Chemical and Microbiological Changes of Expired Platelet Concentrate.

Background: Platelets are a commonly used blood component to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. They are stored at room temperature (22-24°C) for five days unless specific measures are taken to extend the shelf life to seven days or more. After five days, this study evaluated platelet units' biochemical changes and bacterial growth. Study Design and Methods: Platelet concentrate was collected from 30 random donors: 8 females and 22 males. The collected samples were then placed on an agitator at room temperature and tested for their pH, protein content, and glucose levels using Roche Combur 100 Test® Strips. The Haemonetics eBDS™ System was used for bacterial detection. The measurements were taken on day five as the control and then repeated on days 7, 9, and 11 to observe any changes. On days 5 and 7, all parameters remained unchanged. However, glucose levels significantly changed (p=<0.0001) on days 9 and 11. Regarding pH, a significant change was observed on day 9 (p=0.033) and day 11 (p=0.0002). Results: There were no significant changes in all parameters on days 5 and 7. However, glucose was substantially changed (p=<0.0001) on days 9 and 11. For pH, there was a significant change in pH on day 9 (p=0.033) and day 11 (p=0.0002). Discussions: Our study found that platelet concentrate extension is possible for up to seven days. However, further studies are needed to evaluate platelet function during expiry time and to assess the stability of platelet morphology and function.

ABO, Rh, and Kell Blood Group Antigen Frequencies in Blood Donors of Taif City, Saudi Arabia.

BACKGROUND: The ABO, Rh, and Kell blood group antigens are clinically significant. Knowledge of antigen frequencies is important to assess the risk of alloimmunization and to guide the probability of finding antigen-negative donor blood. Patients that lack such antigens may produce antibodies that may cause transfusion reaction. The frequencies of ABO, Rh, and Kell antigens in Taif city, Saudi Arabia have not yet been determined. This study aims to assess the frequencies of ABO, Rh, and Kell blood group antigens among Saudi donors in Taif city, Saudi Arabia. METHODS: A retrospective study was conducted on 2,073 Saudi blood donors of both genders from May 2016 to May 2019. The data were collected, and calculations were done to determine the frequencies of ABO, Rh, and Kell blood group antigens. RESULTS: Of the 2,073 donors, the ABO blood groups of the donors were O (53.8%), A (24.9%), B (16.4%), and AB (4.6%). Rh-positive samples were (87.8%) and (12.1%) were Rh-negative. The most common Rh antigen was e (95.8%), followed by the c and C antigens (81.7% and 62.3%, respectively). The lowest Rh antigen frequency was E (31.3%). DCce was the most prevalent phenotype (29.5%). The KEL1 (K) antigen was determined in (22.1%) of the donors. CONCLUSIONS: This is the first study conducted in Taif city to assess the frequency of ABO, Rh, and Kell antigens among Saudi blood donors. This study provides the first step to create a regional donor database to obtain negative antigen blood units for patients with unexpected antibodies and to offer compatible bloods for multi-transfused cases by designing red cell panels.

Deferasirox versus deferoxamine in managing iron overload in patients with Sickle Cell Anaemia: a systematic review and meta-analysis.

OBJECTIVES: To examine the efficacy of deferasirox (DFX) by comparison with deferoxamine (DFO) in managing iron overload in patients with sickle cell anaemia (SCA). METHODS: Online databases were systematically searched for studies published from January 2007 to July 2022 that had investigated the efficacy of DFX compared with DFO in managing iron overload in patients with SCA. RESULTS: Of the 316 articles identified, three randomized clinical trials met the inclusion criteria. Meta-analysis of liver tissue iron concentration (LIC) showed that iron overload was not significantly higher in the DFX group compared with DFO group (WMD, -1.61 mg Fe/g dw (95% CI -4.42 to 1.21). However, iron overload as measured by serum ferritin was significantly lower in DFO compared with DFX group (WMD, 278.13 µg/l (95% CI 36.69 to 519.57). Although meta-analysis was not performed on myocardial iron concentration due to incomplete data, the original report found no significant difference between DFX and DFO. CONCLUSION: While limited by the number of studies included in this meta-analysis, overall, the results tend to show that DFX was as effective as DFO in managing iron overload in patients with SCA.

