Publications by authors named "Michelle Stewart"

105 Publications

A machine learning model for identifying patients at risk for wild-type transthyretin amyloid cardiomyopathy.

Nat Commun 2021 05 11;12(1):2725. Epub 2021 May 11.

Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Transthyretin amyloid cardiomyopathy, an often unrecognized cause of heart failure, is now treatable with a transthyretin stabilizer. It is therefore important to identify at-risk patients who can undergo targeted testing for earlier diagnosis and treatment, prior to the development of irreversible heart failure. Here we show that a random forest machine learning model can identify potential wild-type transthyretin amyloid cardiomyopathy using medical claims data. We derive a machine learning model in 1071 cases and 1071 non-amyloid heart failure controls and validate the model in three nationally representative cohorts (9412 cases, 9412 matched controls), and a large, single-center electronic health record-based cohort (261 cases, 39393 controls). We show that the machine learning model performs well in identifying patients with cardiac amyloidosis in the derivation cohort and all four validation cohorts, thereby providing a systematic framework to increase the suspicion of transthyretin cardiac amyloidosis in patients with heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-22876-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113237PMC
May 2021

Health Impact of Tafamidis in Transthyretin Amyloid Cardiomyopathy patients: An Analysis from the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT) and the Open-Label Long-term Extension Studies.

Eur Heart J Qual Care Clin Outcomes 2021 Apr 24. Epub 2021 Apr 24.

The Amyloidosis Center, Knight Cardiovascular Institute, Oregon Health & Science University, Portland, Oregon.

Background: The ATTR-ACT trial showed that tafamidis reduced all-cause mortality and cardiovascular-related hospitalizations in patients with transthyretin amyloid cardiomyopathy (ATTR-CM). This study aimed to estimate the impact of tafamidis on survival and quality-adjusted life-years (QALY).

Methods: A multi-state, cohort, Markov model was developed to simulate the disease course of ATTR-CM throughout a lifetime. For survival extrapolation, survival curves were fitted by treatment arm and New York Heart Association (NYHA) class I/II (68% of patients) and NYHA class III (32% of patients) cohorts using the individual patient-level data from both the ATTR-ACT and the corresponding long-term extension study. Univariate and multivariate sensitivity analyses were conducted.

Results: The predicted mean survival for the total population (NYHA class I/II+III) was 6.73 years for tafamidis and 2.85 years for the standard of care (SoC), resulting in an incremental mean survival of 3.88 years (95% CI 1.32-5.66). Of the 6.73 life-years, patients on tafamidis spend, on average, 4.82 years in NYHA class I/II, while patients on standard of care (SoC) spend an average of 1.60 life-years in these classes. The combination of longer survival in lower NYHA classes produced a QALY gain of 5.39 for tafamidis and 2.11 for SoC, resulting in 3.29 incremental QALYs (95% CI 1.21-4.74) in favor of tafamidis.

Conclusions: Based on the disease simulation model results, tafamidis is expected to more than double the life expectancy and QALYs of ATTR-CM patients compared to SoC. Longer-term follow-up data from the ATTR-ACT extension study will further inform these findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/ehjqcco/qcab031DOI Listing
April 2021

Impact of Delayed Diagnosis and Misdiagnosis for Patients with Transthyretin Amyloid Cardiomyopathy (ATTR-CM): A Targeted Literature Review.

Cardiol Ther 2021 Jun 20;10(1):141-159. Epub 2021 Apr 20.

University of Utah Health Sciences Center and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA.

Introduction: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive, fatal and under-recognized disease. This targeted literature review assessed the extent and consequences of diagnostic delay and misdiagnosis in ATTR-CM.

Methods: The Embase database was searched together with proceedings of eight cardiology conferences to identify publications or abstracts on ATTR-CM. Outcomes of interest were time from symptom onset to diagnosis, rates of delayed diagnosis and misdiagnosis, and costs, healthcare resource use or clinical outcomes whilst undiagnosed/misdiagnosed.

