Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI-110 (G > A) β-Thalassemia.

Petros Patsali,Maria Sitarou,Soteroulla Christou,Panayiota Papasavva,Carsten W Lederer,Marina Kleanthous,Michael N Antoniou

doi:10.3390/ijms21186671

Abstract

The β-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced β-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI−110(G > A) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI−110(G > A) β-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for β-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing.

Highlights

The HBBIVSI−110(G > A) β-thalassemia mutation (HBBIVSI−110 ) is classified as a Mediterranean mutation, but through migration has spread globally, with peak relative carrier frequencies in endemic countries, such as Cyprus (76%), Greece (42%), Turkey and North Macedonia and Egypt (37%), and lower frequencies in target countries of mixed migration, such as Germany (25%) and the United Kingdom (5%)
This study introduces a sensitive and accurate duplex reverse-transcription quantitative PCR (RT-qPCR) assay for the quantification of aberrant and normal HBBIVSI−110 -derived mRNA species
We applied this assay to evaluate for the first time absolute and relative HBBIVSII−654(C > T) (HBB) mRNA quantities in primary HBBIVSI−110 -homozygous thalassemic cells

Summary

Introduction

The HBBIVSI−110(G > A) β-thalassemia mutation (HBBIVSI−110 ) is classified as a Mediterranean mutation, but through migration has spread globally, with peak relative carrier frequencies in endemic countries, such as Cyprus (76%), Greece (42%), Turkey and North Macedonia (both 38%) and Egypt (37%), and lower frequencies in target countries of mixed migration, such as Germany (25%) and the United Kingdom (5%) (source: ITHANET [1]). The mutation introduces an aberrant splice site in the HBB gene, leading to integration of 19 nucleotides of intron 1, including an in-frame stop codon, into the corresponding abnormal mRNA. The latter is assumed to be an efficient target for nonsense-mediated decay [2,3]. For relative quantities of normal to aberrant HBB mRNA and based on semi-quantitative RT-PCR assessment, it appears that in reticulocytes and peripheral blood of patients homozygous for the HBBIVSI−110 mutation, normal HBB mRNA predominates by far [4]. Two other research groups instead investigated liquid cultures of primary hematopoietic cells from different HBBIVSI−110 patients, reporting relative quantities of normal transcripts between 60% and 100% in the one case [5] and of approximately 50%

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Conclusion