Genetic Restoration Research Articles

Connexin43 as a Therapeutic To Restore Cardiac Structure in Arrhythmogenic Heart Disease

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is termed a fatal “disease of the desmosome” as 40–50% of patients harbor mutations in genes associated with the desmosome, a mechanical based cell‐cell junction structure found between cardiac muscle cells. Connexin43, the most dominant gap junction protein in ventricles, has been classically associated with electrical functions between adjacent cardiac cells, is consistently downregulated and considered an early disease hallmark of ARVD/C. However, limited efforts have focused on dissecting the role of connexin43 in ARVD/C, especially during end‐stage cardiac disease, where structural alterations are integral in facilitating ventricular failure, arrhythmias and premature death. Here we show that genetic restoration of connexin43 in human and mouse models of ARVD/C is sufficient to bypass desmosomal structural deficits as well as circumvent disease and premature death. In vitro studies demonstrated the ability of human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes to recapitulate classic variations in desmosomal structural defects and reveal connexin43 loss as a molecular predictor of physiological severity within ARVD/C hearts in a donor‐specific manner. Adenoviral restoration of connexin43 could rescue baseline and catecholamine induced electrical and contractile physiological deficits in ARVC hiPSC derived cardiomyocytes in absence or presence of desmosomal structural alterations. Furthermore, in vivo cardiac‐specific connexin43 gene therapy could prolong lifespan and restore cardiac structure (desmosomal protein expression) in a mouse model with end‐stage ARVC, in a dose‐dependent manner. Our results provide evidence for non‐canonical and independent functions for connexin43 in mechanical modulation of junctions. We anticipate that our findings will have broad implications in exploiting connexin43 as a viable therapeutic target in advanced diseases associated with underlying structural defects.Support or Funding InformationDepartment of Defense (PR170125); National Institutes of Health (NIH R01 HL142251‐A1); American Heart Association Postdoctoral Fellowship (18POST34060228)

Restoration of TET2 deficiency inhibits tumor growth in head neck squamous cell carcinoma.

BackgroundTet methylcytosine dioxygenase 2 (TET2) has been increasingly recognized as an important tumor suppressor involved in tumorigenesis. Here, we aimed to explore the expression pattern of TET2, its clinical significance as well as functional roles in head neck squamous cell carcinoma (HNSCC).MethodsBoth mRNA and protein levels of TET2 in primary HNSCC samples were detected via immunohistochemistry and qRT-PCR, respectively. Correlations between TET2 expression with multiple clinicopathological parameters and patient survival were determined. The biological roles of TET2 in HNSCC were assessed via a gain-of-function approach and in 4-nitroquinoline-1-oxide (4NQO)-induced HNSCC model. Restoration of TET2 by chemicals including 5-Aza-2'-deoxycytidine (5-AZA), metformin or Vitamin C (VC) to inhibit tumor growth was determined in vitro and in a xenograft animal model.ResultsReduced TET2 expression was found in a large fraction of HNSCC samples. Downregulated TET2 significantly correlated with larger tumor size, advanced clinical stage and inferior prognosis. Reduced TET2 and 5-hydroxymethylcytosine (5hmC) were observed along with disease progression in the 4NQO-induced HNSCC model. Enforced TET2 overexpression significantly inhibited cell proliferation, migration and enhanced the chemosensitivity of cisplatin in HNSCC cells. Restoration of TET2 following 5-AZA, metformin or VC exposure impaired cell proliferation and migration in vitro. Moreover, VC alone or in synergistic with cisplatin potently inhibited tumor growth in vivo.ConclusionsOur data reveal that reduced TET2 associates with tumor aggressiveness and reduced survival in HNSCC. Genetic or pharmacological restoration of TET2 might be a viable therapeutic strategy for HNSCC patients with TET2 deficiency.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson’s disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Hypothalamic POMC deficiency increases circulating adiponectin despite obesity

