Smt. Kishoritai Bhoyar College of Pharmacy, Kamptee, Nagpur, Maharashtra-441002
Thalassemia, a genetic hematologic disorder, is a huge global public health concern, impacting about 360 million carriers globally. The disease is notably prevalent in the Middle East, Southeast Asia, and certain African and Asian countries. The most severe form, ?-thalassemia major, requires regular blood transfusions for survival. The costly nature of treatment poses a considerable hardship, especially in developing nations. The condition also has a considerable influence on the emotional well-being of affected persons, particularly youngsters. A range of novel approaches have been presented for the detection and management of thalassemia. Recent advances in nanotechnology have involved the utilization of nanomaterials in colorimetric assays, the integration of nanopore sequencing with bioinformatics analysis, the assessment of cell-free fetal DNA through digital PCR for non-invasive prenatal screening, the application of electrochemical biosensors, and the implementation of diverse automated detection systems. The ideas, recommendations, and practical implementations of these new technologies are completely evaluated, revealing their capacity to enhance precision, sensitivity, and early detection in contrast to present techniques. Key features are user-friendly operation, cost-effectiveness, quantitative evaluative capabilities, and adaptability for quick implementation. Yet difficulties remain about scale, lack of resources, and incorporation into therapeutic environments. Survey broadens to emergent fields such as artificial intelligence to promote better data analysis and careful variation analysis. These novel advancements indicate tremendous potential in enhancing thalassemia screening approaches, facilitating rapid cures, and decreasing the global disease burden. The ongoing research efforts aiming at developing and validating these approaches have the potential to bring about considerable progress in the diagnosis and treatment of thalassemia on a worldwide basis.
Thalassemia affects a major global public health due to its hereditary origin (1).The following disorder is characterized by mutations in genes that are necessary for the transfer of oxygen throughout the entire human body. Individuals who are diagnosed with thalassemia generally experience a decrease in the production of Hemoglobin (Hb), a crucial component that improves the ability of red blood cells to transport oxygen (2).Factors such as family history, diverse ancestral origins, and certain dietary ingredients influence susceptibility to thalassemia. The degree of thalassemia is determined by the existence of mutations in ?-globin or ?-globin genes, with a specific quantity of these genes being absent (four for ?-globin and two for ?-globin). Both alpha (?)- and beta (?)-thalassemia are typically inherited in an autosomal recessive pattern, with the initial instances of dominant hereditary transmission documented in an Irish family. For an affected child to be born, both parents must carry the disease-causing trait, and if both parents are carriers of a hemoglobinopathy trait, the likelihood of having an affected child is 25% in each pregnancy (3). The prevalence of hereditary hemoglobin disorders presents a growing concern on a global scale, with approximately 320,000 infants worldwide being born with hemoglobin diseases annually. Alarmingly, around 80% of these cases occur in developing nations (4). Current estimations indicate that at least 5.2% of the world's population, totaling more than 360 million individuals, possess a significant hemoglobin variant (5) over 100 million people affected by ?-thalassemia alone (6). The global prevalence of ?-thalassemia stands at 1.5%, highlighting the substantial impact of these conditions on a large segment of the population (6). Thalassemia represents a diverse array of hereditary conditions distinguished by the diminished production of one or more typical hemoglobin chains, resulting in an imbalance in globin chain synthesis and subsequent microcytic anemia (7).Thalassemia, a hereditary illness, demands the presence of a carrier parent and manifests due to either a genetic change or the removal of certain gene segments. The pathogenesis of Thalassemias originates in mutations occurring in the ? (HBA1/HBA2) and ? globin (HBB) genes, often following an autosomal recessive pattern of inheritance (8). The differentiation of thalassemia into alpha-thalassemia and beta-thalassemia is established based on genetic factors (9). The etiology of alpha-thalassemia involves a deficit in alpha-globin chains, whereas beta-thalassemia is attributed to a shortfall in beta-globin chains (10). The ?-thalassemias are equally heterogeneous. The milder forms (termed ?-thalassemia 2 or ?+ thalassemias) result from one ? globin gene deletion, produce a mild anemia in their homozygous states (11). While ?-thalassemia 1 or ?0-thalassemia is associated with an absence of ? globin chain synthesis because of the deletion of the two ? globin genes on the same chromosome. In homozygous states, it results in the most severe form of thalassemia, namely, Hb Bart’s hydrops fetalis (12). The compound heterozygous states for ? thalassemia 2 and ?