Molecular analysis of β-globin gene mutations in UKMMC using multiplex
amplification refractory mutation system and flow-through hybridization kit: A
comparison of two methods.
NORUNALUWAR Jalil BSc (HONS)1, Khairiliah AUSIKHIN Khairuman3, RUSILAWATY
Abdullah3, Raja Zahratul AZMA MBBS, MPATH2, EMIDA Mohamed PhD3, HAFIZA
Alauddin MBBS, MPATH2, AZLIN Ithnin MBBchBAO, MPATH2, ZARINA Abdul Latiff
MBBS, MMed (Paed)3, HAMIDAH Alias MD, MMed (Paed)3, RAFEAH Tumian MD,
MMed4, Noor FARISAH Abdul Razak BSc (HONS)1, MALISA Mohd Yusoff Dip MLT1 and
AINOON Othman MBBS, Dr Med Sc1,6
Departments of 1Laboratory Diagnostic Services and 2Pathology, UKM Medical Centre,
Kuala Lumpur.
3
Department of Medical Laboratory Technology, Universiti Teknologi MARA, Puncak Alam.
4
Departments of Paediatric & 5Medicine, UKM Medical Centre, Kuala Lumpur.
6
Department of Medical Sciences II, Faculty of Medicine, USIM, K. Lumpur.
Abstract
Introduction: β-thalassaemia causes expression defects mainly by point mutations on
chromosome 11. The aim of this study was to characterize β-globin gene mutations by using
multiplex ARMS polymerase chain reaction (MARMS-PCR) as compared to flow-through
hybridization (FTH) kit. Method: 56 β-thalassaemia cases were selected based on
hypochromic microcytic red cell indices and raised HbA2 level. Mutations analysis was
performed using nine primers of common β-globin gene mutations for MARMS-PCR and
correlated with 25 mutations on designed FTH kit. Results: MARMS-PCR successfully
detected β-globin gene mutations in 49/56 (87.5%) of samples. 24/49 (49.0%) were due to the
common IVS 1-5 and Cd 41/42 mutations. The rest were IVS 1-1, Cd 17, Cd 26, IVS 2-654,
Cd 8/9 and -28. There was one patient with compound heterozygous for Cd 41/42 and Cd 26
mutation. Seven samples are negative for the mutations tested. FTH results were in
agreement with MARMS-PCR in 47 cases, except for the two cases where Cd 8/9 mutation
was not part of FTH panel. FTH detected additional mutations (51/56 cases (91.1%)) as the
panel incorporated more mutational primers in. These comprised del45, 619bp del and polyA mutation. However, 619bp del was needed for a further reference test which is sequencing
as a confirmation since it was not common. Some of these mutations were seen to be
compoundly inherited with other more common β-globin gene mutations hence more severe
phenotypes of the affected patients. Discussion: Both MARMS-PCR and FTH techniques are
reliable and laborious, but FTH is able to detect 25 mutations in the single test for one
patient. However, hybridization is costly for large sample sizes. Thus, MARMS-PCR is more
cost effective to characterize the spectrum of β-thalassaemia mutations.
Keywords: ß-thalassaemia mutations, MARMS-PCR, Flow-through hybridization