Appendix e-1: Detailed description of the material and methods used for the mutation analysis

Sequence analysis

For sequence analysis of the exons and exon-intron boundaries of STXBP1, we used flanking primers designed with the software tool SNP box e1 (Table e-1). Resulting PCR fragments were sequenced with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosytems, Foster City, CA). Sequences were analyzed on an ABI3730 automated sequencer using Sequencing Analysis 5.0 software (Applied Biosytems, Foster City, CA). Automated variation (single nucleotide variations and indels) discovery was performed using novoSNP e2. Presence of mutations in patients and absence from parents and control individuals was confirmed using the MassARRAY system, following the provided protocol (Sequenom, San Diego, CA).

Copy number analysis

The genomic region containing STXBP1 was screened for copy number variations by use of the in-house developed technique Multiplex Amplicon Quantification (MAQ) (www.multiplicon.com). The assay comprises a multiplex PCR amplification of fluorescently labeled target and reference amplicons, followed by fragment analysis on the ABI3730 e3. The MAQ-S software compares the normalized peak areas of the patient with the average of five control individuals (www.multiplicon.com/tools/MAQ-S). Resulting target amplicon doses indicate the copy number of the amplicon (Figure e-1). The multiplex PCR reaction consists of 14 test amplicons located in the genomic region of STXBP1 and six reference amplicons randomly located on different chromosomes. Twelve of the 14 test amplicons are distributed in the region from exon 2 to 20 with an average distance of 3.4 kb and the two additional amplicons are located around the center of the large first intron (Figure 1). The region surrounding exon 1 was not investigated since the high GC content of this region interfered with its amplification in a MAQ assay.

To refine the size of the identified microdeletions we used a SYBR® Green real-time PCR assay. Test amplicons were selected within the region and the reference amplicon was located in the third exon of B2M on chromosome 15q21-q22. Primers were designed with Primer Express Software. Real-time PCR assays were performed on the ABI PRISM 7900HT Sequence Detection System using 2x Power SYBR® Green PCR Master Mix. Reactions were performed in duplicate on DNA of the test individual and five control individuals.

Haplotype analysis

Genotyping of short tandem repeat (STR) markers was performed on genomic DNA of the probands carrying the microdeletion and their parents. We selected two STR markers localized in the region surrounding the STXBP1 gene (based on the UCSC Genome Browser: http://genome.ucsc.edu).

We tested paternity and maternity by genotyping 26 STR-markers located on 17 different chromosomes. PCR amplification of fragments was performed with fluorescently-labeled primers. Next, fragments were size fractionated on the ABI3730 and genotypes were analyzed with the in-house developed software Local Genotype Viewer (LGV).

mRNA studies

Total RNA of the patient with the intronic mutation c.1029+1G>T and a control individual was extracted from lymphocytes using the RNeasy kit (Qiagen, Venlo, The Netherlands). After synthesis of first-strand cDNA with Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA), we used exonic primers annealing in STXBP1 exon 11 and 13 (Table e-1). Gel electrophoresis was used to size fractionate the resulting PCR fragments. The different fragments were excised from agarose gels, purified using the QIAquick Gel Extraction kit (Qiagen, Venlo, The Netherlands) and subsequently sequenced.

A RNA sample of the patient carrying the intronic mutation c.429+1G>A could not be obtained. The on-line available splice sites prediction programs: Splice-view (http://bioinfo.itb.cnr.it/oriel/splice-view.html) and Genesplicer (http://www.cbcb.umd.edu/software/genesplicer) e4 were used to investigate the possible effect of this mutation.

e-References

e1. Weckx S, De Rijk P, Van Broeckhoven C, Del-Favero J. SNPbox: a modular software package for large-scale primer design. Bioinformatics 2005;21:385-387.

e2. Weckx S, Del-Favero J, Rademakers R, et al. novoSNP, a novel computational tool for sequence variation discovery. Genome Res 2005;15:436-442.

e3. Suls A, Claeys KG, Goossens D, et al. Microdeletions involving the SCN1A gene may be common in SCN1A-mutation-negative SMEI patients. Hum Mutat 2006;27:914-920.

e4. Pertea M, Lin X, Salzberg SL. GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res 2001;29:1185-1190.

