Beta-Thalassemia and Hemoglobinopathy via the HBB Gene

Beta-thalassemia (BT) affects about 1 in 100,000 people and is caused by a reduction or absence of the beta chain in hemoglobin, resulting in anemia. There are three subclasses of BT. BT major, also known as Cooley Anemia, is the most severe form of BT affecting infants. Symptoms include severe anemia, feeding problems, splenomegaly, diarrhea, complications related to iron overload, and death. BT intermedia is clinically heterogeneous and may include symptoms such as severe anemia, feeding problems, splenomegaly, diarrhea, and complications related to iron overload (Cao et al. 2013). Patients with major and intermedia forms of BT may require regular blood transfusions and iron chelators to survive. BT minor (carrier state) is typically asymptomatic, yet blood smear analysis reveals microcytosis, hypochromia and marked variations in size and shape of red blood cells (Cao and Galanello 2010; Rachmilewitz and Giardina 2011).

Sickle cell disease (SCD) is predominantly found in African American populations with an incidence of 1 in 400 (Piel et al. 2010). Patient red blood cells have hallmark sickle morphology. Average age of onset is 6 months. Initial symptoms include anemia while progressive symptoms include recurrent vaso-occulsive episodes leading to damage of a variety of organs. Secondary symptoms from vaso-occulsion may include monocular blindness, central neurologic complications, impaired pulmonary function, gallstone development, hyposthenuria, acute pain episodes and death. Supportive care may be employed to limit secondary symptoms. Stem cell transplantation is currently the only curative therapy available for patients with SCD (Ashley-Koch et al 2000; Sheth et al. 2013).

Hemoglobin C Disease is often asymptomatic but can present with mild hemolytic anemia and splenomegaly. Peripheral blood smears display reticulocytosis, irregularly contracted cells, and rod shaped crystals, but treatment is typically unnecessary. Disease is more severe in compound heterozygous states with individuals also being carriers for BT or SCD alleles (Aster et al. 2013).

Hemoglobin E Disease is often asymptomatic but can present with mild anemia. No treatment is required. Peripheral blood smears may display anisopoikilocytosis with basophilic stippling. Disease is more severe in compound heterozygous states with individuals also being carriers for BT or SCD alleles (Vichinsky 2007).

BT is an autosomal recessive disease with more than 200 disease causing mutations being documented solely in the HBB gene. Affected individuals are predominantly of Mediterranean, southeastern Asian and African descent. BT is primarily defined as a disease of decreased beta-globin production. β° alleles are characterized by deletion, frameshift, nonsense, and missense mutations at the start codon or at splice-site junctions, ultimately leading to a complete loss of HBB protein. β+ alleles are caused by point mutations found in the 5’UTR, 3’UTR and coding regions leading to markedly reduced HBB protein. Deep intronic mutations have also been reported to alter splicing leading to BT (Dobkin et al. 1983; Cheng et al. 1984). In rare cases, asymptomatic individuals may have β+/β+ or β°/β+ genotype with single allele defects in the alpha globin gene HBA2. Silent mutations within the 5’ and 3’ UTRs and large deletions have been documented as causative (Giardine et al. 2013). Disease penetrance is reflective of the imbalance of beta to alpha globin gene expression as excess globin chains precipitate, eventually leading to hemolysis (Cao and Galanello 2010; Cao et al. 2013). The three subclasses of BT are genotypically defined as β°/β° for major, β°/β+ or β+/ β+ for intermedia, and β/β° or β/β+ for minor. Together, two alpha and two beta globin proteins plus four heme molecules form a tetramer called hemoglobin A. This metalloprotein is required for transport of oxygen to and carbon dioxide away from tissues.

SCD is an autosomal recessive disease primarily defined by c.20A>T mutation resulting in a p.Glu6Val substitution (Piel et al. 2010). The c.20A>T variant has also been referenced as HbS and p.Glu6Val using legacy nomenclature. This founder mutation is particularly prevalent as heterozygous individuals are more resistant to malaria infections which are endemic to African populations. When hemoglobin in sickle cell disease individuals is deoxygenated, it polymerizes in a kinked fashion reducing red blood cell plasticity resulting in a distorted sickle cell morphology (Aster et al. 2013). These cells fail to return to normal shape when oxygen levels normalize and premature hemolysis occurs. Hemoglobin C disease is an autosomal recessive disease defined by c. 19G>A (p.Glu7Lys). This variant has also been referenced as HbC and p.Glu6Lys using legacy nomenclature. This founder mutation is primarily found in central West African populations. Approximately 2-3% of African Americans are HbC carriers. HbC mutations result in impaired plasticity of red blood cells with homozygous individuals exhibiting mild symptoms. Symptoms are greatly exacerbated in compound heterozygous states (Weatherall 2010).

Hemoglobin E disease is an autosomal recessive disease defined by c.77G>A (p.Glu27Lys). This variant has also been referenced as HbE and p.Glu26Lys using legacy nomenclature. The founder mutation is most commonly found in the Southeast Asian population with carrier frequencies approaching 60% (Vichinsky 2007).

While diagnosis of these diseases individually may not be problematic, often clinical pictures may be complicated by co-inheritance of alleles that modify the disease. Several compound heterozygous states are prevalent including HbE/β-thalassemia, HbS/ β-thalassemia, HbS/HbE, and HbS/HbC (Lionnet et al. 2012; Weatherall 2010). It is important to distinguish between hemoglobin disorders because different clinical courses may be employed depending on the patient’s genotype. For more information on compound heterozygous diseases due to mutation in the HBB gene please refer to Weatherall 2010.

Mutations in HBB account for nearly 100% case of beta-thalassemia.

This test provides full coverage of all coding exons of the HBB gene, plus ~10 bases of flanking noncoding DNA, ~260bp upstream of exon 3, and ~100 bp upstream of the start codon and downstream of the stop codon. We define full coverage as >20X NGS reads or Sanger sequencing.