Development of a denaturing high-performance liquid chromatography screening method for SMAD4 in juvenile polyposis syndrome.
BACKGROUND
Juvenile polyposis syndrome is an inherited condition associated with the development of gastrointestinal polyps and subsequent cancer. An efficient denaturing high-performance liquid chromatography (DHPLC) screening method has been developed to detect mutations in SMAD4, which account for around 20% of reported cases.


METHODS
The nucleotide sequences of polymerase chain reaction (PCR)amplified SMAD4 exons were examined using WAVEMAKER software to determine suitable DHPLC conditions. Multiple wild-type controls were characterized by DHPLC, and known mutations analysed where available.


RESULTS
DHPLC elution profiles for wild-type and variant SMAD4 alleles were successfully characterized across a range of column oven temperatures.


CONCLUSIONS
DHPLC analysis of SMAD4 has been refined to an efficient screening protocol that uses the minimal number of column oven temperatures for reliable mutation detection.
Hatch
Sarah L
SL
Clinical Biochemistry, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, UK. sarah.hatch@rlbuht.nhs.uk
Trewick
Anne L
AL
Payne
Stewart J
SJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D044483:Intestinal Polyposis / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D051901:Smad4 Protein / Q000235:genetics*
D051901:Smad4 Protein