Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia

Nakamura, Kotoka; Du, Liutao; Tunuguntla, Rashmi; Fike, Francesca; Cavalieri, Simona; Morio, Tomohiro; Mizutani, Shuki; Brusco, Alfredo; Gatti, Richard A.

doi:10.1002/humu.21632

Cited by 43 publications

(36 citation statements)

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“…12,13 In particular, some of these mutations have been demonstrated to occur in deep intronic regions that are associated with the retention of intronic sequences in the mRNAs and AONs have been already been designed to successfully abrogate these ATM mutations. 5,14 In the current study, we studied a new deep-intronic mutation detected in an A-T patient using a NGS strategy to resequence the entire 160-kb ATM genomic region, after standard mutation detection techniques (DHPLC, MLPA and cDNA sequencing), failed to identify the second mutation. Our strategy to sequence the proband and the transmitting parent, in our case the mother, was very successful.…”

Section: Discussionmentioning

confidence: 99%

“…Conventional methods for mutation detection are on the basis of PCR amplification of ATM exons, accompanied by MLPA to detect large genomic deletions or duplications. 4,5 This multistep approach identified 495% of the mutations. The application of the next-generation DNA sequencing technology (NGS) is becoming an important tool to identify additional rare mutations.…”

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Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

et al. 2012

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Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C4T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5 0 ) splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3 0 ) splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C4T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction. Keywords: ATM; ataxia-telangiectasia; next-generation sequencing; antisense oligonucleotide; deep intronic mutations INTRODUCTION Ataxia-telangiectasia (A-T) is a rare autosomal recessive neurodegenerative disorder (A-T; MIM# 208900) characterized by progressive cerebellar degeneration, oculocutaneous telangiectasia, immunodeficiency, increased cancer risk, sensitivity to ionizing radiation, and chromosomal instability. 1 A-T is caused by mutations in the ATM gene (MIM#607585) that encodes a 370-kDa ubiquitous protein. ATM is a serine/threonine kinase involved in cell cycle checkpoints, repair of double-strand DNA breaks, response to oxidative stress, and apoptosis. 2,3 More than 600 unique ATM mutations have been identified worldwide (www.LOVD.nl/ATM). The majority lead to absence of ATM protein and loss of its kinase function. Conventional methods for mutation detection are on the basis of PCR amplification of ATM exons, accompanied by MLPA to detect large genomic deletions or duplications. 4,5 This multistep approach identified 495% of the mutations. The application of the next-generation DNA sequencing technology (NGS) is becoming an important tool to identify additional rare mutations. Here, we report the study of an A-T patient with a nonsense mutation in exon 45 and a novel pseudoexon-retaining deep-intronic mutation, detected by genomic resequencing. With the use of antisense morpholino oligonucleo...

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Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

et al. 2012

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“…This can been achieved with the use of the nonsense-codon suppressors Ataluren, read-through compound (RTC)13, Amlexanox, synthetic aminoglycosides and nonaminoglycosides (Du et al, 2008(Du et al, , 2009Gonzalez-Hilarion et al, 2012;Nakamura et al, 2012;Shalev and Baasov, 2014;Welch et al, 2007), although their lack of drug-target specificity is a great concern, given the large number of physiological cellular mRNAs that are NMD targets. Among these compounds, Ataluren (Translarna) is farthest along the drug-development pipeline, as it has shown the greatest effectiveness in treating Duchenne muscular dystrophy caused by nonsense mutations, but at doses that do not detectably inhibit NMD, consistent with its mild effects on cellular metabolism (Finkel et al, 2013;Welch et al, 2007).…”

Section: Therapeutic Approaches For Ptc-associated Diseasesmentioning

confidence: 99%

Nonsense-mediated mRNA decay in humans at a glance

Kurosaki

Maquat

2016

Journal of Cell Science

320

290

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Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that typifies all eukaryotes examined to date. NMD surveys newly synthesized mRNAs and degrades those that harbor a premature termination codon (PTC), thereby preventing the production of truncated proteins that could result in disease in humans. This is evident from dominantly inherited diseases that are due to PTC-containing mRNAs that escape NMD. Although many cellular NMD targets derive from mistakes made during, for example, pre-mRNA splicing and, possibly, transcription initiation, NMD also targets ∼10% of normal physiological mRNAs so as to promote an appropriate cellular response to changing environmental milieus, including those that induce apoptosis, maturation or differentiation. Over the past ∼35 years, a central goal in the NMD field has been to understand how cells discriminate mRNAs that are targeted by NMD from those that are not. In this Cell Science at a Glance and the accompanying poster, we review progress made towards this goal, focusing on human studies and the role of the key NMD factor upframeshift protein 1 (UPF1).

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“…Mutation-targeted therapies, such as read-through compounds (RTCs) for nonsense mutations [10][11][12] and antisense morpholino oligonucleotides for splicing mutations [13][14][15] , have restored functional expression of the ATM protein in A-T patient-derived cells. Several leading RTCs from our previous studies induced expression of functional ATM protein in human nonsense mutation-containing lymphoblastoid cell lines (LCLs) and fibroblasts derived from A-T patients 11 .…”

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SMRT compounds abrogate cellular phenotypes of ataxia telangiectasia in neural derivatives of patient-specific hiPSCs

et al. 2013

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Ataxia telangiectasia is a devastating neurodegenerative disease caused primarily by loss of function mutations in ATM, a hierarchical DNA repair gene and tumour suppressor. So far, murine models of ataxia telangiectasia have failed to accurately recapitulate many aspects of the disease, most notably, the progressive cerebellar ataxia. Here we present a model of human ataxia telangiectasia using induced pluripotent stem cells, and show that small molecule read-through compounds, designed to induce read-through of mRNA around premature termination codons, restore ATM activity and improve the response to DNA damage. This platform allows for efficient screening of novel compounds, identification of target and off-target effects, and preclinical testing on relevant cell types for the pathogenic dissection and treatment of ataxia telangiectasia.

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Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia

Cited by 43 publications

References 54 publications

Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

Nonsense-mediated mRNA decay in humans at a glance

SMRT compounds abrogate cellular phenotypes of ataxia telangiectasia in neural derivatives of patient-specific hiPSCs

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