Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations

Pros, Eva; Fernández-Rodríguez, Juana; Canet, Belén; Benito, Llúcia; Sánchez, Aurora; Benavides, Ana Margarita Silva; Ramos, Feliciano J.; López-Ariztegui, María Asunción; Capellà, Gabriel; Blanco, Ignacio; Serra, Eduard; Lázaro, Conxi

doi:10.1002/humu.20933

Cited by 48 publications

(43 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…19 Later this technology was used successfully for other diseases like cystic fibrosis 20 or more recently for NF1. 21 Today, different pre-clinical and clinical splice-modulating therapies using AONs, like restoring open reading frames, influencing alternative splicing or inducing exon inclusion, have been successfully used for many different genetic disorders. 16,17 In this study, we report a NF2 patient with bilateral vestibular schwannoma who was found to bear a deep intronic mutation in the NF2 gene.…”

Section: Introductionmentioning

confidence: 99%

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

et al. 2012

Self Cite

View full text Add to dashboard Cite

on behalf of the NF2 Multidisciplinary Clinics HUGTiP-ICO-IMPPC Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, B25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T4A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2. INTRODUCTIONNeurofibromatosis type 2 (NF2; MIM ID#101000) is an autosomaldominant cancer syndrome caused by mutations in the NF2 gene, located on chromosome 22q12. NF2 has an incidence of 1 in 33 000 live births showing a wide phenotypic variability and a nearly complete penetrance by the age of 60. 1 The NF2 gene codes for the tumorsuppressor protein merlin (69 kDa). Merlin regulates cellular processes that are found to be altered in tumorigenesis including: cell-cell adhesion, cytoskeletal architecture and membrane protein organization. 2,3 NF2 patients are predisposed to develop lesions of the nervous system, eyes and skin. The presence of bilateral vestibular nerve schwannomas is the most distinctive feature of NF2, but patients can develop other clinical manifestations such as schwannomas in other cranial, spinal and peripheral nerves, and also other types of tumors, like meningiomas (both intracranial and intraspinal) and ependymomas (low-grade central nervous system malignancies). Affected individuals can also show peripheral neuropathies (independently of compressive tumors), cataracts, epiretinal membranes, retinal hamartomas and cutaneous tumors, usually schwannomas. 4 Over 50% of patients are familial cases and the other 50% bear de novo mutations. A minimum of 25-33% of these NF2 sporadic cases are mosaic, 5,6 which complicates clinical diagnostics and genetic testing. 7

show abstract

Section: Introductionmentioning

confidence: 99%

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…Alleles with intronic mutations activating cryptic splicing sites are potentially correctable with antisense oligonucleotides to prevent the splicing machinery from recognizing the cryptic exon and promote normal splicing, as successfully shown in several genetic disorders (42)(43)(44). Similar antisense oligonucleotides may also apply to GS patients bearing deep intronic mutations and can be first tested on the leukocytes expressing NCC mRNA.…”

Section: Discussionmentioning

confidence: 99%

Recurrent Deep Intronic Mutations in the SLC12A3 Gene Responsible for Gitelman's Syndrome

Nozu

Iijima

et al. 2011

Clinical Journal of the American Society of Nephrology

View full text Add to dashboard Cite

SummaryBackground and objectives Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder caused by mutations in the SLC12A3 gene encoding the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC). Despite meticulous sequencing of genomic DNA, approximately one-third of GS patients are negative or heterozygotes for the known mutations.Design, Setting, Participants, & Measurements Because blood leukocytes express NCC mRNA, we evaluate whether deep intronic mutations contribute to GS patients with uniallelic or undetectable SLC12A3 mutations. Twenty-nine patients with GS (men/women ϭ 16/13), including eight negative and 21 uniallelic SLC12A3 mutations from 19 unrelated families, and normal controls were enrolled in an academic medical center. Analysis of cDNA from blood leukocytes, sequencing of the corresponding introns of genomic DNA for abnormal transcript, and analysis of NCC protein expression from renal biopsy were performed. Results We identified nine Taiwan aboriginal patients carrying c.1670 -191C3 T mutations in intron 13and 10 nonaboriginal patients carrying c.2548ϩ253C3 T mutations in intron 21 from 14 families (14/ 19). These two mutations undetected in 100 healthy subjects created pseudoexons containing new premature termination codons. Haplotype analysis with markers flanking SLC12A3 revealed that both mutations did not have founder effects. Apical NCC expression in the DCT of renal tissue was markedly diminished in two patients carrying deep intronic mutations.Conclusions Deep intronic mutations in SLC12A3 causing defective NCC expression can be identified with the RNA-based approach in patients with GS. c.1670 -191C3 T and c.2548ϩ253C3 T are hot spot mutations that can be screened in GS patients with uniallelic or negative SLC12A3 mutations.

show abstract

“…Once regarded as junk DNA, intronic and intergenic sequences are now perceived to contain crucial splicing machinery elements and other gene expression regulating motifs. Recently, several studies have shown that mutations disturbing these important elements can lead to disease [34][35][36][37]. Therefore, we expect that high throughput sequencing of the entire 3.3 Mb candidate region might reveal a more complex mutation associated with DLB in family DR246.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Genetic and Mutation Analysis of Familial Dementia with Lewy Bodies Linked to 2q35-q36

Meeus

Nuytemans

Crosiers

et al. 2010

JAD

View full text Add to dashboard Cite

Abstract. The second most frequent form of neurodegenerative dementia after Alzheimer's disease is dementia with Lewy bodies (DLB). Since informative DLB families are scarce, little is presently known about the molecular genetic etiology of DLB. We recently mapped the first locus for DLB on chromosome 2q35-q36 in a multiplex Belgian family, DR246, with autopsy-proven DLB pathology in a region of 9.2 Mb. Here, we describe the ascertainment of additional DR246 family members and significant finemapping of the DLB locus to 3.3 Mb based on informative meiotic recombinants. Extensive sequencing of the 42 positional candidate genes within the DLB region did not identify a simple pathogenic mutation that co-segregated with disease in family DR246. Also high resolution analysis of copy number variations in the DLB locus did not provide evidence for a complex mutation. In conclusion, we confirmed the DLB locus at 2q35-q36 as a genetic entity but candidate gene-based sequencing and copy number variation analysis did not identify the pathogenic mutation in family DR246. Other detection strategies will be needed to reveal the underlying mutation explaining the linkage of DLB to 2q35-q26. Possibly the disease mutation in this family acts through a more complex mechanism than generally envisaged for monogenic disorders. Nevertheless, identifying the first familial DLB gene is likely to contribute an entry point into the pathogenic cascades underlying DLB pathology.

show abstract

Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations

Cited by 48 publications

References 38 publications

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

Recurrent Deep Intronic Mutations in the SLC12A3 Gene Responsible for Gitelman's Syndrome

Comprehensive Genetic and Mutation Analysis of Familial Dementia with Lewy Bodies Linked to 2q35-q36

Contact Info

Product

Resources

About