Results: Twenty-three articles were included. Weighted means of reported mean and median diagnostic delays were 6.1 and 3.4 years for wild-type (ATTRwt-CM) and 5.7 and 2.6 years for hereditary (ATTRv-CM). Misdiagnosis occurred in 34-57% of patients when reported. Evaluation and misdiagnosis by multiple healthcare providers before receiving an ATTR-CM diagnosis was common, and there was evidence that patients undergo unnecessary or inappropriate evaluations or treatments while misdiagnosed. Diagnostic "red flags" were reported to be underused. Data on the consequences of delay for patients and health systems were sparse, but given the progressive nature of ATTR-CM, delay is likely to have adverse consequences.

Conclusion: ATTR-CM patients commonly experience diagnostic delay and misdiagnosis. Efforts are required to provide timely diagnosis so that patients can benefit from earlier access to new disease-modifying therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40119-021-00219-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126532PMC
June 2021

Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2.

Hum Mol Genet 2021 May;30(10):880-892

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, UK.

Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2β2, AP2μ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1-3 (FHH1-3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddab076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165646PMC
May 2021

Estimating the annual economic burden for the management of patients with transthyretin amyloid polyneuropathy in Spain.

Expert Rev Pharmacoecon Outcomes Res 2021 Mar 24:1-7. Epub 2021 Mar 24.

Patient & Health Impact, Pfizer Inc, Capelle Aan Den IJssel, The Netherlands.

: Transthyretin amyloid polyneuropathy (ATTR-PN) is a fatal disease associated with substantial burden of illness. Three therapies are approved by the European Medicines Agency for the management of this rare disease. The aim of this study was to compare the total annual treatment specific cost per-patient associated with ATTR-PN in Spain.: An Excel-based patient burden and cost estimator tool was developed to itemize direct and indirect costs related to treatment with inotersen, patisiran, and tafamidis in the context of ATTR-PN. The product labels and feedback from five Spanish ATTR-PN experts were used to inform resource use and cost inputs.: Marked differences in costs were observed between the three therapies. The need for patisiran- and inotersen-treated patients to visit hospitals for pre-treatment, administration, and monitoring was associated with increased patient burden and costs compared to those treated with tafamidis. Drug acquisition costs per-patient per-year were 291,076€ (inotersen), 427,250€ (patisiran) and 129,737€ (tafamidis) and accounted for the majority of total costs. Overall, the total annual per-patient costs were lowest for patients treated with tafamidis (137,954€), followed by inotersen (308,358€), and patisiran (458,771€).: Treating patients with tafamidis leads to substantially lower costs and patient burden than with inotersen or patisiran.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/14737167.2021.1900738DOI Listing
March 2021

A novel knockout mouse for the small EDRK-rich factor 2 (Serf2) showing developmental and other deficits.

Mamm Genome 2021 04 13;32(2):94-103. Epub 2021 Mar 13.

Department of Neuromuscular Diseases, Queen Square Institute of Neurology, London, UK.

The small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2 mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2 null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00335-021-09864-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012326PMC
April 2021

Maternal and offspring high-fat diet leads to platelet hyperactivation in male mice offspring.

Sci Rep 2021 Jan 14;11(1):1473. Epub 2021 Jan 14.

Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading - Harborne Building, Reading, RG6 6AS, UK.

Maternal over-nutrition increases the risk of diabetes and cardiovascular events in offspring. While prominent effects on cardiovascular health are observed, the impact on platelet physiology has not been studied. Here, we examined whether maternal high-fat diet (HF) ingestion affects the platelet function in lean and obese offspring. C57BL6/N mice dams were given a HF or control (C) diet for 8 weeks before and during pregnancy. Male and female offspring received C or HF diets for 26 weeks. Experimental groups were: C/C, dam and offspring fed standard laboratory diet; C/HF dam fed standard laboratory diet and offspring fed HF diet; HF/C and HF/HF. Phenotypic and metabolic tests were performed and blood collected for platelet studies. Compared to C/C, offspring HF groups were obese, with fat accumulation, hyperglycaemia and insulin resistance. Female offspring did not present platelet hyperactivity, hence we focused on male offspring. Platelets from HF/HF mice were larger, hyperactive and presented oxidative stress when compared to C/C. Maternal and offspring HF diet results in platelet hyperactivation in male mouse offspring, suggesting a novel 'double-hit' effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-80373-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809045PMC
January 2021

Impact of Tafamidis on Health-Related Quality of Life in Patients With Transthyretin Amyloid Cardiomyopathy (from the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial).

