Control of TGFß signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12.

Transforming growth factor ß (TGFß) pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFß signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFß signalling may help better understand the underlying causes of some of those conditions. Here, we show that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFß signalling in multiple models. Interestingly, TRIP12 control of TGFß signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12-/- cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFß signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFß signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFß mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFß induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. Our work establishes TRIP12 as an evolutionary conserved modulator of TGFß signalling in health and disease.

The link between glycemic control measures and eye microvascular complications in a clinical cohort of type 2 diabetes with microRNA-223-3p signature.

BACKGROUND: Type 2 diabetes (T2D) is a critical healthcare challenge and priority in Qatar which is listed amongst the top 10 countries in the world, with its prevalence presently at 17% double the global average. MicroRNAs (miRNAs) are implicated in the pathogenesis of (T2D) and long-term microvascular complications including diabetic retinopathy (DR). METHODS: In this study, a T2D cohort that accurately matches the characteristics of the general population was employed to find microRNA (miRNA) signatures that are correlated with glycemic and ß cell function measurements. Targeted miRNA profiling was performed in (471) T2D individuals with or without DR and (491) (non-diabetic) healthy controls from the Qatar Biobank. Discovery analysis identified 20 differentially expressed miRNAs in T2D compared to controls, of which miR-223-3p was significantly upregulated (fold change:5.16, p = 3.6e-02) and positively correlated with glucose and hemoglobin A1c (HbA1c) levels (p-value = 9.88e-04 and 1.64e-05, respectively), but did not show any significant associations with insulin or C-peptide. Accordingly, we performed functional validation using a miR-223-3p mimic (overexpression) under control and hyperglycemia-induced conditions in a zebrafish model. RESULTS: Over-expression of miR-223-3p alone was associated with significantly higher glucose (42.7 mg/dL, n = 75 vs 38.7 mg/dL, n = 75, p = 0.02) and degenerated retinal vasculature, and altered retinal morphology involving changes in the ganglion cell layer and inner and outer nuclear layers. Assessment of retinal angiogenesis revealed significant upregulation in the expression of vascular endothelial growth factor and its receptors, including kinase insert domain receptor. Further, the pancreatic markers, pancreatic and duodenal homeobox 1, and the insulin gene expressions were upregulated in the miR-223-3p group. CONCLUSION: Our zebrafish model validates a novel correlation between miR-223-3p and DR development. Targeting miR-223-3p in T2D patients may serve as a promising therapeutic strategy to control DR in at-risk individuals.

CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII.

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

Transcriptome Profile Identifies Actin as an Essential Regulator of Cardiac Myosin Binding Protein C3 Hypertrophic Cardiomyopathy in a Zebrafish Model.

Variants in cardiac myosin-binding protein C (cMyBP-C) are the leading cause of inherited hypertrophic cardiomyopathy (HCM), demonstrating the key role that cMyBP-C plays in the heart's contractile machinery. To investigate the c-MYBPC3 HCM-related cardiac impairment, we generated a zebrafish mypbc3-knockout model. These knockout zebrafish displayed significant morphological heart alterations related to a significant decrease in ventricular and atrial diameters at systolic and diastolic states at the larval stages. Immunofluorescence staining revealed significant hyperplasia in the mutant's total cardiac and ventricular cardiomyocytes. Although cardiac contractility was similar to the wild-type control, the ejection fraction was significantly increased in the mypbc3 mutants. At later stages of larval development, the mutants demonstrated an early cardiac phenotype of myocardium remodeling, concurrent cardiomyocyte hyperplasia, and increased ejection fraction as critical processes in HCM initiation to counteract the increased ventricular myocardial wall stress. The examination of zebrafish adults showed a thickened ventricular cardiac wall with reduced heart rate, swimming speed, and endurance ability in both the mypbc3 heterozygous and homozygous groups. Furthermore, heart transcriptome profiling showed a significant downregulation of the actin-filament-based process, indicating an impaired actin cytoskeleton organization as the main dysregulating factor associated with the early ventricular cardiac hypertrophy in the zebrafish mypbc3 HCM model.

Analysis of incidental findings in Qatar genome participants reveals novel functional variants in LMNA and DSP.

In order to report clinically actionable incidental findings in genetic testing, the American College of Medical Genetics and Genomics (ACMG) recommended the evaluation of variants in 59 genes associated with highly penetrant mutations. However, there is a lack of epidemiological data on medically actionable rare variants in these genes in Arab populations. We used whole genome sequencing data from 6045 participants from the Qatar Genome Programme and integrated it with phenotypic data collected by the Qatar Biobank. We identified novel putative pathogenic variants in the 59 ACMG genes by filtering previously unrecorded variants based on computational prediction of pathogenicity, variant rarity and segregation evidence. We assessed the phenotypic associations of candidate variants in genes linked to cardiovascular diseases. Finally, we used a zebrafish knockdown and synthetic human mRNA co-injection assay to functionally characterize two of these novel variants. We assessed the zebrafish cardiac function in terms of heart rate, rhythm and hemodynamics, as well as the heart structure. We identified 52 492 novel variants, which have not been reported in global and disease-specific databases. A total of 74 novel variants were selected with potentially pathogenic effect. We prioritized two novel cardiovascular variants, DSP c.1841A > G (p.Asp614Gly) and LMNA c.326 T > G (p.Val109Gly) for functional characterization. Our results showed that both variants resulted in abnormal zebrafish heart rate, rhythm and structure. This study highlights medically actionable variants that are specific to the Middle Eastern Qatari population.

Functional Characterization of the MYO6 Variant p.E60Q in Non-Syndromic Hearing Loss Patients.

Hereditary hearing loss (HHL) is a common genetic disorder accounting for at least 60% of pre-lingual deafness in children, of which 70% is inherited in an autosomal recessive pattern. The long tradition of consanguinity among the Qatari population has increased the prevalence of HHL, which negatively impacts the quality of life. Here, we functionally validated the pathogenicity of the c.178G>C, p.E60Q mutation in the MYO6 gene, which was detected previously in a Qatari HHL family, using cellular and animal models. In vitro analysis was conducted in HeLa cells transiently transfected with plasmids carrying MYO6WT or MYO6p.E60Q, and a zebrafish model was generated to characterize the in vivo phenotype. Cells transfected with MYO6WT showed higher expression of MYO6 in the plasma membrane and increased ATPase activity. Modeling the human MYO6 variants in zebrafish resulted in severe otic defects. At 72 h post-injection, MYO6p.E60Q embryos demonstrated alterations in the sizes of the saccule and utricle. Additionally, zebrafish with MYO6p.E60Q displayed super-coiled and bent hair bundles in otic hair cells when compared to control and MYO6WT embryos. In conclusion, our cellular and animal models add support to the in silico prediction that the p.E60Q missense variant is pathogenic and damaging to the protein. Since the c.178G>C MYO6 variant has a 0.5% allele frequency in the Qatari population, about 400 times higher than in other populations, it could contribute to explaining the high prevalence of hearing impairment in Qatar.

A recessive variant in SIM2 in a child with complex craniofacial anomalies and global developmental delay.

Rare deletions and duplications on the long arm of Chromosome 21 have previously been reported in many patients with craniofacial and developmental phenotypes. However, this Down Syndrome Critical Region (DSCR) contains multiple genes, making identifying a single causative gene difficult. Here, we report a case of a boy with bicoronal craniosynostosis, facial dysmorphism, developmental delay, and intellectual impairment who was found by whole genome sequencing to have a homozygous missense mutation in the Single-Minded Homolog 2 (SIM2) gene (c.461 A > G, p.Tyr154Cys) within the DSCR. SIM2 encodes an essential bHLH and PAS domain transcription factor expressed during fetal brain development and acts as a master regulator of neurogenesis. This variant is globally very rare, segregates in the family, and is predicted to be highly deleterious by in silico analysis, 3D molecular modeling of protein structure, and functional analysis of zebrafish models. Zebrafish expressing the human SIM2p.Y154C variant displayed a progressed microcephaly-like phenotype and head shape abnormalities. When combined with careful phenotyping of the patient vis-à-vis previously reported cases harboring structural variants in this critical 21q22 region, the data support a pathogenic role of SIM2 in this complex syndrome and demonstrates the utility of next-generation sequencing in prioritizing genes in contiguous deletions/duplications syndromes and diagnosing microarray-negative patients in the craniofacial clinic.

Microinjection quality control in zebrafish model for genetic manipulations.

