June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Impact of CHM Mutations at the mRNA level
Author Affiliations & Notes
  • Markus Preising
    Department of Ophthalmology, Justus-Liebig-University, Giessen, Germany
  • Nayyir Alavi
    Department of Ophthalmology, Justus-Liebig-University, Giessen, Germany
  • Birgit Lorenz
    Department of Ophthalmology, Justus-Liebig-University, Giessen, Germany
  • Footnotes
    Commercial Relationships Markus Preising, None; Nayyir Alavi, None; Birgit Lorenz, Optos (F)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1330. doi:
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      Markus Preising, Nayyir Alavi, Birgit Lorenz; Impact of CHM Mutations at the mRNA level. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1330.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Purpose: Pathogenic mutations underlying choroideremia fall into four general groups (1) big deletions covering the whole CHM gene or major parts of it, and preterm translation stops by (2) either intragenic deletions or insertions causing frameshifts, (3) splice site mutations causing exon skipping, or (4) nonsense mutations. Therefore, a protein truncation test and western blots were proposed as general diagnostic tools to identify mutations in CHM. Such a general loss of the functional gene product would not be in line with the phenotypical heterogeneity seen in patients. In a previous pilot study on lyonization in female carriers we recognized an escape of frameshifting mutations of CHM from nonsense-mediated decay (NMD). Therefore, we sought to identify the effect of CHM mutations at the mRNA level in males to explain the phenotypical heterogeneity.

Methods: Material and Methods: We generated EBV-immortalized lymphocyte cultures from 11 male index cases with frameshifting mutations (intragenic small deletions (n=3), small insertions (n=4), splice site mutations (n=1)), and nonsense mutations (n=3) in the CHM-gene. Whole RNA was extracted using the Qiagen Whole Blood Mini DNA/RNA Kit. The RNA was subjected to RT-PCR (Qiagen one-step RT-PCR Kit) using mutation-specific primers spanning 3 or more flanking exons. The PCR-products were analyzed on capillary-gel electrophoresis.

Results: Results: The presence of CHM transcripts was confirmed by successful RT-PCR of all mutations tested excluding deleted regions.

Conclusions: Discussion: Here we confirmed the escape from NMD by preterm translation stops created by frameshifting mutations in males in CHM. Therefore, at least 2 types of mutations in CHM have to be considered: (1) mutations producing a translatable mRNA and (2) mutations preventing the production of a translatable mRNA (extended deletions). The effect of post-translational quality control mechanisms must be subject of further studies.

Keywords: 604 mutations • 494 degenerations/dystrophies • 533 gene/expression  
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