ObjectiveThe steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc−/−) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc−/− mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels. MethodsCirculating adiponectin was measured in obese ArcPomc−/− mice at ages 4–52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc−/− mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc−/− mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc−/− mice. Finally, we generated compound Adiopoq−/−:ArcPomc−/− mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls. ResultsObese ArcPomc−/− female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc−/− female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc−/− female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc−/− mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc−/− female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc−/− mice did not exacerbate their metabolic abnormalities. ConclusionIn summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc−/− mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Abstract C021: Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach

Abstract New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally, confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in the non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wild type variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about “drug-ability” of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development. Citation Format: Laure Grandmoursel, Lieke Geerts, Geraldine Servant, Miranda van der Ham, Armin Maier, Jamil Aarbiou, Marijn Vlaming, Jeroen DeGroot, Julia Schuler, Ian Waddell, Anne-Marie Zuurmond. Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C021. doi:10.1158/1535-7163.TARG-19-C021

De Novo Assembly and Annotation from Parental and F1 Puma Genomes of the Florida Panther Genetic Restoration Program.

In the mid-1990s, the population size of Florida panthers became so small that many individuals manifested traits associated with inbreeding depression (e.g., heart defects, cryptorchidism, high pathogen-parasite load). To mitigate these effects, pumas from Texas were introduced into South Florida to augment genetic variation in Florida panthers. In this study, we report a de novo puma genome assembly and annotation after resequencing 10 individual genomes from partial Florida-Texas-F1 trios. The final genome assembly consisted of ∼2.6 Gb and 20,561 functionally annotated protein-coding genes. Foremost, expanded gene families were associated with neuronal and embryological development, whereas contracted gene families were associated with olfactory receptors. Despite the latter, we characterized 17 positively selected genes related to the refinement of multiple sensory perceptions, most notably to visual capabilities. Furthermore, genes under positive selection were enriched for the targeting of proteins to the endoplasmic reticulum, degradation of mRNAs, and transcription of viral genomes. Nearly half (48.5%) of ∼6.2 million SNPs analyzed in the total sample set contained putative unique Texas alleles. Most of these alleles were likely inherited to subsequent F1 Florida panthers, as these individuals manifested a threefold increase in observed heterozygosity with respect to their immediate, canonical Florida panther predecessors. Demographic simulations were consistent with a recent colonization event in North America by a small number of founders from South America during the last glacial period. In conclusion, we provide an extensive set of genomic resources for pumas and elucidate the genomic effects of genetic rescue on this iconic conservation success story.

Genetic diversity and population structure of the Sapsaree, a native Korean dog breed

BackgroundThe Sapsaree is a breed of dog (Canis familiaris) native to Korea, which became perilously close to extinction in the mid-1980s. However, with systematic genetic conservation and restoration efforts, this breed was rescued from extinction and population sizes have been gradually increasing over the past few decades. The aim of this study was to ascertain novel information about the genetic diversity, population structure, and demographic history of the Sapsaree breed using genome-wide single nucleotide polymorphism data. We characterized the genetic profile of the Sapsaree breed by comparison with seven foreign dog breeds with similar morphologies to estimate genetic differentiation within and among these breeds.ResultsThe results suggest that Sapsarees have higher genetic variance compared with the other breeds analyzed. The majority of the Sapsarees in this study share a discrete genetic pattern, although some individuals were slightly different, possibly as a consequence of the recent restoration process. Concordant results from analyses of linkage disequilibrium, effective population size, genetic diversity, and population structural analyses illustrate a relationship among the Sapsaree and the Tibetan breeds Tibetan terrier and Lhasa Apso, and a small genetic introgression from European breeds. The effective population size of the Sapsaree has contracted dramatically over the past generations, and is currently insufficient to maintain long-term viability of the breed’s genetic diversity.ConclusionsThis study provides novel insights regarding the genetic diversity and population structure of the native Korean dog breed Sapsaree. Our results suggest the importance of a strategic and systematic approach to ensure the genetic diversity and the authenticity of the Sapsaree breed.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p<.05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p<.05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p<.05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

Abstract 1265: Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach

Abstract New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wildtype variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about “drug-ability” of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development. Citation Format: Lieke Geerts, Laure Grandmoursel, Jamil Aarbiou, Jeroen DeGroot, Julia Schüler, Ian Waddell, Anne-Marie Zuurmond. Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1265.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

