Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Genotype-phenotype correlations in five patients harboring SERPINA1 mutations and previously showing a severe COVID-19 infection

Background/Objectives: During COVID-19 pandemic, it is essential to detect patients potentially at risk of life-threatening complications, due to possible specific genetic mutations. The aim of our work is to show a practical application of genetic testing, allowing a diagnosis of alpha 1 antitrypsin deficiency in cases with a severe clinical course during COVID-19 infection. Method(s): During hospitalization for COVID-19, we identified 5 patients (3 female, 2 males from two different families, age range 18-47 years) with a severe course of COVID-19 infection, requiring high pressure ventilation with high volume oxygen supply. Two months after discharge, those patients were reevaluated with respiratory function tests, biochemical tests, genetic counselling and genetic testing. A peripheral blood sampling for SERPINA1 genetic testing has been performed, using Sanger sequencing. Result(s): Two months after discharge, in all 5 patients respiratory function tests were consistent with a dysventilatory obstructive syndrome, in contrast with usual findings related to COVID-19 infection. Blood test still showed increase plasmatic transaminase concentration in 3 out of 5 patients, one having increased serum bilirubin as well. We performed SERPINA1 genetic testing showing homozygosity for SERPINA1 pathogenic mutations (c.193del and c.875C>T, respectively) in all 5 patients. Conclusion(s): These cases showed the importance of genetic testing for patients with unexplained severe COVID-19 infection. Genetic testing allowed the diagnosis of cases affected by alpha 1 antitrypsin deficiency, associated with dysventilatory obstructive syndrome, that may worsen the short and long term prognosis of COVID-19.

A Rapid Point-of-Care Dipstick Assay for Differentiation of Sars-Cov-2 Variants in Covid-19 Patients

Intro: Delta and Omicron variants of SARS-CoV-2 are highly contagious, currently dominating the globe and recognized as variant of concern (VOC). The transmissibility efficiency of viruses, disease symptoms, and severity of COVID- 19 is highly heterogeneous. Therefore, testing at the community level is essential to identify the infected people at an early stage-carrying VOC to reduce the spread of virus and combat the pandemic. Method(s): In this study, we analysed thousands of genome sequences representing 30 different SARS-CoV-2 variants and identified Delta and Omicron variants specific nucleic acid signatures in the spike gene. Based on the variant specific nucleic acid sequences we synthesized different oligos and optimized a mPCR assay that can specifically differentiate the Delta and Omicron variants. We further translated our work into a dipstick assay (Tohoku Bio-array, Japan) by adding tag linker sequence to 5' end of the forward primer and adding biotin in 3' end of the oligos. Finding(s): A total of 250 samples were subjected to WGS using MiSeq platform and these confirmed samples were processed for validation of our specific designed probes using PCR assay and the readout was found to be 100% specific to Delta, BA.1 & BA.2 of SARS-CoV-2 variants which were further confirmed by Sanger sequencing. The dipstick was used to screen these samples, and specific signals were observed. WGS and Sanger sequencing were used to validate our PCR and dipstick assay results, and the readout was found to be 100% specific. The results can be visualised by the naked eye and interpreted easily. Conclusion(s): This study developed a rapid point-of-care test of SARS-CoV-2 patients, which can differentiate Delta, BA.1 and BA.2 variants at the same time of confirmation of the infection in patient. The current nucleic-acid chromatography-based dipstick assay is highly specific and can work even in the case of low viral load as well.Copyright © 2023

Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger.

INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

Molecular diagnosis of rhino-orbital mucormycosis in a COVID-19 setting.

