Thalassaemia screening programme was conducted to reduce the burden of the disease [1]. Here, we describe one unexpected discovery in a 33-year-old gentleman and also the importance of DNA analysis in detecting the globin gene mutation.
The aims of this study are to identify and characterize the Haemoglobin G Makassar. Haemoglobin G Makassar was identified in Makassar, Sulawesi (Celebes), Indonesia in 1969 and has been reported in a family of Thai origin in 2002. Haemoglobin G Makassar was found to share identical properties with haemoglobin S in routine haemoglobin separation by cation-exchange HPLC. It is therefore, patients with Haemoglobin G Makassar and Haemoglobin S may sometimes be mistakenly identified for each other.
There were four cases identified from year 2015 to 2016 in Peninsular Malaysia by Molecular Genetics Laboratory, Institute for Medical Research. All patients were asymptomatic with mild hypochromic microcytic anaemia. All patients were analysis with Haemoglobin S trait. Analysis by Capillary Electrophoresis showed that these patients had 39.9 to 44.0% of haemoglobin variant in zone S. Alpha and Beta globin gene analysis were performed on these samples.
DNA sequence analysis, revealed a single nucleotide substitution GAG to GCG at codon 6 of the beta-globin gene (Glu>Ala), indicating of Haemoglobin G Makassar for all the patients (Fig. 1). All patients were positive with Haemoglobin S trait. Multiple Amplification Refractory Mutation System (MARMS) PCR for Haemoglobin S was negative in all cases. However alpha-globin gene analysis showed that two of them had single alpha deletion (α3.7). The mean reading for HGB is 11.95 g/dL, for MCV is 72.1 fL and for MCH is 23.65 pg which all are lower than normal peoples.
The screening method may mistakenly identify Haemoglobin G Makassar as Haemoglobin S. Therefore identification and characterization of Haemoglobin G Makassar by several molecular methods such as polymerase chain reaction (PCR) and sequence analysis are necessary for confirmation of the diagnosis.
Mutations in the δ globin gene are not pathologically significant [1]. However, coinheritance of β and δ thalassaemia can mask the diagnosis of β thalassaemia trait as it causes HbA2 level to be lowered [2,3]. Here, we reported 5 unrelated cases of compound heterozygous β0 Filipino ~ 45 kb deletion and codon 67 (GTG>ATG) HbA2 Deventer in Sabahan population.
Cases of β°-thalassemia traits with unusual low HbA2 were reviewed. These cases were initially referred to our laboratory for definitive diagnosis of β-thalassemia trait. Haematological parameters and Hb analysis were carried out at the referral hospital. Genomic DNA was extracted from the peripheral blood. Multiplex ARMS and Gap PCR were done to detect common point mutations and deletions for both alpha and beta globin genes. Sanger sequencing was performed to detect mutations in delta globin gene.
Patients’ consist of 4 males and 1 female aged between 25-38 years old. All of them are indigenous Sabahan (2 Kadazans, 1 Murut, 1 Dusun and 1 Sungai). Their haemoglobin level ranges between 10.8 – 12.8g/dl. Hb analysis findings of HbA2 and HbF level ranges between 2.9 – 4.0 and 2.2 – 9.4g/dl respectively. Molecular findings revealed heterozygous state of (β)º-thal, Filipino ~45Kb deletion, NG_000007.3:g.[66258_184734del];[66258_184734=] and heterozygous state of Codon 67 [GTG>ATG] Hb A2-Deventer mutation, NG_000007.3:g.[63512G>A];[63512G=] (Figure 1 and 2).
Detection of 5 unrelated cases of HbA2 Deventer may suggest that this delta variant is common among indigenous Sabahan. Since beta thalassaemia is also common in the population, more attention should be paid during diagnosis. Identification of delta variant in beta thalassaemia carrier is important because coinheritance of beta and delta thalassaemia results in a less elevated HbA2 level. Therefore, molecular testing of thalassemia carrier state in the case of borderline HbA2 is warranted to avoid misdiagnosis of beta thalassaemia carriers.
The most common inherited monogenic disorders in the world are the haemoglobinopathies and thalassaemia. Thalassaemia is a heterogeneous group of genetic disorders of haemoglobin synthesis, characterised by a reduction in the production of one or more of the subunits of haemoglobin chains [1]. Haemoglobin A2 (HbA2) level is an important parameter in thalassaemia diagnosis. High HbA2 level (≥4.0) detected in Hb analysis, points to the diagnosis of beta thalassaemia and other haemoglobinopathies. However, in some cases, the HbA2 levels are apparently normal or borderline high despite abnormal haematological profile. In these cases, further testing is required to confirm the diagnosis. The aim of this study is to examine any abnormality at molecular level in cases of Hb analysis results with normal or borderline high HbA2 level.