title: Efficient detection of frequent eIF2B mutations for the rapid molecular diagnosis of CACH/VWM syndrome.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: CHU Clermont-Ferrand, Molecular Biology Laboratory, Biochemistry Department, Clermont-Ferrand, France; GReD, UMR INSERM 931, CNRS 6247, Faculty of Medicine, Clermont-Ferrand, France. Electronic address: ferreira.marieceleste@gmail.com.
authors.forename: Marie-Céleste
authors.initials: MC
authors.lastname: Ferreira
authors.affiliation: Inserm U1141, Paris Diderot University, Sorbonne Paris Cité, DHU PROTECT, Robert Debré Hospital, Paris, France. Electronic address: imen.dorboz@inserm.fr.
authors.forename: Imen
authors.initials: I
authors.lastname: Dorboz
authors.affiliation: Inserm U1141, Paris Diderot University, Sorbonne Paris Cité, DHU PROTECT, Robert Debré Hospital, Paris, France; Reference Center For Leukodystrophies, Department of Neuropediatrics and Metabolic Diseases, Robert Debré Hospital, AP-HP, Paris, France. Electronic address: odile.boespflug-tanguy@rdb.aphp.fr.
authors.forename: Odile
authors.initials: O
authors.lastname: Boespflug-Tanguy
mesh_terms: D000483:Alleles; D015415:Biomarkers / Q000378:metabolism; D017931:DNA Primers / Q000138:chemical synthesis; D020717:Eukaryotic Initiation Factor-2B / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D056784:Leukoencephalopathies / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009154:Mutation; D021122:Protein Subunits
chemical_list: D015415:Biomarkers; D017931:DNA Primers; D020717:Eukaryotic Initiation Factor-2B; D021122:Protein Subunits
keywords: CACH/VWM syndrome; Multiplex single-nucleotide primer extension; eIF2B mutations