Unveiling immune checkpoint regulation: exploring the power of in vivo CRISPR screenings in cancer immunotherapy.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer immunotherapy by reinvigorating antitumor immune responses, but their efficacy remains limited in most patients. To address this challenge and optimize Immune check inhibitor treatment, understanding the underlying molecular intricacies involved is crucial. The emergence of CRISPR-Cas9 technology has empowered researchers to precisely investigate gene function and has introduced transformative shifts in identifying key genes for various physiological and pathological processes. CRISPR screenings, particularly in vivo CRISPR screenings, have become invaluable tools in deciphering molecular networks and signaling pathways governing suppressive immune checkpoint molecules. In this review, we provide a comprehensive overview of in vivo CRISPR screenings in cancer immunotherapy, exploring how this cutting-edge technology has unraveled potential novel therapeutic targets and combination strategies. We delve into the latest findings and advancements, shedding light on immune checkpoint regulation and offering exciting prospects for the development of innovative and effective treatments for cancer patients.

Thalidomide-Revisited: Are COVID-19 Patients Going to Be the Latest Victims of Yet Another Theoretical Drug-Repurposing?

The coronavirus disease 2019 (COVID-19) pandemic is a worldwide threatening health issue. The progression of this viral infection occurs in the airways of the lungs with an exaggerated inflammatory response referred to as the "cytokine storm" that can lead to lethal lung injuries. In the absence of an effective anti-viral molecule and until the formulation of a successful vaccine, anti-inflammatory drugs might offer a complementary tool for controlling the associated complications of COVID-19 and thus decreasing the subsequent fatalities. Drug repurposing for several molecules has emerged as a rapid temporary solution for COVID-19. Among these drugs is Thalidomide; a historically emblematic controversial molecule that harbors an FDA approval for treating erythema nodosum leprosum (ENL) and multiple myeloma (MM). Based on just one-case report that presented positive outcomes in a patient treated amongst others with Thalidomide, two clinical trials on the efficacy and safety of Thalidomide in treating severe respiratory complications in COVID-19 patients were registered. Yet, the absence of substantial evidence on Thalidomide usage in that context along with the discontinued studies on the efficiency of this drug in similar pulmonary diseases, might cause a significant obstacle for carrying out further clinical evaluations. Herein, we will discuss the theoretical effectiveness of Thalidomide in attenuating inflammatory complications that are encountered in COVID-19 patients while pinpointing the lack of the needed evidences to move forward with this drug.

Post-lingual non-syndromic hearing loss phenotype: a polygenic case with 2 biallelic mutations in MYO15A and MITF.

BACKGROUND: Hearing loss (HL) represents the most common congenital sensory impairment with an incidence of 1-5 per 1000 live births. Non-syndromic hearing loss (NSHL) is an isolated finding that is not part of any other disorder accounting for 70% of all genetic hearing loss cases. METHODS: In the current study, we reported a polygenic mode of inheritance in an NSHL consanguineous family using exome sequencing technology and we evaluated the possible effect of the detected single nucleotide variants (SNVs) using in silico methods. RESULTS: Two bi-allelic SNVs were detected in the affected patients; a MYO15A (. p.V485A) variant, and a novel MITF (p.P338L) variant. Along with these homozygous mutations, we detected two heterozygous variants in well described hearing loss genes (MYO7A and MYH14). The novel MITF p. Pro338Leu missense mutation was predicted to change the protein structure and function. CONCLUSION: A novel MITF mutation along with a previously described MYO15A mutation segregate with an autosomal recessive non-syndromic HL case with a post-lingual onset. The findings highlight the importance of carrying whole exome sequencing for a comprehensive assessment of HL genetic heterogeneity.

The Digenic Causality in Familial Hypercholesterolemia: Revising the Genotype-Phenotype Correlations of the Disease.

Genetically inherited defects in lipoprotein metabolism affect more than 10 million individuals around the globe with preponderance in some parts where consanguinity played a major role in establishing founder mutations. Mutations in four genes have been so far linked to the dominant and recessive form of the disease. Those players encode major proteins implicated in cholesterol regulation, namely, the low-density lipoprotein receptor (LDLR) and its associate protein 1 (LDLRAP1), the proprotein convertase substilin/kexin type 9 (PCSK9), and the apolipoprotein B (APOB). Single mutations or compound mutations in one of these genes are enough to account for a spectrum of mild to severe phenotypes. However, recently several reports have identified digenic mutations in familial cases that do not necessarily reflect a much severe phenotype. Yet, data in the literature supporting this notion are still lacking. Herein, we review all the reported cases of digenic mutations focusing on the biological impact of gene dosage and the potential protective effects of single-nucleotide polymorphisms linked to hypolipidemia. We also highlight the difficulty of establishing phenotype-genotype correlations in digenic familial hypercholesterolemia cases due to the complexity and heterogeneity of the phenotypes and the still faulty in silico pathogenicity scoring system. We finally emphasize the importance of having a whole exome/genome sequencing approach for all familial cases of familial hyperlipidemia to better understand the genetic and clinical course of the disease.

Correction to: A Novel Somatic Variant in HEY2 Unveils an Alternative Splicing Isoform Linked to Ventricular Septal Defect.

The original version of this article unfortunately contained a mistake in the author name. The first author name should be Manal Fardoun instead of Manal Fardon. The original article has been corrected.

