Detection of Coronaviruses in Bats in Lebanon during 2020.

Bats are considered the main reservoir of coronaviruses (CoVs), and research evidence suggests the essential role of bats in the emergence of Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoV) and SARS-CoV-2. SARS-CoV-like viruses have been recently detected in bats in different countries. In 2020, we conducted surveillance for CoVs among six different bat species in Lebanon. Of 622 swab specimens taken, 77 tested positive. Alpha- and Beta- CoVs were identified in samples collected from different species. Our results show that SARS-like coronaviruses circulate in bats in this region, and we provide new data on their genetic diversity. The interaction between the spike of the detected SARS-CoV-like viruses and the human angiotensin-converting enzyme 2 (hACE2) receptor could be crucial in understanding the origin of the epidemic. The 3D protein structure analysis revealed that the receptor-binding domains of the SARS-like virus identified in Lebanon bind to the hACE2 protein more efficiently than to the spike of the SARS-CoV-2 strain. The spike of the detected SARS-CoV-like viruses does not contain the recognition site of furin at the cleavage site. Thus, our study highlights the variety of bat coronaviruses in Lebanon and suggests the zoonotic potential for other SARS-CoV-like viruses.

SARS-CoV-2 Variants in Lebanon: Evolution and Current Situation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seen a worldwide spread since its emergence in 2019, including to Lebanon, where 534,968 confirmed cases (8% of the population) and 7569 deaths have been reported as of 14 May 2021. With the genome sequencing of strains from various countries, several classification systems were established via genome comparison. For instance, the GISAID clades classification highlights key mutations in the encoded proteins that could potentially affect the virus' infectivity and transmission rates. In this study, 58 genomes of Lebanese SARS-CoV-2 strains were analyzed, 28 of which were sequenced for this study, and 30 retrieved from the GISAID and GenBank databases. We aimed to classify these strains, establish their phylogenetic relationships, and extract the mutations causing amino acid substitutions within, particularly, the structural proteins. The sequenced Lebanese SARS-COV-2 strains were classified into four GISAID clades and 11 Pango lineages. Moreover, 21 uncommon mutations in the structural proteins were found in the newly sequenced strains, underlining interesting combinations of mutations in the spike proteins. Hence, this study constitutes an observation and description of the current SARS-CoV-2 genetic and clade situation in Lebanon according to the available sequenced strains.

Increasing knowledge in IGF1R defects: lessons from 35 new patients.

BACKGROUND: The type 1 insulin-like growth factor receptor (IGF1R) is a keystone of fetal growth regulation by mediating the effects of IGF-I and IGF-II. Recently, a cohort of patients carrying an IGF1R defect was described, from which a clinical score was established for diagnosis. We assessed this score in a large cohort of patients with identified IGF1R defects, as no external validation was available. Furthermore, we aimed to develop a functional test to allow the classification of variants of unknown significance (VUS) in vitro. METHODS: DNA was tested for either deletions or single nucleotide variant (SNV) and the phosphorylation of downstream pathways studied after stimulation with IGF-I by western blot analysis of fibroblast of nine patients. RESULTS: We detected 21 IGF1R defects in 35 patients, including 8 deletions and 10 heterozygous, 1 homozygous and 1 compound-heterozygous SNVs. The main clinical characteristics of these patients were being born small for gestational age (90.9%), short stature (88.2%) and microcephaly (74.1%). Feeding difficulties and varying degrees of developmental delay were highly prevalent (54.5%). There were no differences in phenotypes between patients with deletions and SNVs of IGF1R. Functional studies showed that the SNVs tested were associated with decreased AKT phosphorylation. CONCLUSION: We report eight new pathogenic variants of IGF1R and an original case with a homozygous SNV. We found the recently proposed clinical score to be accurate for the diagnosis of IGF1R defects with a sensitivity of 95.2%. We developed an efficient functional test to assess the pathogenicity of SNVs, which is useful, especially for VUS.

Transcriptional profiling at the DLK1/MEG3 domain explains clinical overlap between imprinting disorders.

