), cardioembolic (CE), or undetermined events were derived, including adjustment for cardiovascular risk factors. Relationships were characterized by mixed linear models. Results: In 909 eligible patients, MCA-PI, PWV, and SBPV were all positively skewed. Mean values were greatest in LAS than CE and lowest in SVD (p < 0.001). However, after adjustment for age, sex, and risk factors, PI was greatest in LAS and lowest in CE stroke, whilst PWV was greatest in SVD and undetermined stroke (p < 0.001). In multivariate linear models, age was more strongly associated with PWV and PI in patients with small vessel stroke than other aetiologies, particularly under the age of 65, but SBPV was only weakly associated with demographic indices in all stroke subtypes. Conclusions: Intermediate cardiovascular phenotypes of vascular ageing had similar demographic associations between stroke aetiologies, but these were particularly strong in patients with small vessel stroke under the age of 65, implying a potential role of these phenotypes in increasing stroke risk in this patient group.

Lipidomics-Assisted GWAS (lGWAS) Approach for Improving High-Temperature Stress Tolerance of Crops.

High-temperature stress (HT) over crop productivity is an important environmental factor demanding more attention as recent global warming trends are alarming and pose a potential threat to crop production. According to the Sixth IPCC report, future years will have longer warm seasons and frequent heat waves. Thus, the need arises to develop HT-tolerant genotypes that can be used to breed high-yielding crops. Several physiological, biochemical, and molecular alterations are orchestrated in providing HT tolerance to a genotype. One mechanism to counter HT is overcoming high-temperature-induced membrane superfluidity and structural disorganizations. Several HT lipidomic studies on different genotypes have indicated the potential involvement of membrane lipid remodelling in providing HT tolerance. Advances in high-throughput analytical techniques such as tandem mass spectrometry have paved the way for large-scale identification and quantification of the enormously diverse lipid molecules in a single run. Physiological trait-based breeding has been employed so far to identify and select HT tolerant genotypes but has several disadvantages, such as the genotype-phenotype gap affecting the efficiency of identifying the underlying genetic association. Tolerant genotypes maintain a high photosynthetic rate, stable membranes, and membrane-associated mechanisms. In this context, studying the HT-induced membrane lipid remodelling, resultant of several up-/down-regulations of genes and post-translational modifications, will aid in identifying potential lipid biomarkers for HT tolerance/susceptibility. The identified lipid biomarkers (LIPIDOTYPE) can thus be considered an intermediate phenotype, bridging the gap between genotype–phenotype (genotype–LIPIDOTYPE–phenotype). Recent works integrating metabolomics with quantitative genetic studies such as GWAS (mGWAS) have provided close associations between genotype, metabolites, and stress-tolerant phenotypes. This review has been sculpted to provide a potential workflow that combines MS-based lipidomics and the robust GWAS (lipidomics assisted GWAS-lGWAS) to identify membrane lipid remodelling related genes and associations which can be used to develop HS tolerant genotypes with enhanced membrane thermostability (MTS) and heat stable photosynthesis (HP).

A-173 Grey Matter Volume, Psychotic Disorders, And Heredity of Alcohol Use Disorder: Reconceptualization by B-SNIP Biotypes

Abstract Objective: Bipolar and Schizophrenia Network for Intermediate Phenotypes (B-SNIP) researchers have proposed three Biotypes that show advantages of a dimensional approach to classification, revealing different levels of dysfunction severity through EEG and various forms of cognitive testing. Although preliminary work has been done showing neuroanatomical differences among the biotypes and patterns of heredity, more research is required to investigate the contribution of significant co-morbid factors such as alcohol misuse. Objective. Neuroanatomical data were used to further understand the biomarker of grey matter volume (GMV) in relation to the B-SNIP Biotypes and the interacting influence of family history of alcohol use disorder (FH-AUD). Method. Biotype, FH-AUD, and demographic variables (age, sex, education, and ethnicity) were used in hierarchical regression analyses for prediction of total volume and two key regions (superior parietal cortex and posterior cingulate cortex) in stable probands (n = 347) and their healthy first-degree relatives (n = 346) separately. Prediction was also analyzed in the context of conventional diagnosis of psychotic disorders (schizophrenia, schizoaffective disorder, and bipolar disorder with psychotic features). Structural data were analyzed using FreeSurfer. Results. Biotype was shown to be a more robust predictor of GMV than diagnosis both generally and specifically in relevant regions (p &amp;lt; .05). A pattern of GMV reduction based on severity was observed both in probands and relatives. Effects of FH-AUD and its interaction with Biotype were occasionally observed. Conclusions. This study contributes to the efforts of psychobiological research to establish a more valid conceptualization of psychological disorders through biological mechanisms.

