Loss Of Gene Function Research Articles

Histocompatibility and Reproduction: Lessons from the Anglerfish.

Reproduction in certain deep-sea anglerfishes involves the permanent attachment of dwarf males to much larger females and fusion of their tissues leading to the establishment of a shared circulatory system. This unusual phenomenon of sexual parasitism enables anglerfishes to maximize reproductive success in the vast and deep oceans, where females and males otherwise rarely meet. An even more surprising phenomenon relates to the observation that joining of genetically disparate male and female anglerfishes does not evoke a strong anti-graft immune rejection response, which occurs in vertebrates following allogeneic parabiosis. Recent studies demonstrated that the evolutionary processes that led to the unique mating strategy of anglerfishes coevolved with genetic changes that resulted in loss of functional genes encoding critical components of the adaptive immune system. These genetic alterations enabled anglerfishes to tolerate the histoincompatible tissue antigens of their mate and prevent the occurrence of reciprocal graft rejection responses. While the exact mechanisms by which anglerfishes defend themselves against pathogens have not yet been deciphered, it is speculated that during evolution, anglerfishes adopted new immune strategies that compensate for the loss of B and T lymphocyte functions and enable them to resist infection by pathogens.

Degron Tagging Using mAID and SMASh Tags in RPE-1 Cells.

Degron tags allow the precise and well-controlled analysis of essential genes by rapidly inducing degradation of the protein of interest. This is critical when the consequences of loss of gene function needs to be analyzed in a strictly defined time window such as a specific cell cycle phase. We have recently published the successful application of degron tags to analyze cell cycle genes such as CDC6, CCNA2, and CCNB1. A critical aspect of our approach was to combine two tags to generate a synergy in the degradation dynamics. Here we outline our approach and describe some of the essential steps to generate double-degron-tagged genes in RPE-1 cells. Similar procedures can easily be applied to other cell lines.

Distribution of bacteriocin genes in the lineages of Lactiplantibacillus plantarum

Lactiplantibacillus plantarum, previously named “Lactobacillus plantarum,” is found in a wide variety of environments exhibiting a high level of intraspecies genetic diversity. To investigate the strain diversity, we performed comparative genomic analyses of the 54 complete genome sequences. The results revealed that L. plantarum subsp. plantarum was split into three lineages, A, B and C. Of the genes beneficial for probiotic activity, only those associated with the biosynthesis of plantaricin (Pln), an L. plantarum-specific bacteriocin, were found to be significantly different among the lineages. The genes related to the biosynthesis of plnE/F were conserved throughout the three lineages, whereas the outgroups did not possess any Pln-producing genes. In lineage C, the deepest and ancestral type branch, plnE/F genes, were well conserved. In lineage B, loss of gene function was observed due to mobile elements in the pln loci. In lineage A, most strains were predicted to produce more than one type of Pln by possessing diverse Pln-encoding genes. These results showed the presence of functional diversity arising from the trifurcating evolution in L. plantarum subsp. plantarum and demonstrated that Pln is an indicator for differentiating the three lineages.

MIA3 Splice Defect in Cane Corso Dogs with Dental-Skeletal-Retinal Anomaly (DSRA).

We investigated a hereditary syndrome in Cane Corso dogs. Affected dogs developed dental-skeletal-retinal anomaly (DSRA), clinically characterized by brittle, discolored, translucent teeth, disproportionate growth and progressive retinal degeneration resulting in vision loss. Combined linkage and homozygosity mapping delineated a 5.8 Mb critical interval. The comparison of whole genome sequence data of an affected dog to 789 control genomes revealed a private homozygous splice region variant in the critical interval. It affected the MIA3 gene encoding the MIA SH3 domain ER export factor 3, which has an essential role in the export of collagen and other secreted proteins. The identified variant, XM_005640835.3:c.3822+3_3822+4del, leads to skipping of two exons from the wild type transcript, XM_005640835.3:r.3712_3822del. Genotypes at the variant were consistent with monogenic autosomal recessive mode of inheritance in a complete family and showed perfect genotype-phenotype association in 18 affected and 22 unaffected Cane Corso dogs. MIA3 variants had previously been shown to cause related phenotypes in humans and mice. Our data in dogs together with the existing functional knowledge of MIA3 variants in other mammalian species suggest the MIA3 splice defect and a near complete loss of gene function as causative molecular pathomechanism for the DSRA phenotype in the investigated dogs.