-thalassemia 1 result in Hb H disease which varies in severity; at the more severe end, it may be a TDT. The thalassemias are extremely heterogeneous at the molecular level; over 200 different mutations of the ? globin genes have been found in patients with ?-thalassemia, and the ?-t thalassemias are almost as varied in their molecular pathology (13). However, global population seems to carry a few common mutations that are unique to a particular area, together with varying numbers of rare ones. Traditional diagnostic methods for thalassemia have limitations in terms of sensitivity, specificity, and scalability. However, recent advancements in detection technologies offer promising solutions to overcome these challenges This comprehensive review is intended to furnish an extensive and detailed examination of the innovative detection technologies developed for thalassemia. These technologies encompass a wide spectrum of methodologies including colorimetric assays, nanopore sequencing, digital PCR, electrochemical sensors, and bioinformatics pipelines. The utilization of these sophisticated methodologies has revolutionized the techniques employed in the identification, quantification, and execution of prenatal screening for alpha-thalassemia and beta-thalassemia mutations, resulting in a notable enhancement in accuracy and efficiency. The main purpose of this is to give a structured review of the basic ideas, methods, and real-life uses of these new systems. Its objective is to illuminate the potential ramifications of these methodologies in the diagnosis and management of thalassemia. Moreover, extensive investigation is carried out to tackle the challenges and opportunities linked to the adoption of these approaches in healthcare environments. This investigation underscores topics like scalability, cost-effectiveness, and appropriateness for resource-limited settings. The adaptation of these revolutionary detection technologies has the capability to substantially improve patient outcomes, facilitate timely intervention, and mitigate the global impact of thalassemia. The main objective of this review is to contribute to the ongoing discussion about the management of thalassemia and to encourage more research efforts focused on addressing this significant public health problem.
Novel advances in thalassemia detection technologies:
Molecular diagnosis of ? -thalassemias by the colorimetric nanogold:
This research presents a new utilization of gold nanoparticles (AuNPs) in a colorimetric approach for the molecular detection of alpha-thalassemia 1 (SEA deletion). Alpha-thalassemia 1 is a grave hereditary condition commonly found in Southeast Asia, specifically in Thailand, causing notable levels of illness and death (14). Polymerase chain reaction (PCR) and agarose gel electrophoresis appear to be two frequently conducted more complex testing methodologies that may exhibit limitations in efficacy, especially within resource-limited settings. Here, we reported the development and evaluation of a simple, rapid, and cost-effective nanogold colorimetric method for identifying abnormal alpha-globin genes causing alpha-thalassemia 1. Thalassemia is a group of inherited blood disorders characterized by abnormal hemoglobin production, leading to anemia. Alpha-thalassemia, namely alpha-thalassemia 1 with SEA deletion, constitutes a major health concern, particularly widespread in the Southeast Asian region (15). Present diagnostic procedures usually need complex technology equipment and are not easily available in regions with restricted resources. The application of gold nanoparticles (AuNPs) gives an appropriate choice for molecular diagnosis due to their particular optical features and easy properties. DNA probes developed for the SEA deletion associated to alpha-thalassemia 1 were constructed, functionalized with thiol groups, and adsorbed onto the surface of gold nanoparticles (AuNPs). Simultaneously, the target DNA sequences were reproduced and connected to the probes attached to the AuNPs. Introduction of a saline solution generated aggregation of the unattached nanogold probes, resulting in visible colour changes (16). Samples from people identified of alpha-thalassemia 1 were evaluated utilizing the nanogold SEA-probe and compared with traditional agarose gel electrophoresis. The nanogold colorimetric method accurately differentiated samples with normal alpha-globin genes from those with the SEA deletion, in carriers as well as illness situations. Findings focused on the precision, specificity, and sensitivity of the nanogold colorimetric assay for identifying alphathalassemia1. The use of the nanogold SEA-probe offers the following benefits such as simplicity, speed, and cost-efficiency. The results were observed via direct observation thus reducing the demand for advanced apparatus. The following method shows positive results for its possible application in point-of-care testing (POCT) and investigations within the field of population genetics, particularly in locations with a lack of resources of molecular diagnostic facilities. The utilization of nanogold colorimetric technology signifies a significant advancement in the identification of molecules responsible for alpha-thalassemia. The device's ease of use, accuracy, and cost efficiency render it highly beneficial for healthcare practitioners, particularly in regions with constrained resources.