Impact of COVID-19 on Blood Donation and Supply: A Multicenter Cross-Sectional Study from Saudi Arabia.

BACKGROUND: The coronavirus disease-19 (COVID-19) pandemic caused a major impact on blood donation process and supply globally. A lockdown management procedure was launched nationally in Saudi Arabia to manage this global health crisis. The main aim of this study was to determine the effect of COVID-19 lockdown on blood donation services and supply in different regions of Saudi Arabia. Study Design and Methods. A retrospective cross-sectional study was conducted in the blood bank centers of 5 major cities including Riyadh, Jeddah, Dammam, Hail, and Jizan in Saudi Arabia. Demographic and blood characteristics were retrieved from the first 6 months of 2019 (January-June) and compared to the same period of 2020. RESULTS: Our findings showed variation in the characteristics of blood donation and supply among the centers surveyed, as some of these centers were adversely affected, while others showed an increase in the availability of blood products during the pandemic. For example, Jeddah's center was significantly affected by COVID-19 lockdown whereas Hail's center showed a significant increase in the analyzed characteristics of blood donation services in 2020 compared to 2019. Overall, there was no major difference among the surveyed centers between 2020 and 2019, and this might be due to the effective management of blood supply and transfusion. Discussion. Although blood supply and transfusion practice was slightly affected at various degree among the surveyed centers, the whole process did not show a significant effect on the overall outcome. This is in fact due to the proper preparedness, management of blood requirements and supplies, and efficient response of the surveyed centers in Saudi Arabia.

The association between different blood group systems and susceptibility to COVID-19: a single center cross-sectional study from Saudi Arabia.

Background: Since the beginning of COVID-19 pandemic, many associated factors have been investigated to clarify the susceptibility and severity among the affected individuals. Biological markers can play an important role in identification of individual susceptibility to such pandemic. Growing evidence suggest the influence of different blood group systems on susceptibility to COVID-19 virus, with a particular blood type conferring selection advantage. Objectives: The study aimed to determine the association of ABO, Rhesus (D) and P1 blood groups with COVID-19 susceptibility in Taif city, Western Saudi Arabia. Methods: ABO, D and P1 blood antigens were determined in 104 blood samples of COVID-19 patients versus 100 control samples using either automated immunohematology analyser or test tube method. Statistical differences between patients and control samples were calculated based on p-value where results of ≤ 0.05 were considered significant. Results: O+ve blood group constituted the predominant type among the studied samples. Determination of P1 antigen showed significant association where Anti-P1 was positive in 76.9% of patients compared to 61.0% of controls with a P value of 0.01 conferring the susceptibility of P1+ve patients to COVID-19. Conclusion: Although our study showed no significant association between ABO and D, and susceptibility to COVID-19, there was a significant association between P1+ve and COVID-19. P1+ve participants were 2.131 times more associated with the risk of COVID-19 infection than those with Anti P1-ve. Thus, P1 antigen can be used as a biological marker for identification of individuals susceptibility to COVID-19. It is strongly advised that such individuals should consider extra protective measures. Further studies on other contributing factors should also be considered for more scientific clarity.

Prevalence of Red Blood Cell Alloimmunization Among Beta-Thalassemia and Sickle Cell Anemia Patients: a Study from Saudi Arabia.

