ADGRL1 is a glucose receptor involved in mediating energy and glucose homeostasis.

AIMS/HYPOTHESIS: The brain is a major consumer of glucose as an energy source and regulates systemic glucose as well as energy balance. Although glucose transporters such as GLUT2 and sodium-glucose cotransporter 2 (SGLT2) are known to regulate glucose homeostasis and metabolism, the identity of a receptor that binds glucose to activate glucose signalling pathways in the brain is unknown. In this study, we aimed to discover a glucose receptor in the mouse hypothalamus. METHODS: Here we used a high molecular mass glucose-biotin polymer to enrich glucose-bound mouse hypothalamic neurons through cell-based affinity chromatography. We then subjected the enriched neurons to proteomic analyses and identified adhesion G-protein coupled receptor 1 (ADGRL1) as a top candidate for a glucose receptor. We validated glucose-ADGRL1 interactions using CHO cells stably expressing human ADGRL1 and ligand-receptor binding assays. We generated and determined the phenotype of global Adgrl1-knockout mice and hypothalamus-specific Adgrl1-deficient mice. We measured the variables related to glucose and energy homeostasis in these mice. We also generated an Adgrl1Cre mouse model to investigate the role of ADGRL1 in sensing glucose using electrophysiology. RESULTS: Adgrl1 is highly expressed in the ventromedial nucleus of the hypothalamus (VMH) in mice. Lack of Adgrl1 in the VMH in mice caused fasting hyperinsulinaemia, enhanced glucose-stimulated insulin secretion and insulin resistance. In addition, the Adgrl1-deficient mice had impaired feeding responses to glucose and fasting coupled with abnormal glucose sensing and decreased physical activity before development of obesity and hyperglycaemia. In female mice, ovariectomy was necessary to reveal the contribution of ADGRL1 to energy and glucose homeostasis. CONCLUSIONS/INTERPRETATION: Altogether, our findings demonstrate that ADGRL1 binds glucose and is involved in energy as well as glucose homeostasis in a sex-dependent manner. Targeting ADGRL1 may introduce a new class of drugs for the treatment of type 2 diabetes and obesity.

Current and Stray Flux Combined Analysis for Sparking Detection in DC Motors/Generators Using Shannon Entropy.

Brushed DC motors and generators (DCMs) are extensively used in various industrial applications, including the automotive industry, where they are critical for electric vehicles (EVs) due to their high torque, power, and efficiency. Despite their advantages, DCMs are prone to premature failure due to sparking between brushes and commutators, which can lead to significant economic losses. This study proposes two approaches for determining the temporal and frequency evolution of Shannon entropy in armature current and stray flux signals. One approach indirectly achieves this through prior analysis using the Short-Time Fourier Transform (STFT), while the other applies the Stockwell Transform (S-Transform) directly. Experimental results show that increased sparking activity generates significant low-frequency harmonics, which are more pronounced compared to mid and high-frequency ranges, leading to a substantial rise in system entropy. This finding enables the introduction of fault-severity indicators or Key Performance Indicators (KPIs) that relate the current condition of commutation quality to a baseline established under healthy conditions. The proposed technique can be used as a predictive maintenance tool to detect and assess sparking phenomena in DCMs, providing early warnings of component failure and performance degradation, thereby enhancing the reliability and availability of these machines.

Gßγ mediates activation of Rho guanine nucleotide exchange factor ARHGEF17 that promotes metastatic lung cancer progression.

Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gßγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression-based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein-coupled LPA receptors via the Gßγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gßγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.

Preliminary chemical characterization of ethanolic extracts from Colombian plants with promising anti - Trypanosoma cruzi activity.

Chagas disease is caused by Trypanosoma cruzi, and it is an important cause of morbidity and mortality in Latin America. There are no vaccines, and the chemotherapy available to treat this infection has serious side effects. In a search for alternative treatments, we determined the in vitro susceptibility of epimastigote and trypomastigote forms of T. cruzi and the cytotoxic effects on peripheral blood mononuclear cells (PBMCs) of ethanolic extracts obtained from six different plant species. The ethanolic extracts of Ageratina vacciniaefolia, Clethra fimbriata and Siparuna sessiliflora showed antiprotozoal activity against epimastigotes and low cytotoxicity in mammalian cells. However, only the ethanolic extract of C. fimbriata showed activity against T. cruzi trypomastigotes, and it had low cytotoxicity in PBMCs. An analysis on the phytochemical composition of C. fimbriata extract showed that its metabolites were primarily represented by two families of compounds: flavonoids and terpenoids. Lastly, we analyzed whether the A. vacciniaefolia, C. fimbriata, or S. sessiliflora ethanolic extracts induced IFN-γ or TNF-α production. Significantly, ethanolic extracts of C. fimbriata induced TNF-α production and S. sessiliflora induced both cytokines. In addition, C. fimbriata and S. sessiliflora induced the simultaneous secretion of IFN-γ and TNF-α in CD8+ T cells. The antiprotozoal and immunomodulatory activity of C. fimbriata may be related to the presence of flavonoid and triterpene compounds in the extract. Thus, these findings suggest that C. fimbriata may represent a valuable source of new bioactive compounds for the therapeutic treatment of Chagas disease that combines trypanocidal activity with the capacity to boost the immune response.

