SARS-CoV-2 spike gene Sanger sequencing methodology to identify variants of concern.

The global demand for rapid identification of circulating SARS-CoV-2 variants of concern has led to a shortage of commercial kits. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol to identify circulating SARS-CoV-2 (variants of concern). Sets of primers flanking the SARS-CoV-2 spike gene were designed, verified and then validated using 282 nasopharyngeal positive samples for SARS-CoV-2. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole-genome sequencing of the same samples. Out of 282 samples, 123 contained the alpha variant, 78 beta and 13 delta, which were indicted using in-house primers and next-generation sequencing; the numbers of variants found were 100% identical to the reference genome. This protocol is easily adaptable for detection of emerging variants during the pandemic.

Gene Therapy Knowledge and Attitude Among Healthcare Professionals: A Cross-Sectional Study.

This study aimed to assess healthcare professionals' knowledge, attitudes, and concerns toward gene therapy in Saudi Arabia. We conducted an online cross-sectional survey via convenience sampling during the period from December 2018 to March 2019. A total of 419 (358 pharmacists and 61 physicians) responded to our questionnaire. Three hundred and nine (73.7%) were male, and the mean (±SD) age of 32.0 ± 7.7 years. The mean knowledge scores of all participants, pharmacists, and physicians were 3.8 ± 1.9, 3.8 ± 1.9, and 3.7 ± 1.9, respectively (P = 0.73). Higher knowledge score was associated with younger age (Coefficient: -0.03; P = 0.02), male (Coefficient: 0.57; P = 0.01), master's degree (Coefficient: 0.93; P = 0.003) and Ph.D. holders (Coefficient: 1.10; P = 0.01), and participants graduated from Canada (Coefficient: 2.10; P = 0.01).Moreover, about half of the respondents (55%) were concerned about gene therapy, and genetics training at college was considered the best gene therapy education method by 69.4%. Attitude score was not significantly associated with the profession (P = 0.88) but positively correlated with the knowledge score (rho= 0.4; P < 0.001). In conclusion, pharmacists and physicians showed limited knowledge with a positive attitude toward gene therapy. Therefore, educational programs on gene therapy need to be considered, focusing primarily on the safety, and social acceptance of such new therapeutic management.

Development and validation of an in-house, low-cost SARS-CoV-2 detection assay.

BACKGROUND: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. METHODS: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. RESULTS: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89). CONCLUSIONS: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.

Striatin translocates to the cytosol of apoptotic cells and is proteolytically cleaved in a caspase 3-dependent manner.

Striatin (STRN) is a multivalent protein holding great therapeutic potentials in view of its interaction with dynamic partners implicated in apoptosis. Although striatin-3 and striatin-4, that share high structural similarities with STRN, have been linked to apoptosis, the dynamics of STRN in apoptotic cells remain unclear. Herein, we report that the amount of STRN (110 kDa) is reduced in apoptotic cells, in response to various chemotherapeutic agents, thereby yielding a major polypeptide fragment at ~65 kDa, and three minor products at lower molecular weights. While STRN siRNA reduced the 65 kDa derivative fragment, the overexpression of a Myc-tagged STRN precipitated a novel fragment that was detected slightly higher than 65 kDa (due to the Myc-DDK tag on the cleaved fragment), confirming the cleavage of STRN during apoptosis. Interestingly, STRN cleavage was abrogated by the general caspase inhibitor Z-VAD.fmk. Cell fractionation revealed that the STRN pool, mainly distributed in the non-cytosolic fragment of naïve cells, translocates to the cytosol where it is proteolytically cleaved during apoptosis. Interestingly, the ectopic expression of caspase 3 in MCF-7 cells (deprived of caspase 3) induced STRN cleavage under apoptotic conditions. Inhibition of caspase 3 (Ac-DEVD-CHO) conferred a dose-dependent protection against the proteolytic cleavage of STRN. Collectively, our data provide cogent proofs that STRN translocates to the cytosol where it undergoes proteolytic cleavage in a caspase 3-dependent manner during apoptosis. Thus, this study projects the cleavage of STRN as a novel marker for apoptosis to serve pharmacological strategies targeting this particular form of cell death.

A study of the role of DIO1 and DIO2 polymorphism in thyroid cancer and drug response to therapy in the Saudi population.

BACKGROUND: Deiodinases comprise a group of selenoproteins that regulate the bioavailability of active thyroid hormones (TH) in a time and tissue specific fashion. They increase the hormonal activity by metabolizing their inactive precursors to active forms or terminate their activity by deactivating active hormones. The role of the deiodinase (DIO) gene polymorphisms in thyroid cancer is not fully understood yet. This study evaluated the potential association of the DIO1 and DIO2 genes with differentiated thyroid cancer and differential thyroxine dose requirement in thyroidectomized patients in a Saudi cohort. METHODS: We selected four variants (one DIO1 and three DIO2) for the association studies using Taqman assays in 507 DTC patients undergoing treatment with thyroxin against 560 disease-free individual, all of Saudi Arab origin. RESULTS: None of the studied variants was linked to differentiated thyroid cancer. The rs1388378_G > T was initially linked to thyroxine dose requirement (p = 0.035) when all patients were considered together, but this association was lost when the patients were classified into either near suppressed (0.1 ≤ TSH < 0.5) or suppressed (TSH < 0.1) TSH group. DISCUSSION: Although the results suggest only a weak relationship with differentiated thyroid cancer, they strongly indicate that the DIO2 polymorphism influences the hormonal dose requirement in patients undergoing treatment with thyroxine. This probably points to a distinction in the way this gene influences disease as compared to therapy thereof.

SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium.

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Cardiac striatin interacts with caveolin-3 and calmodulin in a calcium sensitive manner and regulates cardiomyocyte spontaneous contraction rate.

Impaired cardiomyocyte contraction rate is detrimental to cardiac function and often lethal. Despite advancements in the field, there is a paucity of information regarding the coordination of molecules implicated in regulating the heart rate. Striatin (STRN) is a dynamic protein with binding domains to calmodulin (CaM) and caveolin (Cav), both of which are regulators of myocardial function. However, its role in cardiomyocyte contraction is not yet determined. Herein, we show that STRN is expressed in cardiomyocytes and is more abundant in atrial myocardium than in ventricles. Cardiac expression of STRN (protein and mRNA) was developmentally regulated with the highest expression being at neonatal stage (day one) and the lowest in adult rats (13 weeks). CaM pulldown assay indicated that the interaction of cardiac STRN with CaM and caveolin-3 (Cav-3) was calcium sensitive. Interestingly, the overexpression of STRN induced an increase (∼2-fold) in the rate of the spontaneous contraction of cultured cardiomyocytes, while the knockdown of STRN reduced their contraction rate (∼40%). The expression level of STRN was inversely proportional to the interaction of Cav-3 with the CaM/STRN complex. Collectively, our data delineate a novel role for STRN in regulating cardiomyocyte spontaneous contraction rate and the dynamics of the STRN/Cav-3/CaM complex.

Screening for Mutations in ABCC8 and KCNJ11 Genes in Saudi Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI) Patients.

The autosomal recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is associated with mutations in either ABCC8 or KCNJ11 genes. In the present study, we describe the clinical features and results of genetic analysis of 13 Saudi Arabian patients with PHHI. Clinically, most patients presented with infantile seizures and/or developmental delay, with a subset of patients who were also found to have abnormal brain imaging and electrophysiological studies. Interestingly no coding pathogenic mutations were identified in these two genes by direct sequencing. However, two splice variants were identified in ABCC8 gene in two patients, and a large deletion of exons 1-22 of the ABCC8 gene was identified in three patients. Our data shows that large deletions in ABCC8 gene are the common genetic mechanism in the Saudi population.

Genotyping of CYP2C19 polymorphisms and its clinical validation in the ethnic Arab population.

OBJECTIVES: The drug-metabolizing enzymes and transporters (DMET) Plus microarray and x-Tag assays have recently been developed for genotyping individuals in personalized medicine. Furthermore, the cytochrome 450-2C19 (CYP2C19) is a key metabolic enzyme encoded by a polymorphic gene commonly associated with diminished metabolism and variable clinical responses to several drugs in an ethnicity-dependent fashion. Therefore, validation of these clinical procedures as well as knowledge of the ethnic-specific incidences of these gene variants is prerequisite for determining their clinical relevance in any given population. METHODS: We determined the distribution of familiar CYP2C19 variants by the DMET Plus chip in 600 candidates and replicated the findings by the Affymetrix Axiom Genome-Wide Asian Structure Identification Array in 5413 individuals, all Saudis of ethic Arab origin. We then tested the robustness of employing the Luminex xMAP system clinically by comparing the results of genotyping 500 Saudi individuals visiting the Blood Bank of our institution with the findings of the two platforms. KEY FINDINGS: The DMET Plus genotyping revealed that eight of the CYP2C19 variants showed some changes. Thereby, the CYP2C19*17 exhibited the highest minor allele frequency (MAF) of 0.256, followed by the CYP2C19_801 (frequency = 0.055). Six other variants, including the CYP2C19*3, showed MAF in the range of 0.001-0.002. We replicated the frequencies of the CYP2C19*17 and CYP2C19*3, and additionally established that of the CYP2C19*2 (0.099) using the Axiom platform. The xTag genotyping also indicated that 0.834 of the 500 Saudi individuals were extensive metabolizers (*1/*1), 0.158 carried the *1/*2 genotype, 0.01% carried *2/*2 (poor metabolizers) and one each (0.2%) harboured the *1/*8, *2/*3 (intermediate metabolizers) and *8/*8 (poor metabolizers) genotypes. CONCLUSIONS: The results showed reproducible genotyping of the CYP2C19 variants in the Saudi Arab population using two Affymetrix platforms and phenotyping using the Luminex xTag assay. The prevalence of two clinically relevant genotypes (CYP2C19*2 and CYP2C19*3) were similar to other ethnic groups, while that of the CYP2C19*17 was comparably higher.