Human Beta-globin Gene Research Articles

Effect of human β-Globin second intron on transient gene expression in mammalian cell lines

Exogenous protein expression in mammalian cells is necessary to produce therapeutic proteins and modern medical applications like developing DNA vaccines and gene therapy. This study examines the human-Globin (hBG) second intron's capacity for intron-mediated enhancement (IME) in various mammalian cell lines. Our study's main aim is to investigate the effect of the incorporation and arrangement of the second intron of the human Beta-globin gene into the pVAX-1 expression cassette on improving the expression of foreign genes. Two plasmids were constructed, one with the hBG second intron positioned upstream and the other downstream in the expression cassette. EGFP expression was evaluated at the mRNA and protein levels after transfection using Lipofectamine 2000 using One-way ANOVA analysis. Results showed that the pVAX-1 harbouring the hBG second intron did not lead to enhanced transient EGFP expression and did not exhibit Intron Mediated Enhancement (IME) in tested mammalian cell lines. Further investigations are necessary to understand factors contributing to the lack of enhancement and explore alternative intron options for optimizing foreign gene expression in cell lines.

Synthesis, characterization, anti-cancer evaluation, and DNA-binding study of new bay-substituted perylene derivatives

Two new perylene derivatives 1,7-di(3,5-diamino-pyrimidoxyl) perylene-3,4,9,10- tetracarboxylic acid bisanhydride (4) and 1,7-di(2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl]) ethoxyperylene-3,4,9,10-tetracarboxylic bisanhydride (6) have been synthesized. We aimed to study their interactions with G-quadruplex (G4) structures as potent G4 ligands and telomerase inhibitors. We used a PCR-amplified guanine-rich region from the human beta-globin gene, oligonucleotide from human telomeres (a-coreTT), an oncogene (c-kit), and SK-HEP-1 adenocarcinoma cells to characterize those compounds’ binding and stabilizing abilities to G4 structures and anti-cancer potential. All results obtained through UV-visible and fluorescence spectroscopies, agarose gel electrophoresis, and MTT assay on SK-HEP-1 adenocarcinoma cells were in good agreement. Compounds 4 and 6 are promising DNA-binding and cytotoxic compounds with a relatively antiproliferative effect on the selected tumour. In all studies, the formal positive charge carrier, compound 6, showed higher activity in terms of anti-cancer effects. These results may help elucidate the feasibility of the perylene derivatives as future chemo-therapeutic agents.

Comparison of Lysis Buffers for Saliva DNA Extraction using Modified Magnetic-Silica Nanoparticle Beads

Our previous study revealed that modified MAGSi (magnetic-silica nanopartivel beads) had various sizes and shapes that affected the DNA extraction process. In this study, we combined our MAGSi with several kinds of lysis buffer for DNA extraction to analyze the result. Human saliva DNA was extracted using three kinds of MAGSi. Each of them was made differently. We used ATL buffer, Triton Buffer, and without buffer for cell lysis. The concentration and purity of the extracted DNA were analyzed using spectrophotometry. Extracted DNA also was used as template DNA for DNA amplification using beta globin gene. This study revealed that our MAGSi-1 provided the best result for DNA concentration and purity. This study also revealed that DNA extraction using lysis buffer produced less concentration yield and purity value but it was more consistent. The concentration and purity value were different but consistent. All the extraction can be used as DNA template for amplification of human beta globin gene. Further studies are still needed to determine the effectiveness and consistency of the lysis buffer and our MAGSi.