BACKGROUND: The periodic transfusion of red blood cells (RBCs) is an essential supportive therapy for beta-thalassemia and sickle cell anemia (SCA) patients. Nevertheless, this therapy can cause some adverse effects, such as RBC alloimmunization. This study aimed to determine the prevalence of RBC's alloimmunization and frequency of Rhesus (Rh) and Kell (K) antigens among SCA and beta-thalassemia patients in Al-Madinah region of Saudi Arabia. METHODS: A cross-sectional study was conducted on 137 multiple transfusion patients with SCA and beta-thalassemia, who attended two of the largest hospitals located in Al-Madinah, Saudi Arabia. Demographic and medical data were collected from medical files of patients. Blood samples were collected from patients and Rh and Kell typing were done. Also, antibody screening and identification tests were performed. RESULTS: There were 66 (48.18%) pediatric patients with mean age of 7.8 years, while adult patients were 71 (51.82%) with mean age of 24.5 years. RBC alloantibodies were found in 9 patients (6.57%), and the most frequent alloantibody was anti-K. Antigen e (99.05%) was the most common antigen, while the least common was the antigen K (3.81%). The association between age (pediatric and adult) and alloimmunization rates was insignificant. CONCLUSIONS: Although the RBC alloimmunization rate among SCD and beta-thalassemia patients in this study is low compared to other studies in Saudi Arabia and countries all over the world, it still warrants more attention. Phenotyping of donors/recipients' RBCs for K and Rh subgroups may reduce the risk of alloimmunization and increase the efficiency of blood transfusion.

Angiogenesis Biomarkers in Ischemic Stroke Patients.

INTRODUCTION: Stroke is a global health issue, and ischemic stroke is among the most common strokes affecting many people worldwide. Throughout ischemic stroke, various immune cells counter its effect by releasing cytokines, chemokines, and angiogenic molecules. These molecules can work as potential biomarkers in the diagnosis and monitoring of the progress of ischemic stroke. The current study investigated the use of angiogenic molecules as biomarkers in ischemic stroke patients. METHODS: The samples were obtained from twenty healthy subjects and nineteen patients with ischemic stroke. Multiplex assay was used to measure the serum levels of angiogenic biomarkers, including endoglin, VEGF-A, endothelin-1, G-CSF, and angiopoietin-2. All data were analyzed using an unpaired Student's t-test. Correlations between measured parameters were made using Pearson correlations. RESULTS: Angiopoietin-2, VEGF-A, endothelin-1, and endoglin levels in stroke patients were significantly higher compared to healthy controls. Nevertheless, G-CSF level showed a non-significant increase in patients compared to controls. The correlation coefficient of measured angiogenic biomarkers among patients showed significant correlations between endoglin, angiopoietin, VEGF-A, and endothelin-1. DISCUSSION: The angiogenic factors were significantly increased in patients with ischemic stroke, which may help in the early detection of ischemic stroke and consequently prompt treatment and better prognosis.

Human Wharton's Jelly Stem Cell Secretions Inhibit Human Leukemic Cell Line K562 in vitro by Inducing Cell Cycle Arrest and Apoptosis.

Emerging resistance to the tyrosine kinase inhibitors that target the BCR-ABL1 oncoprotein has prompted research for novel therapeutics against chronic myeloid leukemia (CML). Herein, we evaluated the tumor inhibitory properties of the human Wharton's jelly stem cells (hWJSCs) co-culture (hWJSC-CC) and their extracts, namely, the hWJSC-conditioned medium (hWJSC-CM; 100%) and hWJSC-lysate (hWJSC-L; 15 µg/ml), on a CML cell line K562 in vitro. The hWJSCs expressed mesenchymal stem cell (MSC)-related cluster of differentiation (CD) markers and demonstrated mesodermal tissue differentiation potential. The cell metabolic activity showed a mean maximal decrease in the K562 cells by 49.12, 41.98, and 68.80% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, respectively, at 72 h. The sub-G1 population in the cell cycle was decreased by 3.2, 4.5, and 3.8% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, whereas the G2/M cell population was increased by 13.7 and 12.5% with the hWJSC-CM and hWJSC-L, respectively, at 48 h. Annexin V-allophycocyanin (APC) assay showed an increase in the apoptotic cells by 4.0, 3.9, and 4.5% at 48 h. The expression of pro-apoptotic BAX and CASP3 genes were increased, whereas BIRC5 (Survivin) was decreased compared with the control. The pro-inflammation-related genes, namely, IFN-γ, TNF-α, IL-1ß, IL-6, IL-8, and IL-12A, were decreased, whereas the anti-inflammatory genes, namely, IL-4 and IL-10, were increased following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L at 48 h. Multiplex bead-based cytokine assay also demonstrated decreases in the pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, IL-6, and IL-12) and an increase in the anti-inflammatory cytokine (IL-10) compared with the control. The pro-inflammatory cytokine IL-8 showed an increase with the hWJSC-CC and decreases with both the hWJSC-CM and the hWJSC-L. The hWJSCs and their extracts inhibited the K562 cells by causing cell cycle arrest and inducing apoptosis via the soluble cellular factors. However, an in vivo evaluation is necessary to unravel the true potential of the hWJSCs and their extracts before its use in CML inhibition.