Relation between Alpha-Synuclein and Core CSF Biomarkers of Alzheimer's Disease.

Background: Alzheimer's disease (AD) is characterized by the presence of ß-amyloid plaques and neurofibrillary tangles, while Lewy body dementia (LBD) is characterized by α-synuclein (α-syn) inclusions. Some authors examine α-syn protein in the neurodegeneration process of AD and propose to consider cerebrospinal fluid (CSF) α-syn as a possible additional biomarker to the so-called "core" of AD. Objective: To determine whether there is a correlation between α-syn levels and "core" AD biomarkers in patients with mild cognitive impairment (MCI). Materials and methods: In total, 81 patients in the early stages of MCI were selected from the outpatient dementia consultation in Alicante General Hospital. Using a cross-sectional case-control design, patients were analyzed in four groups: stable MCI (MCIs; n = 25), MCI due to AD (MCI-AD; n = 32), MCI due to LBD (MCI-LBD; n = 24) and a control group of patients with acute or chronic headache (Ctrl; n = 18). Correlation between CSF protein levels in the different groups was assessed by the Rho Spearman test. Results: We found positive correlations between T-tau protein and α-syn (ρ = 0.418; p value < 0.05) and p-tau181p and α-syn (ρ = 0.571; p value < 0.05) exclusively in the MCI-AD group. Conclusion: The correlation found between α-syn and tau proteins in the first stages of AD support the involvement of α-syn in the pathogenesis of AD. This result may have clinical and diagnostic implications, as well as help to apply the new concept of "precision medicine" in patients with MCI.

A kidney-hypothalamus axis promotes compensatory glucose production in response to glycosuria.

The kidneys facilitate energy conservation through reabsorption of nutrients including glucose. Almost all of the filtered blood glucose is reabsorbed by the kidneys. Loss of glucose in urine (glycosuria) is offset by an increase in endogenous glucose production to maintain normal energy supply in the body. How the body senses this glucose loss and consequently enhances glucose production is unclear. Using renal Glut2 knockout mice, we demonstrate that elevated glycosuria activates the hypothalamic-pituitary-adrenal axis, which in turn drives endogenous glucose production. This phenotype was attenuated by selective afferent renal denervation, indicating the involvement of the afferent nerves in promoting the compensatory increase in glucose production. In addition, through plasma proteomics analyses we observed that acute phase proteins - which are usually involved in body's defense mechanisms against a threat - were the top candidates which were either upregulated or downregulated in renal Glut2 KO mice. Overall, afferent renal nerves contribute to promoting endogenous glucose production in response to elevated glycosuria and loss of glucose in urine is sensed as a biological threat in mice. These findings may be useful in improving efficiency of drugs like SGLT2 inhibitors that are intended to treat hyperglycemia by enhancing glycosuria, but are met with a compensatory increase in endogenous glucose production.

A kidney-hypothalamus axis promotes compensatory glucose production in response to glycosuria.

The kidneys facilitate energy conservation through reabsorption of nutrients including glucose. Almost all the filtered blood glucose is reabsorbed by the kidneys. Loss of glucose in urine (glycosuria) is offset by an increase in endogenous glucose production to maintain normal energy supply in the body. How the body senses this glucose loss and consequently enhances glucose production is unclear. Using renal Slc2a2 (also known as Glut2) knockout mice, we demonstrate that elevated glycosuria activates the hypothalamic-pituitary-adrenal axis, which in turn drives endogenous glucose production. This phenotype was attenuated by selective afferent renal denervation, indicating the involvement of the afferent nerves in promoting the compensatory increase in glucose production. In addition, through plasma proteomics analyses we observed that acute phase proteins - which are usually involved in the body's defense mechanisms against a threat - were the top candidates which were either upregulated or downregulated in renal Slc2a2 KO mice. Overall, afferent renal nerves contribute to promoting endogenous glucose production in response to elevated glycosuria and loss of glucose in urine is sensed as a biological threat in mice. These findings may be useful in improving the efficiency of drugs like SGLT2 inhibitors that are intended to treat hyperglycemia by enhancing glycosuria but are met with a compensatory increase in endogenous glucose production.