In-silico structural and phylogenetic analysis of human beta globin gene family

There are 5 members of the human beta globin gene family. They have blood transportation function. There are some diseases related to the mutation in these gene. the common disease related to mutation these genes are sickle cell anemia, thalassemia etc. The structural and phylogenetic analysis is performed by using some bioinformatics tools. The gene sequence is retrieved and run under some tools to perform the specific analysis. The analysis performed include are similarity with other organism, determination of conserved domains and motifs, multiple sequence alignment, generation of phylogenetic tree, identification of exons and introns and determination of restriction sites. And the tools used for these analyses are BLASTP, pfam, clustal omega, MEGA7, gene structure display tool and serial cloner respectively. This study came out with a result that the genes diverged due to duplication and then mutation. The gene contain mostly non coding portion but there are 3 exons and 2 introns. The length of 2 introns is far more than that of 3 exons. In future the techniques like CRISPR/cas gene editing tool will be used to treat the disease related the beta globin gene family. Key words:human beta globin gene, bioinformatics study of HBB, genome wise study of HBB, Beta subunit, hemoglobin beta subunit.

Engineered Stem Cell Antibody Paired Evasion-2 (ESCAPE-2): Paired HSC Epitope Engineering and Direct Editing of Sickle Allele for Antibody-Mediated Autologous Hematopoietic Stem Cell Therapy Conditioning for the Treatment of Sickle Cell Disease

Introduction of the naturally-occurring Hb G-Makassar variation in the human betaglobin gene (HBB) through base editing to eliminate the polymerizing sickle protein, HbS, the major molecular driver for sickle cell disease (SCD), represents a promising new paradigm for the potential treatment of individuals with this disease. While several ex vivo delivery of gene editing technologies for SCD are advancing in the clinic, several challenges remain for this potentially transformative cell therapy, namely the requirement of myeloablative conditioning necessary for autologous hematopoietic stem cell transplant (HSCT) to be fully effective. Aiming to address this, we have developed a strategy whereby a monoclonal antibody (mAb) that binds to CD117, a critical receptor on HSPCs essential for viability, is coupled with multiplexed engineered HSCs (eHSCs). Our eHSCs are designed to both evade mAb binding and harbor the Makassar therapeutic edit. Our engineered stem cell antibody paired evasion (ESCAPE) strategy is designed to provide a non-genotoxic alternative to current conditioning regimens and a potential curative therapy for the treatment of SCD. In our ESCAPE-1 approach, we demonstrated that we could successfully engineer epitopes of the stem cell marker and receptor, CD117, by introducing missense mutations in the ligand binding domain that preserved normal CD117 function and prevented the binding of a high affinity antibody against wildtype (WT) CD117. We leveraged this selective advantage and screened guides that could install missense mutations that covered the entirety of the CD117 ectodomain and were compatible in a multiplexed edited HSC with a next-generation adenine base editor that could install the therapeutic Hb G-Makassar edit. We generated a library of CD117 variants via highly-efficient base editing of the CD117 gene, in mobilized peripheral blood CD34+ HSPCs and then counter-screened these variants against a library of mAbs that bound to wild-type CD117. We identified an antagonist mAb that interacts in the ligand binding domain of CD117, blocking normal SCF ligand binding to the WT receptor, but was unable to bind eHSCs. In vitro biological de-risking of this engineered epitope showed no significant changes in receptor function in response to SCF, or during in vitro differentiation and colony forming unit (CFU) assays when compared to WT CD117. Furthermore, we could demonstrate that this engineered epitope allowed for evasion of cytotoxicity in vitro when edited HSCs were exposed to increasing concentrations of the mAb. We next optimized the multiplex editing conditions in HSCs and achieved outcomes where all cells harboring a CD117 edit, also included at least one Makassar edit. We previously demonstrated that sickle cells harboring even a single Makassar edit exhibited reduced sickling when exposed to hypoxia in vitro. Subsequent Fc receptor silencing modifications were also optimized to reduce any mast cell degranulation that could be the result of antibody-based binding to Fc-receptor on mast cells. Altogether, our ESCAPE-2 strategy is compatible with the installation of the therapeutic Makassar edit that, when combined with a mAb as a conditioning agent, represents a promising potential alternative to busulfan-based myeloablative regimens in an autologous HSCT setting for the treatment of SCD.

