Objectives: The aim of this study was to develop a reliable, rapid and cost-effective molecular diagnostic assay allowing widespread routine investigation of eIF2B-related disorders (CACH/VWM syndrome). This heterogeneous disease is caused by autosomal recessive mutations in the genes encoding the five subunits of the translation-initiation factor eIF2B. Such a diagnostic method would be particularly adapted to the apparently acute presentation of the disease.
Design and methods: We developed a multiplex PCR amplification method for 7 genomic regions of the eIF2B genes in a single run. This method targeted the 8 most frequent mutations representing 61.4% of all mutations identified to date in our laboratory. These mutations affected eIF2B2 exon 5, eIF2B3 exon 2, eIF2B4 exons 8 and 11 and eIF2B5 exons 5, 7 and 8. PCR products were then pooled and subjected to a primer-extension assay validated using previously genotyped samples.
Results: The results were compared to screening and/or direct sequencing methods: 100% agreement between methods confirmed equivalent sensitivity and specificity. The new assay was highly superior in terms of cost, time to results and robustness despite sample heterogeneity.
Conclusions: This genotyping strategy allows the detection of all eIF2B mutations targeted. A second multiplex primer-extension assay is in development to detect the 11 next-most frequent mutations, thus raising the global detection rate to 76.8%. Our approach is widely applicable as it involves standard techniques and equipment. Moreover, it can easily be further adapted to the clinical and genetic heterogeneity of eIF2B-related disorders by including or excluding mutations.
Keywords: CACH/VWM syndrome; Multiplex single-nucleotide primer extension; eIF2B mutations.
Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.