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Report: Neurofibromatosis type 2

Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy Neurofibromatose de type 2 : analyse de la cohorte franc¸aise par une stratégie exhaustive d’analyse moléculaire du gène NF2 E. Pasmant a,b , C. Louvrier b , A. Luscan a,b , J. Cohen b , I. Laurendeau a , M. Vidaud a,b , D. Vidaud a,b , S. Goutagny c , M. Kalamarides d , B. Parfait a,b,∗ a

EA7331, faculté de pharmacie de Paris, université Paris Descartes, Sorbonne Paris Cité, Paris, France Service de biochimie et de génétique moléculaire, hôpital Cochin, AP–HP, Paris, France c Service de neurochirurgie, hôpital Beaujon, AP–HP, Clichy, France d Service de neurochirurgie, hôpital Pitié-Salpétrière, AP–HP, Paris, France b

a r t i c l e

i n f o

Article history: Received 7 October 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxx Keywords: Neurofibromatosis type 2 NF2 Manchester criteria Multiplex ligation-dependent probe amplification MLPA Somatic mosaicism

a b s t r a c t Objective. – Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. Material and methods. – We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. Results. – A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). Conclusion. – High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis. © 2015 Elsevier Masson SAS. All rights reserved.

r é s u m é Mots clés : Neurofibromatose de type 2 NF2 Critères de Manchester Multiplex ligation-dependent probe amplification MLPA Mosaïque somatique

Objectif. – La neurofibromatose de type 2 (NF2) atteint environ une personne sur 25 000 à 40 000. La plupart des patients atteints de NF2 présentent des mutations privées de type perte-de-fonction, qui sont réparties sur l’ensemble du gène NF2. Nous présentons ici notre stratégie d’analyse moléculaire du gène NF2. Patients et méthodes. – Une analyse moléculaire exhaustive du gène NF2 a été réalisée chez 221 patients franc¸ais atteints de NF2 : 134 patients non apparentés « typiques » répondant aux critères de Manchester et 87 patients non apparentés présentant des symptômes évocateurs de NF2, répondant seulement partiellement aux critères de Manchester. Résultats. – Une mutation de NF2 a été identifiée chez 56 des 221 patients, soit un taux global de détection de 25 %. Ce taux atteignait 37 % (49/134) pour les patients « typiques » répondant aux critères de Manchester et seulement 8 % (7/87) pour les patients avec un phénotype évocateur de NF2. Six de ces sept patients étaient âgés de moins de 25 ans. Dans notre approche moléculaire, 77 % des variants de NF2 ont été retrouvés par séquenc¸age des régions codantes du gène. La MLPA a permis la détection de

∗ Corresponding author. E-mail address: [email protected] (B. Parfait). http://dx.doi.org/10.1016/j.neuchi.2015.01.004 0028-3770/© 2015 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Pasmant E, et al. Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy. Neurochirurgie (2015), http://dx.doi.org/10.1016/j.neuchi.2015.01.004

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réarrangements de NF2 (23 % des variants identifiés de NF2 correspondaient à des délétions complètes ou à des délétions/duplications partielles de NF2). Conclusion. – Un très bon taux de détection de mutation peut être obtenu chez des patients correctement phénotypés et par une étude moléculaire incluant des techniques complémentaires et optimisées. La détection des mosaïques somatiques peut en particulier être améliorée en analysant des échantillons tumoraux congelés. © 2015 Elsevier Masson SAS. Tous droits réservés.

