title: Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome.
abstract: Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA-ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge-Weber syndrome (SWS) using ddPCR and PNA-ddPCR. Importantly, among three patients previously found to be mutation negative by next-generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.|Department of Medical and Clinical Science, Gunma University Graduate School of Medicine, Gunma, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Uchiyama
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Nakashima
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Neurosurgery, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Miyajima
authors.affiliation: Department of Biostatistics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Taguri
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Miyatake
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Miyake
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Hirotomo
authors.initials: H
authors.lastname: Saitsu
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mishima
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kinoshita
authors.affiliation: Department of Neurosurgery, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Arai
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan.
authors.forename: Ko-ichiro
authors.initials: K
authors.lastname: Yoshiura
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Naomichi
authors.initials: N
authors.lastname: Matsumoto
mesh_terms: D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics*; D005787:Gene Frequency; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013341:Sturge-Weber Syndrome / Q000175:diagnosis*
chemical_list: C554061:GNAQ protein, human; D050505:Mutant Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11