Genomic editing of metformin efficacy-associated genetic variants in SLC47A1 does not alter SLC47A1 expression

Several pharmacogenetics studies have identified an association between a greater metformin-dependent reduction in HbA1c levels and the minor A allele at rs2289669 in intron 10 of SLC47A1, encoding multidrug and toxin extrusion 1 (MATE1), a presumed metformin transporter. It is currently unknown if the rs2289669 locus is a cis-eQTL, which would validate its role as predictor of metformin efficacy. We looked at association between common genetic variants in the SLC47A1 gene region and HbA1c reduction after metformin treatment using locus-wise meta-analysis from the MetGen consortium. CRISPR-Cas9 was applied to perform allele editing of, or genomic deletion around, rs2289669 and of the closely linked rs8065082 in HepG2 cells. The genome-edited cells were evaluated for SLC47A1 expression and splicing. None of the common variants including rs2289669 showed significant association with metformin response. Genomic editing of either rs2289669 or rs8065082 did not alter SLC47A1 expression or splicing. Experimental and in silico analyses show that the rs2289669-containing haploblock does not appear to carry genetic variants that could explain its previously reported association with metformin efficacy.Peer reviewe

Solid Organ Transplantation During COVID-19 Pandemic: An International Web-based Survey on Resources’ Allocation

Background. Solid organ transplants (SOTs) are life-saving interventions, recently challenged by coronavirus disease 2019 (COVID-19). SOTs require a multistep process, which can be affected by COVID-19 at several phases. Methods. SOT-specialists, COVID-19-specialists, and medical ethicists designed an international survey according to CHERRIES guidelines. Personal opinions about continuing SOTs, safe managing of donors and recipients, as well as equity of resources' allocation were investigated. The survey was sent by e-mail. Multiple approaches were used (corresponding authors from Scopus, websites of scientific societies, COVID-19 webinars). After the descriptive analysis, univariate and multivariate ordinal regression analysis was performed. Results. There were 1819 complete answers from 71 countries. The response rate was 49%. Data were stratified according to region, macrospecialty, and organ of interest. Answers were analyzed using univariate- multivariate ordinal regression analysis and thematic analysis. Overall, 20% of the responders thought SOTs should not stop (continue transplant without restriction); over 70% suggested SOTs should selectively stop, and almost 10% indicated they should completely stop. Furthermore, 82% agreed to shift resources from transplant to COVID-19 temporarily. Briefly, main reason for not stopping was that if the transplant will not proceed, the organ will be wasted. Focusing on SOT from living donors, 61% stated that activity should be restricted only to "urgent"cases. At the multivariate analysis, factors identified in favor of continuing transplant were Italy, ethicist, partially disagreeing on the equity question, a high number of COVID-19- related deaths on the day of the answer, a high IHDI country. Factors predicting to stop SOTs were Europe except-Italy, public university hospital, and strongly agreeing on the equity question. Conclusions. In conclusion, the majority of responders suggested that transplant activity should be continued through the implementation of isolation measures and the adoption of the COVID-19-free pathways. Differences between professional categories are less strong than supposed

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p

Structural and Functional Analysis of the ApolipoproteinA-I A164S Variant.

Apolipoprotein A-I (apoA-I) is the main protein involved in the formation of high-density lipoprotein (HDL), it is the principal mediator of the reverse cholesterol transfer (RCT) pathway and provides cardio-protection. In addition to functional wild-type apoA-I, several variants have been shown to associate with hereditary amyloidosis. In this study we have performed biophysical and biochemical analyses of the structure and functional properties of the A164S variant of apoA-I (1:500 in the Danish general population), which is the first known mutation of apoA-I that leads to an increased risk of ischaemic heart disease (IHD), myocardial infarction and mortality without associated low HDL cholesterol levels. Despite the fact that epidemiologically IHD is associated with low plasma levels of HDL, the A164S mutation is linked to normal plasma levels of lipids, HDL and apoA-I, suggesting impaired functionality of this variant. Using biophysical techniques (e.g., circular dichroism spectroscopy and electron microscopy) to determine secondary structure, stability and pro-amyloidogenic property of the lipid free A164S apoA-I variant, our observations suggest similarity in structural properties between apoA-I WT and apoA-I A164S. However, the A164S apoA-I variant exhibits lower binding affinity to lipids but forms similar sized HDL particles to those produced by WT

Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle.

Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined if nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and if a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain specific peptides of apoA-I showed that the lipid-free C-terminal 190-243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190-243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. JLR Discoidal HDL and the 190-243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle and the C-terminal α-helical content of apoA-I may be an important determinant of this effect

Analysis of alpha-helical content and thermal stability of apoA-I A164S.

<p>(A) CD analysis of apoA-I WT and A164S. Alpha-helical content of 0.2 mg/ml of apoA-I was calculated from molar ellipticity at 222 nm (n = 3). (B) Thermal stability of 0.2 mg/ml of apoA-I was assessed from normalized sigmoidal decrease of molar ellipticity at 222 nm. The results are mean ± SEM (n = 3). (C) Stopped-flow coupled to CD: apoA-I WT and A164S (0.5 mg/ml final concentration) were mixed with 100 mM SDS at the volume ratio 1:5 and molar ellipticity at 222 nm was measured. (D). Bar graph shows the t1/2 of apoA-I WT and A164S. Data is mean ±SEM (* = p<0.05, n = 6). (E) Near CD analysis of apoA-I WT and A164S (1.25 mg/ml).</p

Cholesterol efflux from macrophages.

<p>J774 macrophages enriched with ³H-cholesterol were incubated with apoA-I WT or A164S and cholesterol efflux quantified as a function of protein concentration (A) or time (B) by scintillation counting of the resulting treatment media. An ACAT inhibitor and CPT-cAMP were used to prevent formation of cholesteryl esters of the ³H-cholesterol and to induce expression of ABCA1, respectively. Data from concentration gradient experiments were fitted using the Michaelis-Menten equation. Time dependent efflux treatments were performed with 50 μg/ml apoA-I WT or A164S. Each figure represents 3 independent experiments and displays mean±SD. (C) ApoA-I WT or A164S were incubated with rat serum for 2 h followed by incubation with J774 macrophages enriched with ³H-cholesterol for 2 h (10 μg/ml apoA-I). Rat serum was used as control for background efflux. Data is mean ±SEM (**** = p<0.0001, n = 3).</p

Limited proteolysis analysis.

<p>ApoA-I WT, A164S and L178H (5μg) were incubated for indicated times at 37°C in the presence of chymotrypsin. The cleaved products from limited proteolysis from different time points were separated by SDS-PAGE and visualized by coomassie staining. <i>Arrows</i> indicate migration distances of full-length proteins.</p

Lipid clearance assay and native gel analyses of rHDL formation.

<p>(A) ApoA-I WT and A164S were combined with DMPC at a 1:100 molar ratio and lipid binding measured by absorbance at 325nm at indicate times. Readings were fitted to one-way decay of non-linear regression. (B) Bar graph shows the lipid clearance rate described as t1/2 values calculated from panel A. Data are mean ±SEM (* = p<0.05, n = 3). (C) ApoAI-A164S and apoA-I WT were incubated with DMPC lipids at 37°C for indicated times and analyzed by native gel. <i>Arrows</i> indicate apoA-I WT and apoA-I A164S rHDL particles of diameters of approximately 10 nm. Lipid-free (LF) proteins at time 0 h prior to mixing with DMPC lipids are included as controls. (D) Formation of HDL upon incubation of apoA-I WT and A164S with rat serum. 0.75mg/ml of WT or A164S apoA-I was incubated with 200 μl rat serum. Samples were collected at indicated times and equal amounts of protein (0.56μg) separated by blue native PAGE and western blotting for human apoA-I was performed. Lipid-free (LF) proteins (not incubated with serum) and serum without protein (ctrl) are included as controls.</p