Exhaustive characterization of the NF2 gene
in neurofibromatosis type 2 patients

1Laboratoire de génétique des tumeurs, INSERM U434 and ²CEPH/ Fondation Jean Dausset, 27 rue Juliette Dodu, 75010 Paris FRANCE; ³CNRS URA 1342 Orsay FRANCE; ⁴Institut Jacques Monod, Paris FRANCE; ⁵CIRC 69372 Lyon, FRANCE.

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ABSTRACT

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (Ezrin-Radixin-Moesin proteins). Even though penetrance of the disease is more than 95 % and no genetic heterogeneity have been described, point mutations in the NF2 gene have been observed in only 34 to 66 % of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients which express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.

Probes for Hybridization on Southern Blot

All PCR amplifications were performed on a 9600 Perkin ElmerDNA cycler, in a volume of 100 ul (micro-liters) using the followingprocedure: initial denaturation step, 96°C for 90s, 35 cycles with adenaturation step at 94°C for 20 s, annealing at X °C for 20 s, extension at72°C for 2 min, following by a final extension at 72°C for 7 min. Foreach DNA amplification, DNA polymerase Cetus (0,5 units,Perkin-Elmer) was used together with 50 ng of genomic DNA, 400 nM of each specific primer, 0.8 mM of dNTPs, 1,5 mM of MgCl2 in the cetus buffer.

Probe 1* Add 5% DMSO final in the reaction.

Probes for FISH.

* Add 5% DMSO final in the reaction.

Transcribed polymorphisms screening

* Add 5% DMSO final in the reaction.