TALEN-mediated single-base-pair editing identification SEE COMMENTARY of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome Hiroshi Ochiaia,b,1, Tatsuo Miyamotoa,1, Akinori Kanaic, Kosuke Hosobaa, Tetsushi Sakumab, Yoshiki Kudod, Keiko Asamie, Atsushi Ogawae, Akihiro Watanabee, Tadashi Kajiif, Takashi Yamamotob, and Shinya Matsuuraa,2 aDepartment of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; bDepartment of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan; cDepartment of Molecular Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; dDepartment of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan; eDepartment of Pediatrics, Niigata Cancer Center Hospital, Niigata 951-8566, Japan; and fHachioji, Tokyo 192-0023, Japan Edited by Albert de la Chapelle, Ohio State University Comprehensive Cancer Center, Columbus, OH, and approved November 20, 2013 (received for review September 11, 2013) Cancer-prone syndrome of premature chromatid separation with reported monoallelic BUB1B mutations in seven Japanese families mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare au- (2), all of which had one null mutation in the first allele but no tosomal recessive disorder characterized by constitutional aneu- second mutation was found in the opposite allele despite the de- ploidy and a high risk of childhood cancer. We previously reported crease in BUB1B transcripts and a conserved BUB1B haplotype. monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven The molecular basis of the second alleles was therefore unknown. Japanese families with the syndrome. No second mutation was In this study, we searched for the mutation in the second allele and found in the opposite allele of any of the families studied, although identified a unique SNP [ss802470619 (G/A)] in an intergenic BUB1B a conserved haplotype and a decreased transcript were region 44 kb upstream of BUB1B as a candidate mutation. identified. To clarify the molecular pathology of the second allele, To prove that this is the disease-causing mutation, we used GENETICS we extended our mutational search to a candidate region sur- transcription activator-like effector nuclease (TALEN)-medi- BUB1B > rounding . A unique single nucleotide substitution, G A ated single-base-pair editing to establish biallelically SNP-modified at ss802470619, was identified in an intergenic region 44 kb up- disease model cells for functional cytological assays. A TALEN stream of a BUB1B transcription start site, which cosegregated with consists of a customizable DNA binding domain and a DNA the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease–mediated cleavage domain and offers the advantage of simple and con- two-step single-base pair editing strategy and biallelically introduced venient design and construction compared with other engineered this substitution into cultured human cells. The cell clones showed endonucleases (EENs) such as zinc-finger nuclease (ZFN). TALENs reduced BUB1B transcripts, increased PCS frequency, and MVA, can introduce DNA double-stranded breaks (DSBs) into a spe- which are the hallmarks of the syndrome. We also encountered a case cific genomic site in pairs and induce the DNA damage response of a Japanese infant with PCS (MVA) syndrome carrying a homozy- to mend such breaks (4–7). In mammalian cells, DSBs are mainly gous single nucleotide substitution at ss802470619. These results repaired by nonhomologous end-joining in which the two ends suggested that the nucleotide substitution identified was the causal are processed and ligated together in a way that is frequently mutation of PCS (MVA) syndrome. accompanied by nucleotide insertions and deletions (6–8). The spindle assembly checkpoint | genome editing Significance udding uninhibited by benzimidazole-related 1 (BUBR1) is A single nucleotide substitution in an intergenic region upstream Ban important protein of the spindle assembly checkpoint. of BUB1B (encoding BUBR1) was identified as a candidate mu- Constitutional mutations in the BUB1B (budding uninhibited by tation for premature chromatid separation with mosaic varie- benzimidazoles 1 homolog beta) gene encoding BUBR1 cause gated aneuploidy [PCS (MVA) syndrome], a cancer-prone genetic the rare human disorder premature chromatid separation (PCS) disorder. To prove that this is the causal mutation, we designed syndrome [Mendelian Inheritance in Man (MIM) 176430], also a unique genome editing strategy, transcription activator-like known as mosaic variegated aneuploidy (MVA) syndrome (MIM effector nuclease–mediated two-step single-base-pair editing, to 257300). PCS (MVA) syndrome is characterized by PCS in >50% biallelically introduce this substitution into cultured human cells. metaphase cells and a variety of mosaic aneuploidies (1, 2). The cell clones showed chromosomal instability in the form of Patient clinical findings include Dandy–Walker complex, post- PCS and MVA, which are cellular hallmarks of the syndrome, cerebellar cysts, hypoplasia of the cerebellar vermis, lissencephaly, suggesting that this is indeed the underlying mutation. This cataracts, uncontrollable clonic seizures, polycystic kidneys, in- single-base-pair editing technique will be useful for investigations of noncoding variants of unknown functional relevance. fantile obesity, and a high risk of malignancy including Wilms’ tumor and rhabdomyosarcoma (3). Author contributions: H.O., T.M., and S.M. designed research; H.O., T.M., A.K., K.H., T.S., Both biallelic and monoallelic mutations of BUB1B have been and S.M. performed research; Y.K., K.A., A.O., A.W., and T.K. provided clinical information/ identified in individuals with the syndrome (1, 2). Biallelic analytic tools; and H.O., T.M., T.Y., and S.M. wrote the paper. mutations were previously found in five of eight families (1), The authors declare no conflict of interest. each of which had one null mutation in the first allele and an- This article is a PNAS Direct Submission. other missense mutation in the second (opposite) allele. The null See Commentary on page 1233. mutations result in a 50% reduction of BUBR1 function, 1H.O. and T.M. contributed equally to this work. whereas the missense mutations partially disrupt BUBR1 protein 2To whom correspondence should be addressed. E-mail: [email protected]. > functions. It was therefore deduced that a 50% reduction of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BUBR1 function is involved in the syndrome. We previously 1073/pnas.1317008111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1317008111 PNAS | January 28, 2014 | vol. 111 | no. 4 | 1461–1466 Downloaded by guest on September 23, 2021 cointroduction of EENs with a targeting vector and subsequent Identification of a Single Nucleotide Substitution in an Intergenic antibiotic selection previously enabled efficient homologous Region Upstream of BUB1B by Deep Sequencing. We speculated recombination-mediated genome editing to be performed in mam- that an unidentified mutation could be embedded around the malian cells (9–11). Moreover, the introduction of a specific nu- BUB1B locus of the second allele. To search for this, we analyzed cleotide substitution was demonstrated using ZFNs and either the haplotypes of a 1-Mb region in Japanese families and Cre/loxP or piggyBac technology (9, 11), as well as EENs and either ascertained the common ancestral haplotype within the 200-kb plasmid donor vectors (11, 12) or single-stranded oligodeox- interval from microsatellite marker 1790GT to 901722 (Fig. 1 C ynucleotides (ssODNs) (11, 13–16) without antibiotic selection. and D). We therefore determined the nucleotide sequences of In this study, we describe the successful use of a selection-based the 200-kb region in a patient with PCS (MVA) syndrome TALEN-mediated two-step single-base pair editing strategy in (PCS1, family 1) who had a truncating BUB1B mutation plus establishing model cells of PCS (MVA) syndrome. 6G3 haplotype. A total of 186 known and 5 unknown SNPs were identified in this region. The frequencies of all known SNPs were Results >1% in the general population and were excluded as candidates PCS (MVA) Syndrome Infant with No BUB1B Exonic Mutations. We for the mutation. Of the five unique SNPs, four did not cose- studied a Japanese infant (KH01) with PCS (MVA) syndrome gregate with the disorder so were excluded as candidates. Only who developed rhabdomyosarcoma of the urinary bladder, but SNP, ss802470619 (G/A), located 44 kb upstream of a BUB1B no microcephaly or Dandy–Walker anomalies, and had PCS in transcription start site, cosegregated with the disease (Fig. 1 C 67% of lymphocytes and mosaic aneuploidy in 17% (Fig. 1A). and D). The second hypomorphic alleles in Japanese families Western blot analysis revealed significant reduction of BUBR1 with PCS (MVA) syndrome all carried the nucleotide sub- expression compared with normal individuals (Fig. 1B). How- stitution G > A at ss802470619, and the infant (KH01) was ever, no BUB1B mutations were found in coding regions, intron- homozygous for the nucleotide substitution (Fig. 1 D and E). The exon boundaries, promoter regions, and untranslated regions. frequency of