PB2212: VARIANT HAEMOGLOBIN ASSESSMENT AND CORRELATION WITH PHENOTYPIC PRESENTATION AT DIAGNOSIS IN SICKLE CELL DISEASE: A SINGLE CENTRE RETROSPECTIVE ANALYSIS OF CENTRAL GUJARAT COHORT

Background: Sickle cell disease (SCD) is caused by heritable point mutation at codon 6 of the human beta-globin gene widely distributed around the world. According to 2011 census, in India, there is a large tribal community of about 180 million. Among them18 million people carry the sickle cell trait and ~140,000 suffer from SCD. Gujarat State of India has a population of about 62 million, there are ~8.9 million tribal populations, among whom ~9, 00,000 people with Sickle cell Trait and ~70,000 people with SCD are expected. Though phenotypic heterogeneity of SCD is well known across the India, there is limited data on phenotypic presentation with variant haemoglobin correlation on large cohort from Central Gujarat. Aims: To correlate variant haemoglobin pattern with phenotypic presentation at diagnosis in SCD. Identify laboratory and clinical parameters to predict risk of complications in SCD. Methods: Five Hundred and seventy three patients registered at Haemato Oncology Care Centre (HOCC), Vadodara, Gujarat between August 2012 to December 2021, and diagnosed to have SCD were included in this study. This cohort represents tribal belt covering Bharuch,Narmada,Vadodara,Panchmahal,Dahod,Sabarkantha and Banaskantha districts of Central Gujarat. Diagnosis was based on clinical profile, complete blood counts (CBC) and peripheral smear evaluation along with variant haemoglobin assessment by High Performance Liquid chromatography / Capillary electrophoresis. Relevant Biochemistry investigations were done along with radiological assessment of hip joints in patients with specific symptoms.Osteonecrosis of Femur head (ONFH) was diagnosed according to Ficat and Arlet classification for ONFH. Recurrent Vaso occlusive episode(R-VOE) was labelled if there are >3 VOE/yr. Occasional VOE (O-VOE) labelled if <3 VOE/yr.Blood transfusion dependency was labelled if > 3 transfusion /yr required to maintain HB > 7 gm%.Splenomegaly was noted as palpable spleen below costal margin. Results: A total 573 SCD patients were evaluated. The median age was 20 years (range: 1-74) with 328 (57%) males and 245 (43%) females.HB was < 10 gm% in 378 (66%),HB <7 gm% in 78(14%),between7gm% to 10 gm% in 300(52.4%).315/378 (83%) had HBA <5%,Only 5/378 (1.32%)had Ferritin <20ng/ml.None had high indirect bilirubin with low HB. Transfusion dependency was found in 28(5%),HBA was 0%,<5%,6 to 10%,>10% in 12/28 (43%),7/28 (25%),6/28(21%),3/28(11%) respectively. R-VOE observed in 207 (36%),O-VOE in 259(45%),No VOE in 107(19%).Splenomegaly was found in 268(47%).HBF <10% in 86 (15%),10 to 20% in 258(45%)>20% in 229(40%).All Patients with no VOE 107(19%) had HBF >10% with palpable splenomegaly.ONFH was diagnosed on imaging in 108 (19%).101/108(93.5%) had HBF >10%.,25/108 (23%)had palpable splenomegaly. Summary/Conclusion: In our cohort High HBF with Splenomegaly was associated with no VOE. No protection for ONFH seen with high HBF or palpable spleen Regional increased prevalence of coinheritance of Thalassemia in SCD play important role in influencing clinical presentation and progression of complication like transfusion dependency and ONFH. In LMIC, Variant haemoglobin assessment with CBC, Retic count values can be used to predict probable coinheritance of Thalassemia; however DNA Mutation analysis on larger cohort can be done for validation. Region specific risk adapted optimization of treatment can modify natural history of disease

Modified bactofection for efficient and functional DNA delivery using invasive E. coli DH10B vector into human epithelial cell line