Selection on genes associated with the evolution of divergent life histories: Gamete recognition or something else?

Gamete compatibility, and fertilization success, is mediated by gamete-recognition genes (GRGs) that are expected to show genetic evidence of a response to sexual selection associated with mating system traits. Changes in the strength of sexual selection can arise from the resolution of sperm competition among males, sexual conflicts of interest between males and females, or other mechanisms of sexual selection. To assess these expectations, we compared patterns of episodic diversifying selection among genes expressed in the gonads of Cryptasterina pentagona and C. hystera, which recently speciated and have evolved different mating systems (gonochoric or hermaphroditic), modes of fertilization (outcrossing or selfing), and dispersal (planktonic larvae or internal brooding). Cryptasterina spp. inhabit the upper intertidal of the coast of Queensland and coral islands of the Great Barrier Reef. We found some evidence for positive selection on a GRG in the outcrossing C. pentagona, and we found evidence of loss of gene function in a GRG of the self-fertilizing C. hystera. The modification or loss of gene functionality may be evidence of relaxed selection on some aspects of gamete interaction in C. hystera. In addition to these genes involved in gamete interactions, we also found genes under selection linked to abiotic stress, chromosomal regulation, polyspermy, and egg-laying. We interpret those results as possible evidence that Cryptasterina spp. with different mating systems may have been adapting in divergent ways to oxidative stress or other factors associated with reproduction in the physiologically challenging environment of the high intertidal. RESEARCH HIGHLIGHTS: Recent speciation between two sea stars was unlikely the result of selection on gamete-recognition genes annotated in this study. Instead, our results point to selection on genes linked to the intertidal environment and reproduction.

Phylogeny and sex chromosome evolution of Palaeognathae

Many paleognaths (ratites and tinamous) have a pair of homomorphic ZW sex chromosomes in contrast to the highly differentiated sex chromosomes of most other birds. To understand the evolutionary causes for the different tempos of sex chromosome evolution, we produced female genomes of 12 paleognathous species and reconstructed the phylogeny and the evolutionary history of paleognathous sex chromosomes. We uncovered that Palaeognathae sex chromosomes had undergone stepwise recombination suppression and formed a pattern of “evolutionary strata”. Nine of the 15 studied species' sex chromosomes have maintained homologous recombination in their long pseudoautosomal regions extending more than half of the entire chromosome length. We found that in the older strata, the W chromosome suffered more serious functional gene loss. Their homologous Z-linked regions, compared with other genomic regions, have produced an excess of species-specific autosomal duplicated genes that evolved female-specific expression, in contrast to their broadly expressed progenitors. We speculate such “defeminization” of Z chromosome with underrepresentation of female-biased genes and slow divergence of sex chromosomes of paleognaths might be related to their distinctive mode of sexual selection targeting females rather than males, which evolved in their common ancestors.

The timing of genetic degeneration of sex chromosomes.

Genetic degeneration is an extraordinary feature of sex chromosomes, with the loss of functions of Y-linked genes in species with XY systems, and W-linked genes in ZW systems, eventually affecting almost all genes. Although degeneration is familiar to most biologists, important aspects are not yet well understood, including how quickly a Y or W chromosome can become completely degenerated. I review the current understanding of the time-course of degeneration. Degeneration starts after crossing over between the sex chromosome pair stops, and theoretical models predict an initially fast degeneration rate and a later much slower one. It has become possible to estimate the two quantities that the models suggest are the most important in determining degeneration rates-the size of the sex-linked region, and the time when recombination became suppressed (which can be estimated using Y-X or W-Z sequence divergence). However, quantifying degeneration is still difficult. I review evidence on gene losses (based on coverage analysis) or loss of function (by classifying coding sequences into functional alleles and pseudogenes). I also review evidence about whether small genome regions degenerate, or only large ones, whether selective constraints on the genes in a sex-linked region also strongly affect degeneration rates, and about how long it takes before all (or almost all) genes are lost. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

TDP43 Exacerbates Atherosclerosis Progression by Promoting Inflammation and Lipid Uptake of Macrophages