PURPOSE: Mucormycosis is a severe fungal infection caused by species of the order Mucorales. Early and accurate diagnosis is a prerequisite in the management of the disease. In the present study, we evaluated and compared two PCR-based techniques for the diagnosis and identification of mucormycosis in patients with rhino-orbital mucormycosis (ROM) post-COVID-19. METHODS: Diagnosed clinically and radiologically, 25 patients of ROM were included in the study and endoscopically or blind collected nasal swabs or orbital tissues were submitted for microbiological evaluation (direct microscopy + culture) and PCR using primers targeting two different loci (ITS and 28S rDNA region) for diagnosis. All PCR products were further processed for species identification using Sanger sequencing whenever possible. RESULT: Of the 25 samples included in the study, 16 samples were positive for presence of fungal filaments by Smear suggestive of Mucorales sp., but only 7/25 grew in culture. ITS-based PCR was able to identify mucormycosis in 7/25 (28%) samples and 28S rDNA PCR showed positivity for 19/25 (76%) samples. Rhizopus oryzae was found to be the predominant species in our study. The sensitivity and specificity of 28S rDNA PCR compared to culture were found to be 85.71% and 27.78%, respectively, while for ITS-based PCR, they were 42.86% and 77.78%, respectively. CONCLUSIONS: 28S rDNA-based PCR is a reliable and sensitive method for early diagnosis of mucormycosis. Molecular techniques have shown a promising future to provide quick and effective treatment by accurately identifying the aetiologic agent.

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene.

Variants of concern (VOCs) of SARS-CoV-2 are viral strains that have mutations associated with increased transmissibility and/or increased virulence, and their main mutations are in the receptor binding domain (RBD) region of the viral spike. This study aimed to characterize SARS-CoV-2 VOCs via Sanger sequencing of the RBD region and compare the results with data obtained via whole genome sequencing (WGS). Clinical samples (oro/nasopharyngeal) with positive RT-qPCR results for SARS-CoV-2 were used in this study. The viral RNA from SARS-CoV-2 was extracted and a PCR fragment of 1006 base pairs was submitted for Sanger sequencing. The results of the Sanger sequencing were compared to the lineage assigned by WGS using next-generation sequencing (NGS) techniques. A total of 37 specimens were sequenced via WGS, and classified as: VOC gamma (8); delta (7); omicron (10), with 3 omicron specimens classified as the BQ.1 subvariant and 12 specimens classified as non-VOC variants. The results of the partial Sanger sequencing presented as 100% in agreement with the WGS. The Sanger protocol made it possible to characterize the main SARS-CoV-2 VOCs currently circulating in Brazil through partial Sanger sequencing of the RBD region of the viral spike. Therefore, the sequencing of the RBD region is a fast and cost-effective laboratory tool for clinical and epidemiological use in the genomic surveillance of SARS-CoV-2.

Sequencing directly from antigen-detection rapid diagnostic tests in Belgium, 2022: a gamechanger in genomic surveillance?

BackgroundLateral flow antigen-detection rapid diagnostic tests (Ag-RDTs) for viral infections constitute a fast, cheap and reliable alternative to nucleic acid amplification tests (NAATs). Whereas leftover material from NAATs can be employed for genomic analysis of positive samples, there is a paucity of information on whether viral genetic characterisation can be achieved from archived Ag-RDTs.AimTo evaluate the possibility of retrieving leftover material of several viruses from a range of Ag-RDTs, for molecular genetic analysis.MethodsArchived Ag-RDTs which had been stored for up to 3 months at room temperature were used to extract viral nucleic acids for subsequent RT-qPCR, Sanger sequencing and Nanopore whole genome sequencing. The effects of brands of Ag-RDT and of various ways to prepare Ag-RDT material were evaluated.ResultsSARS-CoV-2 nucleic acids were successfully extracted and sequenced from nine different brands of Ag-RDTs for SARS-CoV-2, and for five of these, after storage for 3 months at room temperature. The approach also worked for Ag-RDTs for influenza virus (n = 3 brands), as well as for rotavirus and adenovirus 40/41 (n = 1 brand). The buffer of the Ag-RDT had an important influence on viral RNA yield from the test strip and the efficiency of subsequent sequencing.ConclusionOur finding that the test strip in Ag-RDTs is suited to preserve viral genomic material, even for several months at room temperature, and therefore can serve as source material for genetic characterisation could help improve global coverage of genomic surveillance for SARS-CoV-2 as well as for other viruses.

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.

