We studied 13 of 19 carefully characterized schwannomatosis kindreds described previously (6
) and four additional familial probands. Lymphoblastoid cell lines were established from peripheral blood samples as described previously (17
). High-molecular-weight DNA was extracted from peripheral blood leukocytes, cultured lymphoblasts, frozen pulverized tumor, cultured tumor and normal tissues obtained at autopsy using a PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA). This study was approved by the Institutional Review Board of Partners HealthCare and informed consent was obtained from the patients participating in the study. For patients who had died, an autopsy permit was used as consent.
Multiplex ligation-dependent probe amplification
Multiplex ligation-dependent probe amplification was carried out as described by Boyd et al
), using a SALSA multiplex ligation-dependent probe amplification P258 SMARCB1 kit (MRC-Holland, Amsterdam, the Netherlands). Briefly, 20–500 ng DNA were used for hybridization, ligation and amplification of the SMARCB1
exon probes according to the manufacturer's instructions. The amplification products were analyzed using an ABI 3730 DNA Analyzer, with Biomek FX robotics and with GeneScan 500 LIZ (Applied Biosystems, Foster City, CA, USA) as the internal size standard. Relative probe signals were calculated by dividing each measured peak by the sum of all peak areas for that sample. DNA from four unaffected individuals was used for control samples.
mRNA was extracted from frozen cell pellets of established lymphoblastoid cell lines for each kindred, using the PolyATract mRNA extraction kit (Promega, Madison, WI, USA). Poly(A)+ mRNA was reverse transcribed to cDNA with oligo(d)T primers, using the Superscript III cDNA first strand synthesis kit (Invitrogen, Carlesbad, CA, USA). Full-length SMARCB1 and three overlapping fragments of the SMARCB1 transcript were PCR amplified from the cDNAs using nested primers. For the full-length transcript, the primers 5′-CAGCCCTCCTGATCCCT-3′ and 5′-CCCAATCTTCTGAGATGCTC-3′ were used. The reverse primer 5′-ACAAATGGAATGTGTGCCGG-3′ was used when the 3′UTR SNPs were amplified. Exons 1 through 4 were amplified with primers 5′-CAGCCCTCCTGATCCCT-3′ and 5′-TCACAGCTGGGTCATGGTC-3′, exons 3–6 were amplified by 5′-CACGGATACACGACTCTAGC-3′ and 5′-CACTCAAACTGGTCCACC-3′ and for exons 5–9, 5′-CCATGCTCCACAACCATC-3′ and 5′-CCCAATCTTCTGAGATGCTC-3′were used. PCR products were electrophoresed on 2% agarose gels, with a normal control. Aberrantly sized fragments were excised and analyzed by direct sequencing on an ABI Prism 3730 DNA analyzer.
Quantitative analysis of the c.*82C > T mutation (previously denoted c.1240C > T) was carried out using the Mutation Surveyor program v3.20 (Softgenetics LLC, State College, PA, USA) by comparison of relative levels of C and T alleles in cDNA with reference to levels in genomic DNA.
Construction of wild-type and mutant SMARCB1 expression vectors
Mutagenic primers were designed to introduce the c.41C > A and c.158G > T point mutations identified in our cohort into the wild-type human SMARCB1 sequence obtained from Origene (Rockville, MD, USA). Site-directed mutagenesis was carried out using the Quick-change site-directed mutagenesis kits (Stratagene, Carlesbad, CA, USA). Mutagenic primers for c.41C > A were GACCTTCGGGCAGAAGCACGTGAAGTTCCAGCTGG and CCAGCTGGAACTTCACGTGCTTCTGCCCGAAGGTC. Mutagenic primers used for c.158G > T were CCCTCACTCTGGAGGCTACTAGCCACTGTGGAAG and CTTCCACAGTGGCTAGTAGCCTCCAGAGTGAGGG.