ObjectiveAtherosclerosis (AS), characterized by cholesterol overloaded-macrophages accumulation and plaque formation in blood vessels, is the major cause of cardiovascular disease. Transactive response DNA-binding protein∼43 kDa (TDP43) has recently been identified as an independent driver of neurodegenerative diseases through triggering inflammatory response. This study investigated whether TDP43 is involved in AS development, especially in macrophages-mediated-foam cell formation and inflammatory responses.MethodsTransactive response DNA-binding protein∼43 kDa expressions in oxidized low-density lipoprotein (oxLDL)-treated macrophages and peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD) were detected by real time-polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence. Gene gain or loss of function was used to investigate the effects of TDP43 on macrophages-mediated lipid untake and inflammation with ELISA, protein immunoprecipitation, RT-PCR, Western blot, and immunofluorescence. Macrophage TDP43 specific knockout mice with ApoE–/– background were fed with western diet for 12 weeks to establish AS model, and used to explore the role of TDP43 on AS progression.ResultsTransactive response DNA-binding protein∼43 kDa expression increases in oxLDL-treated macrophages and PBMCs from patients with CAD. Furthermore, we find that TDP43 promotes activation of NF-κB to increase inflammatory factor expression in macrophages through triggering mitochondrial DNA release to activate cGAS-STING signaling. Moreover, TDP43 strengthens lipid uptake of macrophages through regulating β-catenin and PPAR-γ complex to promote scavenger receptor gene CD36 transcription. Finally, using macrophage TDP43 specific knockout mice with ApoE–/– background fed with western diet for 12 weeks to establish AS model, we find that specific knockout of TDP43 in macrophages obviously alleviates western diet-induced AS progression in mice.ConclusionsTransactive response DNA-binding protein∼43 kDa exacerbates atherosclerosis progression by promoting inflammation and lipid uptake of macrophages, suggesting TDP43 as a potential target for developing atherosclerotic drug.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Constitutional mismatch repair deficiency is a childhood cancer predisposition syndrome associated with an increased risk of high grade paediatric brain tumours: report of 3 cases and review of the literature

Background: Constitutional mismatch repair deficiency syndrome (CMMRD) represents a biallelic (homozygous or compound heterozygous) mutation in one of the mismatch repair genes (MLH1, MSH2, MSH6 or PMS2), resulting in a recessively inherited childhood cancer predisposition syndrome. A broad range of tumours in infants and children have been described in the CMMRD spectrum, most commonly brain tumours, intestinal tumours, and haematological malignancies. This contrasts with the monoallelic mismatch repair defects (MMR) of Lynch syndrome, in which there is an increased risk of development of malignancies in particular involving the gastrointestinal and genitourinary tract, with onset in adulthood. Phenotypic features of CMMRD overlap with neurofibromatosis, with café-au-lait macules a common finding in this cohort. Aims: To highlight an increasingly recognised childhood cancer predisposition syndrome, and the importance of identification of these cases given significant implications for family counselling. There is an increased cancer risk in siblings, as well as for the parents, who may not yet be aware of their own associated monoallelic MMR defect (Lynch syndrome). Methods: We present three cases of childhood primary brain tumours that were subsequently confirmed in the setting of CMMRD. The histological, immunohistochemical and genetic findings of these cases are discussed, with focus on diagnostic pathologic features that suggest an underlying CMMRD. Results and conclusions: Three high grade brain tumours described include two high grade gliomas (a glioblastoma multiforme and a high grade diffuse intrinsic pontine glioma, H3K27M negative), and a medulloblastoma. These examples all showed complete absence of PMS2 staining on immunohistochemistry in both tumour cells and normal ‘internal control’ tissues of the patient, correlating with biallelic loss of gene function. Review of the literature shows brain tumours as the most frequent presentation in children with CMMRD, with an increased risk of gastrointestinal tumours (including colonic carcinoma) and haematological malignancies also reported.

Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury.

Although mesenchymal stromal (stem) cell (MSC) administration attenuates sepsis-induced lung injury in pre-clinical models, the mechanism(s) of action and host immune system contributions to its therapeutic effects remain elusive. We show that treatment with MSCs decreased expression of host-derived microRNA (miR)-193b-5p and increased expression of its target gene, the tight junctional protein occludin (Ocln), in lungs from septic mice. Mutating the Ocln 3' untranslated region miR-193b-5p binding sequence impaired binding to Ocln mRNA. Inhibition of miR-193b-5p in human primary pulmonary microvascular endothelial cells prevents tumour necrosis factor (TNF)-induced decrease in Ocln gene and protein expression and loss of barrier function. MSC-conditioned media mitigated TNF-induced miR-193b-5p upregulation and Ocln downregulation in vitro When administered in vivo, MSC-conditioned media recapitulated the effects of MSC administration on pulmonary miR-193b-5p and Ocln expression. MiR-193b-deficient mice were resistant to pulmonary inflammation and injury induced by lipopolysaccharide (LPS) instillation. Silencing of Ocln in miR-193b-deficient mice partially recovered the susceptibility to LPS-induced lung injury. In vivo inhibition of miR-193b-5p protected mice from endotoxin-induced lung injury. Finally, the clinical significance of these results was supported by the finding of increased miR-193b-5p expression levels in lung autopsy samples from acute respiratory distress syndrome patients who died with diffuse alveolar damage.

Sequence modification on demand: search and replace tools for precise gene editing in plants.

Until recently, our ability to generate allelic diversity in plants was limited to introduction of variants from domesticated and wild species by breeding via uncontrolled recombination or the use of chemical and physical mutagens-processes that are lengthy and costly or lack specificity, respectively. Gene editing provides a faster and more precise way to create new variation, although its application in plants has been dominated by the creation of short insertion and deletion mutations leading to loss of gene function, mostly due to the dependence of editing outcomes on DNA repair pathway choices intrinsic to higher eukaryotes. Other types of edits such as point mutations and precise and pre-designed targeted sequence insertions have rarely been implemented, despite providing means to modulate the expression of target genes or to engineer the function and stability of their protein products. Several advancements have been developed in recent years to facilitate custom editing by regulation of repair pathway choices or by taking advantage of alternative types of DNA repair. We have seen the advent of novel gene editing tools that are independent of DNA double-strand break repair, and methods completely independent of host DNA repair processes are being increasingly explored. With the aim to provide a comprehensive review of the state-of-the-art methodology for allele replacement in plants, I discuss the adoption of these improvements for plant genome engineering.

198-LB: A Novel Heterozygous Mutation in Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) Gene in a Woman with Familial Partial Lipodystrophy

Background: The nuclear peroxisome proliferator-activated receptor gamma (PPAR-γ) plays a role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, distribution and function. Heterogeneous mutations of PPAR-γ can cause loss of gene function or reduce its expression leading to a clinical variety of phenotypes that characterize type 3 familial partial lipodystrophy syndrome. The most common features of this syndrome are abnormal body fat accumulation, muscle hypertrophy, insulin resistance, hypertriglyceridemia, polycystic ovary syndrome and hepatic steatosis. Case report: A 32-year-old-woman was referred to the Endocrinology Division of PUC-SP for treatment of a severe hypertriglyceridemia diagnosed 4 years ago with recurrent episodes of acute pancreatitis and eruptive xanthomas since then. Also, diabetes mellitus (DM) was diagnosed 1 year ago with poor glycemic control. Her sister also had severe hypertriglyceridemia, recurrent episodes of acute pancreatitis and died in 2016. No DM was reported in her family. On physical examination, we found the presence of lipodystrophy, acanthosis nigricans on the neck and eruptive xanthomas on trunk, upper limbs, and thighs. BMI=24.9kg/m2; waist circumference=96cm. Abdominal CT scan revealed splenomegaly and fatty liver disease. Genetic sequencing showed a heterozygous (autosomal dominant inheritance) in the PPAR-γ of a Chr3 variant: 12.416.957 T> C (or alternatively c.1073T> C - ENST000002287820), promoting the substitution of leucine in codon 358 by proline (p.Leu358Pro). Conclusion: The substitution of the amino acid leucine in codon 358 for proline is highly conserved in several species, and computer programs for predicting in silico of pathogenicity, suggesting that this new heterozygous mutation in the PPAR-γ gene is probably related to the type 3 familial partial lipodystrophy syndrome. Disclosure P. V. Wanderley: None. J. C. Dias: None. M. W. Vieira: None. G. Garcia: None. I. T. Filho: None. R. F. Chamma: None. L. S. Gugoni: None. R. B. Giorgi: None. C. R. G. Barcellos: None.