1. Introduction Neurofibromatosis type 2 (NF2, MIM #101000) is an autosomal dominant disorder with an estimated birth incidence of between 1 in 25,000 and 1 in 40,000 [1,2]. Almost half of NF2 patients are sporadic cases. The disorder has wide phenotypic variability and nearly 100% penetrance by 60 years of age [1]. NF2 is characterized by the development of multiple benign tumors of the nervous system. The hallmark of NF2 is the development of bilateral vestibular schwannomas. The other main tumors are schwannomas of the other cranial, spinal and peripheral nerves, intracranial and intraspinal meningiomas, and low-grade central nervous system malignancies (ependymomas and gliomas). Clinical diagnosis of NF2 is based on the Manchester criteria (Table 1) [3]. Identification of a mutation in the NF2 gene can confirm the NF2 diagnosis. NF2 is caused by dominant loss-of-function mutations of the tumor suppressor gene NF2 (MIM #607379), located at 22q12.1 and containing 17 coding exons over ∼95 kb. NF2 is expressed in nearly all tissues. The NF2 gene encodes the FERM (4.1 protein/ezrin/radixin/moesin) domain protein, termed Merlin or Schwannomin [4,5]. Merlin regulates cell proliferation in response to adhesive signaling, contributes to the formation of cell junctions, activates anti-mitogenic signaling and inhibits oncogenic gene expression [6]. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. A huge number of different constitutional heterozygous NF2 mutations have been identified in NF2 patients [7]. As NF2 is a tumor suppressor gene, tumorigenesis occurs when a second somatic mutation disrupts the remaining functional copy of the gene. Consistent with Knudson’s two hit hypothesis, the somatic alteration of the second wild type NF2 allele can be found in NF2-associated tumors. NF2 patients show marked interfamilial differences in disease severity and tumor susceptibility [1,8,9]. A more severe phenotype has been described in patients with protein-truncating mutations of the NF2 gene compared with patients having single codon alterations [9–11]. Interestingly, a high frequency of somatic mosaic mutations is observed in NF2 patients with de novo mutations. Pedigree analysis and mutation studies in blood and tumor specimens have indicated that up to 25–30% of NF2 sporadic cases are mosaic, with the mutation often detected only in tumor samples and not in lymphocyte DNA [1,11–13]. This condition may also account for a milder disease course in mosaic patients [14].

Table 1 Manchester criteria for neurofibromatosis type 2 diagnosis. Critères de Manchester pour le diagnostic de la neurofibromatose de type 2. NF2 Manchester criteria A. Bilateral vestibular schwannomas (VS) B. Family history of NF2 plus unilateral VS or any two of: meningioma, glioma, neurofibroma, schwannoma, posterior subcapsular lenticular opacities C. Unilateral VS plus any two of: meningioma, glioma, neurofibroma, schwannoma, and posterior subcapsular opacities D. Multiple meningioma (two or more) plus unilateral VS or any two of: glioma, neurofibroma, schwannoma, and cataract

NF2 mutation identification in both familial and sporadic NF2 cases is a challenge for genetic counseling. Because early diagnosis improves clinical care, it is important to provide a presymptomatic genetic testing for the offsprings and relatives of NF2 index cases. Prenatal and pre-implantation diagnoses can be proposed if the constitutional NF2 alteration is identified in index cases. The aim of the present study was to perform a comprehensive NF2 mutation screening in 221 index cases with: • a clinical diagnosis of NF2 fulfilling Manchester criteria • clinical symptoms suggestive of NF2. Our study underlines the need of frozen stored tumor samples for somatic mosaicism identification. We also point out the need of exhaustive clinical descriptions in order to extend the molecular screening to other genes in NF2-negative patients. 2. Materials and methods 2.1. Patients The study population consisted of 221 unrelated patients whose samples were sent to our laboratory (Genetics Department, Cochin hospital, Paris, France) between 2008 and 2013 for NF2 molecular analysis. All patients gave their written informed consent. Eleven frozen and three formalin-fixed and paraffin-embedded (FFPE) tumor tissues were also obtained from 13 patients. Molecular diagnosis was performed only when clinical information was obtained. For each patient, the phenotypic information was recorded in a standardized questionnaire including NF2 symptoms according to the Manchester criteria: presence/absence of bilateral or unilateral vestibular schwannomas, of meningiomas, gliomas, neurofibromas, schwannomas and/or posterior subcapsular lenticular opacities, and family history of NF2. 2.2. Nucleic acids extraction DNA was isolated from peripheral blood leucocytes, or tumor tissue samples, using standard procedures. Peripheral blood leukocyte DNA was used to investigate NF2 germline mutations and tumor tissue DNA was examined for NF2 somatic mutations and mosaic identification. RNA was extracted from PAXgeneTM whole blood samples (Beckton Dickinson) or lymphoblastoid cell lines using standard procedures. 2.3. Microsatellites genotyping Two NF2 intragenic polymorphic microsatellites were genotyped (GT32.6 and GT74.9 located in introns 1 and 10, respectively). After amplification with a 5 -end-labeled primer, followed by the addition of an internal size standard, PCR products were separated on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). Results were analysed with the Genemapper software v4.0 (Applied Biosystems). Primer sequences are available on request.