Am J Cardiol 2021 02 19;141:98-105. Epub 2020 Nov 19.

Columbia University Vagelos College of Physicians and Surgeons, New York, New York.

In the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial, tafamidis significantly reduced all-cause mortality and cardiovascular-related hospitalizations in patients with transthyretin amyloid cardiomyopathy (ATTR-CM). ATTR-CM is associated with a significant burden of disease; further analysis of patient-reported quality of life will provide additional data on the efficacy of tafamidis. In the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial, 441 adult patients with ATTR-CM were randomized (2:1:2) to tafamidis 80 mg, tafamidis 20 mg, or placebo for 30 months, with pooled tafamidis (80 mg and 20 mg) compared with placebo. Change in Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) domain scores, EQ-5D-3L scores, and patient global assessment, were prespecified exploratory end points. A greater proportion of patients improved KCCQ-OS score at month 30 with tafamidis (41.8%) versus placebo (21.4%). Tafamidis significantly reduced the decline in all 4 KCCQ-OS domains (p <0.0001 for all), and in EQ-5D-3L utility (0.09 [confidence interval 0.05 to 0.12]; p <0.0001) and EQ visual analog scale (9.11 [confidence interval 5.39 to 12.83]; p <0.0001) scores at month 30 versus placebo. A larger proportion of tafamidis-treated patients reported their patient global assessment improved at month 30 (42.3% vs 23.8% with placebo). In conclusion, tafamidis effectively reduced the decline in patient-reported outcomes, providing further insight into its efficacy in health-related quality of life in patients with ATTR-CM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.amjcard.2020.10.066DOI Listing
February 2021

Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model.

JBMR Plus 2020 Oct 7;4(10):e10402. Epub 2020 Sep 7.

Academic Endocrine Unit, Radcliffe Department of Medicine Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford Oxford UK.

Calcilytics are calcium-sensing receptor (CaSR) antagonists that reduce the sensitivity of the CaSR to extracellular calcium. Calcilytics have the potential to treat autosomal dominant hypocalcemia type 1 (ADH1), which is caused by germline gain-of-function CaSR mutations and leads to symptomatic hypocalcemia, inappropriately low PTH concentrations, and hypercalciuria. To date, only one calcilytic compound, NPSP795, has been evaluated in patients with ADH1: Doses of up to 30 mg per patient have been shown to increase PTH concentrations, but did not significantly alter ionized blood calcium concentrations. The aim of this study was to further investigate NPSP795 for the treatment of ADH1 by undertaking in vitro and in vivo studies involving mice, which have hypocalcemia in association with a gain-of-function CaSR mutation, Leu723Gln. Treatment of HEK293 cells stably expressing the mutant (Gln723) CaSR with 20nM NPSP795 decreased extracellular Ca-mediated intracellular calcium and phosphorylated ERK responses. An in vivo dose-ranging study was undertaken by administering a s.c. bolus of NPSP795 at doses ranging from 0 to 30 mg/kg to heterozygous and to homozygous mice, and measuring plasma PTH responses at 30 min postdose. NPSP795 significantly increased plasma PTH concentrations in a dose-dependent manner with the 30 mg/kg dose causing a maximal (≥10-fold) rise in PTH. To determine whether NPSP795 can rectify the hypocalcemia of and mice, a submaximal dose (25 mg/kg) was administered, and plasma adjusted-calcium concentrations measured over a 6-hour period. NPSP795 significantly increased plasma adjusted-calcium in mice from 1.87 ± 0.03 mmol/L to 2.16 ± 0.06 mmol/L, and in mice from 1.70 ± 0.03 mmol/L to 1.89 ± 0.05 mmol/L. Our findings show that NPSP795 elicits dose-dependent increases in PTH and ameliorates the hypocalcemia in an ADH1 mouse model. Thus, calcilytics such as NPSP795 represent a potential targeted therapy for ADH1. © 2020 The Authors. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm4.10402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574706PMC
October 2020

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

Proc Natl Acad Sci U S A 2020 07 30;117(29):17195-17203. Epub 2020 Jun 30.