A Novel Somatic Variant in HEY2 Unveils an Alternative Splicing Isoform Linked to Ventricular Septal Defect.

Congenital heart defects (CHDs) are the leading cause of death in infants under 1 year of age. Aberrations in the expression and function of cardiac transcription factors (TFs) are a major contributor to CHDs. Despite the numerous studies undertaken to functionally characterize these TFs, their exact role in different stages of cardiogenesis is still not fully elucidated. Here we focused on HEY2, a basic helix loop helix transcriptional repressor, and its potential role in human ventricular septal defects. Genetic analysis was performed based on sequencing of DNA and cDNA obtained from post-operational cardiac tissues and blood of 17 Lebanese patients with various CHDs. The screen covered the entire coding regions of the GATA4, NKX2.5, TBX5, TBX20 and HEY2 genes. Our results revealed two novel somatic mutations, namely p.Ala229Thr and p.161_190 del, affecting HEY2 in the diseased cardiac tissues of two patients with VSD. These results suggest a potential role of HEY2 in regulating ventricular septation in humans.

A Novel Role for CSRP1 in a Lebanese Family with Congenital Cardiac Defects.

Despite an obvious role for consanguinity in congenital heart disease (CHD), most studies fail to document a monogenic model of inheritance except for few cases. We hereby describe a first-degree cousins consanguineous Lebanese family with 7 conceived children: 2 died in utero of unknown causes, 3 have CHD, and 4 have polydactyly. The aim of the study is to unveil the genetic variant(s) causing these phenotypes using next generation sequencing (NGS) technology. Targeted exome sequencing identified a heterozygous duplication in CSRP1 which leads to a potential frameshift mutation at position 154 of the protein. This mutation is inherited from the father, and segregates only with the CHD phenotype. The in vitro characterization demonstrates that the mutation dramatically abrogates its transcriptional activity over cardiac promoters like NPPA. In addition, it differentially inhibits the physical association of CSRP1 with SRF, GATA4, and with the newly described partner herein TBX5. Whole exome sequencing failed to show any potential variant linked to polydactyly, but revealed a novel missense mutation in TRPS1. This mutation is inherited from the healthy mother, and segregating only with the cardiac phenotype. Both TRPS1 and CSRP1 physically interact, and the mutations in each abrogate their partnership. Our findings add fundamental knowledge into the molecular basis of CHD, and propose the di-genic model of inheritance as responsible for such malformations.

Regulation of de novo ceramide synthesis: the role of dihydroceramide desaturase and transcriptional factors NFATC and Hand2 in the hypoxic mouse heart.

We have previously shown that ceramide, a proapoptotic molecule decreases in the mouse heart as it adapts to hypoxia. We have also shown that its precursor, dihydroceramide, accumulates with hypoxia. This implicates the enzyme dihydroceramide desaturase (DHC-DS), which converts dihydroceramide to ceramide, in a potential regulatory checkpoint in cardiomyocytes. We hypothesised that the regulation of de novo ceramide synthesis plays an important role in the cardiomyocyte adaptation to hypoxia. We used an established mouse model to induce acute and chronic hypoxia. Cardiac tissues were extracted and quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression levels of DHC-DS. Electrophoretic Mobility Shift Assays (EMSAs) and qRT-PCR were used to evaluate the activity and expression levels of an array of transcription factors that might regulate DEGS1 gene expression. We demonstrated that DEGS1 mRNA levels decrease with time in hypoxic mice concurrent with the decrease in HAND2 transcripts. Interestingly, the DEGS1 promoter harbors overlapping sites for Hand2 and Nuclear Factor of Activated T-cells (NFATC) transcription factors. We have demonstrated a physical interaction between NFATC1 and the E-Box proteins with EMSA and coimmunoprecipitation assays. The regulation of de novo ceramide synthesis in response to hypoxia and this newly described interaction between E-box and NFATC transcription factors will pave the way to identify new pathways in the adaptation of the cardiomyocyte to stress. The elucidation of these pathways will in the long-term provide insights into potential targets for novel therapeutic regimens.

Two heterozygous mutations in NFATC1 in a patient with Tricuspid Atresia.

Tricuspid Atresia (TA) is a rare form of congenital heart disease (CHD) with usually poor prognosis in humans. It presents as a complete absence of the right atrio-ventricular connection secured normally by the tricuspid valve. Defects in the tricuspid valve are so far not associated with any genetic locus, although mutations in numerous genes were linked to multiple forms of congenital heart disease. In the last decade, Knock-out mice have offered models for cardiologists and geneticists to study the causes of congenital disease. One such model was the Nfatc1(-/-) mice embryos which die at mid-gestation stage due to a complete absence of the valves. NFATC1 belongs to the Rel family of transcription factors members of which were shown to be implicated in gene activation, cell differentiation, and organogenesis. We have previously shown that a tandem repeat in the intronic region of NFATC1 is associated with ventricular septal defects. In this report, we unravel for the first time a potential link between a mutation in NFATC1 and TA. Two heterozygous missense mutations were found in the NFATC1 gene in one indexed-case out of 19 patients with TA. The two amino-acids changes were not found neither in other patients with CHDs, nor in the control healthy population. Moreover, we showed that these mutations alter dramatically the normal function of the protein at the cellular localization, DNA binding and transcriptional levels suggesting they are disease-causing.