The attempts to attain satisfactory gene delivery ability from various engineered or non-engineered bacterial vectors have been reported by several groups. We have successfully developed a modified bactofection method for large-sized DNA delivery into human cell lines.Applying the same method of delivery, this study for the first time demonstrates an efficient and functional delivery of an 80-kb BAC clone carrying human beta-globin gene into HT1080 cell line via invasive E. coli DH10B vector. As a resultant, the modified bactofection method attained high DNA delivery even at low MOI and showed ∼5 folds increased delivery over the classic bactofection method. Upon comparing delivery efficiencies of the classic bactofection, modified bactofection, and Lipofectamine mediated transfection (10 μg/μl) at 1000 MOI, it showed 10%, 60%, and 14% GFP expression, respectively. Furthermore, functionality of the delivered BAC DNA was assessed and confirmed by RT-PCR analysis. To sum up, it can be envisioned that with careful study the modified bactofection method could successfully be employed for delivering various large-sized genes into human cells, be it for therapeutic purposes or studying their functional aspects. Besides, applying the modified bactofection method to discrete bacterial vector systems could help to achieve high efficiency DNA delivery of the large-sized genes.

Studies on molecular spectrum of beta thalassemia among residents of Chennai

&lt;abstract&gt; &lt;p&gt;Beta thalassemia is caused by a mutation in the human beta globin gene. More than 400 causative mutations have been characterized in the Hemoglobin Subunit Beta (HBB) gene. These causative mutations are present in the beta globin gene or the regulatory region. Though more than 400 causative mutations of HBB region have been described, rare and novel mutations are being reported in studies indicating the need for characterization of mutations in all regions and information regarding the same should be made available for successful implementation of prenatal diagnosis. The study aims to characterize the spectrum of beta thalassemia mutations in beta thalassemia heterozygous among residents of Chennai. A total of 5,207 cases were screened for beta thalassemia heterozygous by HPLC method. 387 beta thalassemia heterozygous identified by HPLC method were subjected to molecular DNA analysis by ARMS PCR technique and DNA Sanger sequencing for the characterization of causative beta thalassemia mutations. In the present study molecular characterization of beta thalassemia mutations revealed 30 different mutations with a high prevalence of IVS 1-5 (G-C) mutation, five new rare mutations viz., IVS II-1 (G&amp;gt;T), CD 37 TGG-TGA, IVS II 781 (C-G), CD114 CTG-CCG and Poly A (A-G) were diagnosed and reported first in India. One novel beta thalassemia mutation HBB.c319DelC was detected in the study. The diagnostic outcome of detecting the causative mutations for beta thalassemia imposes strong resources for developing easy and cheaper methods for prenatal diagnosis which will reduce the burden of disease.&lt;/p&gt; &lt;/abstract&gt;

Rapid Near Point-of-Care Assay for HLA-B*57:01 Genotype Associated with Severe Hypersensitivity Reaction to Abacavir.

The nucleoside reverse transcriptase inhibitor abacavir (ABC) is used commonly to treat young children with HIV infection and is a component of the fixed-dose-combination Triumeq®. ABC can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for HLA-B*57:01. Testing for HLA-B*57:01 before ABC initiation is standard-of-care in high-resource settings, but current tests are costly or difficult to access in resource-limited settings. To address these gaps, we developed an inexpensive simple-to-use rapid assay to detect HLA-B*57:01. We designed and optimized a multiplexed polymerase chain reaction (PCR) to amplify HLA-B*57 subtypes and the human beta-globin gene; employed probes and ligation to specifically tag the HLA-B*57:01 allele with biotin. Tagged-ligated products were detected by immunocapture in an enzyme-linked immunosorbent assay plate or lateral flow strip. Cell lines with known HLA genotypes were used to optimize the assay. The optimized assay was then compared with genotypes of clinical specimens (n = 60) determined by sequencing, with specimens enriched for individuals with HLA-B*57:01. The optimized assay utilizes 40-min 35-cycle multiplex PCR for B*57 and beta-globin; 20-min ligation reaction; and 15-min detection. Evaluation of the HLA-B*57:01 oligonucleotide ligation assay using clinical specimens had a sensitivity of 100% (n = 27/27 typed as B*57:01) and specificity of 100% (n = 33/33 typed as non-B*57:01) by visual interpretation of lateral flow strips. The cost is US$5.96/specimen. This rapid and economical assay accurately detects HLA-B*57:01 in clinical specimens. Use of this assay could expand access to HLA-B*57:01 genotyping and facilitate safe same-day initiation of ABC-based treatment.

