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Identification of a mutation that perturbs NF1 agene splicing using genomic DNA samples and a minigene assay
  1. M Baralle1,
  2. D Baralle2,3,
  3. L De Conti1,
  4. C Mattocks2,
  5. J Whittaker2,
  6. A Knezevich1,
  7. C ffrench-Constant2,3,
  8. F E Baralle1
  1. 1International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italy
  2. 2Department of Medical Genetics, Addenbrooke’s Hospital, Cambridge, UK
  3. 3Department of Pathology, University of Cambridge, Cambridge, UK
  1. Correspondence to:
 Dr D Baralle, Department of Medical Genetics, Addenbrooke’s Hospital, Box 134, Hills Road, Cambridge CB2 2QQ, UK;
 db314{at}cam.ac.uk

https://doi.org/10.1136/jmg.40.3.220

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    Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disease. In recent studies on the neurofibromatosis type 1 (NF1) gene neurofibromin, splicing abnormalities were seen in 30-50% of cases when RNA taken from cell lines was analysed.1,2 Unlike mutations that alter critical amino acids or generate premature stop codons, splicing abnormalities can be very hard to predict from sequence analysis alone. Apart from the two base pairs 5′ and 3′ of each exon, few of the nucleotides in regions critical for splicing are absolutely conserved. As a consequence, it can be very difficult to conclude that a sequence variation found in a patient will alter splicing and so represents a pathogenic mutation.

    Key points

    • Abnormalities of pre-mRNA splicing represent an important mechanism by which gene mutations cause disease.

    • Effects on splicing can be predicted from genomic DNA sequence analysis if mutations alter highly conserved canonical splicing signals. However, it is extremely difficult to predict the effects of changes in intronic and exonic sequences not obviously involved in the splicing process.

    • We present here an efficient and simple test using genomic DNA to construct a minigene and analyse the effect on splicing of sequence variations.

    • Using this system, we describe the identification of one such sequence variation in the NF1 gene as the disease causing mutation. The aberrant splicing can then be rescued by coexpression of an altered U1 snRNA that restores normal base pairing.

    This difficulty is well illustrated by a family with NF1 in which we recently identified a sequence variation. The three generation family is from the UK and meets NIH diagnostic criteria. The index case, at the age of 82, has classical features of NF1 including multiple café au lait macules, neurofibromas, and axillary and inguinal freckling. Her son was similarly affected and died …

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