Please cite this article in press as: Pasmant E, et al. Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy. Neurochirurgie (2015), http://dx.doi.org/10.1016/j.neuchi.2015.01.004

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2.4. Real-time PCR-based gene dosage We quantified NF2 exon targets by determining the threshold cycle (Ct) number at which the increase in the signal associated with exponential growth of PCR products begins, as previously described [15]. Primer sequences for real-time PCR-based gene dosage are available on request. We also quantified the ALB gene (encoding albumin and mapping to chromosome region 4q11-q13) as an endogenous copy number control, and each sample was normalized on the basis of its ALB content. The relative copy number of the NF2 exon targets was also normalized to a calibrator, consisting of genomic DNA from a normal subject. Final results, expressed as N-fold differences in the NF2 exon targets copy number relative to the ALB gene and the calibrator were determined as follows: Nfold = 2(Ctsample − Ctcalibrator) , where Ct values of the sample and calibrator are determined by subtracting the average Ct value of the NF2 exon target from the average Ct value of the ALB gene. Given the NF2 exon target, samples with N-fold values of 1.3 were considered deleted, or duplicated, respectively. 2.5. Multiplex ligation-dependent probe amplification (MLPA) analysis Single and multi-exon deletions/duplications screening was performed by MLPA analysis using the SALSA MLPA kits P044 NF2 as recommended by the manufacturer (MRC Holland, Amsterdam, Netherlands). The probe mix contained probes for all constitutive NF2 exons. Two probes were also present in NF2 promoter. In addition two probes were present in the NIPSNAP1 and CABP7 genes, located 22 kb upstream and 26 kb downstream of the NF2 gene, respectively. 2.6. NF2 mutation analysis by DNA sequencing Mutation screening was performed using bidirectional DNA sequencing of the purified PCR products of the 17 NF2 coding exons and exon/intron boundaries. Sequencing analysis was then performed on purified PCR fragments using upper/reverse primers with the ABI BigDye terminator sequencing kit v1.1 (Applied Biosystems) on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). The primer sequences for DNA sequencing are available on request. Sequences were aligned with Seqscape analysis software v2.5 (Applied Biosystems) and were compared with the corresponding genomic DNA reference sequence NC 000022.11 (nt 22:29603556 to 29698600 on Human dec. 2013 GRCh38 Assembly). 2.7. NF2 mutation analysis by cDNA sequencing RNA samples were reverse transcribed and the full cDNA was then amplified in four overlapping fragments ranging from 496 to 770 bp, using ampli Taq Gold® DNA polymerase (Applied Biosystems). Sequencing analysis was then performed on purified RT-PCR fragments using RT-PCR and upper/reverse primers with the ABI BigDye terminator sequencing kit v1.1 (Applied Biosystems) on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). Sequences were aligned with Seqscape analysis software v2.5 (Applied Biosystems) and were compared with the corresponding cDNA reference sequence NM 000268.3. The primer sequences for cDNA sequencing are available on request. 3. Results A comprehensive mutation screening of the NF2 gene was performed in 221 index cases through intragenic NF2 microsatellites analysis, real-time PCR-based gene-dosage, and NF2 sequencing at