Cedar Trial in Progress: A First in Human, Phase 1/2 Study of the Correction of a Single Nucleotide Mutation in Autologous HSCs (GPH101) to Convert HbS to HbA for Treating Severe SCD

Cedar Trial in Progress: A First in Human, Phase 1/2 Study of the Correction of a Single Nucleotide Mutation in Autologous HSCs (GPH101) to Convert HbS to HbA for Treating Severe SCD

Preclinical Development of HBB Gene Correction in Autologous Hematopoietic Stem and Progenitor Cells to Treat Severe Sickle Cell Disease

Sickle Cell Disease (SCD) is an autosomal recessive hemoglobinopathy affecting 70-100,000 people in the United States, and millions of people globally. The disease is caused by an adenine to thymidine single nucleotide variant in codon 6 in the human beta-globin gene (HBB gene) that leads to the substitution of glutamic acid by valine (“E6V”) in the beta-globin protein ultimatelyresulting in the formation of sickle beta-globin protein (HgbS) in red cells. While there are therapies like hydroxyurea and red blood cell transfusions that modify the course of the disease, the only curative treatment is allogeneic hematopoietic stem cell transplantation (allo-HSCT), which are available for only 15-20% of the SCD patients, severely limiting the application of allo-HSCT for patients who need it the most. Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for patients with SCD. An alternative, potentially curative, treatment strategy is to perform gene-editing by homologous recombination (HR) to directly correct the E6V mutation in the HBB gene in autologous hematopoietic stem and progenitor cells (HSPC) from sickle cell patients and then reinfuse these modified cells post-myeloablation. Here we present our IND-enabling preclinical development for CRISPR-Cas9 and rAAV6-mediated HBB genome editing of the E6V mutation in SCD-derived HSPCs as well as in plerixafor mobilized HSPCs from multiple healthy individuals. We demonstrate 30-75% E6V conversion in edited SCD-HSPCs that when differentiated into reticulocytes in vitro, corresponds in the change from HgbS to HgbA tetramers. HBB edited SCD-HSPCs displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 16 weeks post-transplant, where we identified that ~50% (range of 30-78%) of human cells were edited at the E6V position. Furthermore, by using a recombinant high fidelity Cas9 (HiFi), we were able to eliminate off-target activity as much as 20 fold in edited SCD-HSPCs while maintaining robust on-target activity. Because GCSF use is contraindicated in SCD patients in view of case reports of mortality and that there are ongoing clinical trials using plerixafor to mobilize HSPCs in SCD patients for gene therapy, we also performed IND-enabling HBB genome editing studies in plerixafor mobilized HSPCs from several healthy individuals. We achieved median gene targeting frequencies of ~25% (range of 17-27%) while maintaining high purity (CD34+ percentage >95%) and viability (>80%) across multiple independent experiments. CRISPR-Cas9 and rAAV6- HBB edited plerixafor-HSPCs displayed high potency as evidenced by multilineage methylcellulose progenitor colony formation and importantly, long-term multilineage B, myeloid, and T/NK cell engraftment in the bone marrow of NSG mice at 16-20 weeks post transplant, where a median of ~5% (range of 1-15%) of human alleles were modified at the E6V position. Moreover, In vitro erythroid differentiated human CD34+ HSPCs from NSG mouse bone marrow displayed editing frequencies of 2-15%. These results suggest long-term maintenance of HBB gene targeting in tri-lineage (myeloid/lymphoid/erythroid) plerixafor mobilized repopulating human hematopoietic stem cells. Further optimization of HSPC culture conditions resulted in 2-3 fold higher gene targeting frequencies (up to 70%) and ongoing NSG transplant studies are underway to determine whether the increases in HR frequencies also occur in long-lived repopulating hematopoietic stem cells. Altogether, these preclinical IND-enabling studies outline the framework for the manufacturing of an investigational cellular product consisting of autologous CD34+ HSPC that have been targeted for HBB gene correction of the SCD-causing E6V point mutation for the treatment of severe SCD. DisclosuresVakulskas:IDT: Employment. Behlke:IDT: Employment. Porteus:CRISPRTx: Equity Ownership.