Table e-1: Primers used for the mutation analysis of STXBP1

Exon / Orientation / Sequence Primer
Primers for sequence analysis of exons and intron-exon boundaries of STXBP1
STXBP1 exon1 / Forward / GAGTGGGTGACGCCAACG
Reverse / GACCCCACGGACCAGGAG
STXBP1 exon2 / Forward / GCCTAAGGTGGGAAATGACC
Reverse / TCCAACTCCATCCCCTTCTG
STXBP1 exon3 / Forward / CGTGGGCTTCTGGTCCATAG
Reverse / TGGTGCTAGGGATTCAGTGATG
STXBP1 exon4 / Forward / TGGAATTCACTCCCAAGGAAC
Reverse / CACCCCATTGTGGATTGC
STXBP1 exon5 / Forward / TGGCTCCAAAGAACTGATACTGTC
Reverse / AACCAGCAGATCGGGACAAG
STXBP1 exon6 / Forward / TGACAGCAGGTGCCTATAATCC
Reverse / TTTGGCAATTCAGAGCATGG
STXBP1 exon7 / Forward / ACATGGGCACATGGTAGGC
Reverse / AAAACGGTAGGCAGCCTGAG
STXBP1 exon8 / Forward / TTATGGTCCAGGCACTGAGC
Reverse / AGAGCTGCAACCAGGAAAGC
STXBP1 exon9 / Forward / CAGTAGACTTCTGTGCTGCGTTAG
Reverse / CACTCCCGCTTGGGTGAC
STXBP1 exon10 / Forward / CCTGACTTCATGCCCTGGAC
Reverse / GCTCAAGCTCAGAGAACAGAAGG
STXBP1 exon11 / Forward / GGTGATCTGCCCACCTTG
Reverse / CACCTCAGCCTGGGAAAAAG
STXBP1 exon12 / Forward / AGCCAGAGGCCTGGAAATC
Reverse / GCCAACAGCAAGGGAATAGG
STXBP1 exon13 / Forward / TGTGGTTTGTCCCTCTCTTTTG
Reverse / AAACGGCACAGCATTCCAG
STXBP1 exon14 / Forward / GATGCTGTGGGAGACAATGC
Reverse / CACAAGGCCAAGCAGCTTTC
STXBP1 exon15 / Forward / GAGGTGGGGGAGCAATGTC
Reverse / GGACCCCTGTTATGGTTTGG
STXBP1 exon16 / Forward / CCCCCACCATTACTCTTTGC
Reverse / CAAATAGATCTGGGGCTGTGC
STXBP1 exon17 / Forward / TGCTATAACTGAGCCCTGTGC
Reverse / ATGGCTGATGCAGAGTGAGC
STXBP1 exon18 / Forward / CCTGGGAACCAATCCACTTG
Reverse / TGCCTTCCATTGAGAGATGC
STXBP1 exon19 / Forward / TATATCGGGAGGTGGGAACC
Reverse / CGGTGTTGAGAACTGCAAGC
STXBP1 exon20a / Forward / AATCGAGAAGCGCAGGAAAG
Reverse / TCACCCAGGGTTATGTACAAGG
Exon / Orientation / Sequence Primer
Primers for sequence analysis of exons and intron-exon boundaries of STXBP1
STXBP1 exon20b / Forward / ATGTGCAATTGGTGCACAGC
Reverse / ATGATGCCTGGGGACAGAAG
STXBP1 exon20c / Forward / CTGTGATCCCGAGGGAAGTC
Reverse / GGGAGCAGGCTCTGAAAGG
STXBP1 exon20d / Forward / CTCCTGCAGGCCATGTGTG
Reverse / GGGTCCAGAAGACTCCATGC
Primers for analysis of the aberrant transcripts caused by the mutation c.1029+1G>T
STXBP1 exon11 / Forward / GAAGTCACCCGGTCTCTGAA
STXBP1 exon13 / Reverse / CCACTCGGCAGAGTTTGTCT