Interactive Component in Oral Narratives of Preschool Children: on the Formulation of Hypothesis

The article presents an effort to elaborate the hypothesis for the experimental research devoted to interactive component in the oral narratives of preschool children. It is vital for psycholinguistics to reach a close approach to understanding of the essence of narrative processes which have in ontogenesis their own specifics, and according to some researches, these specifics are presented in indirect character of the process of narrative creation by child in the form of his verbal interaction with an adult.&#x0D; We assume that in order to reveal the ontogenetic cognitive discursive mechanisms involved in the process of narration, it is necessary to correlate the regularities of the verbal interaction of the participants in the narrative situation with the overall narrative tasks coming up in narrative process.&#x0D; Mеthodology, purpose and tasks of the research are determined by experimentally-genetic method (L.S. Vygotsky) put forward as main requirements «differentiation of the analysis of a thing and analysis of the process», «genetic restoration of all moments of the process development». The following of these requirements means a close following of the process of narrative development in its dynamics and the peculiarities of its formation at different stages of child’s development, paying attention to frequency facts as well as facts overcoming the limits of «narrow» phenomenon – narrative text.&#x0D; To work out the hypothesis the narrative material of the earlier made experiment with a group of 3 to 7 age children was used in which an adult took a passive part. The task was to tell the story. The material was not subjected to any analysis for the reason of the adult’s presence during the storytelling. The analysis of oral histories with interactive component included the following tasks: a) choose regular sequences of utterances in the dialogic interaction of child and adult; b) define the functional purpose of the adult’s utterance in the course in the child’s storytelling.&#x0D; The obtained results show that the adult practically at every step of the child’s age is oriented on the child’s comprehension and analyzing episodes of the history, that is on child’s formation of his mental image of structural and event sequence in history. The results of the analysis enabled us to propose a hypothesis for the next research step in the field. Along the line of this hypothesis, in the verbal ontogenesis the very nature of oral story as a multifaceted phenomenon of the discursive activity of man brings up to the surface the inner mechanisms of narration which have dialogue basis.

Genetic Restoration of Heme Oxygenase-1 Expression Protects from Type 1 Diabetes in NOD Mice.

Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was induced in DCs exhibited a lower Type 1 Diabetes (T1D) incidence and a reduced insulitis compared to non-induced mice. Upregulation of HO-1 in DCs also prevented further increase of glycemia in recently diabetic NOD mice. Altogether, our data demonstrated the potential of induction of HO-1 expression in DCs as a preventative treatment, and potential as a curative approach for T1D.

Identification of female sex-specific DNA marker in a dioecious rattan (Calamus travancoricus Bedd. ex Becc.)

Calamus travancoricus Bedd. ex Becc. is a dioecious rattan species of commercial importance used in the furniture and handicraft industry. In order to develop genetic improvement and restoration programmes, it is essential to identify sex at seedling stage. In the present study, we have identified molecular markers linked to sex determination using RAPD markers. Of the twenty random primers standardised, the primer OPAU 02 yielded a unique amplicon specific to female genotypes. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving further breeding programs in C. travancoricus.

The Genomic Footprints of the Fall and Recovery of the Crested Ibis

SummaryHuman-induced environmental change and habitat fragmentation pose major threats to biodiversity and require active conservation efforts to mitigate their consequences. Genetic rescue through translocation and the introduction of variation into imperiled populations has been argued as a powerful means to preserve, or even increase, the genetic diversity and evolutionary potential of endangered species [1, 2, 3, 4]. However, factors such as outbreeding depression [5, 6] and a reduction in available genetic diversity render the success of such approaches uncertain. An improved evaluation of the consequence of genetic restoration requires knowledge of temporal changes to genetic diversity before and after the advent of management programs. To provide such information, a growing number of studies have included small numbers of genomic loci extracted from historic and even ancient specimens [7, 8]. We extend this approach to its natural conclusion, by characterizing the complete genomic sequences of modern and historic population samples of the crested ibis (Nipponia nippon), an endangered bird that is perhaps the most successful example of how conservation effort has brought a species back from the brink of extinction. Though its once tiny population has today recovered to >2,000 individuals [9], this process was accompanied by almost half of ancestral loss of genetic variation and high deleterious mutation load. We furthermore show how genetic drift coupled to inbreeding following the population bottleneck has largely purged the ancient polymorphisms from the current population. In conclusion, we demonstrate the unique promise of exploiting genomic information held within museum samples for conservation and ecological research.