Imprinting disorders (IDs) often affect growth in humans, leading to diseases with overlapping features, regardless of the genomic region affected. IDs related to hypomethylation of the human 14q32.2 region and its DLK1/MEG3 domain are associated with Temple syndrome (TS14). TS14 is a rare type of growth retardation, the clinical signs of which overlap considerably with those of Silver-Russell syndrome (SRS), another ID related to IGF2 down-regulation at 11p15.5 region. We show that 14q32.2 hypomethylation affects expression, not only for genes at this locus but also for other imprinted genes, and especially lowers IGF2 levels at 11p15.5. Furthermore, expression of nonimprinted genes is also affected, some of which are also deregulated in SRS patients. These findings highlight the epigenetic regulation of gene expression at the DLK1/MEG3 domain. Expression profiling of TS14 and SRS patients highlights common signatures, which may account for the clinical overlap observed between TS14 and SRS.

Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome.

Context: Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap. Objective: To describe the clinical overlap between SRS and TS and extensively study the molecular aspects of TS. Patients: We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted region, identified in our center, and performed extensive molecular analysis. Results: Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients (72.7%) had a Netchine-Harbison SRS clinical scoring system ≥4/6, and consistent with a clinical diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5% had premature pubarche. The body mass index of all patients increased before pubarche and/or the onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients. We identified four potentially damaging genetic variants in genes encoding proteins involved in the establishment or maintenance of DNA methylation. Conclusions: Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS. These clinical data suggest similar management of patients with TS and SRS, with special attention to their young age at the onset of puberty and early increase of body mass index.

Genetic disruption of the oncogenic HMGA2-PLAG1-IGF2 pathway causes fetal growth restriction.

PurposeFetal growth is a complex process involving maternal, placental and fetal factors. The etiology of fetal growth retardation remains unknown in many cases. The aim of this study is to identify novel human mutations and genes related to Silver-Russell syndrome (SRS), a syndromic form of fetal growth retardation, usually caused by epigenetic downregulation of the potent fetal growth factor IGF2.MethodsWhole-exome sequencing was carried out on members of an SRS familial case. The candidate gene from the familial case and two other genes were screened by targeted high-throughput sequencing in a large cohort of suspected SRS patients. Functional experiments were then used to link these genes into a regulatory pathway.ResultsWe report the first mutations of the PLAG1 gene in humans, as well as new mutations in HMGA2 and IGF2 in six sporadic and/or familial cases of SRS. We demonstrate that HMGA2 regulates IGF2 expression through PLAG1 and in a PLAG1-independent manner.ConclusionGenetic defects of the HMGA2-PLAG1-IGF2 pathway can lead to fetal and postnatal growth restriction, highlighting the role of this oncogenic pathway in the fine regulation of physiological fetal/postnatal growth. This work defines new genetic causes of SRS, important for genetic counseling.

11p15 ICR1 Partial Deletions Associated with IGF2/H19 DMR Hypomethylation and Silver-Russell Syndrome.

The 11p15 region harbors the IGF2/H19 imprinted domain, implicated in fetal and postnatal growth. Silver-Russell syndrome (SRS) is characterized by fetal and postnatal growth failure, and is caused principally by hypomethylation of the 11p15 imprinting control region 1 (ICR1). However, the mechanisms leading to ICR1 hypomethylation remain unknown. Maternally inherited genetic defects affecting the ICR1 domain have been associated with ICR1 hypermethylation and Beckwith-Wiedemann syndrome (an overgrowth syndrome, the clinical and molecular mirror of SRS), and paternal deletions of IGF2 enhancers have been detected in four SRS patients. However, no paternal deletions of ICR1 have ever been associated with hypomethylation of the IGF2/H19 domain in SRS. We screened for new genetic defects within the ICR1 in a cohort of 234 SRS patients with hypomethylated IGF2/H19 domain. We report deletions close to the boundaries of ICR1 on the paternal allele in one familial and two sporadic cases of SRS with ICR1 hypomethylation. These deletions are associated with hypomethylation of the remaining CBS, and decreased IGF2 expression. These results suggest that these regions are most likely required to maintain methylation after fertilization. We estimate these anomalies to occur in about 1% of SRS cases with ICR1 hypomethylation.

Liver X Receptors differentially modulate central myelin gene mRNA levels in a region-, age- and isoform-specific manner.