A-174 Measurement invariance reveals Brief Assessment of Cognition in Schizophrenia (BACS) Token Motor subtest differs in U.S.-based black and white samples

Abstract Objective: The Brief Assessment of Cognition in Schizophrenia (BACS) is widely used to assess cognition in schizophrenia spectrum disorders (SZ). However, norms were developed using a sample of 85% white and 15% black participants. Validation of the BACS across cultures have yielded mixed results suggesting that subtests may function differently. Measurement invariance can be used to assess item consistency across groups. The present study examined the measurement invariance of six BACS subtests using models with increasing constraints across 2 different U.S.-based cultural groups (i.e., black and white). Methods: Participants were 429 black and 475 white individuals diagnosed with SZ from the Bipolar Schizophrenia Network for Intermediate Phenotypes consortium. Results: The Comparative Fit Index (CFI) was used to assess model fit. A change in CFI between models of increasing constraint (i.e., &amp;lt;/= 0.01) is acceptable (Cheung &amp; Rensvold, 2002). Baseline configural invariance (CFI=.983) was achieved. The CFI of the weak model (CFI=.967) exceeded the cut-off difference suggesting subtests do not achieve weak invariance. Partial invariance tests revealed that the Token Motor subtest was not equivalent across groups, leading to a 5-subtest partial weak invariance model. Conclusions: With the exception of the Token Motor subtest, findings suggest that subtests function similarly across these two racial groups. The Token Motor subtest has both the lowest internal consistancy and factor loadings on the BACS and has been removed from the BACS composite score. Future research should examine the utility of the Token Motor test across clinical and cultural groups.

Integrative Bayesian models using Post-selective inference: A case study in radiogenomics.

Integrative analyses based on statistically relevant associations between genomics and a wealth of intermediary phenotypes (such as imaging) provide vital insights into their clinical relevance in terms of the disease mechanisms. Estimates for uncertainty in the resulting integrative models are however unreliable unless inference accounts for the selection of these associations with accuracy. In this paper, we develop selection-aware Bayesian methods, which (1) counteract the impact of model selection bias through a "selection-aware posterior" in a flexible class of integrative Bayesian models post a selection of promising variables via ℓ1 -regularized algorithms; (2) strike an inevitable trade-off between the quality of model selection and inferential power when the same data set is used for both selection and uncertainty estimation. Central to our methodological development, a carefully constructed conditional likelihood function deployed with a reparameterization mapping provides tractable updates when gradient-based Markov chain Monte Carlo (MCMC) sampling is used for estimating uncertainties from the selection-aware posterior. Applying our methods to a radiogenomic analysis, we successfully recover several important gene pathways and estimate uncertainties for their associations with patient survivaltimes.

Applicability of the IrisPlex system for eye color prediction in an admixed population from Argentina.

Eye color prediction based on an individual's genetic information is of interest in the field of forensic genetics. In recent years, researchers have studied different genes and markers associated with this externally visible characteristic and have developed methods for its prediction. The IrisPlex represents a validated tool for homogeneous populations, though its applicability in populations of mixed ancestry is limited, mainly regarding the prediction of intermediate eye colors. With the aim of validating the applicability of this system in an admixed population from Argentina (n=302), we analyzed the six single nucleotide variants used in that multiplex for eye color and four additional SNPs, and evaluated its prediction ability. We also performed a genotype-phenotype association analysis. This system proved to be useful when dealing with the extreme ends of the eye color spectrum (blue and brown) but presented difficulties in determining the intermediate phenotypes (green), which were found in a large proportion of our population. We concluded that these genetic tools should be used with caution in admixed populations and that more studies are required in order to improve the prediction of intermediate phenotypes.

