Beta-Thalassemia and Hemoglobinopathy via the HBB Gene
- Summary and Pricing
- Clinical Features and Genetics
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Mutations in HBB account for nearly 100% case of beta-thalassemia.
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.
This test involves bidirectional Sanger DNA sequencing of all coding exons of HBB. The entire coding region and ~20 bp of flanking non-coding DNA on each side, 260bp upstream of exon 3, and ~200 bp upstream of the start codon and downstream of the stop codon. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Candidates for this test are patients showing features consistent with BT (elevated LDH, decreased haptoglobin, unconjugated bilirubin, anemia, microcytosis) or SCA (hemoglobin levels between 6 and 9 g/dL, accelerated red blood cell destruction, sickle cell appearance). Family members of patients who have known HBB mutations are the strongest candidates. Symptoms may be overlapping with alpha-thalassemia, iron deficiency anemia, and siderblastic anemias (Aster et al. 2013).
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- Ashley-Koch A, Yang Q, Olney RS. 2000. Sickle hemoglobin (HbS) allele and sickle cell disease: a HuGE review. Am. J. Epidemiol. 151: 839–845. PubMed ID: 10791557
- Aster, JC, Pozdnyakova, O, Kutok, JL. Hematopathology. Philadelphia: Elsevier Saunders, 2013.
- Cao A, Galanello R, Origa R. 2013. Beta-Thalassemia. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301599
- Cao A, Galanello R. 2010. Beta-thalassemia. Genetics in Medicine 12: 61–76. PubMed ID: 20098328
- Cheng TC, Orkin SH, Antonarakis SE, Potter MJ, Sexton JP, Markham AF, Giardina PJ, Li A, Kazazian HH Jr. 1984. beta-Thalassemia in Chinese: use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects. Proc. Natl. Acad. Sci. U.S.A. 81: 2821–2825. PubMed ID: 6585831
- Dobkin C, Pergolizzi RG, Bahre P, Bank A. 1983. Abnormal splice in a mutant human beta-globin gene not at the site of a mutation. Proc. Natl. Acad. Sci. U.S.A. 80: 1184–1188. PubMed ID: 6298782
- Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, Bartsakoulia M, Riemer C, Miller W, Tzimas G, Wajcman H, Hardison RC, et al. 2013. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Research. PubMed ID: 24137000
- Lionnet F, Hammoudi N, Stojanovic KS, Avellino V, Grateau G, Girot R, Haymann J-P. 2012. Hemoglobin sickle cell disease complications: a clinical study of 179 cases. Haematologica 97: 1136–1141. PubMed ID: 22315500
- Piel FB, Patil AP, Howes RE, Nyangiri OA, Gething PW, Williams TN, Weatherall DJ, Hay SI. 2010. Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nat Commun 1: 104. PubMed ID: 21045822
- Rachmilewitz EA, Giardina PJ. 2011. How I treat thalassemia. Blood 118: 3479–3488. PubMed ID: 21813448
- Sheth S, Licursi M, Bhatia M. 2013. Sickle cell disease: time for a closer look at treatment options? Br. J. Haematol. 162: 455–464. PubMed ID: 23772687
- Vichinsky E. 2007. Hemoglobin e syndromes. Hematology Am Soc Hematol Educ Program 79–83. PubMed ID: 18024613
- Weatherall DJ. 2010. The inherited diseases of hemoglobin are an emerging global health burden. Blood 115: 4331–4336. PubMed ID: 20233970
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
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- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
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(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
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