Novel <i>IRF6 </i>mutations in families with Van Der Woude syndrome and popliteal pterygium syndrome from sub-Saharan Africa

Orofacial clefts (OFC) are complex genetic traits that are often classified as syndromic or nonsyndromic clefts. Currently, there are over 500 types of syndromic clefts in the Online Mendelian Inheritance in Man (OMIM) database, of which Van der Woude syndrome (VWS) is one of the most common (accounting for 2% of all OFC). Popliteal pterygium syndrome (PPS) is considered to be a more severe form of VWS. Mutations in the IRF6 gene have been reported worldwide to cause VWS and PPS. Here, we report studies of families with VWS and PPS in sub-Saharan Africa. We screened the DNA of eight families with VWS and one family with PPS from Nigeria and Ethiopia by Sanger sequencing of the most commonly affected exons in IRF6 (exons 3, 4, 7, and 9). For the VWS families, we found a novel nonsense variant in exon 4 (p.Lys66X), a novel splice-site variant in exon 4 (p.Pro126Pro), a novel missense variant in exon 4 (p.Phe230Leu), a previously reported splice-site variant in exon 7 that changes the acceptor splice site, and a known missense variant in exon 7 (p.Leu251Pro). A previously known missense variant was found in exon 4 (p.Arg84His) in the PPS family. All the mutations segregate in the families. Our data confirm the presence of IRF6-related VWS and PPS in sub-Saharan Africa and highlights the importance of screening for novel mutations in known genes when studying diverse global populations. This is important for counseling and prenatal diagnosis for high-risk families

Impact of the 2014–2016 marine heatwave on US and Canada West Coast fisheries: Surprises and lessons from key case studies

Marine heatwaves are increasingly affecting marine ecosystems, with cascading impacts on coastal economies, communities, and food systems. Studies of heatwaves provide crucial insights into potential ecosystem shifts under future climate change and put fisheries social-ecological systems through “stress tests” that expose both vulnerabilities and resilience. The 2014–16 Northeast Pacific heatwave was the strongest and longest marine heatwave on record and resulted in profound ecological changes that impacted fisheries, fisheries management, and human livelihoods. Here, we synthesize the impacts of the 2014–2016 marine heatwave on US and Canada West Coast fisheries and extract key lessons for preparing global fisheries science, management, and industries for the future. We set the stage with a brief review of the impacts of the heatwave on marine ecosystems and the first systematic analysis of the economic impacts of these changes on commercial and recreational fisheries. We then examine ten key case studies that provide instructive examples of the complex and surprising challenges that heatwaves pose to fisheries social-ecological systems. These reveal important insights into improving the resilience of monitoring and management and increasing adaptive capacity to future stressors. Key recommendations include: (1) expanding monitoring to enhance mechanistic understanding, provide early warning signals, and improve predictions of impacts; (2) increasing the flexibility, adaptiveness, and inclusiveness of management where possible; (3) using simulation testing to help guide management decisions; and (4) enhancing the adaptive capacity of fishing communities by promoting engagement, flexibility, experimentation, and failsafes. These advancements are important as global fisheries prepare for a changing oceanWe are grateful to Nate Mantua, Manuel Hidalgo, Kiva Oken, and Cori Lopazanski for feedback on manuscript drafts. We thank Jean Lee for sharing a non-confidential version of the Gulf of Alaska commercial fisheries landings data and Evan Damkjar and John Davidson for sharing non-confidential versions of British Columbia's commercial and recreational fisheries landings data. CMF was funded by The Nature Conservancy, California. BM was partially supported by the Future Seas II project under NOAA's Climate and Fisheries Adaptation Program (NA20OAR431050). The scientific results and conclusions, as well as any views or opinions expressed herein, are those of the author(s) and do not necessarily reflect the views of NOAA or the Department of Commerce.Ye

Optimising experimental design for high-throughput phenotyping in mice: a case study

To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used

S1 Data -

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.</div

Species relationships and phenotypes of the study species.

