title: Rapid genotyping for most common TGFBI mutations with real-time polymerase chain reaction.
abstract: Recent studies of the corneal dystrophies (CDs) have shown that most cases of granular CD, Avellino CD, and lattice CD type I are caused by mutations in the human transforming growth factor beta-induced (TGFBI) gene. The aim of this study was to develop a rapid diagnostic assay to detect mutations in the TGFBI gene. Sixty-six patients from 64 families with TGFBI-associated CD were studied. A primer probe set was designed to examine the genome from exons 4 and 12 of the TGFBI gene in order to identify mutant and wild-type alleles. A region spanning the mutations was amplified by the polymerase chain reaction (PCR) in a commercial cycler. Mutations were then identified by melting curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. Using this system, we clearly distinguished each CD genotype (homozygous and heterozygous 418G-->A, heterozygous 417C-->T, heterozygous 1710C-->T, and wild-type) of all the patients by means of the clearly distinct melting peaks at different temperatures. One thermal cycling took approximately 54 min, and all results were completely in concordance with the genotypes determined by conventional DNA sequencing. Thus, the technique is accurate and can be used for routine clinical diagnosis. We expect that our new method will help in making precise diagnoses of patients with atypical CDs and aid the revision of the clinical classification of inherited corneal diseases based on the genetic pathogenesis.
authors.affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan. yosida@med.kyushu-u.ac.jp
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yoshida
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamaji
authors.forename: Ayako
authors.initials: A
authors.lastname: Yoshida
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Noda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kumano
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Ishibashi
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D003199:Computer Systems; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics*; D015342:DNA Probes; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D016212:Transforming Growth Factor beta / Q000235:genetics*; D000099222:betaIG-H3 Protein
chemical_list: D015342:DNA Probes; D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein
references.citation: Am J Ophthalmol. 2003 Nov;136(5):872-8
references.pmid: 14597039
references.citation: Br J Ophthalmol. 1998 Nov;82(11):1280-4
references.pmid: 9924333
references.citation: Am J Hum Genet. 1998 Feb;62(2):320-4
references.pmid: 9463327
references.citation: Am J Ophthalmol. 2004 Mar;137(3):586-8
references.pmid: 15013897
references.citation: Invest Ophthalmol Vis Sci. 2004 May;45(5):1382-8
references.pmid: 15111592
references.citation: Am J Ophthalmol. 2004 Oct;138(4):670-1
references.pmid: 15488808
references.citation: Clin Chem. 1997 Dec;43(12):2262-7
references.pmid: 9439442
references.citation: Front Biosci. 2003 May 01;8:d687-713
references.pmid: 12700042
references.citation: Arch Ophthalmol. 2004 Aug;122(8):1224-7
references.pmid: 15302666
references.citation: Cornea. 2001 Jul;20(5):525-9
references.pmid: 11413411
references.citation: Cornea. 2000 May;19(3 Suppl):S21-3
references.pmid: 10832717
references.citation: Ophthalmology. 2004 Jun;111(6):1108-14
references.pmid: 15177960
references.citation: Am J Ophthalmol. 2004 Nov;138(5):772-81
references.pmid: 15531312
references.citation: Am J Ophthalmol. 2000 Mar;129(3):411-2
references.pmid: 10755958
references.citation: Am J Ophthalmol. 2004 Apr;137(4):765-7
references.pmid: 15059726
references.citation: Clin Chem. 1999 May;45(5):694-6
references.pmid: 10222362
references.citation: Jpn J Ophthalmol. 2002 Jul-Aug;46(4):469-71
references.pmid: 12225829
references.citation: Eye (Lond). 2005 Feb;19(2):215-7
references.pmid: 15184964
references.citation: Am J Ophthalmol. 2000 May;129(5):665-6
references.pmid: 10844062
references.citation: Arch Ophthalmol. 2001 Jan;119(1):16-22
references.pmid: 11146721
references.citation: J Leukoc Biol. 2003 Jan;73(1):137-44
references.pmid: 12525571
references.citation: J Hum Genet. 2001;46(8):431-5
references.pmid: 11501939
references.citation: Lab Invest. 2004 Jan;84(1):122-30
references.pmid: 14631379
references.citation: Cornea. 1999 Jul;18(4):424-9
references.pmid: 10422854
references.citation: Br J Ophthalmol. 2003 Jun;87(6):686-9
references.pmid: 12770961
references.citation: Am J Ophthalmol. 2000 Oct;130(4):516-7
references.pmid: 11024425
references.citation: Anal Biochem. 1998 Jan 1;255(1):101-7
references.pmid: 9448847
references.citation: Anal Biochem. 1998 Jul 1;260(2):142-8
references.pmid: 9657870
references.citation: Hum Genet. 1998 Sep;103(3):286-9
references.pmid: 9799082
references.citation: Am J Hum Genet. 1997 Dec;61(6):1448-50
references.pmid: 9399907
references.citation: Mol Vis. 2004 Sep 24;10:682-7
references.pmid: 15467524
references.citation: Nat Genet. 1997 Mar;15(3):247-51
references.pmid: 9054935
references.citation: Am J Ophthalmol. 2002 Jun;133(6):830-2
references.pmid: 12036680
references.citation: Ophthalmology. 2002 Apr;109(4):793-7
references.pmid: 11927442
references.citation: Lab Invest. 2003 Jul;83(7):927-38
references.pmid: 12861033
references.citation: Ophthalmology. 2001 Apr;108(4):818-23
references.pmid: 11297504
references.citation: Cornea. 2000 Nov;19(6):842-5
references.pmid: 11095060
references.citation: Eye (Lond). 2004 Jul;18(7):723-8
references.pmid: 15017378
references.citation: Br J Ophthalmol. 2000 Jun;84(6):583-5
references.pmid: 10837380
references.citation: Am J Ophthalmol. 2001 Aug;132(2):255-7
references.pmid: 11476689
references.citation: Cornea. 2002 Aug;21(6):570-3
references.pmid: 12131032
references.citation: Am J Ophthalmol. 1999 Jul;128(1):104-6
references.pmid: 10482106
references.citation: Ophthalmology. 2004 Jul;111(7):1407-9
references.pmid: 15234146
references.citation: Invest Ophthalmol Vis Sci. 1998 Sep;39(10):1947-53
references.pmid: 9727418