Laboratory experience in massive SARS-CoV-2 mutation screening using laboratory-developed tests

Objectives. The main goal of the study was to develop laboratory-developed tests (LDT) for monitoring SARS-CoV-2 variants of concern (VOC) currently present in Latvia. METHODS. We have studied the latest scientific articles to prognose specific mutations that could be indicators of VOC. Mutations responsible for immune escape were chosen as targets for our LDTs. Multiple TaqMan RT-PCR LDTs detecting alfa, beta, gamma, delta and omicron strains in nasopharyngeal swab and saliva samples were developed and validated in our laboratory. RESULTS. More than 15,000 SARS-CoV-2 positive samples were tested. In total, 10,874 different VOCs of SARS-CoV-2 were found by our LDTs. Beta, delta and omicron strains were first detected in Latvia by E. Gulbis Laboratory. Sanger sequencing methods for RT-PCR result confirmation were also developed. The first cases of VOCs detected by the RT-PCR method were also confirmed in our laboratory by Sanger sequencing. Our results were later confirmed by the National Reference Laboratory. CONCLUSIONS. Using our laboratory capacity and intellectual potential, we have developed skills for an urgent response to future VOCs of SARS-CoV-2 or other potentially harmful infectious diseases.

Genomic surveillance of SARS-CoV-2 by sequencing the RBD region using Sanger sequencing from North Kerala.

Next Generation Sequencing (NGS) is the gold standard for the detection of new variants of SARS-CoV-2 including those which have immune escape properties, high infectivity, and variable severity. This test is helpful in genomic surveillance, for planning appropriate and timely public health interventions. But labs with NGS facilities are not available in small or medium research settings due to the high cost of setting up such a facility. Transportation of samples from many places to few centers for NGS testing also produces delays due to transportation and sample overload leading in turn to delays in patient management and community interventions. This becomes more important for patients traveling from hotspot regions or those suspected of harboring a new variant. Another major issue is the high cost of NGS-based tests. Thus, it may not be a good option for an economically viable surveillance program requiring immediate result generation and patient follow-up. The current study used a cost-effective facility which can be set up in a common research lab and which is replicable in similar centers with expertise in Sanger nucleotide sequencing. More samples can be processed at a time and can generate the results in a maximum of 2 days (1 day for a 24 h working lab). We analyzed the nucleotide sequence of the Receptor Binding Domain (RBD) region of SARS-CoV-2 by the Sanger sequencing using in-house developed methods. The SARS-CoV-2 variant surveillance was done during the period of March 2021 to May 2022 in the Northern region of Kerala, a state in India with a population of 36.4 million, for implementing appropriate timely interventions. Our findings broadly agree with those from elsewhere in India and other countries during the period.

Highly Conserve Sequences in Envelope, Nucleoprotein and RNA-Dependent RNA Polymerase of SARS-CoV-2 in Nasopharyngeal Samples of the COVID-19 Patients;a Diagnostic Target for Further Studies

Background: The etiological agent of coronavirus diseases 2019 (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Conventional molecular methods are used to detect viruses in COVID-19 infected patients. This study aimed to investigate escape mutations from molecular detection on SARS-CoV-2 targeted genes, which indicates the importance of mutations in false-negative PCR test results in the detection of virus in clinical specimens of patients with COVID-19. Materials and Methods: The 20 nasopharyngeal swabs samples collected from COVID-19 confirmed patients. The SARS-CoV-2 E, nsp12, and N genetic regions are amplified by RT-PCR assay. PCR products were sequenced using the Sanger sequencing method and Multiple sequence alignment (MSA) to assess the polymorphism and mutations performed using MEGA X software and the Maximum likelihood method for the phylogenetic evaluation. Results: Among all COVID-19 cases, 60% and 40% were male and female, respectively. The MSA showed high conservation between all evaluated samples and VOCs in all N, E, and nsp12 genes. Also, the phylogenetic evaluation by the Maximum likelihood method reported high similarity between all SARS-CoV-2 sequenced samples, VOCs, and Wuhan reference sequences in the evaluated region. Conclusion: Our study results approved the relatively conserved suitability of the E, N, and RdRp-gene regions with no diversity, therefore, making them perfect candidates for first-line screening.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima.

