RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems

Hum Mutat. 2000;15(5):418-29. doi: 10.1002/(SICI)1098-1004(200005)15:5<418::AID-HUMU3>3.0.CO;2-2.


Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea-formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic case.

MeSH terms

  • Amino Acid Substitution
  • Base Sequence
  • DNA Mutational Analysis
  • Drosophila Proteins*
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Variation
  • Glial Cell Line-Derived Neurotrophic Factor
  • Glial Cell Line-Derived Neurotrophic Factor Receptors
  • Hirschsprung Disease / genetics*
  • Humans
  • Molecular Sequence Data
  • Mutation*
  • Mutation, Missense
  • Nerve Growth Factor / genetics
  • Nerve Growth Factors*
  • Nerve Tissue Proteins / genetics*
  • Polymorphism, Genetic
  • Polymorphism, Single-Stranded Conformational
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins c-ret
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Deletion


  • Drosophila Proteins
  • GDNF protein, human
  • Glial Cell Line-Derived Neurotrophic Factor
  • Glial Cell Line-Derived Neurotrophic Factor Receptors
  • Nerve Growth Factors
  • Nerve Tissue Proteins
  • Proto-Oncogene Proteins
  • Nerve Growth Factor
  • Proto-Oncogene Proteins c-ret
  • Receptor Protein-Tyrosine Kinases
  • Ret protein, Drosophila