3

Table 2 Distribution of the different NF2 mutation types we identified in DNA and/or cDNA extracted from blood samples. Distribution des différents types de mutations du gène NF2, identifiées au sein de l’ADN et/ou de l’ADN complémentaire extrait d’échantillons sanguins. a Deux des 15 mutations non-sens étaient à l’état de mosaïque somatique. NF2 mutation type

Number (total: 56)

Repartition (%)

Missense Nonsense Frameshift short deletion and/or insertion Inframe short deletion and/or insertion Splice frameshift Splice inframe Splice (unknown consequence) Deletion of single/multiple exons Duplication of single/multiple exons Deletion of NF2 promoter Complete NF2 gene deletion

3 15 11 1 7 3 3 5 2 3 3

5 27a 20 2 13 5 5 9 4 5 5

a

Two of the 15 nonsense mutations were detected with somatic mosaic evidence.

DNA and/or cDNA levels. MLPA was also performed for NF2 negative patients. The molecular diagnostic strategy used in this study is presented in Fig. 1. This strategy enabled the identification of an NF2 heterozygous mutation in 56 out of 221 (∼25%) index cases. 3.1. Large NF2 complete or partial deletion identification by intragenic microsatellite genotyping (pre-screening), real-time PCR gene dosage (confirmation) and MLPA Analysis of two NF2 intragenic microsatellite markers located in introns 1 and 10 revealed an homozygous haplotype in 10% of cases (18/171), suggesting an NF2 deletion. Among 27% of them (5/18), real-time PCR gene dosage confirmed a complete (n = 3) or partial deletion (n = 2) of the NF2 locus. The 3/5 patients with complete NF2 deletion accounted for ∼5% of NF2-mutated patients. The two partial NF2 deletions encompassed introns 1 to 11 and introns 1 to 3 gene region. Because of high informativity (∼90%) of the combined NF2 intragenic microsatellite markers, haplotyping was also helpful to identify loss of heterozygosity (LOH) when performed simultaneously on tumoral DNA versus constitutional DNA. 3.2. NF2 mutation screening by sequencing at DNA and/or cDNA levels NF2 point mutation screening performed in DNA and/or cDNA extracted from blood samples, identified a NF2 heterozygous alteration in 43 out of 56 NF2-mutated index cases. Table 2 summarizes the distribution of the different types of NF2 mutations. Nonsense mutations corresponded to 27% of identified mutations in our study (n = 15); cDNA analysis was performed for 7 of the 15 variants. Three of them revealed few or no detectable mutated allele suggesting a possible mRNA decay mechanism. Two of the 15 nonsense variants showed an unbalanced mutant/wild-type allele ratio, suggesting a somatic mosaicism at the blood level. Missense variants accounted for 5% (n = 3) of mutations and cDNA sequencing confirmed that the three variants did not alter NF2 transcript maturation. Two of the three missense variants (c.191T > C, p.Leu64Proc.613 A > G, p.Met205Val) were previously reported [7,16]. The functional effect of the remaining variant (c.1195G > C, p.Ala399Pro) is currently unknown. About 22% of reported mutations were small insertions and/or deletions (n = 12), most of them with frameshift consequences (n = 11). Splice alterations accounted for about one fourth (23%, n = 13), with frameshift (n = 7), inframe (n = 3) or unknown (n = 3) consequences. Mutations distribution across the NF2 gene did not exhibit any hot-spot domain. Among