Liver X Receptors (LXRs) α and ß are nuclear receptors able to bind oxidative forms of cholesterol. They play important roles in the central nervous system (CNS), through their implication in a large variety of physiological and pathological processes among which modulation of cholesterol homeostasis and inflammation. Besides, we recently revealed their crucial role in myelination and remyelination in the cerebellum. Given the pleiotropic effects of such receptors on CNS functioning, we studied here the influence of LXRs on myelin gene mRNA accumulation in the major myelinated regions of the CNS in vivo. We show that both LXR isoforms differentially affect mRNA amount of myelin genes (PLP and MBP) in highly myelinated structures such as spinal cord, corpus callosum, optic nerve and cerebellum. In the adult, LXR activation by the synthetic agonist TO901317 significantly increases myelin gene mRNA amount in the cerebellum but not in the other regions studied. Invalidation of the sole LXRß isoform leads to decreased PLP and MBP mRNA levels in all the structures except the spinal cord, while the knock out of both isoforms (LXR dKO) decreases myelin gene mRNA amounts in all the regions tested except the corpus callosum. Interestingly, during myelination process (post-natal day 21), both cerebellum and optic nerve display a decrease in myelin gene mRNA levels in LXR dKO mice. Concomitantly, PLP and MBP mRNA accumulation in the spinal cord is increased. Relative expression level of LXR isoforms could account for the differential modulation of myelin gene expression in the CNS. Altogether our results suggest that, within the CNS, each LXR isoform differentially influences myelin gene mRNA levels in a region- and age-dependant manner, participating in the fine regulation of myelin gene expression.

Extensive investigation of the IGF2/H19 imprinting control region reveals novel OCT4/SOX2 binding site defects associated with specific methylation patterns in Beckwith-Wiedemann syndrome.

Isolated gain of methylation (GOM) at the IGF2/H19 imprinting control region 1 (ICR1) accounts for about 10% of patients with BWS. A subset of these patients have genetic defects within ICR1, but the frequency of these defects has not yet been established in a large cohort of BWS patients with isolated ICR1 GOM. Here, we carried out a genetic analysis in a large cohort of 57 BWS patients with isolated ICR1 GOM and analyzed the methylation status of the entire domain. We found a new point mutation in two unrelated families and a 21 bp deletion in another unrelated child, both of which were maternally inherited and affected the OCT4/SOX2 binding site in the A2 repeat of ICR1. Based on data from this and previous studies, we estimate that cis genetic defects account for about 20% of BWS patients with isolated ICR1 GOM. Methylation analysis at eight loci of the IGF2/H19 domain revealed that sites surrounding OCT4/SOX2 binding site mutations were fully methylated and methylation indexes declined as a function of distance from these sites. This was not the case in BWS patients without genetic defects identified. Thus, GOM does not spread uniformly across the IGF2/H19 domain, suggesting that OCT4/SOX2 protects against methylation at local sites. These findings add new insights to the mechanism of the regulation of the ICR1 domain. Our data show that mutations and deletions within ICR1 are relatively common. Systematic identification is therefore necessary to establish appropriate genetic counseling for BWS patients with isolated ICR1 GOM.

Beckwith-Wiedemann and Russell-Silver Syndromes: from new molecular insights to the comprehension of imprinting regulation.

PURPOSE OF REVIEW: The imprinted human 11p15.5 region encompasses two imprinted domains important for the control of fetal growth: the H19/IGF2 domain in the telomeric region and the KCNQ1OT1/CDKN1C domain in the centromeric region. These two domains are differentially methylated and each is regulated by its own imprinting control region (ICR): ICR1 in the telomeric region and ICR2 in the centromeric region. Aberrant methylation of the 11p15.5 imprinted region, through genetic or epigenetic mechanisms, leads to two clinical syndromes, with opposite growth phenotypes: Russell-Silver Syndrome (RSS; with severe fetal and postnatal growth retardation) and Beckwith-Wiedemann Syndrome (BWS; an overgrowth syndrome). RECENT FINDINGS: In this review, we discuss the recently identified molecular abnormalities at 11p15.5 involved in RSS and BWS, which have led to the identification of cis-acting elements and trans-acting regulatory factors involved in the regulation of imprinting in this region. We also discuss the multilocus imprinting disorders identified in various human syndromes, their clinical outcomes and their impact on commonly identified metabolism disorders. SUMMARY: These new findings and progress in this field will have direct consequence for diagnostic and predictive tools, risk assessment and genetic counseling for these syndromes.