Survey on the Situation of Medical Departments in the Pharmaceutical Industry in Spain.

INTRODUCTION: Medical departments have evolved from a position of support to one of strategic leadership. The number of tasks and the complexity of interactions in which they are involved is increasing. However, the spectrum of their activity in the sector differs significantly from one company to another. Therefore, the aim of this study was to describe their situation within the pharmaceutical industry, analyzing the positions, functions, and profiles of their professionals. METHODS: This study consisted of an online survey containing 25 questions grouped into four blocks (structure, medical direction, training, and activities and responsibilities). Medical departments in the Spanish pharmaceutical industry of different sizes and scope were invited to participate. The survey took place in 2021, with a designated response period of three months. It is important to note that all responses collected during this time were treated as anonymous. RESULTS: Thirty companies participated. A total of 93.3% of respondents worked for an international laboratory, with a size of 0-5 or 11-20 people (20.7%). For 27.6% of the companies, the number of medical advisors per medical department was 1 or 4, with varying numbers of medical scientific liaisons (1, 6-10, and > 20). A total of 56.7%, 33.3%, and 6.7% indicated that the country manager, head of regional medical affairs, and head of global medical affairs, respectively, had a solid-line reporting relationship with the medical directorate. Medical directors were mostly graduates in medicine (86.2%) with a doctorate (34.5%), and medical managers were mainly graduates in medicine (77.8%) and pharmacy (66.7%). CONCLUSIONS: This study reveals that respondents predominantly work in internationally focused laboratories, with professionals ranging from experienced medical directors to managers with 6-20 years of experience, each with distinct roles.

Clethra fimbriata hexanic extract triggers alteration in the energy metabolism in epimastigotes of Trypanosoma cruzi.

Chagas disease (ChD), caused by Trypanosoma cruzi, is endemic in American countries and an estimated 8 million people worldwide are chronically infected. Currently, only two drugs are available for therapeutic use against T. cruzi and their use is controversial due to several disadvantages associated with side effects and low compliance with treatment. Therefore, there is a need to search for new tripanocidal agents. Natural products have been considered a potential innovative source of effective and selective agents for drug development to treat T. cruzi infection. Recently, our research group showed that hexanic extract from Clethra fimbriata (CFHEX) exhibits anti-parasitic activity against all stages of T. cruzi parasite, being apoptosis the main cell death mechanism in both epimastigotes and trypomastigotes stages. With the aim of deepening the understanding of the mechanisms of death induced by CFHEX, the metabolic alterations elicited after treatment using a multiplatform metabolomics analysis (RP/HILIC-LC-QTOF-MS and GC-QTOF-MS) were performed. A total of 154 altered compounds were found significant in the treated parasites corresponding to amino acids (Arginine, threonine, cysteine, methionine, glycine, valine, proline, isoleucine, alanine, leucine, glutamic acid, and serine), fatty acids (stearic acid), glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine), sulfur compounds (trypanothione) and carboxylic acids (pyruvate and phosphoenolpyruvate). The most affected metabolic pathways were mainly related to energy metabolism, which was found to be decrease during the evaluated treatment time. Further, exogenous compounds of the triterpene type (betulinic, ursolic and pomolic acid) previously described in C. fimbriata were found inside the treated parasites. Our findings suggest that triterpene-type compounds may contribute to the activity of CFHEX by altering essential processes in the parasite.

Lupeol Acetate and α-Amyrin Terpenes Activity against Trypanosoma cruzi: Insights into Toxicity and Potential Mechanisms of Action.

BACKGROUND: Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi. There is growing scientific interest in finding new and better therapeutic alternatives for this disease's treatment. METHODS: A total of 81 terpene compounds with potential trypanocidal activity were screened and found to have potential T. cruzi cysteine synthase (TcCS) inhibition using molecular docking, molecular dynamics, ADME and PAIN property analyses and in vitro susceptibility assays. RESULTS: Molecular docking analyses revealed energy ranges from -10.5 to -4.9 kcal/mol in the 81 tested compounds, where pentacyclic triterpenes were the best. Six compounds were selected to assess the stability of the TcCS-ligand complexes, of which lupeol acetate (ACLUPE) and α-amyrin (AMIR) exhibited the highest stability during 200 ns of molecular dynamics analysis. Such stability was primarily due to their hydrophobic interactions with the amino acids located in the enzyme's active site. In addition, ACLUPE and AMIR exhibited lipophilic characteristics, low intestinal absorption and no structural interferences or toxicity. Finally, selective index for ACLUPE was >5.94, with moderate potency in the trypomastigote stage (EC50 = 15.82 ± 3.7 µg/mL). AMIR's selective index was >9.36 and it was moderately potent in the amastigote stage (IC50 = 9.08 ± 23.85 µg/mL). CONCLUSIONS: The present study proposes a rational approach for exploring lupeol acetate and α-amyrin terpene compounds to design new drugs candidates for Chagas disease.