Natural variants suppress mutations in hundreds of essential genes.

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, and lethal in another. The extent to which mutant phenotypes are malleable, the architecture of modifiers and the identities of causal genes remain largely unknown. Here, we measure the fitness effects of ~ 1,100 temperature‐sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds and map the modifiers for 19 combinations. Altogether, fitness defects for 149 of the 580 tested genes (26%) could be suppressed by genetic variation in at least one yeast strain. Suppression was generally driven by gain‐of‐function of a single, strong modifier gene, and involved both genes encoding complex or pathway partners suppressing specific temperature‐sensitive alleles, as well as general modifiers altering the effect of many alleles. The emerging frequency of suppression and range of possible mechanisms suggest that a substantial fraction of monogenic diseases could be managed by modulating other gene products.

Attaining the promise of plant gene editing at scale

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

Evolution of dosage compensation does not depend on genomic background.

Organisms have evolved various mechanisms to cope with the differences in the gene copy numbers between sexes caused by degeneration of Y and W sex chromosomes. Complete dosage compensation or at least expression balance between sexes has been reported predominantly in XX/XY systems, but rarely in ZZ/ZW systems. However, this often-reported pattern is based on comparisons of lineages where sex chromosomes evolved from nonhomologous genomic regions, potentially differing in sensitivity to differences in gene copy numbers. Here we document that two reptilian lineages (XX/XY iguanas and ZZ/ZW softshell turtles), which independently co-opted the same ancestral genomic region for the function of sex chromosomes, evolved different gene dose regulatory mechanisms. The independent co-option of the same genomic region for the role of sex chromosomes as in the iguanas and the softshell turtles offers great opportunity for testing evolutionary scenarios on sex chromosome evolution under the explicit control of the genomic background and gene identity. We show that the parallel loss of functional genes from the Y chromosome of the green anole and the W chromosome of the Florida softshell turtle led to different dosage compensation mechanisms. Our approach controlling for genetic background thus does not support that the variability in the regulation of gene dose differences is a consequence of ancestral autosomal gene content.

PERK Pathway and Neurodegenerative Disease: To Inhibit or to Activate?

With the extension of life span in recent decades, there is an increasing burden of late-onset neurodegenerative diseases, for which effective treatments are lacking. Neurodegenerative diseases include the widespread Alzheimer’s disease (AD) and Parkinson’s disease (PD), the less frequent Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS) and also rare early-onset diseases linked to mutations that cause protein aggregation or loss of function in genes that maintain protein homeostasis. The difficulties in applying gene therapy approaches to tackle these diseases is drawing increasing attention to strategies that aim to inhibit cellular toxicity and restore homeostasis by intervening in cellular pathways. These include the unfolded protein response (UPR), activated in response to endoplasmic reticulum (ER) stress, a cellular affliction that is shared by these diseases. Special focus is turned to the PKR-like ER kinase (PERK) pathway of the UPR as a target for intervention. However, the complexity of the pathway and its ability to promote cell survival or death, depending on ER stress resolution, has led to some confusion in conflicting studies. Both inhibition and activation of the PERK pathway have been reported to be beneficial in disease models, although there are also some reports where they are counterproductive. Although with the current knowledge a definitive answer cannot be given on whether it is better to activate or to inhibit the pathway, the most encouraging strategies appear to rely on boosting some steps without compromising downstream recovery.

Overall survival of patients with metastatic castrate-resistant prostate cancer (mCRPC) who have PTEN tumor suppressor gene loss of function.

