Rapid and cost-effective high-throughput sequencing for identification of germline mutations of BRCA1 and BRCA2.
Genetic testing for breast cancer predisposing genes, BRCA1 and BRCA2, can take advantage for early identification of carriers with pathogenic germline mutations. However, conventional approaches based on Sanger sequencing are laborious and expensive. Next-generation sequencing technology has a great impact on investigation of medical genomics and now applied clinical genetics. We provide a protocol based on a pool and capture method followed by high-throughput sequencing, which realizes a rapid, high-quality, high-accuracy and low-cost testing for mutations in BRCA1 and BRCA2 by using small amounts of input DNA. Custom capture probes were designed for 195 kb regions encompassing the entire BRCA1 and BRCA2. DNA libraries of 96 samples with distinct indices were pooled before hybridizing to the capture probes, which largely reduced labor and cost. The captured library was run on the Illumina MiSeq sequencer. We applied the method to 384 Japanese individuals including 11 patients with breast cancer whose mutation statuses had been determined by standard clinical testing and 373 individuals from a general population. 99.99% of coding exons and their 20 bp flanking regions were covered with a minimum of 20 reads and the average depth was 179.5, supporting confident variant detection. The sequencing method rendered concordant results for 11 patients with breast cancer compared with the standard clinical testing including nine mutations in eight patients. Among 373 individuals from the general population, novel stop gain and frameshift deletion in BRCA2 were identified, which led to truncated protein and were most likely to be pathogenic. The result suggests the importance of a large-scale population-wide screening for carriers of mutations in these genes.
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan.
Somayeh
S
Ahmadloo
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan.
Hirofumi
H
Nakaoka
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan.
Takahide
T
Hayano
Department of Bioinformatics and Genomics, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japan.
Kazuyoshi
K
Hosomichi
Affiliated Hospital of the Academy of Military Medical Sciences, Beijing, China.
Hua
H
You
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Emi
E
Utsuno
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Takafumi
T
Sangai
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.|Department of Molecular Diagnosis, Chiba University Graduate School of Medicine, Chiba, Japan.
Motoi
M
Nishimura
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.|Department of Molecular Diagnosis, Chiba University Graduate School of Medicine, Chiba, Japan.
Kazuyuki
K
Matsushita
Center for Preventive Medical Sciences, Chiba University, Chiba, Japan.|Department of Public Health, Chiba University Graduate School of Medicine, Chiba, Japan.
Akira
A
Hata
Divisions of Clinical Mass Spectrometry and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Fumio
F
Nomura
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan.
Ituro
I
Inoue
D044466:Asian People / Q000235:genetics; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics; D003362:Cost-Benefit Analysis; D005260:Female; D020022:Genetic Predisposition to Disease; D018095:Germ-Line Mutation / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics* / Q000379:methods*; D006801:Humans; D009154:Mutation
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human