Antibody-Functionalized Copper Oxide Nanoparticles with Targeted Antibacterial Activity.

Copper oxide nanoparticles (CuO-NPs) were functionalized with specific antibodies to target their antibacterial activity against Gram-positive or Gram-negative bacteria. The CuO-NPs were covalently functionalized to cover their surface with specific antibodies. The differently prepared CuO-NPs were characterized by X-ray diffraction, transmission electron microscopy and dynamic light scattering. The antibacterial activities of the unmodified CuO-NPs and the antibody-functionalized nanoparticles (CuO-NP-AbGram- and CuO-NP-AbGram+ ) were determined for both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. The antibody-functionalized NPs showed a differential increase of their antibacterial activity according to the specific antibody. The CuO-NP-AbGram- in E. coli showed reduced half maximal inhibitory concentration (IC50 ) and minimum inhibitory concentration (MIC) values when compared with unfunctionalized CuO-NPs. On the other hand, the CuO-NP-AbGram+ also showed reduced IC50 and MIC values in B. subtilis, when compared with non-functionalized CuO-NPs. Thus, the functionalized CuO nanoparticles with specific antibodies showed enhanced specificity of their antibacterial activity. The advantages of "smart" antibiotic nanoparticles are discussed.

CaSR links endocytic and secretory pathways via MADD, a Rab11A effector that activates Rab27B.

Calcium sensing receptor (CaSR), a class C GPCR, regulates essential secretory pathways, involving communication between endocytic and secretory Rab GTPases, via still to be fully defined molecular mechanisms. To address how communication between endocytic and secretory vesicles occurs, we hypothesized that CaSR activates endocytic Rab11A-dependent effector pathways acting upstream of Rab27B-regulated secretion. We found that Rab11A is critical to promote Rab27B-dependent secretion of chemotactic and inflammatory factors, including IL-8, CCL2/MCP-1, and IL1-ß, in response to CaSR stimulation. It also attenuates secretion of IL-6. The process is mediated by endosomal PI3-kinases, Vps34 and PI3KC2α, which promote Rab27B activation. Rab11A interacts with and activates MADD, a guanine exchange factor for Rab3, and Rab27A/B. Mechanistically, CaSR drives Rab11A-dependent coupling of recycling endosomes to secretory-vesicles via endosomal PI3K-mediated activation of a MADD/Rab27B pathway.

Sequestration of aristolochic acid I from Aristolochia pilosa by Mapeta xanthomelas Walker, 1863.

Sequestration of secondary plant chemicals and brightly colored bodies occur in a number of unpalatable insects. The utilization of toxic plant chemicals has been proposed as a strategy of chemical defense, while aposematic coloration may advertise unpalatability. Here, we tested for the presence of aristolochic acid I in leaves of Aristolochia pilosa and female bodies of Mapeta xanthomelas, obtained from larvae feeding on the plant, using high performance liquid chromatography with photodiode array detection and electrospray ionization mass spectrometry. The presence of aristolochic acid I in females of this conspicuous diurnal moth, an oligophagous herbivore of Aristolochia, is the first report of sequestration of aristolochic acids by an herbivore other than a species of Papilionidae.

A terpenoid-rich extract from Clethra fimbriata exhibits anti-Trypanosoma cru zi activity and induces T cell cytokine production.