This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based partial sequencing to the targeted spike gene (nt22,735~nt23,532) using an in-house-developed primer set. The identification of variants was done by unique checkpoints of base nucleotide changes in the targeted spike gene. Moreover, we amplified one full-length genome using Sanger method and an in-house-developed primer library. Using NGS strains of the same sampling period from GISAID, a phylogenetic tree was constructed to examine the distribution pattern of variants in Hiroshima and to validate our Sanger method. The modified primer set provided 100% validation and 99.2% amplification. PANGO Lineage R.1 was detected in late in the third wave, followed by Alpha (B.1.1.7) domination in the fourth wave, Delta (B.1.617.2) domination in the fifth wave, and Omicron (B.1.1.529) domination in the sixth wave, and there was no significant difference in viral copies between variants (p = 0.09). The variants showed different transmission patterns, but the distribution of variants is consistent to that shown by the phylogenetic tree. The Sanger method also provided successful amplification of the full-length genome of the SARS-CoV-2 virus. Our Sanger sequencing strategy was useful for the screening of SASR-CoV-2 variants without the need for full-genome amplification. The modified primer set was validated to use universally, which allows an understanding of the variants' distribution in real time and provides the evidence for policy-making and the formulation or modification of preventive strategies.

Tracking SARS-CoV-2 variants by entire S-gene analysis using long-range RT-PCR and Sanger sequencing.

INTRODUCTION: Genomic surveillance of the SARS-CoV-2 virus is important to assess transmissibility, disease severity, and vaccine effectiveness. The SARS-CoV-2 genome consists of approximately 30 kb single-stranded RNA that is too large to analyze the whole genome by Sanger sequencing. Thus, in this study, we performed Sanger sequencing following long-range RT-PCR of the entire SARS-CoV-2 S-gene and analyzed the mutational dynamics. METHODS: The 4 kb region, including the S-gene, was amplified by two-step long-range RT-PCR. Then, the entire S-gene sequence was determined by Sanger sequencing. The amino acid mutations were identified as compared with the reference SARS-CoV-2 genome. RESULTS: The S:D614G mutation was found in all samples. The R.1 variants were detected after January 2021. Alpha variants started to emerge in April 2021. Delta variants replaced Alpha in July 2021. Then, Omicron variants were detected after December 2021. These mutational dynamics in samples collected in the Chiba University Hospital were similar to those in Japan. CONCLUSION: The emergence of variants of concern (VOC) has been reported by the entire S-gene analysis. As the VOCs have unique mutational patterns of the S-gene region, analysis of the entire S-gene will be useful for molecular surveillance of the SARS-CoV-2 in clinical laboratories.

Analysis of mitochondrial DNA cytochrome-b (CYB) and ATPase-6 gene mutations in COVID-19 patients.

Coronavirus disease of 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Mutations of mitochondrial DNA (mtDNA) are becoming increasingly common in various diseases. This study aims to investigate mutations in the cytochrome-b (CYB) and adenosine triphosphatase-6 (ATPase-6) genes of mtDNA in COVID-19 patients. The association between mtDNA mutations and clinical outcomes is investigated here. In the present study, mutations of the mtDNA genes CYB and ATPase-6 were investigated in COVID-19 (+) (n = 65) and COVID-19 (-) patients (n = 65). First, we isolated DNA from the blood samples. After the PCR analyses, the mutations were defined using Sanger DNA sequencing. The age, creatinine, ferritin, and CRP levels of the COVID 19 (+) patients were higher than those of the COVID-19 (-) patients (p = 0.0036, p = 0.0383, p = 0.0305, p < 0.0001, respectively). We also found 16 different mutations in the CYB gene and 14 different mutations in the ATPase-6 gene. The incidences of CYB gene mutations A15326G, T15454C, and C15452A were higher in COVID-19 (+) patients than COVID-19 (-) patients; p < 0.0001: OR (95% CI): 4.966 (2.215-10.89), p = 0.0226, and p = 0.0226, respectively. In contrast, the incidences of A8860G and G9055A ATPase-6 gene mutations were higher in COVID-19 (+) patients than COVID-19 (-) patients; p < 0.0001: OR (95%CI): 5.333 (2.359-12.16) and p = 0.0121 respectively. Yet, no significant relationship was found between mtDNA mutations and patients' age and biochemical parameters (p > 0.05). The results showed that the frequency of mtDNA mutations in COVID-19 patients is quite high and it is important to investigate the association of these mutations with other genetic mechanisms in larger patient populations.

Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants.