A Cross-Sectional Study of Detection of Beta Globin (HBB) Haplotypes Among Beta Thalassemia Patients.

Introduction Beta-thalassemia is among the most common monogenic disorders in the Arabian Peninsula. This study aimed to investigate the ß-globin (HBB) haplotypes among ß-thalassemia patients in Saudi cohort which have potential implications in understanding the clinical care of patients and population genetic factors associated with ß-thalassemia. Methods We analyzed 60 ß-thalassemia patients. Male/female distribution for ß-thalassemia was 58.33%/41.66%. Results of hematological parameters and indices were obtained from the database. HBB haplotyping assay was performed for four specific loci of the HBB gene cluster using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results HBB haplotyping assay identified three novel patterns namely haplotype 1, haplotype 2, and haplotype 3 and three common African haplotypes including Benin, Senegal, and Cameron. The frequency of haplotype 1 was the highest among the studied samples (62%, n = 37) with 56.76% (n = 21) observed in males compared to 43.24% (n = 16) in females. This was followed by Senegal, haplotype 2, Benin and haplotype 3 with similar percentage, and Cameron haplotype with 18%, 12%, 3% and 2%, respectively. The relationship between these haplotypes and various hematological parameters was calculated and our study found no significant relationship (p-value >0.05). Conclusion Our study indicated the importance of finding out types of ß-globin haplotypes as novel types being discovered. Though no statistically significant association was identified among all the haplotypes in terms of hematological parameters, Cameroon or Benin haplotypes had the mildest form because they have the highest means among all parameters. Further studies need to be carried out on a larger population to detect the frequency of each specific mutation in each haplotype among ß-thalassemia patients. This would help to re-address the question of the origin(s) of the ß-thalassemia.

Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and Other Coronaviruses: A Genome-wide Comparative Annotation and Analysis.

Novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) causes mild to severe respiratory illness. The early symptoms may be fever, dry cough, sour throat, and difficulty in breathing which may lead to death in severe cases. Compared to previous outbreaks like SARS-CoV and Middle East Respiratory Syndrome (MERS), SARS-CoV2 disease (COVID-19) outbreak has been much distressing due to its high rate of infection but low infection fatality rate (IFR) with 1.4% around the world. World Health Organization (WHO) has declared (COVID-19) a pandemic on March 11, 2020. In the month of January 2020, the whole genome of SARS-CoV2 was sequenced which made work easy for researchers to develop diagnostic kits and to carry out drug repurposing to effectively alleviate the pandemic situation in the world. Now, it is important to understand why this virus has high rate of infectivity or is there any factor involved at the genome level which actually facilitates this virus infection globally? In this study, we have extensively analyzed the whole genomes of different coronaviruses infecting humans and animals in different geographical locations around the world. The main aim of the study is to identify the similarity and the mutational adaptation of the coronaviruses from different host and geographical locations to the SARS-CoV2 and provide a better strategy to understand the mutational rate for specific target-based drug designing. This study is focused to every annotation in a comparative manner which includes SNPs, repeat analysis with the different categorization of the short-sequence repeats and long-sequence repeats, different UTR's, transcriptional factors, and the predicted matured peptides with the specific length and positions on the genomes. The extensive analysis on SNPs revealed that Wuhan SARS-CoV2 and Indian SARS-CoV2 are having only eight SNPs. Collectively, phylogenetic analysis, repeat analysis, and the polymorphism revealed the genomic conserveness within the SARS-CoV2 and few other coronaviruses with very less mutational chances and the huge distance and mutations from the few other species.

