Autosomal Dominant Spastic Paraplegia (SPG XX) with Sensory Deficits and Muscle Waisting

Schüle et al., Autosomal dominant spastic paraplegia with peripheral neuropathy maps to chr12q23-24.

Appendix e-1: Detailed Experimental Procedures

Genotyping using 10K SNP microarrays

SNP genotyping was undertaken using the GeneChip Mapping 10Kv2.0 Xba Array containing 10204

SNP markers following the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Briefly,

250 ng of genomic DNA from each sample was digested with the restriction endonuclease XbaI for

2.5 h. Digested DNA was mixed with Xba adapters and ligated using T4 ligase for 2.5 h. Ligated DNA was added to four separate polymerase chain reactions (PCR), amplified, pooled and purified to remove unincorporated dNTPs. The purified PCR product was then fragmented with DNase1, end- labeled with biotin and hybridized to an array for 18 h in an Affymetrix 640 hybridization oven.

Arrays were then washed and stained using an Affymetrix Fluidics Station F450. The arrays were scanned using an Affymetrix GeneArray scanner 3000. The raw microarray feature intensities (RAS scores) were processed using the Affymetrix Genotyping Tools software package GCOS/GTYPE

(Affymetrix) to derive SNP genotypes, marker order and linear chromosomal location.

Microsatellite genotyping and linkage analysis

Microsatellite markers were amplified with fluorescent primers. PCR products were analyzed on an

ABI 3100 automated sequencer with a fluorescence detection system.

Candidate gene analysis

All exons and exon-intron boundaries of selected candidate genes were sequenced in two affected family members using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems, Darmstadt, Germany). Sequencing products were resolved on an ABI 3100 automated sequencer (Applied Biosystems) and analyzed using SeqPilot (JSI medical systems, Kippenheim,

Germany). Primer sequences and reaction conditions can be obtained upon request.