Molecular Detection of alpha Thalassemia: A Review of Prevalent Techniques

Abstract

Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger sequencing is widely used to detect non-deletion mutations of α-thalassemia. Recently, next-generation sequencing was introduced in the diagnosis of both deletion and point mutations of α-thalassemia. Despite the advantages and disadvantages of different techniques, the routine method employed in the laboratory should be based on the facility, expertise, available equipment, and economic conditions.

The need to perform α‐thalassemia genetic testing in Italian patients with β‐thalassemia trait: A case report

“…Therefore, the choice of the appropriate genetic testing depends on laboratory's equipment, know‐how and cost involved. 8 …”

“…This could lead to conclude the genetic diagnosis with a single test reducing the turn around time (TAT) and optimizing the cost. Therefore, the choice of the appropriate genetic testing depends on laboratory's equipment, know‐how and cost involved 8 …”

“…Therefore, the choice of the appropriate genetic testing depends on laboratory's equipment, know-how and cost involved. 8 In our laboratory, we performed sanger sequencing for single nucleotide variants (SNVs) detection and MLPA technique for copy number variants (CNVs). To date, few studies about the utility of MLPA in routine clinical laboratory settings have been conducted.…”

Applications of next generation sequencing in the screening and diagnosis of thalassemia: A mini-review

“…WGS amplifies the entire gene in the genome while WES and TCS amplify only the protein-coding regions (exons) or a group of selected genes, respectively. Amplified products are then loaded into a chosen sequencing platform to generate millions of short-read sequences, and the sequencing data are processed and analyzed in various bioinformatic pipelines (9,19,20). In short, the downstream analysis involves the reads mapped to a known reference genome followed by the detection and classification of variants in compressed data outputs called variant calling format (VCF) files (21).…”

“…The initial detection of thalassemia is based on low hemoglobin levels and abnormal red cell indices: mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). This is followed by biochemical analysis using Hb electrophoresis, high-performance liquid chromatography (HPLC), or capillary electrophoresis (CE) ( 9 ). Subsequently, based on the initial results, routine genetic testing such as Gap-PCR, Reverse dot blot (RDB), multiplex ligation-dependent probe amplification (MLPA), or direct Sanger sequencing, among others, will be adopted as a confirmatory test ( Figure 1 ).…”