(A) The African clawed frog Xenopus laevis (Pipidae) is a model organism for laboratory studies and for developing in vivo techniques for testing gene function. Images by JD, the adult was photographed near Hluhluwe, KwaZulu-Natal, South Africa. (B) The glassfrog Hyalinobatrachium fleischmanni (Centrolenidae) exhibits transparent tissues, through which many organs are visible during embryonic, tadpole and adult life-stages. Fathers provide parental care to developing eggs and embryos can time hatching in response to parenting [28–31]. Images by JD, the adult was photographed near San Gabriel Mixtepec, Oaxaca, Mexico. (C) Allobates femoralis is a cryptic poison frog (Aromobatidae) whose adult behavior is well studied in the context of spatial cognition and parental behavior [32–35]. Images by DS (tadpole) and Andrius Pašukonis (adult from Nouragues Nature Reserve, French Guiana). (D) Poison frogs of the Dendrobatidae are well known for their aposematic coloration, chemical defenses, and diverse social behaviors [25–27, 36]. Images are of Dendrobates tinctorius by DS (captive animals) and Ranitomeya imitator, by DS (tadpole) and Evan Twomey (adult from near San Jose, San Martin, Peru).</p

Step-by-step protocol, also available on protocols.io.

Step-by-step protocol, also available on protocols.io.</p

Tadpole mortality and GFP expression in brain cells.

(A) Tadpole mortality increased with pulse number during brain electroporation. Smaller circles show tadpoles that survived (0) or died (1), larger circles and error bars show the mean ± 95% CI of the proportion of tadpoles that died (estimated from a binomial GLMM). (B) The proportional area of transfected brain cells increased with pulse number across all species. However, the magnitude of treatment effects varied by species. Smaller circles show the proportional area of individual brains with GFP-positive cells, larger circles and error bars show the mean ± 95% CI (estimated from a beta-binomial GLMM). In both plots, data points are jittered to show stacked points. (C) Images of dissected brains (top row) and live tadpoles (bottom row) showing GFP-positive brain cells (green; from fluorescence microscopy). (D) An image from two-photon microscopy showing GFP-labeled cells in vivo within the hindbrain of an R. imitator tadpole.</p

Electroporation set-up.

(A) We placed the tadpole on a platform of modeling clay and positioned it under a dissecting scope to target the tissue for injection and electroporation. We positioned micromanipulators on both sides of the platform, mounted with either the electrode/s (L) or the microinjector (R). (B) Muscle electroporation in a Ranitomeya imitator tadpole. (C) Brain electroporation in a Ranitomeya imitator tadpole. Note that the clay has been molded to counteract the pressure and maintain head shape during injection and electroporation. (D) In tadpoles that are soft and globular, platinum needles are recommended instead of platinum foil sheets.</p

GFP expression in muscle cells in tadpole tails.

(A) The number of transfected myocytes increased with the volume of plasmid DNA across all 4 species. Smaller circles show individual data points, larger circles and error bars show mean ± 95% CI (estimated from a quasi-Poisson GLMM). Datapoints are jittered to show stacked points. (B) Images of live tadpoles showing GFP-positive myocytes (green; from fluorescence microscopy) in their tail myomeres. Note that specie’s differences in the extent of skin pigmentation and muscle-tissue opacity made counting cells difficult from images alone (i.e., adjusting exposure to capture specific cells can overexpose others and vice-versa depending on cell depth, position, and expression levels). Therefore, we counted myocytes directly under a dissecting (fluorescent) microscope, where we could adjust light levels, the animal’s position, and use forceps and probes to delimit the edges of individual myocytes at different depths. (C) An image showing the targeted region of muscle tissue in a tadpole’s tail. Inset: myomeres are blocks of muscle tissue that are arranged in sequence across the tadpole’s tail. Note the individual myocytes arranged in parallel within myomeres, which are large cylindrical cells. (D) Brightfield (left) and fluorescent (right) images showing GFP-labeled myocytes within and delimited by the boarders of the myomeres.</p

Protocol video.

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.</div