Hemoglobinopathies are a group of inherited disorders in which there is abnormal production or structure of the globin moiety of the hemoglobin molecule. Hemoglobinopathies, which include the thalassemias and structural hemoglobin (Hb) variants, are the most common group of autosomal recessively inherited monogenic disorders of Hb production and pose a
significant health burden in India.(1,2)
There are thousands of genetic abnormalities possible in the globin genes, many of which are asymptomatic. Proper and timely diagnosis is imperative to ensure optimal treatment and also to offer genetic counselling to those who may be carriers. Hemoglobinopathies can be broadly classified into:
These are caused by a qualitative defect in the genetic code that leads to structural change in the hemoglobin molecule. Most alpha and beta globin chain variants are clinically silent and are discovered incidentally or during screening of family members of a patient.(3) A few variant hemoglobins are capable of causing severe disease, especially in homozygous state (e.g. HbS) or when inherited in conjunction with another variant or a thalassemia mutation. Common examples of variant hemoglobins in India include HbS, HbE and HbD. These hemoglobinopathies are prevalent in certain geographies and communities, e.g. HbE in the north eastern states of India.(4)
These are quantitative defects of Hb resulting in reduced levels of a globin chain in red cells. As a result, one or the other globin chains will accumulate, form aggregates and then precipitate, leading to premature intramedullary or intravascular destruction of RBCs.
Mutations in gamma and delta globin genes can also cause thalassemic disorders ranging from clinically silent (Hereditary persistence of fetal hemoglobin – HPFH) to symptomatic (delta-beta thalassemia). Rarely, thalassemic mutations can result in altered hemoglobin structure (eg: Hb Lepore). An example of delta globin chain mutation not uncommonly seen in India is HbA2 Saurashtra.(6)
The laboratory diagnosis of hemoglobinopathies has evolved quite a bit over the years, progressing from gel electrophoresis all the way to next generation sequencing.
“In most cases, a well elicited family history and parental testing in combination with HPLC is sufficient for definitive diagnosis of common Hb variants.”(4)
There are 2 broad categories of tests for diagnosis/screening of hemoglobinopathies:(4)
These are the methods of choice in the present day, with more advanced DNA based tests being used only for cases which cannot be resolved using protein-based methods. The routinely used protein-based methods are as follows:(4)
Table 1 – Comparison of Protein-Based Methods(4)
Advantages(4) | Disadvantages(4) | |
High performance liquid chromatography (HPLC) |
• Fully automated • High precision • Rapid, high throughput • Only a very small sample is required • Can identify and quantify many variant hemoglobins as well as normal hemoglo-bins.• Information from the pattern gives the possible diagnosis of variant peaks and guides further testing |
• Interpretation of results requires technical skill, experience • Interference /interpretation of results af- fected by prematurity (in newborn), recent transfusion • Cannot distinguish Hb E and Hb Korle Bu |
Isoelectric focusing (IEF) |
• Excellent resolution of common Hb vari- ants and Hb Barts • Distinguishes Hb E from Hb O and Hb S from Hb D and Hb G |
• Cannot precisely quantify Hb variants • Bands on gel are less sharp with automat- ed compared with manual IEF |
Capillary electrophoresis | • Fully automated • High-throughput • Identifies common Hb variants • Separates Hb A2 from Hb E (unlike HPLC) • Precise quantification of hemoglobins • Information from zone pattern of variants guides further testing • Complementary to HPLC |
• Capital cost (equipment, reagents) |
Electrophoresis (cellulose acetate, citrate agar) |
• Low cost • Separation of major Hbs (Hb A, Hb F, Hb S/D, Hb C/E/O-Arab) and several less common Hb variants • Acid pH (citrate agar) resolves Hb S and Hb C, and Hb E and Hb D Iran |
• Poor separation of Hbs other than HbS A, F, S, C • Cannot distinguish Hb E from Hb O, or Hb D from Hb G • Labor intensive |
These are almost never used as first-line tests. They do have immense significance and potential in prenatal and newborn screening. The commonly used methods are:(4)
Table 2 – Comparison of DNA-Based Methods (4)
Advantages(4) | Disadvantages(4) | |
Gap polymerase chain reaction (PCR) |
• Identification of common alpha thalas-semia deletions
• Identification of beta thalassemia and HPFH deletions • Identification of alpha globin gene dupli-cations • Can be multiplexed to test several dele- |
• Specific probes required for known dele- tional lesions of globin chains • Testing is limited to selected/pre-de- fined deletions (cannot detect unknown deletions) |
Traditional DNA sequenc- ing (Sanger) |
• Identifies a spectrum of point mutations and small insertions and deletions (in-dels) that result in a hemoglobinopathy (thalassemia, Hb variants) |
• Cannot identify large deletions or dupli- • Only one DNA sequence (up to 1 kb) |
Multiplex ligation-depen- dent probe amplification(MLPA) |
• Detects large deletions and duplications (alpha or beta genes) |
• Capital cost, kits, reagents, software |
Next-generation DNA se- quencing (NGS) | • Simultaneous identification of complete spectrum of deletions, duplications, point mutations in alpha and beta globin genes (single parallel assay) • Requires very small sample (less than that for Sanger sequencing) • Rapid • High accuracy, reliability |
• Capital cost, reagents (but less expen-sive than Sanger sequencing) |
It is recommended that screening for hemoglobinopathies should be done in the following situations with the method of choice being HPLC:(1)