Cryptic intronic NBAS variant reveals the genetic basis of recurrent liver failure in a child

BackgroundIn almost half of patients with acute liver failure the cause is unknown, making targeted treatment and decisions about liver transplantation a challenge. Monogenic disorders may contribute to a significant proportion of these undiagnosed patients, and so the incorporation of technologies such as next generation sequencing (NGS) in the clinic could aid in providing a definitive diagnosis. However, this technology may present a major challenge in interpretation of sequence variants, particularly those in non-coding regions. ResultsIn this report we describe a case of Infantile liver failure syndrome 2 (ILFS2; MIM 616483) due to novel bi-allelic variants in the NBAS gene. A missense variant NM_015909.3(NBAS):c.2617C > T, NP_056993.2(NBAS):p.(Arg873Trp) was identified by whole genome sequencing (WGS). By combining WGS and reverse transcription-polymerase chain reaction (RT-PCR) we were able to identify a novel deep intronic variant, NM_015909.3(NBAS):c.2423 + 404G > C, leading to the inclusion of a pseudo-exon. This mechanism has not been described previously in this syndrome. ConclusionsThis study highlights the utility of analyzing NGS data in conjunction with investigating complementary DNA (cDNA) using techniques such as RT-PCR for detection of variants that otherwise would be likely to be missed in common NGS bioinformatic analysis pipelines. Combining these approaches, particularly when the phenotype match is strong, could lead to an increase in the diagnostic yield in acute liver failure and thus aid in targeted treatment, accurate genetic counseling and restoration of reproductive confidence.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Genetic decline, restoration and rescue of an isolated ungulate population.

Isolation of small populations is expected to reduce fitness through inbreeding and loss of genetic variation, impeding population growth and compromising population persistence. Species with long generation time are the least likely to be rescued by evolution alone. Management interventions that maintain or restore genetic variation to assure population viability are consequently of significant importance. We investigated, over 27 years, the genetic and demographic consequences of a demographic bottleneck followed by artificial supplementation in an isolated population of bighorn sheep (Ovis canadensis). Based on a long‐term pedigree and individual monitoring, we documented the genetic decline, restoration and rescue of the population. Microsatellite analyses revealed that the demographic bottleneck reduced expected heterozygosity and allelic diversity by 6.2% and 11.3%, respectively, over two generations. Following supplementation, first‐generation admixed lambs were 6.4% heavier at weaning and had 28.3% higher survival to 1 year compared to lambs of endemic ancestry. Expected heterozygosity and allelic diversity increased by 4.6% and 14.3% after two generations through new alleles contributed by translocated individuals. We found no evidence for outbreeding depression and did not see immediate evidence of swamping of local genes. Rapid intervention following the demographic bottleneck allowed the genetic restoration and rescue of this bighorn sheep population, likely preventing further losses at both the genetic and demographic levels. Our results provide further empirical evidence that translocation can be used to reduce inbreeding depression in nature and has the potential to mitigate the effect of human‐driven environmental changes on wild populations.

Phylogeography of a widely distributed species reveals a cryptic assemblage of distinct genetic lineages needing separate conservation strategies