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia

Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.

In silico analysis of missense Single Nucleotide Variants (SNVs) in HBB gene associated with the β-thalassemia

Backgroundβ-Thalassemia is a rapidly prevailing autosomal recessive disorder throughout the world. The human beta-globin gene (HBB) is responsible for the synthesis of the β-globin chains of hemoglobin, and numerous mutations in this gene, are correlated with the β-thalassemia. Among these mutations, Single Nucleotide Variations (SNVs) are the primary genetic factors behind the etiology of β-thalassemia. ObjectiveThis study aims to identify the pathogenic missense mutations from the conserved regions of β-globin, decreasing protein stability and altering molecular interactions and three-dimensional folding of the mutated proteins. The nominated mutations would, therefore, more likely to associate with the incidence of this disease. MethodsComputational tools based on sequence homology (SIFT, PROVEAN, PANTHER & Mutation Assessor), supervised learning (SNAP2, PhD-SNP, SNP&GO, SuSpect & MutPred), and consensus-based algorithms (PredictSNP & PON-P2) pinpoints the potentially pathogenic missense variants from the HBB gene. Furthermore, the ConSurf tool screens mutations, specifically from the evolutionarily conserved regions of the protein. Finally, the mutations decreasing protein stability were compared with the native proteins' model to highlight the differences in 3D structure, molecular interactions, and protein folding. ResultsA total of 12 pathogenic missense mutations including, G25V, G25D, G25R, G65D, T39P, P37H, P37T, H93P, H93D, L97P, D100V, and P101R, are recognized from the beta-globin protein. These mutations were disturbing the molecular interactions of mutated proteins, decreasing the stability of this protein. ConclusionsThe identified mutations could be the potential genetic aberrations responsible for the beta-thalassemia, all these mutations, however, need experimental validations before their use for the screening and management of the β-thalassemia.

Frequency of hereditary hemochromatosis gene mutations and their effects on iron overload among beta thalassemia patients of Chennai residents

&lt;abstract&gt; &lt;p&gt;Hereditary Hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism associated with &lt;italic&gt;HFE&lt;/italic&gt; gene mutations, characterized by increased iron absorption and accumulation leading to multi-organ damage caused by iron overload toxicity. Beta thalassemia is caused by a mutation in the human beta globin gene. Imbalanced production of globin chain results in beta thalassemia, where the unpaired alpha chains precipitates in red cell precursors leading to ineffective erythropoiesis and reduced RBC survival. Both HH and beta thalassemia condition results in rapid accumulation of iron lead to iron overload in tissues and organs. The study aims to analyze the frequency of &lt;italic&gt;HFE&lt;/italic&gt; variants among beta thalassemia cases and their effect on iron overload. The frequency of three &lt;italic&gt;HFE&lt;/italic&gt; variants C282Y, H63D, S65C was analyzed by PCR RFLP method among Beta Thalassemia Trait (BTT) (n = 203), Beta Thalassemia Major (BTM) (n = 19) and age and sex-matched control samples (n = 200). The present study furnished allele frequency of H63D variant in BTT, BTM and controls 8.13, 15.8 and 6% respectively. Ten out of 33 heterozygous H63D variants exhibited iron overload with higher ferritin levels indicating &lt;italic&gt;HFE&lt;/italic&gt; variant might aggravate the absorption of iron. The C282Y variant was present in heterozygous state in 1 case among beta thalassemia carriers. The C282Y variant was absent among BTM and control cases. S65C &lt;italic&gt;HFE&lt;/italic&gt; variant was absent in the present study. Iron overload was completely absent in the control cases among all three &lt;italic&gt;HFE&lt;/italic&gt; genotypes. Hence it is inferred from the present investigation, analysis of &lt;italic&gt;HFE&lt;/italic&gt; genes and iron status will remarkably help to reason out the probable reason behind the iron status and support in proper management of beta thalassemia cases.&lt;/p&gt; &lt;/abstract&gt;