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Fig. 1. Flow chart for NF2 comprehensive mutation screening. Pre-screening of large NF2 deletions by two intragenic microsatellites genotyping leads to a complete homozygosity situation in only 10% of the cases (step 1). Around one fourth of them harbour a complete or partial deletion of the NF2 locus, confirmed by real-time PCR-based gene dosage. Multiple ligation-dependent probe amplification (MLPA) enables the accurate characterization of these deletions. NF2 complete and large partial deletions were observed in 5 and 4% of NF2-positive patients studied in our laboratory, respectively. In absence of large deletion, molecular investigation of NF2 is achieved by a coding exons Sanger sequencing approach (step 2). Point mutations identified by this approach are observed in 77% of the NF2-positive patients. In front of a negative screening for large deletion or point mutations, a quantitative analysis of all the coding exons on genomic DNA is performed by MLPA analysis leading to the identification of single or multi-exon deletions/duplications in 14% of the NF2-positive patients (step 3). This comprehensive mutation screening enabled us to identify a NF2 mutation in 25% of the NF2 patients in our experience. In case of a negative result and when a somatic mosaicism is suspected, NF2 molecular investigation is performed at the tumor level when −80 ◦ C frozen samples are available (step 4). Logigramme d’analyse moléculaire exhaustive du gène NF2. Le pré-criblage des grandes délétions du gène NF2 par le génotypage de deux microsatellites intragéniques identifie une homozygotie complète dans seulement 10 % des cas (étape 1). Environ un quart de ces patients présente une délétion totale ou partielle du locus NF2, confirmée par dosage génique par PCR en temps réel. Une approche de multiple ligation-dependent probe amplification (MLPA) permet la caractérisation précise de ces délétions. Des délétions totales ou partielles du gène NF2 ont été observées respectivement chez 5 et 4 % des patients NF2 génotypés dans notre étude. En l’absence de grande délétion, l’analyse moléculaire de NF2 est complétée par le séquenc¸age de l’ensemble des exons codants du gène par méthode de Sanger (étape 2). Cette approche permet l’identification de mutations ponctuelles chez 77 % des patients présentant une altération du gène NF2. En l’absence de l’identification de grandes délétions ou de mutations ponctuelles, une analyse quantitative de chacun des exons codants est réalisée sur l’ADN génomique par MLPA. Des délétions ou duplications d’un ou plusieurs exons sont ainsi retrouvées chez 14 % des patients présentant une altération de NF2 (étape 3). Cet algorithme moléculaire exhaustif a permis d’identifier une altération du gène NF2 chez 25 % des patients atteints de NF2. En cas de résultat négatif et quand un mosaïcisme somatique est suspecté, l’analyse moléculaire du gène NF2 est effectuée au niveau somatique quand des échantillons tumoraux congelés à −80 ◦ C sont disponibles (étape 4).

the 38 distinct point mutations, only three were recurrent. The most recurrent mutation (c.169C > T, p.Arg57*) was identified in four unrelated patients. This mutation is localised in NF2 exon 2 in a CpG motif. 3.3. NF2 mutation screening by MLPA analysis NF2 single and multi-exon deletions/duplications screening performed by MLPA analysis confirmed the five large deletions identified by microsatellites and real time PCR studies. It also identified NF2 partial deletions/duplications in eight out of 56 NF2-mutated index cases (∼14%). Among these eight deletions/duplications, only three affected one or two exons and were detected by cDNA analysis. The five remaining deletions/duplications could not be detected by cDNA analysis because they were too large or they were located in the NF2 promoter region. The 13 NF2 partial or large deletions/duplications are detailed in Fig. 2.

3.4. Mosaic detection NF2 screening did not allow identification of a constitutional mutation in 165 cases. Eighty-four of these 165 cases presented typical NF2 fulfilling the Manchester criteria. In order to identify somatic mosaic events in case of typical NF2 patients, we performed the NF2 molecular analysis at the tumor level in 14 tumors from 13 patients. One tumor sample was available for 12 patients and two distinct tumors samples were available for one patient. We failed to screen the entire NF2 coding sequence in the three FFPE tumor samples. In six of the 11 frozen tumor tissues (55%), two NF2 hits were identified: one point mutation plus LOH in one sample from two patients, one common point mutation plus LOH in two distinct tumor samples from one patient, two distinct point mutations in one tumor sample from two patients. In two of the 11 frozen tumor tissues, only one NF2 hit could be identified at the tumor level. For the three remaining frozen tumor tissues, no NF2 coding sequence abnormality was identified.