Warp Drive Bio, Inc., Redwood City, CA 94063;

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2006560117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382241PMC
July 2020

The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities.

PLoS One 2020 15;15(6):e0230162. Epub 2020 Jun 15.

Mary Lyon Centre, MRC Harwell Institute, Oxfordshire, United Kingdom.

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230162PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295225PMC
August 2020

Extrapolation of Survival Benefits in Patients with Transthyretin Amyloid Cardiomyopathy Receiving Tafamidis: Analysis of the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial.

Cardiol Ther 2020 Dec 10;9(2):535-540. Epub 2020 Jun 10.

Pfizer, Groton, CT, USA.

Introduction: In the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT; ClinicalTrials.gov number NCT01994889), tafamidis reduced the risk of all-cause mortality in patients with transthyretin amyloid cardiomyopathy (ATTR-CM) by 30% versus placebo. Median overall survival was not achieved in either treatment arm (57.1 and 70.5% of patients in the placebo and tafamidis groups, respectively, survived at 30 months), limiting assessment of the potential survival benefits of treatment.

Methods: A survival extrapolation analysis was conducted following technical support guidelines from the National Institute for Health and Care Excellence. Multiple models (i.e., exponential, Weibull, gamma, log-logistic, log-normal, Gompertz, generalized gamma, and generalized F) were applied to systematically fit different candidate curves to existing patient-level data from the 30-month treatment period in ATTR-ACT. The relative goodness-of-fit for each candidate curve was then tested by Akaike's and Bayesian information criteria to select a single model that was fitted to the placebo and pooled tafamidis treatment arms.

Results: A gamma distribution was selected as best fitting model and fitted to both treatment arms. The resulting estimated median overall survival was 35.16 months for placebo and 52.64 months for tafamidis (difference 17.48 months).

Conclusions: This extrapolation of survival data from ATTR-ACT further supports the efficacy of tafamidis in patients with ATTR-CM. Owing to the limitations of this analysis, these survival estimates should be interpreted with caution; however, they are consistent with recently presented findings from a combined analysis of data from ATTR-ACT and interim data from an ongoing long-term extension study (median follow-up 36 months; ClinicalTrials.gov number NCT02791230).

Trial Registration: ClinicalTrials.gov: NCT01994889.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40119-020-00179-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584686PMC
December 2020

Extrapolation of Survival Benefits in Patients with Transthyretin Amyloid Cardiomyopathy Receiving Tafamidis: Analysis of the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial.

Cardiol Ther 2020 Dec 10;9(2):535-540. Epub 2020 Jun 10.

Pfizer, Groton, CT, USA.

Introduction: In the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT; ClinicalTrials.gov number NCT01994889), tafamidis reduced the risk of all-cause mortality in patients with transthyretin amyloid cardiomyopathy (ATTR-CM) by 30% versus placebo. Median overall survival was not achieved in either treatment arm (57.1 and 70.5% of patients in the placebo and tafamidis groups, respectively, survived at 30 months), limiting assessment of the potential survival benefits of treatment.

Methods: A survival extrapolation analysis was conducted following technical support guidelines from the National Institute for Health and Care Excellence. Multiple models (i.e., exponential, Weibull, gamma, log-logistic, log-normal, Gompertz, generalized gamma, and generalized F) were applied to systematically fit different candidate curves to existing patient-level data from the 30-month treatment period in ATTR-ACT. The relative goodness-of-fit for each candidate curve was then tested by Akaike's and Bayesian information criteria to select a single model that was fitted to the placebo and pooled tafamidis treatment arms.

Results: A gamma distribution was selected as best fitting model and fitted to both treatment arms. The resulting estimated median overall survival was 35.16 months for placebo and 52.64 months for tafamidis (difference 17.48 months).

Conclusions: This extrapolation of survival data from ATTR-ACT further supports the efficacy of tafamidis in patients with ATTR-CM. Owing to the limitations of this analysis, these survival estimates should be interpreted with caution; however, they are consistent with recently presented findings from a combined analysis of data from ATTR-ACT and interim data from an ongoing long-term extension study (median follow-up 36 months; ClinicalTrials.gov number NCT02791230).