In silico study on RNA structures of intronic mutations of beta-globin gene

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G&gt;T) and 147 (IVS1nt5G&gt;C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and 3dRNA v2.0, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G&gt;T and IVS1nt5G&gt;C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

In silico study on RNA structures of intronic mutations of beta-globin gene

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G&gt;T) and 147 (IVS1nt5G&gt;C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and RNA Composer, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G&gt;T and IVS1nt5G&gt;C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

Analysis of Human Beta Globin Gene and its Sequence Variants

Hemoglobin is a protein commonly found in in red blood cells of all vertebrates and invertebrates. It consists of two chains, mainly alpha and beta chain. The globin genes are used as a model system to study gene expression in various eukaryotic organisms. Moreover, these genes controls the gene regulation process. Mutations in these genes can cause destruction in various regulatory pathways encoded by beta globin genes. The introns in beta globin genes mostly interrupts the sequence between codons 30-31 and between codons 104-105. The study involves a comparative genomics approach for identification and analysis of selected human beta globin gene (HBB) by using various computational tools. The nucleotide sequence of HBB gene was obtained from Genbank-NCBI and analyzed to study the transcripts within the coding region of gene by using ensemble software. These transcript maps will help to understand the regulatory features of genes, splice variants and putative indels in the reading frame. Also, the protein encoded by coding region of HBB gene was further analyzed to study various protein domains, profiles and signatures. This was done by using Interpro and CDD domain analysis software. The results are presented along with the proposed utilization of the studied gene. Comparative approaches are not only useful for evolutionary analysis but also can help to understand the function of various conserved genes.

In silico study on RNA structures of intronic mutations of beta-globin gene

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G&gt;T) and 147 (IVS1nt5G&gt;C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and RNA Composer, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G&gt;T and IVS1nt5G&gt;C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots.

Dried blood spots (DBS) are widely utilized as part of universal newborn screening and as a means of transporting samples from field sites. We use DBS from African field sites to assess for rare maternal-fetal cell exchange during pregnancy known as microchimerism. We aimed to develop a protocol to maximize the quantity of high-quality genomic DNA (gDNA) extracted from DBS. The total gDNA yield obtained from control DBS utilizing a Qiagen-based protocol and a Chelex® 100 resin-based protocol was first compared. Variations of the Chelex® protocol were subsequently tested to develop an optimized protocol. The gDNA was quantified by qPCR targeting the human beta-globin gene. DNA yield for a given experimental condition was normalized to a Chelex® control performed on the same day, and the total yields were compared using a Student’s t-test. The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit . The absolute efficiency of the control Chelex® protocol was 54%, compared to an absolute efficiency of 9% for the QIAamp® DNA Blood Mini Kit. Modification of the Chelex® protocol to include a second heat precipitation from the same DBS increased the gDNA yield by 29% (P < 0.001). Our optimized protocol including this modification increased the absolute efficiency of extraction to 68%. The gDNA extracted using the Chelex® protocol was stable through repeated freeze–thaw cycles. In a mock microchimerism experiment, rare donor alleles at a frequency of 10 in 100 000 could be identified in gDNA from DBS extracted using the optimized Chelex® protocol. Our findings may be of significance for a diverse range of applications that utilize DBS and require high-quality DNA, including newborn screening programs, pathogen and drug resistance screening from remote field sites, forensics, and rare allele detection.