Chagas disease, a worldwide public health concern, is a chronic infection caused by Trypanosoma cruzi. Considering T. cruzi chronic persistence correlates with CD4+ and CD8+ T cell dysfunction and the safety and efficacy profiles of Benznidazol and Nifurtimox, the two drugs currently used for its etiological treatment, are far from ideal, the search of new trypanocidal treatment options is a highly relevant issue. Therefore, the objective of this work was to evaluate the trypanocidal effect and cytokine production induction of three extracts (hexane, dichloromethane and hydroalcoholic) obtained from Clethra fimbriata, a plant traditionally used as a febrifuge in Colombia. Additionally, the extracts' major components with the highest trypanocidal activity were determined. It was evidenced C. fimbriata hexane extract exhibited the highest activity capable of inhibiting the three parasite developmental stages with an IC50/EC50 of 153.9 ± 29.5 (epimastigotes), 39.3 ± 7.2 (trypomastigotes), and 45.6 ± 10.5 (amastigotes) µg/mL, presenting a low cytotoxicity in VERO cells with a selectivity index ranging from 6.49 to 25.4. Moreover, this extract induced trypomastigote apoptotic death and inhibited parasite cell infection. The extract also induced IFN-γ and TNF production in CD4+ and CD8+ T cells, as well as de novo production of the cytotoxic molecules granzyme B and perforin in CD8+ T cells from healthy donors. Fatty acids and terpenes represented C. fimbriata key compounds. Thus, the trypanocidal activity and cytokine production induction of the hexane extract may be associated with terpene presence, particularly, triterpenes.

Calcium sensing receptor stimulates breast cancer cell migration via the Gßγ-AKT-mTORC2 signaling pathway.

Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA-MB-231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gßγ-PI3K-mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.

The calcium sensing receptor (CaSR) promotes Rab27B expression and activity to control secretion in breast cancer cells.

Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1ß, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment.

Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes.

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2.

Use of edoxaban in clinical practice: Comparison of data from the Spanish population in the ETNA-AF-Europe registry.

Aim: To ascertain the clinical profile and management of edoxaban in clinical practice. Materials & methods: Prospective, noninterventional postauthorization study of nonselected patients with atrial fibrillation treated with edoxaban from 12 European countries. Patients' baseline characteristics are presented. Results: A total of 13,638 patients (73.6 ± 9.5 years; 76.6/23.4% edoxaban 60/30 mg; CHA2DS2-VASc 3.1; 838 [6.1%] from Spain) were included. In Spain, the percentage of very elderly and fragile patients was greater and the risk of thromboembolism (CHA2DS2-VASc ≥2, 98.0 vs 87.3%; p < 0.001) and bleeding (HAS-BLED, 3.2 vs 2.7; p < 0.001) was greater in patients treated with edoxaban 30 mg. The proportion of patients taking edoxaban 30 mg was similar than in ENGAGE AF-TIMI 48. Conclusion: In Spain, patients treated with edoxaban were older and fragile.

Protein Purification and Western Blot Detection from Single Zebrafish Embryo.

Characterization of a protein of interest during development is essential for functional studies. A general strategy for understanding the function of a particular protein involves the generation of null mutations, or treatment with drugs, that interfere with its activity. To demonstrate that the synthesis, stability, or activity of a protein has been affected, accurate and efficient detection of low amounts of protein is essential. This can be achieved by immunohistochemistry or by western blot. Here we describe a method for the detection of proteins from single de-yolked zebrafish embryos. This procedure includes a fixation step and the concomitant elimination of lipids from the yolk cell. We show that this approach allows the rapid analysis of proteins in embryos without having to manually remove the yolk. This method provides a convenient alternative for genotyping of mutant embryos as early as the 128 cell stage. In addition, in drug- or morpholino-treated embryos, the correlation between the penetrance of a phenotype and the concentration of a protein present may be established.

Likelihood ratios to assess genetic evidence for clinical significance of uncertain variants: hereditary hemorrhagic telangiectasia as a model.

Clinical laboratories performing gene sequencing discover previously unreported and/or uncharacterized variants. Often these are missense or intronic mutations in which the contribution to disease cannot be predicted, and consequently these mutations are reported as variants of uncertain significance. Follow-up to assess family concordance is recommended by the American College of Medical Genetics to provide genetic evidence for clinical significance. Although family concordance studies show whether a variant segregates with disease in the family, the strength of evidence varies depending on the number and degree of relatedness of family members available for testing. We investigated a statistical model which accounts for the pedigree, inheritance patterns, and penetrance to determine the likelihood of a variant being a causative or deleterious mutation. We used hereditary hemorrhagic telangiectasia (HHT) as a model for an autosomal dominant disease. Pedigree data were transferred to MLINK, and a Bayesian analysis was calculated to determine the likelihood that a variant is causative of disease. In applying this analysis to HHT pedigrees we found Bayes Factors of variants showing odds in favor of causality ranging from approximately 4:1 to over 400:1. These numbers provide an objective measure of the strength of genetic evidence. Other parameters such as amino acid severity predictions, ortholog and paralog comparisons and functional assays can be included in the analysis to increase the evidence of causality.