Hemoglobin Disorders Among Anemic Patients: a Cross-Sectional Study from Jeddah City, Western Saudi Arabia.

BACKGROUND: Hemoglobinopathies and thalassemia are defined as a group of inherited blood disorders characterized by a variable degree of anemia with a wide spectrum of clinical symptoms. They are commonly found in the Mediterranean area, sub-Sahara Africa, Middle East, Central India, and Southeast Asia with an estimation of 400,000 babies born annually with serious hemoglobinopathies. Of those, 90% of the births occur in underdevel-oped or developing countries. This study was undertaken to investigate the prevalence of hemoglobin disorders among anemic patients who visited a tertiary care setting represented by King Abdulaziz University Hospital. METHODS: This is a cross sectional study which investigated blood samples from 668 anemic patients for possible causes of anemia. This investigation involved the use of complete blood count, hemoglobin separation using capillary electrophoresis, and measurement of nutritional elements commonly investigated for anemia. RESULTS: We found that the frequency of different types of hemoglobinopathies and thalassemia among the subjects were as follow; normal (HbAA) 439 (65.7%); Sickle Cell Trait (HbAS) 65 (9.7%); Sickle Cell Anemia (HbSS) 63 (9.4%); ß-thalassemia trait 48 (7.2%); Hb S/ß 27 (4.0%); HbH 7 (1.0%); HbE 6 (0.9%); beta-thalassemia major 6 (0.9%); Hb E/beta-thalassemia 4 (0.6%); HbC 1 (0.1%); HbD 1 (0.1%) and HbSC 1 (0.1%). CONCLUSIONS: The findings of this study emphasize the necessity of increasing public health education, neonatal and adult screening programs, as well as nutritional guidance and plans to start the eradication of this burden.

Knowledge and attitude among healthcare workers towards COVID-19: a cross sectional study from Jeddah city, Saudi Arabia.

INTRODUCTION: The emergence of the new Coronavirus disease 2019 (COVID-19) has caused a major impact on global health system. This cross-sectional study was designed to appraise the knowledge and attitude of healthcare workers towards COVID-19 and find out their understanding about clinical aspects of the infection. METHODOLOGY: A total number of 1023 of healthcare workers responded to an online questionnaire and provided their data between February and March, 2020 in Jeddah city, Western province, Saudi Arabia. The questionnaire was distributed to physicians, nurses, pharmacists, technical staff and administrative staff working in clinical settings. RESULTS: Results revealed that mean scores for knowledge and attitude were 20.793 ± 2.436 and 4.744 ± 0.297 respectively. More than 88% of participants displayed positive knowledge and attitude towards COVID-19. Knowledge data showed that social media and the workplace, were the main sources of information for the majority of respondents. Approximately 99.12% of respondents were aware of the viral pandemic, and the causative agent. Statistically significant association was found when compared the demographic characteristics with the mean knowledge while no statistical significance was observed when compared demographic characteristics with the mean attitude score except with marital status. CONCLUSION: This study showed that healthcare workers had sufficient knowledge and positive attitude towards COVID-19. However, hospital staff should be periodically given sufficient training to effectively cope with such outbreaks in the future.

Dimethoate Induces DNA Damage and Mitochondrial Dysfunction Triggering Apoptosis in Rat Bone-Marrow and Peripheral Blood Cells.

Dimethoate (DM) is an organophosphorus (OP) pesticide with wide use in the pest control. Its persistence in crops and soils could possibly cause adverse health consequences in humans as well as other non-target species. Since molecular studies confirming potential genotoxicity of DM have not been previously reported, the acute in vivo toxicological impact was evaluated in Wistar rats. Significant micronuclei induction and metaphase chromosome abnormalities in bone marrow cells exposed to three different DM doses (20, 40 and 60 mg/kg-bw) at multiple treatment durations (24, 48 and 72 h) indicated positive dose response relationship, confirming its genotoxic and cytotoxic potential. Significant mitotic index decrease was seen in dosed animals compared to vehicle control. The study used peripheral blood comet assay, indicating DM-mediated damage to DNA at all exposure levels in a time responsive manner. These assays were found to be an effective, precise, and fast technique with applied value in biomonitoring studies. Cell cycle and apoptosis along with mitochondrial membrane potential (MMP) in flow cytometric analyses confirmed DM exposure decreased MMP, affected the cell cycle, and inflicted DNA damage, which led to cellular apoptosis of leukocytes culminating into immunotoxic effects. The in silico experiments consequently augmented that DM showed acceptable binding energy value for Cyclin A2, suggesting that it could inhibit the cell cycle progression by inhibiting cyclin A2.

Detection of BCL11A and HBS1L-MYB Genotypes in Sickle Cell Anemia.

Sickle Cell Anemia (SCA) is one of the most common monogenic disorders worldwide. Molecular modifiers of clinical symptoms play an essential role in the amelioration of the effects of the disease. Single Nucleotide Polymorphisms (SNPs) of the BCL11A gene and within the HBS1L-MYB intergenic region, which are located outside the ß-globin locus on chromosome 11, are considered to be genetic modifiers that are associated with elevated levels of foetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin, HbS. The work reported here aimed to detect the most common SNPs of BCL11A and HBS1L-MYB related to HbF in SCA patients and to estimate the frequency of occurrence of these genotypes. A total of 132 SCA patients whose condition was stable were recruited from Jeddah city, Saudi Arabia. SNPs at site locus rs4671393 on BCL11A, and at loci rs28384513 and rs9399137 on HBS1L-MYB were identified using TaqMan genotyping assay. Haematological parameters were analysed based on complete blood count and haemoglobin separation using the capillary electrophoresis technique. Highly significant differences in the diagnostic haematological parameters, including all blood-cell types and HbF, were observed between the study cohort and control groups. We also found that BCL11A rs4671393 genotypes of GG and AG were more likely to show increases in HbF levels than other genotypes. In addition, a strong relationship was found between HBS1L-MYB rs9399137 and rs28384513 genotypes in the cohort, whereas no significant association was observed between BCL11A rs4671393 variant and other variants. Our study highlights the importance of investigating genetic determinants that play roles in the amelioration of the severity of clinical symptoms and complications of SCA.

A comprehensive study of mobile-health based assistive technology for the healthcare of dementia and Alzheimer's disease (AD).

Assistive technology (AT) involvement in therapeutic treatment has provided simple and efficient healthcare solutions to people. Within a short span of time, mobile health (mHealth) has grown rapidly for assisting people living with a chronic disorder. This research paper presents the comprehensive study to identify and review existing mHealth dementia applications (apps), and also synthesize the evidence of using these applications in assisting people with dementia including Alzheimer's disease (AD) and their caregivers. Six electronic databases searched with the purpose of finding literature-based evidence. The search yielded 2818 research articles, with 29 meeting quantified inclusion and exclusion criteria. Six groups and their associated sub-groups emerged from the literature. The main groups are (1) activities of daily living (ADL) based cognitive training, (2) monitoring, (3) dementia screening, (4) reminiscence and socialization, (5) tracking, and (6) caregiver support. Moreover, two commercial mobile application stores i.e., Apple App Store (iOS) and Google Play Store (Android) explored with the intention of identifying the advantages and disadvantages of existing commercially available dementia and AD healthcare apps. From 678 apps, a total of 38 mobile apps qualified as per defined exclusion and inclusion criteria. The shortlisted commercial apps generally targeted different aspects of dementia as identified in research articles. This comprehensive study determined the feasibility of using mobile Health based applications for dementia including AD individuals and their caregivers regardless of limited available research, and these apps have capability to incorporate a variety of strategies and resources to dementia community care.

Computational Analysis of Protein Structure Changes as a Result of Nondeletion Insertion Mutations in Human ß-Globin Gene Suggests Possible Cause of ß-Thalassemia.