Ageing desexualizes the Drosophila brain transcriptome.

General evolutionary theory predicts that individuals in low condition should invest less in sexual traits compared to individuals in high condition. Whether this positive association between condition and investment also holds between young (high condition) and senesced (low condition) individuals is however less clear, since elevated investment into reproduction may be beneficial when individuals approach the end of their life. To address how investment into sexual traits changes with age, we study genes with sex-biased expression in the brain, the tissue from which sexual behaviours are directed. Across two distinct populations of Drosophila melanogaster, we find that old brains display fewer sex-biased genes, and that expression of both male-biased and female-biased genes converges towards a sexually intermediate phenotype owing to changes in both sexes with age. We further find that sex-biased genes in general show heightened age-dependent expression in comparison to unbiased genes and that age-related changes in the sexual brain transcriptome are commonly larger in males than females. Our results hence show that ageing causes a desexualization of the fruit fly brain transcriptome and that this change mirrors the general prediction that low condition individuals should invest less in sexual phenotypes.

Mapping phenotypic heterogeneity in melanoma onto the epithelial-hybrid-mesenchymal axis

Epithelial to mesenchymal transition (EMT) is a well-studied hallmark of epithelial-like cancers that is characterized by loss of epithelial markers and gain of mesenchymal markers. Melanoma, which is derived from melanocytes of the skin, also undergo phenotypic plasticity toward mesenchymal-like phenotypes under the influence of various micro-environmental cues. Our study connects EMT to the phenomenon of de-differentiation (i.e., transition from proliferative to more invasive phenotypes) observed in melanoma cells during drug treatment. By analyzing 78 publicly available transcriptomic melanoma datasets, we found that de-differentiation in melanoma is accompanied by upregulation of mesenchymal genes, but not necessarily a concomitant loss of an epithelial program, suggesting a more “one-dimensional” EMT that leads to a hybrid epithelial/mesenchymal phenotype. Samples lying in the hybrid epithelial/mesenchymal phenotype also correspond to the intermediate phenotypes in melanoma along the proliferative-invasive axis - neural crest and transitory ones. As melanoma cells progress along the invasive axis, the mesenchymal signature does not increase monotonically. Instead, we observe a peak in mesenchymal scores followed by a decline, as cells further de-differentiate. This biphasic response recapitulates the dynamics of melanocyte development, suggesting close interactions among genes controlling differentiation and mesenchymal programs in melanocytes. Similar trends were noted for metabolic changes often associated with EMT in carcinomas in which progression along mesenchymal axis correlates with the downregulation of oxidative phosphorylation, while largely maintaining glycolytic capacity. Overall, these results provide an explanation for how EMT and de-differentiation axes overlap with respect to their transcriptional and metabolic programs in melanoma.

Systematic Functional Analysis of PINK1 and PRKN Coding Variants.

Loss of either PINK1 or PRKN causes an early onset Parkinson’s disease (PD) phenotype. Functionally, PINK1 and PRKN work together to mediate stress-activated mitochondrial quality control. Upon mitochondrial damage, PINK1, a ubiquitin kinase and PRKN, a ubiquitin ligase, decorate damaged organelles with phosphorylated ubiquitin for sequestration and degradation in lysosomes, a process known as mitophagy. While several genetic mutations are established to result in loss of mitophagy function, many others have not been extensively characterized and are of unknown significance. Here, we analyzed a set of twenty variants, ten in each gene, focusing on understudied variants mostly from the Parkinson’s progressive marker initiative, with sensitive assays to define potential functional deficits. Our results nominate specific rare genetic PINK1 and PRKN variants that cause loss of enzymatic function in line with a potential causative role for PD. Additionally, we identify several variants with intermediate phenotypes and follow up on two of them by gene editing midbrain-derived neuronal precursor cells. Thereof derived isogenic neurons show a stability defect of the rare PINK1 D525N mutation, while the common PINK1 Q115L substitution results in reduced kinase activity. Our strategy to analyze variants with sensitive functional readouts will help aid diagnostics and disease treatment in line with current genomic and therapeutic advances.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

