title: Identification of the COL2A1 mutation in patients with type I Stickler syndrome using RNA from freshly isolated peripheral white blood cells.
abstract: Stickler syndrome type I is caused by mutations in the type II collagen gene (COL2A1), which is specifically expressed in cartilage and vitreous humor. We developed a simple and noninvasive strategy for identifying the COL2A1 mutation using RNA from freshly isolated peripheral white blood cells and identified a new 3' splice site mutation in a Japanese family with Stickler syndrome. RNA was isolated from a patient's peripheral white blood cells that had been incubated with cycloheximide, an inhibitor of nonsense-mediated mRNA decay. COL2A1 cDNA fragments covering the entire coding region were obtained by RT-polymerase chain reaction cloning using a high-fidelity DNA polymerase and sequenced. Whole sequencing of the patient's cDNA resulted in identification of a 49-bp deletion in the region corresponding to exon 18. The deletion introduced a premature termination codon. Targeted genome sequencing identified a base substitution at the A (-2) position of the 3' splice acceptor site of intron 17. This mutation led to utilization of the cryptic splice site, which is located 49 bases downstream of the normal splice site and causes aberrant mRNA splicing, resulting in a 49-base deletion in the patient's mRNA. Our method is much easier than conventional genomic screening and provides a simple and noninvasive diagnostic test for patients with Stickler syndrome.
authors.affiliation: Department of Ophthalmology, Kanazawa Medical University School of Medicine, Uchinada, Ishikawa, Japan.
authors.forename: Hiromoto
authors.initials: H
authors.lastname: Yaguchi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ikeda
authors.forename: Hiromi
authors.initials: H
authors.lastname: Osada
authors.forename: Yoshino
authors.initials: Y
authors.lastname: Yoshitake
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sasaki
authors.forename: Hideto
authors.initials: H
authors.lastname: Yonekura
mesh_terms: D000293:Adolescent; D000328:Adult; D001168:Arthritis / Q000235:genetics / Q000473:pathology; D001483:Base Sequence; D003001:Cloning, Molecular; D024043:Collagen Type II / Q000235:genetics*; D003240:Connective Tissue Diseases / Q000235:genetics / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D005190:Family; D006319:Hearing Loss, Sensorineural / Q000235:genetics / Q000473:pathology; D006801:Humans; D007564:Japan; D007962:Leukocytes / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012163:Retinal Detachment / Q000235:genetics / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: C499177:COL2A1 protein, human; D024043:Collagen Type II; D018076:DNA, Complementary; D012333:RNA, Messenger