Noninvasive prenatal testing for ?-thalassemia by targeted nanopore sequencing in addition with relative haplotype dosage (RHDO):
Noninvasive prenatal testing (NIPT) for single gene diseases (SGDs) offers major difficulties due to the need for rapid detection of minor genetic defects inherited from the mother. One promising technique is relative haplotype dosage (RHDO) analysis, which relies on generation of parental haplotypes. Nanopore sequencing, a recent advancement in sequencing technology, gives a direct method for haplotype construction. This study examines the feasibility of combining nanopore sequencing with RHDO analysis for NIPT of ?-thalassemia. Thirteen families at risk for ?-thalassemia were selected. Long-range PCR was applied to amplify targeted regions of parental genomic DNA into 10 kb and 20 kb amplicons. Parental haplotypes were built using nanopore sequencing data and next-generation sequencing (NGS) data. RHDO analysis was carried out using maternal plasma DNA sequencing to determine fetal inheritance of paternal haplotypes. Haplotype phasing took place in 12 families using the 10 kb library, with all 13 families successfully phased using the 20 kb library. Fetal status was accurately classified in 12 out of 13 families, demonstrating the feasibility of targeted nanopore sequencing combined with RHDO analysis for NIPT of ?-thalassemia. This targeted nanopore sequencing approach offers several advantages. It eliminates the need for a proband, reducing analysis costs. Additionally, targeted enrichment reduces sequencing costs, particularly when compared to methods like hybridization. The portable Min ION sequencer used in this study is cost-effective and accessible for research and clinical labs. The study also highlights the potential of nanopore sequencing to generate long reads for reliable SNP phasing. Despite its promise, nanopore sequencing still faces challenges, particularly in SNP resolution. Improvements in base-calling accuracy are needed to enhance reliability. The study also notes ongoing advancements in nanopore sequencing technology, such as the release of the R10 chemistry, which may improve read accuracy. Long-range PCR of 10 kb amplicons was initially used for haplotype construction, followed by optimization to 20 kb amplicons, enabling successful construction of all parental haplotypes. Long-range PCR was crucial for generating sufficient material for sequencing, particularly in diagnostic applications where DNA quantity may be limited (17). The study concludes that targeted nanopore sequencing-based haplotyping, coupled with RHDO analysis, shows promise for NIPT of ?-thalassemia. Further validation with clinical samples is necessary, along with improvements in nanopore sequencing accuracy, to facilitate its clinical use in the future. The methodology employed involved amplification of a ~50 kb targeted region containing the HBB gene, followed by sequencing using nanopore and NGS technologies. Data analysis included haplotype phasing and RHDO analysis to determine fetal inheritance of parental haplotypes. The study also discusses challenges and potential future developments in nanopore sequencing for NIPT of SGDs.
