title: Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.
abstract: BACKGROUND
  Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation.
  
  
  METHODS
  Recombinant wild-type and mutant plasmids of c.3321delA mutation were constructed, heterozygous mutant plasmids were prepared by mixing the mutant plasmids and wild-type plasmids at 1:1 ratio. High-resolution melting analysis (HRMA) coupled with an unlabeled DNA probe was employed to identify the shift in melting temperature of the probe-template complex, which reflects the presence of c.3321delA mutation.
  
  
  RESULTS
  Unlabeled probe based HRMA was able to distinguish all three genotypes (wild-type, heterozygote, and mutant) of c.3321delA mutation. Then, we applied this method to genotype 1,317 clinical samples. Genotyping results obtained from unlabeled probe HRMA were 100% concordant with the results from direct sequencing.
  
  
  CONCLUSIONS
  We developed a fast and high-throughput method to detect the c.3321delA mutation.
authors.affiliation: Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China.|Division of Clinical Medicine, Shantou University Medical College, Shantou, Guangdong, P.R. China.
authors.forename: Wei-Long
authors.initials: WL
authors.lastname: Zhong
authors.affiliation: Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China.
authors.forename: Luo
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China.|Division of Clinical Medicine, Guangzhou Medical University, Guangzhou, Guangdong, P.R. China.
authors.forename: Xia
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China.
authors.forename: Xiao-Fan
authors.initials: XF
authors.lastname: Chen
authors.affiliation: Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. zhangweispace@yeah.net.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China. douxia@medmail.com.cn.|Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. douxia@medmail.com.cn.
authors.forename: Xia
authors.initials: X
authors.lastname: Dou
authors.affiliation: Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, P.R. China. yubomd@hotmail.com.|Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, P.R. China. yubomd@hotmail.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
mesh_terms: D000544:Alzheimer Disease / Q000175:diagnosis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D000091344:Filaggrin Proteins; D005838:Genotype; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008297:Male; D021141:Nucleic Acid Amplification Techniques; D017384:Sequence Deletion / Q000235:genetics*
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins
keywords: atopic dermatitis; c.3321delA mutation; filaggrin gene; skin barrier; unlabeled probe based high-resolution melting analysis