Trial Registration: ClinicalTrials.gov: NCT01994889.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40119-020-00179-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584686PMC
December 2020

Impact of Non-Cardiac Clinicopathologic Characteristics on Survival in Transthyretin Amyloid Polyneuropathy.

Neurol Ther 2020 Jun 31;9(1):135-149. Epub 2020 Mar 31.

Pfizer Inc, Groton, CT, USA.

Introduction: Hereditary (variant) transthyretin amyloidosis (ATTRv) with polyneuropathy (ATTR-PN) is a rare genetic disorder that causes progressive autonomic and sensorimotor neuropathy, severe disability, and death within 10 years of onset. Previous studies have primarily focused on how baseline cardiac characteristics affect mortality, but the impact of non-cardiac baseline characteristics is less defined.

Methods: We systematically searched PubMed/Medline (1990-2019) to identify studies that assessed the impact of baseline ATTR-PN characteristics on survival. Outcomes were first summarized descriptively. Extracted survival data were then disaggregated, and parametric mixture models were used to assess survival differences among patient groups defined by factors known to affect survival.

Results: The search yielded 1193 records, of which 35 were retained for analysis. Median survival ranged from 0.5 to > 25 years. The largest survival differences were between cohorts who underwent liver transplantation (LTx) versus those who did not. Among LTx cohorts, pre-LTx ATTR-PN disease duration ≥ 7 years, poor nutritional status, and late disease onset reduced median survival by 13, 12, and 10 years, respectively. Other prognostic survival factors included non-Val30Met genotype and baseline presence of urinary incontinence, erectile dysfunction, or muscle weakness.

Conclusion: Survival in patients with ATTR-PN is highly variable and affected by non-cardiac baseline characteristics, such as autonomic dysfunction, large fiber involvement, late-onset disease, and non-Val30Met mutation. Careful interpretation of these findings is warranted given that this synthesis did not control for differences between studies. Survival in patients with ATTR-PN remains poor among those who are untreated or with delayed diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40120-020-00183-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229108PMC
June 2020

null C57BL/6N mice develop cardiomyopathy, whereas null C57BL/6J mice do not.

Life Sci Alliance 2020 04 25;3(4). Epub 2020 Mar 25.

Centre for Endocrinology, William Harvey Research Institute, Charterhouse Square, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase () null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 () mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J -null mice do not develop cardiomyopathy; however, we identified a null mutation in as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.26508/lsa.201900593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103425PMC
April 2020

Evaluation of Mortality During Long-Term Treatment with Tafamidis for Transthyretin Amyloidosis with Polyneuropathy: Clinical Trial Results up to 8.5 Years.

Neurol Ther 2020 Jun 27;9(1):105-115. Epub 2020 Feb 27.

Pfizer Inc, New York, NY, USA.

Introduction: The effects of tafamidis on mortality in Val30Met and non-Val30Met patients with transthyretin amyloidosis with polyneuropathy (ATTR-PN) were evaluated.

Methods: The analyses were based on cumulative data from the Val30Met patients in the 18-month double-blind registration study and its 12-month open-label extension study, the non-Val30Met patients of the 12-month open-label study, and both patient groups in the ongoing 10-year extension study. Kaplan-Meier analyses of time to death from first treatment dose were performed. For the Val30Met group, two treatment groups were analyzed: those who received tafamidis in both the parent and extension studies (T-T) and those who received placebo in the parent study and switched to tafamidis in the extension studies (P-T).

Results: Kaplan-Meier estimates (95% confidence interval [CI]) were available up to 9 years for the Val30Met group, at which time 85.9% (53.1-96.4) and 91.1% (77.9-96.6) of the patients in the T-T and P-T groups, respectively, were alive. For the non-Val30Met group, estimates were available up to 8 years from the first dose, and the percentage of patients alive was 75.9% (47.7-90.2).

Conclusion: Long-term tafamidis treatment may confer survival benefit in patients with ATTR-PN.

Trial Registration: ClinicalTrials.gov identifier: NCT00409175, NCT00791492, NCT00630864, and NCT00925002.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40120-020-00180-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229124PMC
June 2020

The impact of clinical heterogeneity on conducting network meta-analyses in transthyretin amyloidosis with polyneuropathy.

Curr Med Res Opin 2020 05 17;36(5):799-808. Epub 2020 Feb 17.

Value & Evidence Division, Marketing and Market Access, Eversana, Sydney, Canada.

The comparative safety and efficacy of tafamidis, patisiran and inotersen treatments for transthyretin amyloidosis with polyneuropathy (ATTR-PN) has not been evaluated in clinical trials. In the absence of head-to-head evidence, indirect treatment comparisons such as network meta-analyses (NMAs) can be performed to evaluate relative effects of treatments. This study aims to assess the feasibility of conducting an NMA of available therapies for ATTR-PN patients. Pivotal trials for three approved ATTR-PN treatments, tafamidis (Fx-005), patisiran (APOLLO) and inotersen (NEURO-TTR), were compared in terms of study design, baseline population characteristics, outcome definitions and baseline risk. These assessments of heterogeneity informed the decision to perform Bayesian NMAs. Despite similar study designs, clear differences in eligibility criteria between trials were accompanied by imbalances in baseline population characteristics considered to be plausible effect modifiers, such as disease stage and previous treatment. Of the outcomes assessed, only quality of life and adverse events were similarly reported in all trials. Neuropathy outcomes were not evaluated consistently between trials. An NMA of ATTR-PN treatments was not feasible, given the observed cross-trial heterogeneity. This decision highlights the importance of careful consideration for clinical heterogeneity that may threaten the validity of indirect comparisons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/03007995.2020.1725742DOI Listing
May 2020

Human and mouse essentiality screens as a resource for disease gene discovery.

Nat Commun 2020 01 31;11(1):655. Epub 2020 Jan 31.

Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14284-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994715PMC
January 2020

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

PLoS Biol 2019 09 3;17(9):e3000414. Epub 2019 Sep 3.

Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.3000414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743795PMC
September 2019

Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP-1.

Obesity (Silver Spring) 2019 08;27(8):1292-1304

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Objective: A previous genome-wide association study linked overexpression of an ATP-binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse.

Methods: A global Abcc5 mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon-like peptide 1 (GLP-1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme-linked immunosorbent assay.

Results: Abcc5 mice had decreased fat mass and increased plasma levels of GLP-1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP-1 release.

Conclusions: ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP-1 secretion from enteroendocrine cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/oby.22521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658130PMC
August 2019

Psychopathology in parents of children with autism spectrum disorder: A systematic review and meta-analysis of prevalence.

Autism 2020 01 9;24(1):26-40. Epub 2019 May 9.

Deakin University, Australia.

Parents of children with autism spectrum disorder appear to experience high levels of psychological distress, yet little is known about the prevalence of psychological disorders in this population. The aim of this systematic review and meta-analysis was to estimate the proportion of these parents who experience clinically significant psychopathology. Articles reporting proportions of psychological disorders in a sample of parents of children with autism spectrum disorder were located. The initial search returned 25,988 articles. Thirty-one studies with a total sample of 9208 parents were included in the final review. The median meta-analytic proportions were 31% (95% confidence interval = [24%, 38%]) for depressive disorders, 33% (95% confidence interval = [20%, 48%]) for anxiety disorders, 10% (95% confidence interval = [1%, 41%]) for obsessive-compulsive disorder, 4% (95% confidence interval = [0%, 22%]) for personality disorders, 2% (95% confidence interval = [1%, 4%]) for alcohol and substance use disorders and 1% (95% confidence interval = [0%, 5%]) for schizophrenia spectrum disorders. Significant heterogeneity was detected in these categories. Further research is needed to gain more insight into variables that may moderate parental psychopathology. This review and meta-analysis is the first to provide prevalence estimates of psychological disorders in parents of children with autism spectrum disorder.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1362361319844636DOI Listing
January 2020

Epidemiological and clinical characteristics of symptomatic hereditary transthyretin amyloid polyneuropathy: a global case series.

