Estratègies terapèutiques per la Neurofibromatosi de tipus 1

Estratègies terapèutiques per la

Neurofibromatosi de tipus 1

Joan Castellsagué Garcia

Aquesta tesi doctoral està subjecta a la llicència Reconeixement 3.0. Espanya de Creative

Commons.

Esta tesis doctoral está sujeta a la licencia Reconocimiento 3.0. España de Creative

Commons.

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?

Human Mutation

B

R

A Mild Neurofibromatosis Type 1 Phenotype Produced

by the Combination of the Benign Nature of a Leaky

NF1

-Splice Mutation and the Presence

of a Complex Mosaicism

Juana Ferna´ndez-Rodrı´guez,1yJoan Castellsague´,1yLlu´cia Benito,2Yolanda Benavente,3,4Gabriel Capella´,1

Ignacio Blanco,2_{Eduard Serra,}5_{and Conxi La´zaro}1

1_{Hereditary Cancer Program, Genetic Diagnosis Unit;}2_{Hereditary Cancer Program, Genetic Counseling Unit;}3_{Unit of Infections and Cancer}

(UNIC), Cancer Epidemiology Research Program, Institut Catala` d’Oncologia (ICO-IDIBELL);4_{CIBER de Epidemiologı´a y Salud Pu´blica}

(CIBERESP), Spain;5_{L’Institut de Medicina Predictiva i Personalitzada del Ca`ncer (IMPPC), Badalona, Barcelona, Spain}

Communicated by Garry R. Cutting

Received 19 November 2010; accepted revised manuscript 24 February 2011.

Published online 2 June 2011 in Wiley Online Library (www.wiley.com/humanmutation). DOI 10.1002/humu.21500

ABSTRACT: Here we analyze the genetic and molecular basis responsible for a very benign phenotype observed in an NF1 patient. Quantification of cells carrying theNF1 mutation in different samples derived from the three embryonic layers revealed mosaicism. Furthermore, the construction of a minigene with patient’s mutation (c.3198314G4A) confirmed its benign nature due to the leakiness of the splicing mechanism that generated a proportion of correctly spliced transcripts. Hence, we concluded that the mild phenotype observed in this patient is the result of the presence of mosaicism together with the benign nature of a leakyNF1-splice mutation. Finally, with the aim of developing a personalized therapeutic approach for this patient, we demonstrated correction of the splicing defect by using specific antisense morpholino oligomers. Our results provide an example of the molecular complexity behind disease phenotypes and highlight the importance of using comprehensive genetic approaches to better assess phenotype–genotype correlations.

Hum Mutat 32:705–709, 2011.&2011 Wiley-Liss, Inc. KEY WORDS:NF1; mosaicism; genotype–phenotype; splicing

Neurofibromatosis type 1 (NF1; MIM]162200) is an autosomal

dominant disorder characterized by an increased predisposition to develop certain types of malignancies as well as by the presence of a wide range of clinical traits involving cells of neural crest origin (reviewed in [Riccardi, 1992]). NF1 is caused by germline

mutations in theNF1gene, which is one of the human genes

with a higher mutation rate. Comprehensive genetic studies identified more than 1,100 disease-causing mutations allowing a

precise depiction of the NF1 germline mutational spectrum

(reviewed in [Messiaen and Wimmer, 2008]). So far, two

constitutiveNF1mutations have been correlated with a particular

NF1 phenotype. Individuals with type 1 NF1-deletions, which

encompass 1.4 Mb of genomic DNA [Kayes et al., 1994; Lopez Correa et al., 1999] and involve several other genes in addition to

theNF1, are characterized by a severe phenotype, consisting of

learning problems, dysmorphic features and a high number of dermal neurofibromas [Mautner et al., 2010; Pasmant et al., 2010].

By contrast, patients with the recurrent c.29702972 delAAT

mutation seem to express a moderate phenotype characterized by the absence of dermal neurofibromas [Upadhyaya et al., 2007].

It has been suggested that a proportion of the new mutations are actually somatic implying than some sporadic patients are mosaics

for a NF1 mutation [Zlotogora, 1993; Kehrer-Sawatzki and

Cooper, 2008]. Depending on the stage during development of the occurrence of the mutation we can distinguish patients showing generalized mosaicism, segmental mosaicism, and gona-dal mosaicism [Ruggieri and Huson, 2001]. Generalized mosaicism cases exhibit typical symptoms of the disease in a mild generalized form, making them very difficult to distinguish from nonmosaic patients. Segmental manifestation show clinical manifestations limited to one or a few areas of the body [Crowe et al., 1956; Moss and Green, 1994; Riccardi, 1982]; this is a rare condition that occurs at around 1:36,000–40,000 individuals [Friedman et al., 1999; Ingordo et al., 1995; Ruggieri and Polizzi, 2000; Wolkenstein et al., 1995]. Gonadal mosaicism is confined to the germline and is extremely uncommon in NF1 [Bottillo et al., 2010; Lazaro et al., 1994]. Identification of somatic mosaicism and assessment of

tissues affected by the NF1somatic mutation is difficult and

represents a challenge because it is especially important for providing accurate genetic counselling to the patient.

For several genetic conditions, genotype–phenotype studies have suggested the importance of mechanisms regulating splicing as modifiers of phenotype in carriers of splicing defects [Nissim-Rafinia and Kerem, 2005]. For instance, a leaky effect of some splicing mutations associated with the production of wild-type transcripts from mutated alleles has been described. In some cases this phenomenon has been associated with a mild phenotype [Beck et al., 1999]. The high number and diversity of splicing

OFFICIAL JOURNAL

www.hgvs.org

&2011 WILEY-LISS, INC.

Contract grant sponsors: Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute and Autonomous Government of Catalonia; Contract grant numbers:

ISCIIIRETIC; RD06/0020/1051; RD06/0020/1050; 2009SGR290; PI10/01422; CA08/00248. Additional Supporting Information may be found in the online version of this article.

_{Correspondence to: Conxi La´zaro, Hereditary Cancer Program, Genetic Diagnosis}

Unit, Laboratori de Recerca Translacional, Institut Catala` d’Oncologia-ICO-IDIBELL,

Hospital Duran i Reynals, Gran Via 199-203, L’Hospitalet de Llobregat, 08907, Spain. E-mail: clazaro@iconcologia.net