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GT74.9

GT32.6 1

NF2

5

2

3

4

5

6

7

8

10

9

11

12

13

14

15

16

17

22q12.1

F1

? ?

F3

F2

F4

Promotor Promotor-1

12

5-6

15-16

N=2

2-11

?

2-17

?

Complete deletion

?

N=3

Fig. 2. Location of the 13 single or multi-exons deletions in the NF2 gene. The 17 coding exons are represented by rectangles (not proportional to their size). Vertical arrows indicate the location of microsatellites used to search large deletion (introns 1 and 10). Horizontal lines F1 to F4 represent the RT-PCR fragments used for the sequencing of the NF2 gene transcript. Duplications and deletions are shown above and below the NF2 gene, respectively. Affected exons are indicated above the corresponding duplicated or deleted segments. Localisation des 13 délétions d’un ou plusieurs exons identifiées dans le gène NF2. Les 17 exons codants sont représentés par des rectangles (taille non proportionnelle). Les flèches verticales indiquent les microsatellites génotypés pour la mise en évidence des grandes délétions (introns 1 et 10). Les lignes horizontales F1 à F4 représentent les fragments de RT-PCR utilisés pour le séquenc¸age du transcrit NF2. Les duplications et délétions sont respectivement indiquées au-dessus et en-dessous du gène NF2. Les exons dupliqués ou délétés sont indiqués au-dessus des segments correspondants.

3.5. Phenotype analysis Clinical data of 221 index patients was obtained via a standardized questionnaire including clinical symptoms of NF2 according to the Manchester criteria. One hundred and thirty-four NF2 index cases fulfilled the Manchester criteria, with 100 patients presenting bilateral vestibular schwannomas. The mean age of the 134 patients at the time of blood sampling was 39 years (range 4–88). Males (46%) and females (54%) were equally represented. For the 134 patients, molecular investigation confirmed the diagnostic of NF2 in 37% of the cases (49/134). The clinical characteristics of the 134 patients are provided in Table 3. The mean age of the NF2positive patients at the time of blood sampling was 30 years (range 4–68). Eighty-seven index cases did not completely fulfil the Manchester criteria but presented symptoms suggestive of NF2. We considered four age groups among these 87 patients. A first group was composed of 17 patients aged between 2 and 20. A second group was composed of 23 patients between 21 and 30. The third group was consisting of 11 patients between 31 and 40 and the remaining fourth group was composed of 36 patients over 40 years of age. For the 87 patients, molecular investigation confirmed the diagnostic of NF2 in ∼8% of cases (7/87). The clinical characteristics of NF2-mutated patients are presented in Table 3. 4. Discussion Here, we report our experience of NF2 mutation screening in 221 NF2 patients between 2008 and 2013. In this study, 134 typical NF2 index cases fulfilling the Manchester criteria and 87 patients presenting NF2-related symptoms were analysed using a comprehensive molecular strategy combining complementary approaches. NF2 heterozygous alterations were identified in 56 of 221 index cases (25%). This rate reached 37% for typical NF2 patients (n = 49/134) and only 8% (n = 7/87) for patients presenting symptoms suggestive of NF2 (not fulfilling Manchester criteria). It is to