Noninvasive prenatal diagnosis of beta thalassemia disease by using digital pcr analysis in maternal plasma of cell-free fetal DNA:
The study focused on revolutionizing prenatal diagnosis for beta-thalassemia, a genetic disorder characterized by reduced hemoglobin production, which can lead to severe anemia and other complications. In previous times, the identification of such issues prior to childbirth frequently required invasive techniques like chorionic villus sampling or amniocentesis, both of which carry a risk of possible miscarriage (18). Nevertheless, the identification of cell-free fetal DNA (cff-DNA) in maternal blood plasma has unveiled significant possibilities for non-invasive prenatal testing. Experts have employed a novel technique called droplet digital PCR (dPCR) to quantify cff-DNA in maternal plasma (19). The subsequent methodology provides heightened sensitivity and specificity when detecting diverse mutations present in small amounts of DNA. The primary objective is to evaluate the efficacy of digital PCR (dPCR) in accurately identifying beta-thalassemia in fetuses at high risk, thus reducing the necessity for invasive procedures (20). A cohort consisting of 35 pairs of individuals deemed to have a higher probability of producing offspring affected by severe beta-thalassemia was selected for participation in the study. Specimens of maternal blood were collected during the 18th week of pregnancy, and cell-free fetal DNA (cff-DNA) was isolated and analyzed using digital polymerase chain reaction (dPCR). Encouraging results surfaced, indicating the effectiveness of dPCR evaluation of cff-DNA in maternal blood plasma for prenatal detection of beta-thalassemia. More specifically, the presence of paternal genetic alterations consistently signalled the transmission of these mutations to the fetus, while their absence negated paternal heritance. Likewise, an exact proportion of mutant to normal DNA effectively proved the presence of maternal mutations. The known aspect of this study is its higher sensitivity and specificity in anticipating fetal alterations related to beta-thalassemia (21).The occurrence of incorrect positive outcomes was dramatically lowered, indicating the capacity to effectively avoid intrusive diagnostic approaches in many settings. The discovery is significant as it has the ability to lessen risks for both the mother and the fetus associated to invasive operations. Notwithstanding these hopeful outcomes, the study did admit significant restrictions, namely the relatively restricted sample size, especially in instances with identical mutations. Further studies with larger cohorts are necessary to validate these findings. Additionally, the gestational age at which the dPCR tests were conducted skewed towards later stages of pregnancy, potentially limiting its applicability in earlier gestational ages. The method also offers a rapid, simple, and relatively cost-effective alternative to traditional invasive procedures. Moreover, it opens avenues for similar noninvasive diagnostic approaches for other single gene disorders. However, additional validation and refinement of the technique are warranted before its widespread clinical implementation. Selective electrochemical sensing of hemoglobin from blood of ?-thalassemia major patients by tellurium nanowires-graphene oxide modified electrode:
The study focuses on addressing the challenges posed by ?-thalassemia, a group of genetic disorders characterized by abnormal hemoglobin production. Individuals suffering from ?-thalassemia may manifest varying levels of severity, spanning from mild anemia (known as ?-thalassemia minor) to critical conditions necessitating consistent medical care, such as blood transfusions (referred to as ?-thalassemia major or intermedia). The long-term survival of individuals with ?-thalassemia is contingent upon effective management strategies, which encompass blood transfusions and iron chelation (7).Complications associated with ?-thalassemia include iron overload, which increases the risk of hepatocellular carcinoma, and transfusional siderosis, which can lead to myocardial diseases and contribute to mortality. Oxidative stress, characterized by the imbalance between reactive oxygen species (ROS) and antioxidant defenses, plays a significant role in ?-thalassemia-related complications, including cell damage and organ dysfunction (22). The electrochemical sensors address diagnostic issues associated with ?-thalassemia, allowing a precise evaluation of hemoglobin levels in individuals with ?-thalassemia. A unique electrochemical device has been developed, combining tellurium nanowires (TeNWs) with graphene oxide (GO) for monitoring hemoglobin levels in persons suffering from ?-thalassemia. The inclusion of TeNWs improves the processes of charge transfer, whereas GO enhances both the surface area and conductivity of the composite material. Specifically built for monitoring hemoglobin levels in blood specimens, this innovative sensor provides a non-invasive and efficient diagnostic equipment for the management of ?-thalassemia (23).The application of this hybrid material on a glassy carbon electrode (TeNWs/GO-GCE) increases the electrochemical characteristics when compared to untreated electrodes. The suggested sensor displays excellent specificity in detecting hemoglobin among numerous interfering chemicals in blood tests. Its considerable robustness is proven by cyclic voltammetry and chronoamperometry evaluations, proving its capacity to sustain several cycles and extended durations. Additionally, the sensor displays excellent recovery, linearity, limit of detection, and limit of quantification. The broad research provides the capabilities of electrochemical sensors, specifical the inclusion of Tellurium Nanowires and Graphene Oxide (TeNWs/GO), for proper detection of hemoglobin levels in persons suffering from ?-thalassemia major. This technique gives a hopeful path for non-invasive, quick, and economical detection and surveillance of ?-thalassemia, thereby boosting the well-being and quality of life of patients.