Reccurence of atypical hemolytic uremic syndrome after COVID-19 vaccination

Background: Atypical Hemolytic Uremic Syndrome (aHUS) is a complement-mediated thrombotic microangiopathy. Pathophysiological mechanism involves uncontrolled complement activation due to a genetic or acquired anomaly coupled with a triggering event. We report a case of aHUS recurrence following COVID-19 vaccination. Material and methods: Whole blood (EDTA) was collected and processed with CD46-PE, CD45-PerCP, isotype control-PE markers. Staining was measured through median fluorescence intensity and expressed as CD46/isotype ratio. Sanger sequencing was used for identification of variants in CD46 gene. All the participants provided informed written consent. Results: Proband (P) is a 39-year-old woman admitted for nausea, vomiting, epigastric pain and haematuria, three days after first dose of ChAdOx1 nCov-19 vaccine. Laboratory testing showed MAHA (Hb:8.8g/dL, Ht:26%), thrombocytopenia (80x109/mm3) and acute kidney injury (Cr:2.15mg/dL, Ur:92mg/dL). P and three of her siblings have experienced recurrent TMA episodes since childhood. In 2019, genetic study from P's sister (S) identified two heterozygous variants in CD46, one pathogenic (Glu179Gln) and one of uncertain significance (Cys94Tyr). We demonstrated that P carries the same variants and observed a 50% decrease of CD46 expression in both P and S (fig.1). Platelet transfusion, corticosteroids and 9 sessions of plasmapheresis contributed to rapid recovery of P. Discussion: Glu179Gln was reported to increase CD46 expression on granulocytes in aHUS patient and to reduce C4b cofactor activity1. We observed that combination of Glu179Gln and Cys94Tyr was associated with low levels of CD46 on cell surface. Conclusion: This case report supports the evidence of COVID-19 vaccine as a precipitating event for aHUS recurrence.

COVID-19 pandemic in patients with familial mediterranean fever;The possible protective role of colchicine in COVID-19 symptoms

Familial Mediterranean Fever (FMF) is the most common autoinflammatory disease characterized by recurrent fever and attacks of polyserositis, arthritis, erysipelas-like erythema. Colchicine;has been the main therapeutic agent in FMF patients since 1972. The Novel Coronavirus Disease (COVID-19) was first identified in a group of patients with respiratory symptoms in Wuhan, China, and affected the whole world.This study aims to evaluate the possible correlation between the frequency and severity of FMF attacks and COVID-19 symptoms after colchicine use in FMF patients with pathogenic/likely pathogenic MEFV mutation(s) during the pandemic. We included FMF patients who applied to Çanakkale Onsekiz Mart University Hospital, Medical Genetics Department between 01.01.2010-15.03.2020 and having variable. Pathogenic/likely pathogenic MEFV mutations detected by variable methods (pyrosequencing, NGS, Sanger sequencing, fragment analysis, real-time PCR, etc.). A 19-question questionnaire was created online via Google Drive and the questionnaire link was sent to patients' mobile phone via SMS. The responses received until 31.12.2020 were evaluated and analyzed with the IBM SPSS Statistics 25. Permission was obtained from the local ethics committee and the ethics committee of the Ministry of Health to conduct this research. We obtained 110 responses that met the research criteria. The number of patients with COVID-19 symptoms was 13(%11.8). While there was no statistically significant difference in frequency and severity of attacks between the groups of patients with and without symptoms of COVID-19, a significant difference was found in the need to increase the colchicine dose. Furthermore, while the rate of patients who had an FMF attack (n=45)(46.4%) and who didn't (n=52)(53.6%) was close to each other in those without symptoms, the rate of patients who had an attack was significantly higher with symptoms (n=12)(92.3%). It is reported that the symptoms in FMF and COVID-19 diseases may have arisen as a result of similar inflammatory responses. Although the frequency and severity of attacks did not change among our patients with COVID-19 symptoms, the need to increase the colchicine dose and the higher rate of individuals who had an attack may be due to this clinical similarity. There are also reports on the use of colchicine in individuals diagnosed with COVID-19. Due to the small number of participants and the inability to clearly evaluate other factors that may affect the course of the disease, more comprehensive studies are needed on this subject.