title: Candidate gene analysis using genomic quantitative PCR: identification of ADAMTS13 large deletions in two patients with Upshaw-Schulman syndrome.
abstract: Direct sequencing is a popular method to discover mutations in candidate genes responsible for hereditary diseases. A certain type of mutation, however, can be missed by the method. Here, we report a comprehensive genomic quantitative polymerase chain reaction (qPCR) to complement the weakness of direct sequencing. Upshaw-Schulman syndrome (USS) is a recessively inherited disease associated with severe deficiency of plasma ADAMTS13 activity. We previously analyzed ADAMTS13 in 47 USS patients using direct sequencing, and 44 of them had either homozygous or compound heterozygous mutations. Then, we sought to reveal more extensive defects of ADAMTS13 in the remaining three patients. We quantified copy numbers of each ADAMTS13 exon in the patients by using genomic qPCR. Each primer pair was designed to contain at least one of the two primers used in direct sequencing, to avoid missing any exonic deletions. The qPCR demonstrated heterozygous loss of exons 7 and 8 in one patient and exon 27 in the other, and further analysis revealed c.746_987+373del1782 and c.3751_3892+587del729, respectively. Genomic qPCR provides an effective method for identifying extensive defects of the target genes.
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center Suita, Osaka, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Eura
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center Suita, Osaka, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kokame
authors.affiliation: Department of Hematology and Clinical Immunology, Nishi-Kobe Medical Center Kobe, Hyogo, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Takafuta
authors.affiliation: Department of Nephrology, Hyogo Prefectural Kobe Children's Hospital Kobe, Hyogo, Japan.
authors.forename: Ryojiro
authors.initials: R
authors.lastname: Tanaka
authors.affiliation: Department of Hematology, Nagano Red Cross Hospital Nagano, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine Matsumoto, Nagano, Japan.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishida
authors.affiliation: Department of Nephrology, Koga General Hospital Miyazaki, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hisanaga
authors.affiliation: Department of Blood Transfusion Medicine, Nara Medical University Kashihara, Nara, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Blood Transfusion Medicine, Nara Medical University Kashihara, Nara, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fujimura
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center Suita, Osaka, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Miyata
keywords: ADAMTS13; Upshaw-Schulman syndrome; genetic analysis; hereditary disease; mutation; quantitative PCR; thrombotic thrombocytopenic purpura