note that five of these latest seven patients were less than 13 years of age. The sixth patient was 21 years of age and the remaining seventh patient was 40. Hence, high mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques (Fig. 1). The first step of the molecular diagnostic strategy was the study of two intragenic microsatellites in NF2 introns 1 and 10. When homozygous haplotypes suggested a NF2 large deletion, gene dosage by real time quantitative PCR of exons 2 and 17 was performed to confirm NF2 large deletion. This led us to identify a whole gene or large partial gene deletion in ∼5% and ∼4% of all NF2 alterations, respectively. When a large NF2 deletion was excluded (with at least one heterozygous microsatellite), the study of the 17 NF2 coding exons was then performed at DNA or cDNA level. Among the 56 NF2 heterozygous mutations, 77% (43/56) were identified in nearly all exons except exons 16 and 17 in accordance with the literature data [7]. Deleterious effect was unclear for only two missense variants and one inframe deletion (9 bp). In order to detect a broader spectrum of mutations and provide a greater understanding of their molecular consequences [17], both DNA and cDNA were screened in 101 index cases. We observed no discrepancies between DNA and cDNA strategies for the 71 negative NF2 screening. Among the 30 positive NF2 screening, three of the 15 nonsense variants detected at DNA level, were not detected at cDNA level, probably due to an mRNA decay mechanism. This disadvantage in cDNA strategy led us to perform the molecular investigation of NF2 at DNA level in a first step. The cDNA study was then only performed when splice alterations were identified at DNA level in order to assess their consequences on NF2 mRNA splicing. When no NF2 mutation was found at DNA level, exons deletion/duplication screening was performed using MLPA (Fig. 1, step 3). Deletion or duplication of single or multiple exons accounted for ∼14% (n = 8) of all NF2 alterations. MLPA allowed the identification of such restricted rearrangements missed by DNA sequencing or microsatellite analysis.

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Table 3 Clinical features of the 56 mutated-NF2-associated patients. Caractéristiques cliniques des 56 patients avec une mutation de NF2. Clinical feature

Number of index cases

Manchester criteria

Study sample (n = 56) (%)

Bilateral VS Family history of NF2 plus unilateral VS Family history of NF2 plus any two of: meningioma, glioma, neurofibroma, schwannoma, posterior subcapsular lenticular opacities Unilateral VS plus any two of: meningioma, glioma, neurofibroma, schwannoma, and posterior subcapsular opacities Multiple meningioma plus unilateral VS Multiple meningioma plus any two of: glioma, neurofibroma, schwannoma, ependymoma Age < 20 and unilateral VS Age < 20 and any two of: meningioma, glioma, neurofibroma, schwannoma, ependymoma Age < 20 and multiple meningiomas Age between 20–30 and Unilateral VS Age between 20–30, meningioma and schwannomas Age between 30–40 and unilateral VS

44 1 –

A B B

78 2 –



C



3 1

D D

5 2

– 4

– –

– 7

1 – 1 1

– – – –

2 – 2 2

VS: vestibular schwannoma. VS: schwannome vestibulaire.

Our comprehensive mutation screening of the NF2 gene failed to identify any mutation in 75% (N = 165/221) cases. Half of these negative cases (84/165) presented typical NF2 fulfilling the Manchester criteria. The first explanation for the negative NF2 screening is somatic mosaicism. Mosaicism could be suspected in NF2-negative index cases showing typical NF2 symptoms (including bilateral vestibular schwannomas) with no family history of NF2 [1,13,18]. In our experience, Sanger sequencing allowed mutation visualization at an allele ratio >15% [19]. When a somatic mosaicism was suspected in familial presentation, patients of the second generation were first tested for molecular investigation in order to avoid a false negative result. In case of a negative result in a sporadic patient with a typical NF2, somatic mosaicism NF2 mutation can be suspected. Molecular screening can therefore be performed on tumors samples. In 13 patients, NF2 molecular screening was performed in tumor DNA extracted from FFPE (three tumors, three patients) and frozen (11 tumors, 10 patients) samples. Our comprehensive screening could not be successfully performed on FFPE DNA. Frozen tumor samples analysis identified a somatic mosaic event in five of the 10 patients. This result led us to propose a genetic counselling, even if blood screening was negative. This result underlines the need of frozen storage after surgery, especially in sporadic NF2 cases. Another explanation for the negative NF2 screening is the presence of an undetected mutation in the NF2 locus. This observation also points out the need for a more comprehensive NF2 molecular diagnostic strategy. For example, a cytogenetic analysis should be performed in negative patients, to exclude potential genomic rearrangements such as translocations or ring chromosome 22 [20,21] that would have been missed. The small size of the families included in this study did not allow performing segregation analysis in familial NF2-negative cases. Genetic heterogeneity could also be considered as a cause of negative screening. Other genes were involved in NF2 overlapping phenotypes: SMARCB1 or LZTR1 in multiple schwannomas [22,23], SMARCE1 in multiple spinal meningiomas [24], and SUFU in a NF2negative multiplex family of multiple meningiomas [25]. In order to perform molecular diagnosis of these overlapping syndromes, simultaneous molecular study of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU genes instead of time-consuming sequential molecular studies has to be considered. We currently develop a targeted nextgeneration sequencing (NGS) of these five genes using a multiplex PCR approach (179 amplicons of ∼100 bp) on a PGM sequencer (Life Technologies). This accurate and fast targeted NGS approach will

