Quantitative assay of deletion or duplication genotype by capillary electrophoresis system: Application in Prader-Willi syndrome and Duchenne muscular dystrophy.
BACKGROUND
Deletions and duplications involving large DNA segments result in underexpression or overexpression, depending on the changes in allele dose, and are known to cause many common disorders. Detection of allele dose variations in the human genome is increasingly important in medical genetic diagnosis.


METHODS
We used multiplex quantitative PCR coupled with capillary electrophoresis for accurate allele dose determination. In cases of Prader-Willi syndrome (PWS), a total of 24 patients with PWS, as well as 205 control individuals from the general population, were analyzed by use of multiplex quantitative PCR to amplify the FGFR2 gene, the KRIT1 gene, and the SNRPN gene simultaneously. In cases of Duchenne muscular dystrophy (DMD), we optimized the multiplex quantitative PCR to amplify 38 exons to analyze the DMD gene for rapid diagnosis of 12 DMD-affected males, 12 obligate carriers from families, and 50 unaffected female controls.


RESULTS
We were able to unambiguously diagnose the deletion genotype in PWS patients and identify all deletion or duplication genotypes and carrier status in DMD-affected cases with 100% sensitivity and specificity.


CONCLUSIONS
This report describes a novel single assay that can rapidly quantify allele dose to provide accurate clinical genetic diagnosis. This technique offers a valuable alternative for the rapid detection of genomic deletions or duplications and decreases costs because it does not require expensive fluorescent reagents.
Hung
Chia-Cheng
CC
Institute of Biomedical Engineering, College of Medicine and College of Engineering, Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chen
Chih-Ping
CP
Lin
Shuan-Pei
SP
Chien
Shu-Chin
SC
Lee
Chien-Nan
CN
Cheng
Wen-Fang
WF
Hsieh
Wu-Shiun
WS
Liu
Ming S
MS
Su
Yi-Ning
YN
Lin
Win-Li
WL
D001324:Autoantigens / Q000235:genetics; D004247:DNA / Q000032:analysis; D016189:Dystrophin / Q000235:genetics; D019075:Electrophoresis, Capillary; D005260:Female; D018628:Gene Dosage; D020440:Gene Duplication; D006801:Humans; D000075927:KRIT1 Protein; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011218:Prader-Willi Syndrome / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D055517:snRNP Core Proteins
D001324:Autoantigens; C484258:DMD protein, human; D016189:Dystrophin; D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D017411:Ribonucleoproteins, Small Nuclear; C526417:SNRPN protein, human; D055517:snRNP Core Proteins; D004247:DNA; C496353:FGFR2 protein, human; D051497:Receptor, Fibroblast Growth Factor, Type 2