Low-cost biosensor-based molecular differential diagnosis of ? -thalassemia (Southeast Asia deletion):
The study attempt was centered on strengthening the detection of individuals who carry ?-thalassemia1 (SEA deletion), a dangerous hereditary blood condition, through the advancement of a quartz crystal microbalance (QCM) technique. This method involved immobilizing a biotinylated probe on a silver electrode of the QCM surface, amplifying the ?-globin gene fragment, and hybridizing it with the probe. Hybridization was detected by changes in quartz oscillation. Notably, each drying step was enhanced by using an air pump for 30 minutes instead of overnight air drying, reducing analysis time. Results demonstrated that the silver QCM effectively identified samples with abnormal ?-globin genes, including homozygous and heterozygous forms, from normal samples. Thirteen out of 70 blood samples were identified as carriers of ?-thalassemia1 (SEA deletion), consistent with standard agarose gel electrophoresis results. Additionally, using silver instead of gold QCM reduced production expenses tenfold, and the air pump drying method decreased analysis time from 3 days to 4 hours (24).The study emphasized the severity of ?-thalassemia1 and the importance of accurately identifying carriers for prevention programs and family planning. While various screening methods exist, definitive diagnosis requires gene-level confirmation. The developed silver QCM method addressed this need, offering a label-free, cost-effective, and rapid technique without carcinogenic staining (25).It demonstrated high potency for one-step molecular diagnosis of ?-thalassemia1 (SEA deletion), being simple, specific, sensitive, affordable, rapid, stable, and suitable for field application.
Overall, the study introduced a promising method for the differential identification of ?-globin genes between normal and ?-thalassemia1 (SEA deletion) variants, offering significant improvements in efficiency and practicality for clinical application. Table 1 describes the various techniques along with their advantages and limitations
Table No: 1 Diagnostics for thalassemia: advantages and limitations of different techniques
A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene:
The study described the development of an electrochemical biosensor for the efficient detection of ?-thalassemia using PCR products from real blood samples. The biosensor utilized a modified carbon paste electrode (MCPE) incorporating silver and platinum nanoparticles, and methylene blue (MB) served as the electroactive indicator. The detection principle relied on the electrochemical transduction of hybridization reactions between short complementary ?-thalassemia oligonucleotides and the PCR products. The biosensor showed improved voltametric indicator peaks after hybridization, reflecting the level of hybridization. The reduction in Methylene Blue (MB) currents during probe presence and post hybridization demonstrated significant modifications. The application of linear sweep voltammetry (LSV) enables for the exact determination of MB reduction to determine the presence of ?-thalassemia PCR products (26). The test of the biosensor's applicability involved investigating its response to non-complementary PCR products, leading to modest reductions in the MB signal, suggesting inadequate hybridization efficiency. simultaneous assessment of PCR samples including both complementary and non-complementary sequences confirmed the biosensor's high discriminatory capability. Nano composites such as silver and platinum nanoparticles using MB as the probe, the biosensor achieved higher hybridization signals and improved sensitivity. The clear differentiation amongst complementary and non-complementary PCR samples shows the potential for developing electrochemical biosensors for the detection and quantification of different biological components. The newly constructed biosensor acts as a potential technology for the exact and discriminating identification of ?-thalassemia utilizing PCR products derived from real blood samples. Its potential applications extend to the detection and quantification of various biocompounds, contributing to advancements in biomedical diagnostics and research.
A label-free electrochemical biosensor for the detection of alpha-thalassemia 1 (SEA deletion) carriers using screen-printed carbon electrodes:
A label-free electrochemical DNA biosensor utilizing electrochemical impedance spectroscopy (EIS) has been extensively researched for diagnosing human genetic diseases. However, its practical application has been largely confined to simulated target DNA. This study aimed to advance EIS biosensor technology to identify carriers of alpha-thalassemia 1 with Southeast Asia (SEA) deletion. The biosensor, integrated with screen-printed carbon electrodes (SPCEs) and Hoechst 33,258 dye, underwent evaluation using forty samples with known DNA genotypes to establish optimal conditions and cutoff criteria. The findings specified a specific threshold value for the detection of carriers of ?-thalassemia 1 (SEA deletion) through the use of negative imaginary impedance (-Z?) and charge transfer resistance (Rct) (27). Comparable sensitivity to traditional gel electrophoresis was observed in the newly created biosensor, without any instances of cross-reactivity with different types of thalassemia. Clinical testing on 81 blood samples demonstrated high sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for Rct measurement, indicating its potential as a screening tool for ?-thalassemia 1 (SEA deletion) carriers. The biosensor offers simplicity, cost-effectiveness, and significantly reduced turnaround time compared to conventional methods, thus facilitating effective management and prevention of severe thalassemia. Additionally, the study proposes future enhancements such as hybridization methods to further improve specificity and field application in remote areas, underscoring the biosensor's promise in population genetic screening (28)(29). Fig No.1 represents the diagrammatic representation of various approaches for thalassemia detection
Fig No. 1: Diagrammatic representation of various approaches for thalassemia detection
Isothermal strand-displacement polymerase reaction for visual detection of the southeast Asian type deletion of a-thalassemia:
Alpha-thalassemia is a diverse inherited disorder prevalent in Southeast Asia and the Indian subcontinent, with a carrier frequency reaching 10% to 15% (30). The condition arises from various genotypes, with southern China alone reporting over 40 distinct types. Typically, alpha-thalassemia stems from large deletions in the alpha-globin gene cluster on chromosome 16p13.3, leading to reduced or absent synthesis of alpha-globin chains, integral components of hemoglobin (Hb). The most prevalent type, Southeast Asian (SEA) alpha-thalassemia, predominantly results from a 20.5 kb deletion and poses risks of severe fetal complications, necessitating accurate diagnostic assays for genetic consultation and prenatal diagnosis (31). In many cases, thalassemia patients hail from developing nations like China, Thailand, and the Philippines. However, implementing molecular testing for SEA alpha-thalassemia in such settings proves challenging due to the complexity and cost of current diagnostic methods, notably the multiplex gap-PCR analysis. To address this, isothermal nucleic acid amplification technologies offer promise, notably the isothermal strand-displacement polymerase reaction (ISDPR). This approach, coupled with lateral flow biosensor (LFB) technology utilizing gold nanoparticles (AuNPs), provides a feasible solution for point-of-care genetic testing (32). The new biosensor, which combines ISDPR and AuNPs, has proven potential in the diagnosis of SEA alpha thalassemia, exhibiting full agreement with PCR data. This technique permits separation between SEA alpha-thalassemia and other thalassemia variants, providing considerable sensitivity (detecting down to 0.01 fmol/L nucleic acid), specificity, user-friendliness, and cost-effectiveness (33). Through harnessing the signal amplification capabilities of ISDPR, the biosensor enables quick and precise detection, eliminating the demand for sophisticated equipment generally required by standard PCR-based approaches. Validation experiments have proven the biosensor's ability to detect synthetic target DNA and human genomic DNA, indicating a limit of detection of 5 ng/mL. Moreover, the biosensor's specificity was proven through its precise recognition of single-base alterations. The linear amplification process of the technology allows for greater sensitivity in a decreased timescale when compared to PCR, rendering it suited for the detection of genetic diseases, including applications at the point of care and screening of populations in resource-constrained contexts. In the domain of clinical studies, the LFB effectively detected SEA alpha-thalassemia in patient specimens, correlating with PCR findings and displaying selectivity towards other kinds of thalassemia. Due to its rapid processing time (<40>
NGS4THAL, a one-stop molecular diagnosis and carrier screening tool for thalassemia and other hemoglobinopathies by next-generation sequencing:
Thalassemia stands out as a prevalent genetic disorder, particularly rampant in tropical and subtropical areas, where carrier rates are notably high across diverse ethnic populations. The condition encompasses a spectrum of inherited disorders characterized by mutations in the hemoglobin genes, presenting a complex landscape of genetic variability. This diversity poses significant challenges to accurate diagnosis, as mutations vary not only between different ethnic groups but also within geographic regions. Traditionally, the diagnostic method often commences with hematological tests aimed at evaluating the mean corpuscular volume and mean corpuscular hemoglobin, which is later followed by the employment of hemoglobin electrophoresis to detect any aberrant hemoglobin variations. Nevertheless, it is crucial to highlight that this progressive methodology may fail to detect certain mutations, particularly in circumstances where these changes do not display normal hematological features. Genetic examinations such as Gap-PCR and MLPA, may present complexity in specific cases and could be relegated to specialized tertiary healthcare centers, hence decreasing its usage. The innovation presents a molecular diagnosis capable of recognizing a broad variety of harmful mutations. The intricate structure of the hemoglobin gene clusters provides considerable analytical hurdles due to a massive number of similar sequences and pseudogenes(34). As a result, the alignment of short NGS sequences often proves unsatisfactory, preventing the exact identification of mutations, particularly those connected to structural variants (SVs) commonly reported in thalassemia cases. NGS4THAL has been created primarily to overcome the issues involved with finding genetic variants in hemoglobin through a bioinformatics analytical technique. The program provides a complete strategy that incorporates different alignment algorithms with SV calls to boost both sensitivity and specificity. By leveraging several alignment algorithms such as unique, best match, and all matches, NGS4THAL effectively tackles the issue of ambiguous read mapping, resulting in a considerable improvement in pinpointing point mutations. With specialized algorithms tuned for detecting Structural Variants (SVs), NGS4THAL accounts for the many types of SVs usually detected in cases of thalassemia. By dividing SVs into separate subtypes and including heterozygous controls, this system ensures reliable identification of compound SVs typically observed in locations with a high frequency of thalassemia. NGS4THAL accelerates the detection of novel genetic variations and variables contributing to the illness, offering useful insights for subsequent clinical assessments. Despite its efficacy, NGS4THAL is continuously developing, with current endeavors centered on enhancing efficiency and scalability, especially for extensive population screening initiatives. By streamlining realignment steps and enhancing computational performance, future iterations of NGS4THAL aim to broaden its applicability and impact in the clinical diagnosis of thalassemia and other hemoglobinopathies.
A sensitive electrochemical genosensor for highly specific detection of thalassemia gene:
The escalating demand for rapid, affordable, and portable testing methods, as opposed to the cumbersome and costly techniques like PCR and FISH, has powered research in DNA sensors or genosensors. These sensors offer a promising alternative for detecting specific nucleic acid sequences, employing optical, piezoelectric, and electrochemical transduction methods. Electrochemical gene sensors, particularly, exhibit desirable features such as high sensitivity, rapid response, and cost-effectiveness, essential for preliminary disease detection, genetic disorder therapy, and infection treatment. These sensors typically involve immobilizing DNA probes onto electrode surfaces, generating electrical signals upon binding to target DNA sequences. Signal amplification in DNA-based sensors often relies on surface modifications, with nanomaterials playing a crucial role. Nanomaterial platforms offer remarkable prospects for creating sensitive biosensors. Graphene oxide (GO) and reduced graphene oxide (rGO) have features relating to conductivity, simple manufacture, broad surface area, and compatibility in biological systems. The addition of gold nanoparticles (AuNPs) boosts the performance of genosensors, owing to their great affinity towards biological entities when paired with rGO, subsequently leading in an augmented surface area and signal amplification. An electrochemical biosensor for DNA detection, which comprises connecting the 4-aminothiophenol monomer and gold nanoparticles to a glassy carbon electrode that has been modified with reduced graphene oxide (rGO/GCE) (35). This biosensor was applied for the detection of minute levels of the ?-globin gene, connected to Thalassemia, an inherited blood condition. The thiolated portion of the ?-thalassemia gene worked as the probe, making a covalent interaction with the gold nanoparticles, permitting accurate identification of the ?-globin gene. A variety of surface modification and DNA hybridization resulted in varied impedance spectra for the changed electrodes. The enhancement of genosensor performance was pursued through the optimization of factors such as the duration of target incubation and the concentration of probe DNA.The genosensor demonstrated excellent analytical performance, with a wide linear range and low detection limits for target DNA concentration. Stability, reproducibility, repeatability and selectivity studies confirmed the reliability and specificity of the genosensor. Real sample analysis using serum samples demonstrated the applicability of the genosensor for detecting ?-thalassemia gene in human serum with high accuracy and precision. Overall, the developed genosensor offers a sensitive and selective tool for detecting low concentrations of the ?-thalassemia gene, presenting significant potential for clinical diagnostics. Based on the technologies discussed in the review article, here are some potential future directions for thalassemia detection:
The use of nanomaterials like gold nanoparticles, graphene, tellurium nanowires etc. shows promise for simple, cost-effective and sensitive detection of thalassemia mutations and quantification of hemoglobin levels. Further optimization and integration into portable devices could enable point-of-care testing.
Next-generation sequencing combined with tailored bioinformatics pipelines like NGS4THAL can provide comprehensive detection of point mutations, indels and structural variants across the globin gene clusters. As sequencing costs drop, this could become an affordable frontline diagnostic approach.
Techniques like nanopore sequencing of cell-free fetal DNA and digital PCR analysis show potential for reliable NIPT for thalassemias. Continued development of these methods aligned with clinical validation studies could make NIPT a standard option.
Integrating multiple detection modalities like electrochemical, optical, mass-based sensors on a single platform could enable parallel screening for different thalassemia mutations and quantitative analysis of disease biomarkers.
Machine learning models trained on multi-modal data from sequencing, biosensors, clinical phenotypes etc. could potentially provide highly accurate detection and disease stratification for thalassemias. Overall, emerging nano biosensors, sequencing technologies, multi-omics integration and AI-based diagnostics could revolutionize thalassemia screening - enabling early, accurate and comprehensive detection from affordable platforms accessible at the point-of-care and prenatal stages.
CONCLUSION:
Thalassemia persists as a notable concern within the realm of global public health, yet recent progressions in the realm of detection technologies present promising pathways for enhanced diagnosis and management of this condition. The examination undertaken in this review delved into a wide array of ground-breaking techniques, encompassing colorimetric assays, nanopore sequencing, digital PCR, electrochemical sensors, and bioinformatics tools among others. These methodologies exhibit substantial potential to transform the landscape of thalassemia diagnosis through the amplification of precision, efficacy, and availability. Despite the distinct advantages inherent in each of these techniques, it is imperative to acknowledge the existence of limitations. Further exploration and enhancement are imperative to guarantee the scalability, cost-efficiency, and appropriateness of these methodologies for settings constrained by limited resources. Nonetheless, the assimilation of these pioneering technologies harbors the capacity to markedly enhance patient outcomes, streamline early intervention practices, and ultimately play a pivotal role in the global mitigation of the burden posed by thalassemia. The overarching objective of this review is to contribute to the continual discourse surrounding thalassemia management, while also serving as a source of inspiration for additional research endeavors aimed at combatting this pressing public health dilemma.
ACKNOWLEDGEMENT:
The authors like to acknowledge the staff and management of the Smt. Kishoritai Bhoyar College of Pharmacy, Kamptee, Nagpur, Maharashtra-441002, India for providing necessary facilities and provisions to prepare this article.
Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest.
List of Abbreviations:
cff-DNA : Cell-Free Fetal DNA
RCT : Charge Transfer Resistance
DNA : Deoxyribonucleic Acid
dPC : Droplet Digital PCR
EIS : Electrochemical Impedance Spectroscopy
FISH: : Fluorescence In Situ Hybridization
Gap-PCR : Gap Polymerase Chain Reaction
MLPA : Multiplex Ligation-Dependent Probe Amplification
TeNWs : Tellurium Nanowire
GCE : Glassy Carbon Electrode
GO : Graphene oxide
rGO : Reduced Graphene Oxide
Hb : Hemoglobin
HBA1 : Hemoglobin Subunit Alpha 1
HBA2 : Hemoglobin Subunit Alpha 2
HBB : Hemoglobin Subunit Beta
ISDPR : Isothermal Strand-Displacement Polymerase Reaction
LFB : Lateral Flow Biosensor
LSV : Linear Sweep Voltammetry
MB : Methylene Blue
MCPE : Modified Carbon Paste Electrode
NPV : Negative Predictive Value
NGS : Next-Generation Sequencing
NIPT : Non-invasive prenatal testing
SGDs : Single Gene Diseases
PCR : Polymerase Chain Reaction
POCT : Point-Of-Care Testing
PPV : Positive Predictive Value
QCM : Quartz Crystal Microbalance
ROS : Reactive Oxygen Species
rGO : Reduced Graphene Oxide
RHDO : Relative Haplotype Dosage
SPCEs : Screen-Printed Carbon Electrodes
SEA : Southeast Asia deletion
SVs : Structural Variants
TDT : Terminal Deoxynucleotidyl Transferase
REFERENCES:
Milind J. Umekar, Tanvi D. Premchandani , Neha S. Raut, Mohammad Qutub, Novel Molecular And Nano Biosensor Approaches For Comprehensive Thalassemia Detection, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 9, 1036-1052. https://doi.org/10.5281/zenodo.13820944
10.5281/zenodo.13820944
