Abi prism sequence detector

To verify the expected deletion in SIM1 exon 9 we used DNA, for which MLPA indicated a homozygous loss of both alleles and performed PCR with four overlapping primers flanking the exon 9 MLPA probe binding site (see Supplemental Figure 1). Genomic DNA was extracted from peripheral leukocytes using a Qiagen DNA extraction kit (Qiagen, Hilden, Germany). The region in SIM1 was amplified by PCR under the following conditions: denaturation at 96 °C for 3 min, then 25 cycles of denaturation at 96 °C for 30 s, annealing at 56 °C for 15 s and elongation at 60 °C for 4 min, in the end followed by elongation at 72 °C for 10 min. We inspected the PCR products for the presence of single bands of the expected size on a 1% agarose gel and extracted them using the QIAquick Gel Extraction Kit (Qiagen).
Sequencing was performed on overlapping fragments (primer see Supplemental Table 2). The detected SIM1 variant was verified by reamplification and resequencing. Sequencing of the purified PCR products was performed using the ABI Prism sequence detector in combination with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).
The rs3734354 genotype data for the extended cohort were derived from the HumanExome BeadChip v1.0 (Illumina, Inc., San Diego, CA, USA) using standard protocols suggested by the manufacturer.