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J Med Genet 48:640-644 doi:10.1136/jmg.2011.089615
  • Molecular mechanisms
  • Short report

Efficiency of translation termination in humans is highly dependent upon nucleotides in the neighbourhood of a (premature) termination codon

  1. Holm Schneider1
  1. 1Division of Molecular Pediatrics, Children's Hospital, University of Erlangen-Nürnberg, Erlangen, Germany
  2. 2Institute of Molecular Biology, Center for Molecular Biosciences, University of Innsbruck, Austria
  3. 3Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, Rome, Italy
  4. 4Department of Dermatology, University Medical Center Freiburg, Germany
  1. Correspondence to Dr Holm Schneider, Children's Hospital, University of Erlangen-Nürnberg, Loschgestr 15, 91054 Erlangen, Germany; holm.schneider{at}uk-erlangen.de
  1. Contributors HS coordinated clinical data collection and follow-up examinations of the patient; GZ established cultures of the patient's keratinocytes; VC and DK performed immunoprecipitation assays and immunofluorescence analyses, respectively; FP planned and carried out experiments and analysed the data; HS supervised the experiments; FP and HS interpreted the data and wrote the manuscript.

  • Received 20 January 2011
  • Revised 17 May 2011
  • Accepted 26 May 2011
  • Published Online First 21 June 2011

Abstract

Background Spontaneous read-through of a premature termination codon (PTC) has so far not been observed in patients carrying nonsense mutations. This report describes a patient with junctional epidermolysis bullosa who was expected to die because of compound heterozygous nonsense mutations in the gene LAMA3 (R943X/R1159X), but was rescued by spontaneous read-through of the R943X allele.

Results and conclusion FACS analysis of cells carrying various PTCs surrounded by their natural neighbouring codons revealed significant reporter gene expression despite the PTC only for this patient's genetic context. Gene expression could be abolished by replacing the first or third nucleotide before, or one of the two nucleotides following the PTC. Site-directed mutagenesis was used to identify genotypes allowing PTC read-through. The genetic context of the LAMA3 mutation R943X is close to a hypothetical consensus sequence for maximum PTC read-through. Bioinformatic analysis showed that this consensus sequence is present in four sequences from the NCBI reference database, each of which contains another in-frame termination codon three or four codons apart. This indicates strong selective pressure against leaky termination codons in the human genome. This patient's mutated full length mRNA escaped nonsense-mediated decay, leading to LAMA3 mRNA levels similar to those of a healthy control, and full length laminin α3 could be detected in culture supernatant of the patient's keratinocytes. Immunofluorescence analyses of skin biopsies and continuous clinical improvement of the patient's condition suggested accumulation of intact laminin-332 in the epidermal basement membrane. These findings provide important clues for the prediction of PTC read-through in human genetic disease.

Footnotes

  • Competing interests None.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.