When a Geneticist Writes DIAGNOSIS OF HAEMOGLOBINOPATHIES: FROM SCREENING TO CONFIRMATION OF GENETIC DEFECT Written by: Prof. Dr. Raja Zahratul Azma Raja Sabudin Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia
H
aemoglobinopathies is one of the most common genetic disorders affecting worldwide. Haemoglobinopathies are broadly classified into thalassaemias (α, β, δβ) and abnormal structural variants. Few structural variants such as Haemoglobin (Hb) Lepore and HbE clinically resemble thalassaemic phenotype. Due to global migration, haemoglobinopathies has emerged as a health problem in developing countries including Malaysia. The prevalence of carriers of haemoglobin disorders in Malaysia is 4.5–11% [1-4]. As such, knowledge of the prevalence and heterogeneity of haemoglobinopathies in a target population is key to the selection of the most suitable laboratory methods to be utilised at screening centres. In Malaysia, thalassaemia screening programmes were implemented in 2005 [4] through the initial approach of antenatal and cascade screening. More recently, a nationwide compulsory screening programme involving form four students in secondary schools in Malaysia was launched by the Ministry of Health [5]. The first line of laboratory screening approach for haemoglobinopathy is the full blood count measurement using fully automated haematology analyser which is highly sensitive for carriers. This screening approach provides measurements of red cell indices such as haemoglobin (Hb), mean corpuscular haemoglobin (MCH) value, mean corpuscular volume (MCV) value, red cell distribution width (RDW) and red cell count (RCC) are helpful in differentiating carriers from patients with underlying iron deficiency anaemia. Patients with haemoglobinopathies showed hypochromia (MCH<27pg) red cells associated with eryhtrocytosis (RBC>4.75x10^12/dl) [6], while iron deficiency anaemia patients tend to have hypochromia with lower RBC [7]. Morphology of red cells in carriers was more homogenous compared to those with iron deficiency [8]. Detection of Haemoglobin H (HbH) inclusion bodies by peripheral blood smear stain with methylene blue has been used to diagnose α-thalassaemia. However, the sensitivity of this test was often unsatisfactory and a study has shown that none of single or two α-gene deletions thalassaemia patients showed positive results in this test [9]. Only three and four gene deletion forms of α-thalassaemia will have positive results with HbH inclusion. Hb analysis using High Performance Liquid Chromatography (HPLC) or Capillary Electrophoresis is the next method in line. They are relatively expensive but have been successfully in detecting majority of β-thalassaemia, three and four gene deletion forms of α-thalassaemia; and some of structural variants such as Hb Constant Spring (HbCS). However, the test is not useful in the detection of α-thalassaemia carriers and most of these cases have been missed for years. Even though HbCS ‘peak’ may appear in Hb analysis, quite a number of heterozygotes cases have been missed using HPLC [10].
The accurate determination of gene abnormalities in haemoglobinopathies is very important. Both α and β-thalassaemias which show heterogenous genetic abnormalities and increasing number of cases now require advanced methods of molecular analysis for confirmation. The molecular defects of α and β- thalassaemia in the major ethnics in Malaysia have been established. The commonest causes of α-thalassaemia are the α-gene deletions (αα/--SEA, αα/-α3.7, α α /- α 4.2) and a non-deletional abnormality i.e a HbCS [HBA2: TAA>CAA] [3,11]. Multiplex polymerase chain reaction (PCR) (GAP and amplification refractory mutation system (ARMS)) allows rapid detection of deletion and non-deletion α-gene abnormalities respectively. However, the applicability requires the definition of the breakpoints limited to known and well defined genetic deletions and mutations. Thus less common but not less important deletional and non-deletional α-gene abnormalities, as well as undiscovered genetic abnormalities could be missed and these could be novel to the Malaysian population. Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a simple technique that is suitable for rapid and mass screening of gene deletions. MLPA has been applied succesfully in a number of genes in which deletions and duplications are common [12]. From our recent study, MLPA was able to detect all deletional gene abnormalities with 95% concordance rate with conventional multiplex ARMS [13]. However, even though MLPA showed 100% sensitivity and specificity in detecting HbCS, the only non-deletional α-thalassaemia available in MLPA method, it was unable to differentiate homozygous from heterozygous states of HbCS [13]. Multiplex ARMS is still the best method for deferentiating the zygosity of HbCS even thouh it requires an additional run for wild type. Another good molecular method available is real time PCR (Taqman@ SNP genotyping assays) where it easily differentiates homozygous from heterozygous states of HbCS, but this method is expensive and laborious [10,11]. Hb Adana [HBA2: c.179G>A] is another non-deletional alpha thalassaemia which is more frequently detected since the introduction of multiplex ARMS for non-deletional α-thalassaemia in molecular laboratories in Malaysia [11,14,15]. Of recent, a case of 1VS-I-1G>A [HBA2: c.95+1G>A] [16], a rare α2-gene mutation has been discovered with the advent of better molecular skills and techniques. The estimated carrier rate for β-thalassaemia in Malaysia is 4.5% predominated by Malays and Chinese [17,18]. The common β-globin gene mutations affecting the Malays are Cd26 (G>A) HbE, IVS1-5 (G>C), IVS 1-1 (G>T), Cd 19 (A>G) Hb Malay and Cd 17 (A>T) while Chinese have Cd41/42 (-TCTT), IVS 2-654 (C>T), -28 (A>G), Cd17 (A>T) and Cd71/72 (+A) mutations [18]. Filipino β0-deletion (45kb deletion) is seen more commonly in indigenous population of Sabah and Sarawak in East Malaysia [19]. β-thalassaemia shows considerable phenotypic variations posing diagnostic difficulties with HPLC or CE. A considerable MyHVP Newsletter | Jan-June 2020| page 4