The splice mutant lacking exon 4 was obtained by PCR amplification of full-length cDNA generated from mRNA of a lymphoblastoid cell line from family 11, using a forward primer containing an NheI restriction site, GCATGCTAGCATGATGATGATGGCGCTGAGC, and a reverse primer containing a HindIII restriction site, TTTAAGCTTCCAGGCCGGGGCCGTGTT. Each mutant transcript was sub-cloned into a pcDNA plasmid, containing a C-terminal GFP tag.
For the cyclin D1 repression experiment, the cyclin D1 promoter region −1745 to +35 was sub-cloned into the pGL3-basic plasmid, which contains a luciferase reporter gene (Promega, Madison, WI, USA).
For the 3′UTR experiment, site-directed mutagenesis was used to generate the c.*82C > T mutant, with primers TGGCAAGGACAGAGGTGAGGGGACAGCCCA and TGGGCTGTCCCCTCACCTCTGTCCTTGCCA. cDNAs representing the wild-type and c.*82C > T mutant 3′UTRs were each sub-cloned into the pGL3-promoter vector, downstream of the luciferase coding region.
HEK293T cells were purchased from ATCC (Manassas, VA , USA). MON tumor cells were obtained from the laboratory of Dr Olivier Delattre.
Luciferase reporter assays
For experiments investigating SMARCB1 3′UTR regulation, using a luciferase reporter, HEK293T cells were co-transfected with the pGL3-promoter vector construct with either a wild-type or mutant SMARCB1 3′UTR and an eGFP vector, using Lipofectamine transfection reagent (Invitrogen, Carlesbad, CA, USA). Cells were harvested after 48 h and luciferase activity was measured using One-glow luciferase reagent (Promega, Madison, WI, USA).
For experiments investigating the regulation of cyclin D1, the wild-type SMARCB1 transcript and three mutant transcripts [containing the exon 1 missense mutation (c.41C > A), the exon 2 missense mutation (c.158G > T) or the deletion of exon 4], were subcloned into the pcDNA vector with a C-terminal GFP tag. The cyclin D1 luciferase vector was transfected into MON cells which lack endogenous SMARCB1 along with each of the SMARCB1 constructs, or an empty GFP control vector. Cells were harvested after 48 h and luciferase activity was measured using One-glow luciferase reagent (Promega, Madison, WI, USA). Luciferase levels were normalized to transfection efficiency using GFP fluorescence.
qRT–PCR analysis of luciferase-3′UTR reporters
Total RNA was extracted from transfected cells using the RNeasy kit (Qiagen, Valencia, CA, USA) and treated with DNase (Ambion, Foster City, CA, USA). First-strand cDNA synthesis was performed using random hexamers (GE Healthcare, Piscataway, NJ, USA). Subsequently, mRNA expression levels of luciferase-3′ UTR reporters were assessed using 1 μl of the appropriate cDNA for real-time qRT–PCR using FastStart Universal SyberGreen and a LightCycler 480 machine (Roche, Indianapolis, IN, USA). The forward oligonucleotide primer (5′-GGTCTTACCGGAAAACTCGAC-3′) corresponds to a sequence at the 3′ end of the luciferase cDNA; the reverse primer (5′-CTCTGTCCTTGCCAGAAGATG-3′) is within the 3′UTR of SMARCB1. The results were analyzed using the comparative Ct method (ΔΔCt) and normalized to GAPDH and eGFP expression levels to ensure equal loading and transfection efficiencies.
Formalin-fixed, paraffin-embedded tissue sections from four schwannomas resected from member of family 19 (c.795 + 1G > T) and a single schwannoma from family 9 were immunostained using a commercial SMARCB1 antibody (BD Transduction Laboratories, Franklin Lakes, NJ, USA) along with appropriate positive (normal cortex) and negative controls (RTPS1). Antigen retrieval was achieved by microwaving in a Borg Decloaker RTU for 45 min (primary antibody concentration 1:50).