Vitamin C attenuates predisposition to high-fat diet-induced metabolic dysregulation in GLUT10-deficient mouse model

BackgroundThe development of type 2 diabetes mellitus (T2DM) is highly influenced by complex interactions between genetic and environmental (dietary and lifestyle) factors. While vitamin C (ascorbic acid, AA) has been suggested as a complementary nutritional treatment for T2DM, evidence for the significance and beneficial effects of AA in T2DM is thus far inconclusive. We suspect that clinical studies on the topic might need to account for combination of genetic and dietary factors that could influence AA effects on metabolism. In this study, we tested this general idea using a mouse model with genetic predisposition to diet-induced metabolic dysfunction. In particular, we utilized mice carrying a human orthologous GLUT10G128E variant (GLUT10G128E mice), which are highly sensitive to high-fat diet (HFD)-induced metabolic dysregulation. The genetic variant has high relevance to human populations, as genetic polymorphisms in glucose transporter 10 (GLUT10) are associated with a T2DM intermediate phenotype in nondiabetic population.ResultsWe investigated the impacts of AA supplementation on metabolism in wild-type (WT) mice and GLUT10G128E mice fed with a normal diet or HFD. Overall, the beneficial effects of AA on metabolism were greater in HFD-fed GLUT10G128E mice than in HFD-fed WT mice. At early postnatal stages, AA improved the development of compromised epididymal white adipose tissue (eWAT) in GLUT10G128E mice. In adult animals, AA supplementation attenuated the predisposition of GLUT10G128E mice to HFD-triggered eWAT inflammation, adipokine dysregulation, ectopic fatty acid accumulation, metabolic dysregulation, and body weight gain, as compared with WT mice.ConclusionsTaken together, our findings suggest that AA has greater beneficial effects on metabolism in HFD-fed GLUT10G128E mice than HFD-fed WT mice. As such, AA plays an important role in supporting eWAT development and attenuating HFD-induced metabolic dysregulation in GLUT10G128E mice. Our results suggest that proper WAT development is essential for metabolic regulation later in life. Furthermore, when considering the usage of AA as a complementary nutrition for prevention and treatment of T2DM, individual differences in genetics and dietary patterns should be taken into account.

Cut-off values for diagnosis of G6PD deficiency by flow cytometry in Thai population

In heterozygous females, X-inactivation causes a change in glucose-6-phosphate dehydrogenase (G6PD) activity from normal to deficient. Most G6PD screening tests are used to accurately diagnose hemizygous males, but they are less reliable for diagnosing heterozygous females. This study established flow cytometric cut-off values for screening of G6PD deficiency in hemizygous males and heterozygous or homozygous females. We studied 205 (125 females, 80 males) leftover blood samples from quantitative methemoglobin reduction (MR) screening. G6PD gene mutations determined by multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing were used as the gold standard reference. Accuracy of the test, including the sensitivity, specificity, and positive and negative predictive values, was analyzed using MedCalc software. The optimal cut-off values for classification of %red blood cells with normal G6PD activity or %bright cells into homozygous normal, heterozygous, and homozygous deficiency in females were 85.4–100%, 6.3–85.3%, and 0–6.2%, respectively (sensitivity 93.2%, specificity 100%). The cut-offs for classification into hemizygous normal and hemizygous deficiency in males were 76.5–100% and 0–76.4%, respectively (sensitivity 100%, specificity 96.5%). Flow cytometry can be used to differentiate heterozygous females with intermediate phenotype from homozygous females, but cannot distinguish between heterozygous females with extreme phenotype and homozygous females. By flow cytometry, heterozygous and homozygous deficiency was detected in 29.6% and 3.2% of females, respectively. Among males, hemizygous deficiency was found in 31.3%. Flow cytometry can be used to screen patients with G6PD deficiency, and reliably and efficiently identify heterozygous and homozygous females, and hemizygous males based on cellular G6PD activity.