Alpha Thalassemia Deletion/Duplication and Constant Spring Panel

Hemoglobin A, the main form of hemoglobin, is a polypeptide comprised of two alpha and two beta chains. Alpha chains are encoded through the HBA1 and HBA2 genes. Defects in these genes result in alpha thalassemia, which is a common hemoglobin disorder commonly found in African and Asian populations. Carrier frequencies are often >1% due to a selective advantage of malaria resistance in these individuals. There are four clinical conditions ranging in severity from asymptomatic to hydrops fetalis: silent carrier, alpha thalassemia trait, hemoglobin H (HbH), and hemoglobin Bart hydrops fetalis (Hb Bart). Disease severity is correlated to the degree of impaired alpha chain production (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641).

The two main clinical forms of alpha thalassemia are Hb Bart and HbH. Hb Bart syndrome is a neonatal lethal disease characterized by pleural and pericardial effusions, severe hypochromic anemia, ascites, and generalized edema. HbH disease, also referred to as alpha thalassemia intermedia, is characterized by microcytic hypochromic anemia, splenomegaly, jaundice, and in some cases skeletal changes primarily affecting facial features. Onset for HbH ranges from first years of life to adulthood. HbH individuals may also develop gallstones, have iron overload, or acute hemolytic episodes during infections or in response to oxidant drugs. In more severe forms HbH individuals may require blood transfusions. Patients with alpha thalassemia trait may have mild anemia, but are largely symptomatic (Origa and moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).

Two neighboring genes with high homology, HBA1 and HBA2, comprise the 4 alpha globin alleles. Classification of the different forms of alpha thalassemia is determined by the loss or inactivation of the alpha globin alleles.

Deletions in the HBA1 and HBA2 genes are found in over 90% of alpha thalassemia cases with seven founder mutations accounting for ~85% of all alpha thalassemia cases: -α3.7, -α4.2, -(α)20.5, --SEA, --MED, --FIL, and –THAI. The -α3.7 and -α4.2 deletions result in loss of a single alpha globin gene whereas the -(α)20.5, --SEA, --MED, --FIL, and -–THAI deletions encompass both HBA1 and HBA2 genes. Several other loss of function variants including splicing alterations, insertions/deletions, and nonsense changes have been reported in both the HBA1 and HBA2 genes. Patients with non-deletional forms of alpha thalassemia often present with more severe disease. The most common point variant found in Asian populations is Hemoglobin Constant Spring (HbCS) which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln) (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).

The -α^3.7, -α^4.2, -(α)^20.5, --SEA, --MED, --FIL, and –THAI deletions account for ~85% of all pathogenic variants detected in alpha thalassemia patients. Other full gene deletions encompassing the HBA1 and/or HBA2 genes are found in ~<5% of cases. Pathogenic variants detectable by sequencing analysis are found in ~15% of cases (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs, 2010. PubMed ID: 20507641). The most common pathogenic variant involving one or a few nucleotides found in Asian populations is Hemoglobin Constant Spring (HbCS) which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln).

Multiplex Ligation-Dependent Probe Amplification (MLPA) enables the detection of deletion and duplications of single and multiple exons within a given gene (Eijk-Van Os and Schouten 2011). This test involves analysis only of the specific gene(s) of interest for each patient. The MLPA test is designed to have coverage for all exons for each targeted gene. It is a semi-quantitative technique to determine relative copy number using a multiplex PCR-based reaction. Only hybridized and ligated adjacent probe oligonucleotides of approximately 60 nucleotides in length are amplified using PCR and thus are specific for the sequence of interest. A stuffer sequence attached to the probe ensures a particular length for deciphering the probe target. Therefore, MLPA enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. For additional information please see www.mlpa.com.

This MLPA test is also capable of detecting Hemoglobin Constant Spring (HbCS), a common structural variant found in Asian populations is which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln) (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs, 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).