Genetic structure in broadly distributed herbs may be shaped by past climate events, leading to distinct genetic lineages, and possibly to different Evolutionary Significant Units (ESUs). We identified the potential glacial refugia, postglacial (re)colonization routes, contact zones and ESUs in the widespread but locally rare Silene nutans to propose appropriate conservation strategies related to migration history-related genetic structure. We sequenced four plastid DNA markers from 136 populations covering S. nutans European distribution range. We built a network on concatenated sequences using statistical parsimony and performed Bayesian and Maximum Likelihood analyses to build a phylogenetic tree. Haplotype genetic diversity and between-population differentiation (GST, NST) were calculated. Based on 23 plastid haplotypes, populations were ascribed to seven strongly differentiated genetic lineages. Potential refugia were identified: in the Iberian and Italian Peninsulas, expanding to south-western France and the Alps, respectively; in south-central France, reaching England and Belgium; an eastern refugium, following a north-westward expansion; in the Balkans, expanding to north-eastern Italy, and in the Carpathians. Several contact zones were identified. Silene nutans consists of a cryptic assemblage of genetically distinct taxa, with at least seven ESUs. Evaluating the distribution range, the abundance, and the conservation status of each ESU is necessary for the preservation of the S. nutans species complex at European and regional levels. Ex situ conservation and genetic restoration involving plant translocations must consider the phylogeographic history and the distinct genetic lineages to avoid the introduction of poorly adapted plants and outbreeding depression leading to non-viable hybrid progeny, especially in contact zones.

English

Seed bank (henceforth referred to as SB) refers to viable seed which is present on or in soil and associated with litter/humus. Under different land use/land cover systems and climatic regions, density of seeds in the soil is variable both in space and time. SB density decreases with increasing depth and age of seeds in the soil. Smaller seeds are more easily incorporated, moved to deeper soil layers and persist longer in the soil, whereas large sized seeds without hard seed coats lack persistence in the soil. Moreover, small and elongated seeds are more persistent than large and round seeds. In most vegetation types, grass and herbs have denser seeds in the soil than woody species. Due to shade intolerant properties, density of grasses and forbs are also higher in forest gaps and farmlands than under shade of trees. In most of vegetation types and under medium disturbances e.g. under grazing, density and diversity of species are higher than that of lower and higher disturbance intensities. Tillage practices also have negative impacts on density, composition and abundance of SB. Hence, seeds decline under conventional tillage than under conservational tillage practices. In several studies, seeds in the SB are not similar to above ground vegetation. Similarly, density and diversity of seeds in a SB exceeds that of standing vegetation. Persistence seeds in soil are essential in maintaining individual species and the plant community, conservation of genetic biodiversity and restoration of plant communities of degraded lands after disturbances under harsh environmental conditions, especially in arid regions. As a result, SB characteristics are used to elucidate seed dynamics in various vegetation types. Overall, scientific knowledge of SB is used for land use planning, to make recommendations for future cost effective measures and to establish policies for conservation or restoration programmes. Keywords: Seed bank (SB), dispersal, persistence, restoration, vegetation type.

Purging of Strongly Deleterious Mutations Explains Long-Term Persistence and Absence of Inbreeding Depression in Island Foxes

The recovery and persistence of rare and endangered species are often threatened by genetic factors, such as the accumulation of deleterious mutations, loss of adaptive potential, and inbreeding depression [1]. Island foxes (Urocyon littoralis), thedwarfed descendants of mainland gray foxes (Urocyon cinereoargenteus), have inhabited California's Channel Islands for >9,000 years [2-4]. Previous genomic analyses revealed that island foxes have exceptionally low levels of diversity and elevated levels of putatively deleterious variation [5]. Nonetheless, all six populations have persisted for thousands of generations, and several populations rebounded rapidly after recent severe bottlenecks [6, 7]. Here,we combine morphological and genomic data withpopulation-genetic simulations to determine the mechanism underlying the enigmatic persistence of these foxes. First, through analysis of genomes from 1929 to 2009, we show that island foxes have remained at small population sizes with low diversity for many generations. Second, we present morphological data indicating an absence of inbreeding depression in island foxes, confirming that they are not afflicted with congenital defects common to other small and inbred populations. Lastly, our population-genetic simulations suggest that long-term small population size results in a reduced burden of strongly deleterious recessive alleles, providing a mechanism for the absence of inbreeding depression in island foxes. Importantly, the island fox illustrates a scenario in which genetic restoration through human-assisted gene flow could be a counterproductive or even harmful conservation strategy. Our study sheds light on the puzzle of island fox persistence, a unique success story that provides a model for the preservation of small populations.