Multiplex ligation-dependent probe amplification (MLPA) assay: a single centre experience of MLPA assay for alpha thalassaemia diagnosis.

Introduction: Individuals with alpha(α)-thalassaemia usually have evidence of microcytosis but showed normal haemoglobin A2 and F, except those with three or four gene deletions or those with abnormal Haemoglobin (Hb) such as Hb Constant Spring (HbCS). Definitive diagnosis requires molecular analysis. Multiplex amplification refractory mutation system (ARMS) and gap PCR are reliable for detecting common α-gene mutations; however, many rare or novel mutations remain unidentified. Using principle of primer-specific amplification, abnormality analysed is primer-dependent. This study aimed to compare the detection of HBA gene rearrangements by multiplex ligation-dependent probe amplification (MLPA) with multiplex PCR (ARMS and Gap).

Materials and methods: MLPA facilitates amplification of multiple nucleic acid sequences with a single primer pair via identical end probe amplification, thus giving wide α-globin analysis in a single experiment to provide high-resolution detection. Amplification products only require capillary electrophoresis separation followed by software analysis. Seventy-three samples that have been analysed by multiplex PCR were selected for this study. Fifty-five confirmed cases of α-thalassaemia and 18 normal samples were tested using MLPA. Discordant cases suspected of α-thalassaemia underwent sequencing analysis.

Results: All normal samples and 50 positive cases showed consistent findings between both methods. MLPA showed 100% sensitivity and specificity in detecting HbCS mutation. However, MLPA could not determine zygosity of three homozygous HbCS cases detected by multiplex PCR. The concordant rate was 93.2% between both methods. MLPA results in five discordant cases.

Conclusion: MLPA is a reliable and accurate technique for characterising HBA gene rearrangements. Overall, both methods showed excellent concordance rate and statistically good agreement. The simplicity of wide α-globin cluster analysis makes MLPA as favourable diagnostic method for the detection both common and unresolved HBA gene abnormalities involving HBA gene cluster.