Beta-thalassemia is described as a group of hereditary blood disorders characterized by abnormalities in the synthesis of beta chains of hemoglobin. These anomalies result in different phenotypes ranging from moderate to severe clinical symptoms to no symptoms at all. Most of the defects in hemoglobin arise directly from the mutations in the structural ß-globin gene (HBB). Recent advances in computational tools have allowed the study of the relationship between the genotype and phenotype in many diseases including ß-thalassemia. Due to high prevalence of ß-thalassemia, these analyses have helped to understand the molecular basis of the disease in a better way. In this direction, a relational database, named HbVar, was developed in 2001 by a collective academic effort to provide quality and up-to-date information on the genomic variations leading to hemoglobinopathies and thalassemia. The database recorded details about each variant including the altered sequence, hematological defects, its pathology, and its occurrence along with references. In the present study, an attempt was made to investigate nondeletion mutations in the HBB picked up from HbVar and their effects using the in silico approach. Our study investigated 12 nucleotides insertion mutations in six different altered sequences. These 12 extra nucleotides led to the formation of a loop in the protein structure and did not alter its function. It appears that these mutations act as 'silent' mutations. However, further in vitro studies are required to reach definitive conclusions.

The Effect of Nonsense Mediated Decay on Transcriptional Activity Within the Novel ß-Thalassemia Mutation HBB: c.129delT.

Premature termination codons (PTCs) are caused by mutations in the coding sequences of functional genes resulting in an incorrect assignment of a stop codon. Abnormal and truncated proteins are prevented from being translated due to the rapid degradation of mRNA carrying these mutations by an RNA surveillance mechanism referred to as nonsense mediated decay (NMD). Recently, a novel mutation in a patient from Thailand with the clinical diagnosis of Hb E (HBB: c.79G > A)/ß(0)-thalassemia (Hb E/ß(0)-thal) and whose molecular analysis demonstrated a novel mutation in the ß-globin gene, HBB: c.129delT, was reported. The result of this deletion is a frameshift (FSC) resulting in a PTC at codon 60. We have analyzed the impact of this mutation on transcription and translation of the affected ß-globin gene using an in vitro model. The quantitative real-time polymerase chain reaction (qReTi-PCR) analysis revealed that this nucleotide mutation resulted in marked mRNA degradation, which we attributed to the NMD mechanism and as such, the expected deleterious truncated HBB was not generated. This result highlights a valuable application of our in vitro gene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations.

Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Boghé (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions.

In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (-55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.

Molecular characterization of Hb Hamilton Hill (HBA2: c.388delC), a novel HBA2 variant generating a premature termination codon and truncated HBA2 chain.

In recent years, the identification of α-thalassemias caused by nondeletional mutations has increased significantly due to the advancement of sensitive molecular genetics tools. We report clinical and experimental data for a novel frameshift mutation caused by a single base deletion at position 388 in exon 3 of the α2-globin gene (HBA2: c.388delC; Hb Hamilton Hill), resulting in the phenotype of α-thalassemia (α-thal). Hb Hamilton Hill was identified in an adult female of unknown ethnicity investigated for unexplained microcytosis. Direct DNA sequencing of the HBA2 gene revealed a heterozygous mutation, HBA2: c.388delC, and the molecular effect of this mutation was assessed experimentally using our previously described in vitro model. The experimental analysis involved transfection of a human bladder carcinoma (5637) cell line with expression vectors carrying either HBA2-wild type (HBA2-WT) or HBA2: c.388delC followed by total RNA purification and cDNA synthesis. Both wild type and mutant gene expression was studied and compared at the transcriptional and translational levels using quantitative real time polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC), respectively. Our experimental data showed a significant reduction by 25.0% (p = 0.04) in the transcriptional activity generated from HBA2: c.388delC compared to HBA2-WT. As a result of this base deletion, a frameshift in the open reading frame generates a premature termination codon (PTC) at codon 132 of exon 3 resulting in the formation of a truncated α-globin chain. The truncated α-globin chain, observed by the IFC technique, is most likely unstable and undergoes a rapid turnover resulting in the thalassemic phenotype.