58 Background: It is estimated that more than 40% of patients with mCRPC have functional loss of phosphatase and tensin homolog (PTEN) tumor suppressor gene, which is associated with unfavorable prognosis and reduced response to androgen receptor-targeting therapy. We describe patient characteristics and survival outcomes by PTEN LOF status among patients with mCRPC in real-world clinical practice. Methods: We conducted a retrospective cohort study using data from the nationwide Flatiron Health-Foundation Medicine mCRPC Clinico-Genomic database (FH-FMI CGDB), a de-identified database linking data derived from electronic health records with genomic data derived from FMI comprehensive genomic profiling (CGP) tests. The study included patients ≥18 years old, with a primary diagnosis of mCRPC between 1/1/2013 and 6/30/2019 who underwent FMI CGP testing and who had a valid PTEN LOF status. Patients were included if their PTEN report date and mCRPC diagnosis date occurred before death or censoring. PTEN LOF status was identified via FMI’s CGP testing. Kaplan-Meier (KM) methods assessed overall survival (OS) by PTEN LOF status from the date of mCRPC diagnosis (later of metastasis and castration resistance) until death or end of study follow-up. A stratified Cox regression model was used to estimate the hazard of death. The Cox model was adjusted for age, race and sequence of metastasis/CRPC diagnoses, and was stratified by the year of mCRPC diagnosis. Adjustments to account for left-truncation and survivorship bias were made in the KM analysis and the Cox regression model. Results: Among the 458 patients who met the eligibility criteria, 174 (38%) had PTEN LOF. The majority of the study sample (76%) was diagnosed with castration-resistance after metastasis. The PTEN LOF group had a higher percentage of white patients (80% vs. 68%; p= 0.01) compared to the PTEN non-LOF group. The mean age of the study sample was 68 years, and there was no difference in mean age at diagnosis by PTEN LOF status ( p= 0.17). Based on the KM estimates adjusted for left-truncation, the median OS was 14.3 months (95% confidence interval [CI]: 11.1-19.7) in the PTEN LOF group compared to 18.3 months (95%CI: 15.5-21.5) in the PTEN non-LOF group (log-rank p= 0.049). In the multivariable Cox model, the PTEN LOF group had numerically 30% higher risk of death compared to the PTEN non-LOF group (hazard ratio = 1.30; 95% CI: 0.99-1.71; p= 0.057). Conclusions: Among real-world patients with mCRPC in the CGDB, PTEN LOF could be associated with poorer survival outcomes, potentially highlighting the unmet need among these patients. Additional studies with larger cohorts are needed to better evaluate the survival outcomes of patients with PTEN LOF. Therapeutic agents acting on the PTEN/PI3K/AKT/mTOR pathway are being tested in clinical trials, and could potentially improve outcomes in this subgroup of patients with mCRPC.

Bicuspid aortic valve and aortopathy: Genetic background through 4 familial case reports

Bicuspid aortic valve (BAV) is the most common congenital heart defect and its prevalence is estimated around 1% of the population. 50 to 70% of BAV are associated with ascending aorta dilatation. The issue of hemodynamic or genetic determinism of both conditions is still a matter of debate. The aim of this study was to describe the genetic profile of 4 different families presenting with BAV and aortopathy (AP). We included 4 families with BAV + AP from a prospective, mono-centric cohort of 381 BAV patients included from January 2014 to January 2020. We performed a whole exome sequencing of all index patients and available relatives in order to identify candidate genes, using the Varaft program. Once identified, polymerase chain reaction (PCR) were carried out in the remaining first-degree relatives to perform a segregation analysis. In case of a novel single nucleotide variant, in vitro models were used to examine its functional effect. Family A includes 2 affected members linked to mutation in one gene implicated in the endothelial to mesenchymal transition (EMT) of the endothelial cells, not found in two unaffected relatives. In vitro culture using fibroblast from index patient confirmed the mutation involvement in loss of gene function. Family B includes 2 affected members with mutation in one gene known to activate the NOTCH signaling pathway implicated in EMT. Family C includes 2 members with a rare stop-gain variant in gene coding for one extracellular matrix protein essential for the structure of the aortic valve and ascending aorta. Family D includes 3 members with mutation in one gene involved in connective tissue disease. Among a French prospective cohort of 381 BAV patients we identify 4 different families with non-syndromic BAV + AP. Whole exome sequencing analysis allows to reveal 4 distinct mutations of genes implicated in the EMT, the extracellular matrix structure and connective tissue disease.

P53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

Background & AimsA coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury.MethodsNitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2.ResultsIn a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response.ConclusionsCharacteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.