Mutation analysis of the RET gene in total intestinal aganglionosis by wave DNA fragment analysis system.
OBJECTIVE
Total intestinal aganglionosis (TIA) extending from the duodenum to the rectum is the most rare form of Hirschprung's disease (HSCR) and usually is fatal. RET is the major gene associated with HSCR, and germline mutations of this gene account for up 50% of familial and up to 15 to 20% of sporadic cases in HSCR. The aim of this study was to investigate DNA variants in the RET gene in TIA patients using the WAVE DNA Fragment Analysis System.


METHODS
Genomic DNA was extracted from whole blood samples from 6 patients with TIA. Polymerase chain reaction (PCR) amplification of the 21 exons of RET was performed using published oligonucleotide primers. Heteroduplexes were followed by the WAVE DNA Fragment Analysis System with the DNASep cartridge.


RESULTS
WAVE system technology detected 16 variants in the RET gene in the 6 patients with TIA. Three patients had a significant mutation in exon 8, 11, and 15, respectively. Thirteen RET polymorphic variants also were detected in the 6 patients, with L746L variant in exon 13 occurring in 4 patients.


CONCLUSIONS
WAVE system technology is an efficient method for the detection of DNA sequence variants. Our findings suggest that not only RET mutations but also RET polymorphic variants may contribute to the occurrence of TIA.
Solari
V
V
Children's Research Centre, Our Lady's Hospital for Sick Children, University College Dublin, Crumlin, Ireland.
Ennis
S
S
Yoneda
A
A
Wong
L
L
Messineo
A
A
Höllwarth
M E
ME
Green
A
A
Puri
P
P
D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005260:Female; D020180:Heteroduplex Analysis; D006627:Hirschsprung Disease / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D007438:Introns / Q000235:genetics; D008297:Male; D020125:Mutation, Missense; D015513:Oncogene Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*
D018389:Codon, Nonsense; D015513:Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases