TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome

Abstract

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome. Both biallelic and monoallelic mutations of BUB1B have been identified in individuals with the syndrome (1, 2). Biallelic mutations were previously found in five of eight families (1), each of which had one null mutation in the first allele and another missense mutation in the second (opposite) allele. The null mutations result in a 50% reduction of BUBR1 function, whereas the missense mutations partially disrupt BUBR1 protein functions. It was therefore deduced that a >50% reduction of BUBR1 function is involved in the syndrome. We previously reported monoallelic BUB1B mutations in seven Japanese families (2), all of which had one null mutation in the first allele but no second mutation was found in the opposite allele despite the decrease in BUB1B transcripts and a conserved BUB1B haplotype. The molecular basis of the second alleles was therefore unknown. In this study, we searched for the mutation in the second allele and identified a unique SNP [ss802470619 (G/A)] in an intergenic region 44 kb upstream of BUB1B as a candidate mutation.To prove that this is the disease-causing mutation, we used transcription activator-like effector nuclease (TALEN)-mediated single-base-pair editing to establish biallelically SNP-modified disease model cells for functional cytological assays. A TALEN consists of a customizable DNA binding domain and a DNA cleavage domain and offers the advantage of simple and convenient design and construction compared with other engineered endonucleases (EENs) such as zinc-finger nuclease (ZFN). TALENs can introduce DNA double-stranded breaks (DSBs) into a spe...

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

“…1h) [92]. The first step included TALEN-mediated insertion of a drug-selectable marker cassette into an SNV flanking region.…”

“…Compared with the CORRECT method, the TALEN-mediated two-step singlebase-pair editing strategy enables more efficient isolation of the edited clones, since it uses an antibiotic resistance marker for screening. Using this approach, we identified a causal mutation of a cancer-prone genetic disorder, premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA)] syndrome [92][93][94]. Both biallelic and monoallelic mutations of the BUB1B gene encoding BubR1 have been reported in patients [95,96].…”

“…No second mutation in exons of the BUB1B gene was found in the opposite allele, although a conserved BUB1B haplotype within a 200-kb interval among the Japanese patients and a reduced transcript level were identified [96]. We thus determined the nucleotide sequence of the 200-kb region in a patient with this syndrome using deep-sequencing analysis and found that a unique SNV in an intergenic region 44-kb upstream of the BUB1B transcription start site was linked to the disease [92]. We used TALEN-mediated single-base-pair editing technology to biallelically introduce this substitution into HCT116 cells.…”

“…We used TALEN-mediated single-base-pair editing technology to biallelically introduce this substitution into HCT116 cells. The genome-edited clones showed reduced BUB1B transcript levels and PCS (MVA) syndrome-specific chromosomal instability, demonstrating that the identified SNV was indeed the causal mutation [92]. The single-base-pair editing technique is thus useful to evaluate nucleotide variants with unknown functional relevance.…”

A practical guide to induced pluripotent stem cell research using patient samples

“…[58][59][60][61][62] TALEN has also been used to generate single base-pair mutations, linking single-nucleotide polymorphisms to specific human disease. 63 Furthermore, TALENs have even been utilized to eliminate the mutant form of mitochondrial DNA from patient-derived cells. 64 Currently, TALEN plasmids targeting 18 740 protein-coding human genes have been assembled using a high-throughput GoldenGate cloning system.…”