Revisiting and updating molecular epidemiology of α-thalassemia mutations in Thailand using MLPA and new multiplex gap-PCR for nine α-thalassemia deletion

Abstract

Abstractα-thalassemia is an inherited blood disorder that is most frequently found in Southeast Asian populations. In Thailand, molecular characterization can diagnose most patients with α-thalassemia; however, several atypical patients are also observed in routine analyses. Here, we characterized α-thalassemia mutations among 137 Hemoglobin H (Hb H) disease patients and three fetuses of Hb Bart’s hydrops, a fatal clinical phenotype of α-thalassemia. Specifically, we performed multiplex ligation-dependent probe amplification (MLPA) followed by direct DNA sequencing. We noticed common genotypes in 129 patients and eight patients had rare Hb H disease caused by compound heterozygous α0-thalassemia (--CR or --SA deletion) with α+-thalassemia (-α3.7/-α4.2/αConstant Springα). Furthermore, two affected fetuses had the --SA/--SEA and one had the --CR/--SEA genotypes. Next, we developed and validated a new multiplex gap-PCR and applied this method to 844 subjects with microcytic red blood cells (RBCs) from various parts of Thailand. The frequency of heterozygous α0-thalassemia was dominated by --SEA 363/844 (43%), followed by --THAI 3/844 (0.4%), --SA 2/844 (0.2%), and --CR 2/844 (0.2%) mutations. These findings suggest that aforementioned four mutations should be routinely applied to increase the effectiveness of diagnosis and genetic counseling in this region.

Characterization of New Alpha Zero (α0) Thalassaemia Deletion (−−GB) among Malays in Malaysian Population

“…Malaysia has a heterogeneous population; thus, the distribution of thalassaemia carriers varies by geographical location and ethnic groups. The distribution of specific genotype determinants has been reported by several local researchers [7][8][9][10]. In Malaysia, most common alpha determinants that were reported are the −α 3 .…”

“…The α 0 -thalassaemia, involves complete loss of both functional α globin Diagnostics 2023, 13, 3286 2 of 11 genes in cis on the same chromosome mainly caused by deletions (−−/αα). The molecular deletions resulting in α 0 -thalassaemia include the −− SEA , −− THAI , −− MED , and −− FIL deletions and are presented with mild hypochromic microcytic anaemia [1,9]. The deletions vary in size and tend to be geographically isolated, with two particularly common ones in the Southeast Asia and Mediterranean region, which are −− SEA and −− MED deletions, respectively.…”

“…The designated α + (alpha plus) thalassaemia is used. At the other extreme there is a four gene deletion with a total lack of α-globin gene chain that causes haemoglobin Bart's hydrops fetalis, a condition that is generally incompatible with life [9,13,14]. Haemoglobin H disease is a clinical syndrome resulting from a variety of genetic abnormalities.…”

“…7 in the Mediterranean area [3,10,15]. When both α-genes on a single chromosome are deleted, the designated α • thalassaemia (alpha zero thalassaemia) is used [9].…”

“…Conventional methods using either capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are unable to definitively detect alpha thalassaemia carriers [11,24]. Although common deletions and non-deletional alpha genes relevant to the Southeast Asian population can be detected by a single multiplex Gap-PCR and ARMS-PCR, the rare and novel deletions depend on advanced techniques for their identification [9]. The multiplex ligation-dependent probe amplification (MLPA) technique has been used for this purpose and was successfully used in this study to detect the molecular alterations responsible for the α-thalassaemia phenotype in 29 individuals of Malay ethnicity that remained uncharacterized after sequential analysis and Gap-PCR for common deletions.…”