Abstract for presentation at 11th International Congress of Human Genetics

Screening for germline mutations in the MEN1 gene by Thermal Gradient Capillary Electrophoresis (TGCE)

  • Ms Cara Reith, Molecular Diagnostic Laboratory, London Health Sciences Centre, Ontario, Canada
  • Mr Matthew Vlasschaert, Molecular Diagnostic Laboratory, London Health Sciences Centre, Ontario, Canada
  • Mr Alan Stuart, Molecular Diagnostic Laboratory, London Health Sciences Centre, Ontario, Canada
  • Dr Jack Jung, Medical Genetics, London Health Sciences Centre, and University of Western Ontario, Canada
  • Dr Peter Ainsworth, Molecular Diagnostic Laboratory, London Health Sciences Centre, and University of Western Ontario, Canada

    • Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by parathyroid hyperplasia and pituitary adenomas, as well as neuroendocrine tumours of the pancreas and duodenum. Germline mutations are reported to be distributed throughout the MEN1 gene demonstrating the need to investigate the entire gene when characterizing new MEN 1 families (1). A PCR-based approach can be used to enable the detection of sequence variants such as frameshifting small deletions or insertions, as well as point mutations that may result in nonsense codons or abnormal RNA splicing. Mutation detection by direct sequence analysis is laborious and costly, however mutation scanning methods including the detection of hetero-duplexes (HD) have proved to be very sensitive and can economize mutation detection. One such approach involves differentiation of thermodynamic stability and mobility of PCR amplified HD’s from heterozygous samples using Thermal Gradient Capillary Electrophoresis (TGCE) (2).
    MEN1 coding sequence was PCR-amplified using DNA from blood samples obtained from a panel of 30 individuals identified to be at risk for the MEN1 syndrome. These were screened by TGCE using a Spectrumedix analyzer with a 24 capillary array, followed by DNA sequence analysis of abnormal samples. The 9 coding exons of the MEN1 gene were amplified by PCR, MEN1 exon 2 being amplified in two overlapping fragments (exon 2A and 2B) and exons 5 and 6 in a single PCR amplicon. A total of 9 individual variants identified by TGCE were characterized, demonstrating 5 apparently deleterious mutations including: EX9 1253_1254delACinsCT:D418A, EX10 1378C>T:R460X, EX9 1306T>C:W436R, an IVS7+2T>G splice jct mutn, and an 8 bp insert in exon 2; as well as 4 apparently benign sequence variants 2 of which were recurrent. These results demonstrated the utility of TGCE as a mutation scanning method in MEN1.
    1 Guo S et al (2001) Mol Endocrinol 15:1653-1664
    2 Chou L et al (2005) J Mol Diagn 7:111-20

    Conference Organiser - ICMS Pty Ltd