Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Du, Liutao; Pollard, Julianne M.; Gatti, Richard A.

doi:10.1073/pnas.0608616104

Cited by 97 publications

(79 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%

“…The AMO was synthesized and purified by Gene Tools (LLC, Philomath, OR, USA). Treatment of LCLs with AMO was performed, as previously described by Du et al 14 Briefly, 5 Â 10 5 LCLs cells were resuspended in 0.5 ml 5% FBS/RPMI 1640 medium and AMO was added directly to the medium at the concentration indicated. Endo-Porter (Gene Tools) was added to the medium to help intracellular corporation of the AMO.…”

Section: Minigene Constructionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Minigene Constructionmentioning

confidence: 99%

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

et al. 2012

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“…It is now also time to look at novel approaches to therapy for PID, based on the disease mechanisms. This is not restricted to gene therapy, but also includes bypassing biochemical and/or cellular defects, and exploiting the use of chemical compounds to allow reading-through nonsense mutations or correction of splice-site mutations [46]. …”

Section: Discussionmentioning

confidence: 99%

Antibody deficiency diseases

Pan‐Hammarström

Hammarström

2008

Eur J Immunol

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Primary immunodeficiency diseases are rare disorders characterized by quantitative or qualitative defects in cells or components in the immune system, resulting in a high degree of susceptibility to various types of infections. During differentiation, stem cells undergo a series of discrete steps, governed by a large number of different genes. Mutations/deletions in these genes will result in a block in differentiation of the affected cell lineage(s), leading to immunodeficiency. To date, more than 150 different types of disorders have been described. In this review, we will focus on novel findings in antibody deficiency syndromes. IntroductionPrimary immunodeficiency diseases (PID) are disorders characterized by quantitative or qualitative defects in cells or components in the immune system, resulting in a high degree of susceptibility to various types of infections. During differentiation, stem cells undergo a series of discrete steps, governed by a large number of different genes. Mutations/deletions in these genes will result in a block in differentiation of the affected cell lineage(s), leading to immunodeficiency. To date, more than 150 different types of diseases have been described and the genetic basis for most of these defects is known (see [1,2] for recent reviews).Principally, PID can be subdivided into T cell, B cell, combined (T+B), phagocyte and complement defects. The combined immunodeficiencies (SCID) are clinically the most severe and will, if undiagnosed, lead to death of the affected child, usually within the first year of life. Other defects, numerically more prevalent and including common variable immunodeficiency (CVID) and IgA deficiency (IgAD), will result in considerable morbidity, but are rarely associated with mortality.The above defects are all "global", i.e. associated with susceptibility to a variety of different pathogens. However, recently, several PID have been described that are characterized by an increased frequency of infections caused by selected pathogens such as mycobacteria, pneumococci [3] and herpes simplex virus [4,5]. Such "holes-in-the-repertoire" defects (for recent review see [6]) are likely to be common in the population and may, taken together, account for a substantial number of, hitherto largely undiagnosed, patients with PID. Furthermore, a recent observation [7] shows that novel mutations in "classical" genes, i.e. those known to be involved in a given PID, may give rise to a different phenotype, suggesting that the field is getting more complex than previously recognized. In addition, the etiology of several phenotypically similar, albeit in some cases not entirely identical, PID have recently been shown to be caused by mutations in other genes than those originally implicated (examples include hyper-IgM (HIGM, see below), X-linked lymphoproliferative syndrome [8], hyper-IgE [9, 10], Omenn syndrome [11] and X-linked susceptibility to mycobacterial infections [12]. Differences in glycosylation pattern may also account for selected disease phenotypes [13] and...

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“…When such a new splice site occurs in an intron in the vicinity of a suitable pseudo splice site, the intervening intronic region can be included in the mRNA as a cryptic exon. This mechanism has been observed in multiple diseases, including cystic fibrosis (22) and ataxia telangiectasia (23,24) among others. In XLA, we have previously identified and described 2 such families (25,26).…”

Section: Introductionmentioning

confidence: 99%

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

Bestas¹,

Moreno²,

Blomberg³

et al. 2014

J. Clin. Invest.

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Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Cited by 97 publications

References 47 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)

Antibody deficiency diseases

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

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