Orphanet J Rare Dis 2019 02 8;14(1):34. Epub 2019 Feb 8.

Pfizer Inc., Collegeville, PA, USA.

We describe 542 cases of symptomatic hereditary transthyretin amyloid polyneuropathy (ATTR-PN) identified through a review of the literature published between 2005 and 2016. Approximately 18% of the cases were from countries where ATTR-PN is traditionally considered to be endemic (i.e., Portugal, Japan, and Sweden). East Asia (Japan, China, Taiwan, and South Korea) contributed a sizeable combined proportion (37.0%, n = 200) with Japan (n = 92) and China (n = 71) being the primary contributors. The most common genotypes among the 65 genotypes represented in the sample were Val30Met (47.6%), Ser77Tyr (10%), Ala97Ser (6.5%), and Phe64Leu (4.4%). Cases with genotypes other than the aforementioned four had the lowest ages at onset (mean 49.2 [standard deviation {SD} 21.0; inter-quartile range {IQR}14.7]) and diagnosis (mean 53.4 [SD 21.0; IQR 14.7]). Conversely, Phe64Leu mean age of onset was 67.5 (SD 8.8; IQR 5.2) and mean age of diagnosis was 71.3 (SD 8.8; IQR 5.4). The prevalence of upper and lower limb involvement at the time of diagnosis (67 and 41%) observed across all cases is consistent with the typical presentation of ATTR-PN. Other notable findings at the time of diagnosis included a high rate of impotence among the Ala97Ser cases versus all others (67% vs. 21%) and a high rate of non-motor visual symptoms (i.e., visual opacities and glaucoma) in the Ser77Tyr cases versus all others (93% vs. 16%). Though comparisons were made descriptively and were hindered by inconsistency of reporting across the cases, these findings support the notion that ATTR-PN is a more phenotypically and geographically variable disease than is typically considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-019-1000-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368811PMC
February 2019

Loss of disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities.

Dis Model Mech 2019 02 22;12(2). Epub 2019 Feb 22.

MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time , how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.036806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398485PMC
February 2019

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Commun Biol 2018 21;1:236. Epub 2018 Dec 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-018-0226-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303268PMC
December 2018

Tafamidis Treatment for Patients with Transthyretin Amyloid Cardiomyopathy.

N Engl J Med 2018 Sep 27;379(11):1007-1016. Epub 2018 Aug 27.

From the Columbia University Vagelos College of Physicians and Surgeons (M.S.M.) and Pfizer (J.H.S., A.I.B., P.H., J.S., M.B.S.), New York; Syneos Health, Raleigh, NC (B.G.); University College London and St. Bartholomew's Hospital, London (P.M.E.); the Amyloidosis Center, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, and the University of Pavia, Pavia (G.M.), and the Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Bologna (C.R.) - both in Italy; the Amyloidosis Center (CEPARM), Federal University of Rio de Janeiro, Rio de Janeiro (M.W-C.); the Amyloidosis Center, Medical University of Heidelberg, Heidelberg, Germany (A.V.K.); the Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (M.G.); Stanford University School of Medicine, Stanford, CA (R.W.); the French Referral Center for Cardiac Amyloidosis, Amyloidosis Mondor Network, GRC Amyloid Research Institute and Department of Cardiology, Assistance Publique-Hôpitaux de Paris, CHU Henri Mondor, and INSERM Unité 955, Clinical Investigation Center 006, and DHU ATVB, Creteil, France (T.D.); Penn Presbyterian Medical Center, University of Pennsylvania Health System, Philadelphia (B.M.D.); the Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago (S.J.S.); Cleveland Clinic, Cleveland (M.H.); the Medical University of South Carolina, Charleston (D.P.J.); and Pfizer, Groton, CT (T.A.P., S.R., M.S.).

Background: Transthyretin amyloid cardiomyopathy is caused by the deposition of transthyretin amyloid fibrils in the myocardium. The deposition occurs when wild-type or variant transthyretin becomes unstable and misfolds. Tafamidis binds to transthyretin, preventing tetramer dissociation and amyloidogenesis.