Microinjection technique is one of the essential methodologies that are used widely in zebrafish research. Microinjection is utilized to perform genetic manipulations within the developing zebrafish model. Further, this technique is used to study a wide range of genetic diseases and gene of interest role in early developmental processes. Thus, quality control for microinjection is an essential factor to ensure experimental reproducibility and consistency. In this technical note, in vitro transcribed synthetic mRNA encoding green fluorescence protein (eGFP), and red fluorescent protein (m-cherry) as well as fluorescein and rhodamine fluorescent dyes were injected into a single-cell zebrafish embryo for volume quality control. Given the importance of having quality control system and methodology to yield similar genetic manipulation within the zebrafish embryo:•We aimed to establish the unified delivery of injected material into zebrafish one cell stage embryo.•We aimed to establish consistency of the injected volume into mineral oil droplets that will serve as a quality control parameter to conforms a quality control practice to ensure the reproducibility of the microinjection technique.•The calibration of microinjection droplet size resulted in the visualization of fluorescent protein and dyes in the zebrafish embryo with precise volumes of delivered materials under the control of needle opening, injection pressure and time.

Frequency and management of medically actionable incidental findings from genome and exome sequencing data: a systematic review.

The application of whole genome/exome sequencing technologies in clinical genetics and research has resulted in the discovery of incidental findings unrelated to the primary purpose of genetic testing. The American College of Medical Genetics and Genomics published guidelines for reporting pathogenic and likely pathogenic variants that are deemed to be medically actionable, which allowed us to learn about the epidemiology of incidental findings in different populations. However, consensus guidelines for variant reporting and classification are still lacking. We conducted a systematic literature review of incidental findings in whole genome/exome sequencing studies to obtain a comprehensive understanding of variable reporting and classification methods for variants that are deemed to be medically actionable across different populations. The review highlights the elements that demand further consideration or adjustment.

Deconstructing the mouse olfactory percept through an ethological atlas.

Odor perception in non-humans is poorly understood. Here, we generated the most comprehensive mouse olfactory ethological atlas to date, consisting of behavioral responses to a diverse panel of 73 odorants, including 12 at multiple concentrations. These data revealed that mouse behavior is incredibly diverse and changes in response to odorant identity and concentration. Using only behavioral responses observed in other mice, we could predict which of two odorants was presented to a held-out mouse 82% of the time. Considering all 73 possible odorants, we could uniquely identify the target odorant from behavior on the first try 20% of the time and 46% within five attempts. Although mouse behavior is difficult to predict from human perception, they share three fundamental properties: first, odor valence parameters explained the highest variance of olfactory perception. Second, physicochemical properties of odorants can be used to predict the olfactory percept. Third, odorant concentration quantitatively and qualitatively impacts olfactory perception. These results increase our understanding of mouse olfactory behavior and how it compares to human odor perception and provide a template for future comparative studies of olfactory percepts among species.

Prevalence and Phylogenetic Analysis of Parvovirus (B19V) among Blood Donors with Different Nationalities Residing in Qatar.

Human parvovirus (B19V) is the causative agent of erythema infectiosum in children and is linked to a wide range of clinical manifestations. Studies related to B19V prevalence in the Middle East and North Africa (MENA) region and other parts of Asia are very scarce. The objectives of this study were to estimate the seroprevalence (anti-B19V IgM and IgG), the viremia rate (B19V DNA), and the circulating genotypes of B19V among blood donors in Qatar. METHODS: Donors' blood samples (n = 5026) from different nationalities, mainly from the MENA region and South East Asia, were collected from 2014-2016. Samples were tested for the B19V DNA using RT-PCR. Furthermore, 1000 selected samples were tested to determine the seroprevalence of B19V antibodies using enzyme-linked immunosorbent assay (ELISA). Genotyping was performed on 65 DNA positive samples by sequencing of nested PCR fragments (NS1-VP1u region, 927 nt). RESULTS: Only 1.4% (70/5026) of the samples had detectible B19V DNA in their blood. B19V DNA prevalence statistically decreased with age (p = 0.03). Anti-B19V IgG was detected in 60.3% (561/930) of the tested samples, while only 2.1% (20/930) were IgM-positive and 1.2% (11/930) were both IgM- and IgG-positive. B19V genotyping showed a predominance of Genotype 1 (100%). Sequence analysis of the NS1-VP1u region revealed 139 mutation sites, some of which were amino acid substitutions. CONCLUSION: Our results indicated a relatively high seroprevalence of B19V in Qatar. Most importantly, B19 DNA was detected among Qatari and non-Qatari blood donors. Therefore, blood banks in Qatar might need to consider screening for B19V, especially when transfusion is intended for high-risk populations, including immunocompromised patients.