also provide quantitative information based on sequencing depth allowing identification of single and multiple exons deletion or duplication. The design is suitable for DNA extracted from frozen as well as FFPE biopsies. Somatic mutation detection sensitivity is expected to be increased [19]. The remaining negative index cases will be candidates for a whole exome sequencing approach looking for causal mutations in other loci in the genome. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Evans DG, Huson SM, Donnai D, Neary W, Blair V, Teare D, et al. A genetic study of type 2 neurofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fitness, and confirmation of maternal transmission effect on severity. J Med Genet 1992;29:841–6. [2] Evans DG, Moran A, King A, Saeed S, Gurusinghe N, Ramsden R. Incidence of vestibular schwannoma and neurofibromatosis 2 in the North West of England over a 10-year period: higher incidence than previously thought. Otol Neurotol 2005;26:93–7. [3] Evans DGR, Huson S, Donnai D, Neary W, Blair V, Newton V, et al. A clinical study of type 2 neurofibromatosis. Q J Med 1992;84:603–18. [4] Rouleau GA, Merel P, Lutchman M, Sanson M, Zucman J, Marineau C, et al. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 1993;363:515–21. [5] Trofatter JA, MacCollin MM, Rutter JL, Murrell JR, Duyao MP, Parry DM, et al. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 1993;75:826. [6] Cooper J, Giancotti FG. Molecular insights into NF2/Merlin tumor suppressor function. FEBS Lett 2014;588:2743–52. [7] Ahronowitz I, Xin W, Kiely R, Sims K, MacCollin M, Nunes FP. Mutational spectrum of the NF2 gene: a meta-analysis of 12 years of research and diagnostic laboratory findings. Hum Mutat 2007;28:1–12. [8] Eldridge R, Parry DM, Kaiser-Kupfer MI. Neurofibromatosis 2 (NF2): clinical heterogeneity and natural history based on 39 individuals in 9 families and 16 sporadic cases. Am J Hum Genet 1991;49:133. [9] Parry DM, Eldridge R, Kaiser-Kupfer MI, Bouzas EA, Pikus A, Patronas N. Neurofibromatosis 2 (NF2): clinical characteristics of 63 affected individuals and clinical evidence for heterogeneity. Am J Med Genet 1994;52:450–61. [10] Ruttledge MH, Andermann AA, Phelan CM, Claudio JO, Han F, Chretien N, et al. Type of mutation in the neurofibromatosis type 2 gene (NF2) frequently determines severity of disease. Am J Hum Genet 1996;59:331–42. [11] Evans DGR, Trueman L, Wallace A, Collins S, Strachan T. Genotype/phenotype correlations in type 2 neurofibromatosis (NF2): evidence for more severe disease associated with truncating mutations. J Med Genet 1998;35:450–5. [12] Kluwe L, Mautner V, Heinrich B, Dezube R, Jacoby LB, Friedrich RE, et al. Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas. J Med Genet 2003;40:109–14. [13] Evans DG, Ramsden RT, Shenton A, Gokhale C, Bowers NL, Huson SM, et al. Mosaicism in neurofibromatosis type 2: an update of risk based on

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Please cite this article in press as: Pasmant E, et al. Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy. Neurochirurgie (2015), http://dx.doi.org/10.1016/j.neuchi.2015.01.004

Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy.

Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along...
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