Methods: In a multicenter, international, double-blind, placebo-controlled, phase 3 trial, we randomly assigned 441 patients with transthyretin amyloid cardiomyopathy in a 2:1:2 ratio to receive 80 mg of tafamidis, 20 mg of tafamidis, or placebo for 30 months. In the primary analysis, we hierarchically assessed all-cause mortality, followed by frequency of cardiovascular-related hospitalizations according to the Finkelstein-Schoenfeld method. Key secondary end points were the change from baseline to month 30 for the 6-minute walk test and the score on the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS), in which higher scores indicate better health status.

Results: In the primary analysis, all-cause mortality and rates of cardiovascular-related hospitalizations were lower among the 264 patients who received tafamidis than among the 177 patients who received placebo (P<0.001). Tafamidis was associated with lower all-cause mortality than placebo (78 of 264 [29.5%] vs. 76 of 177 [42.9%]; hazard ratio, 0.70; 95% confidence interval [CI], 0.51 to 0.96) and a lower rate of cardiovascular-related hospitalizations, with a relative risk ratio of 0.68 (0.48 per year vs. 0.70 per year; 95% CI, 0.56 to 0.81). At month 30, tafamidis was also associated with a lower rate of decline in distance for the 6-minute walk test (P<0.001) and a lower rate of decline in KCCQ-OS score (P<0.001). The incidence and types of adverse events were similar in the two groups.

Conclusions: In patients with transthyretin amyloid cardiomyopathy, tafamidis was associated with reductions in all-cause mortality and cardiovascular-related hospitalizations and reduced the decline in functional capacity and quality of life as compared with placebo. (Funded by Pfizer; ATTR-ACT ClinicalTrials.gov number, NCT01994889 .).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1805689DOI Listing
September 2018

Characterizing the High Disease Burden of Transthyretin Amyloidosis for Patients and Caregivers.

Neurol Ther 2018 Dec 2;7(2):349-364. Epub 2018 Aug 2.

Evidera, Bethesda, MD, USA.

Introduction: Transthyretin amyloidosis (ATTR amyloidosis), whether manifesting as familial amyloid polyneuropathy (ATTR-PN) or cardiomyopathy (ATTR-CM), is a progressive, debilitating, and often fatal, rare disease requiring significant caregiver support. This study aims to better characterize the burden of disease for ATTR amyloidosis patients and caregivers.

Methods: Patients and caregivers in the USA and Spain were recruited through patient advocacy groups to complete a cross-sectional survey. Assessments included the 12-Item Short Form Health Survey, the Work Productivity and Activity Impairment Questionnaire, the Zarit Burden Interview, pain and symptom measures, health care resource use measures, and caregiving burden measures.

Results: Respondents included 60 ATTR amyloidosis patients and 32 caregivers. Patients registered scores up to two standard deviations below normal for physical health, impairment in quality of life, and reduced work productivity. Patients with liver transplant versus without liver transplant reported better overall outcomes, and those without liver transplant reported a greater impact on physical versus mental health. A high rate of health care utilization in the 3 months prior to the survey was reported by patients. Caregivers reported substantial burden, including poor mental health, work impairment, and much time spent providing care (mean 45.9 h/week). Work productivity was impacted for employed patients and caregivers.

Conclusion: ATTR amyloidosis was associated with high levels of impairment in many domains, including physical health, quality of life, and reduced productivity. Providing care for ATTR amyloidosis patients is associated with a negative impact on the mental health of caregivers. These results highlight the substantial burden of ATTR amyloidosis for patients and caregivers.

Trial Registration: ClinicalTrials.gov: NCT01604122.

Funding: Pfizer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40120-018-0106-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283802PMC
December 2018

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

BMC Biol 2018 06 21;16(1):70. Epub 2018 Jun 21.

The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon, OX11 0RD, UK.

Background: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.

Results: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors.

Conclusion: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-018-0530-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011369PMC
June 2018

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

EMBO J 2018 06 15;37(11). Epub 2018 May 15.

UCL Institute of Neurology and Francis Crick Institute, London, UK.

TDP-43 (encoded by the gene ) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous to dissect TDP-43 function at physiological levels both and Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. , this gain-of-function mutation in endogenous causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201798684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983119PMC
June 2018