On line Supplemental DataSamaranch et al, 1

APPENDIX e-1:

Haplotype analysis of the 15q13-15 region

Methods:We performed haplotype analysis of the 15q13-15 region in all the ARHSP-TCC families to identify common disease haplotypes wherein a frequent SPG11 mutation could lay. Although our kindred’ size do not allow enough sensitivity to detect genetic linkage, still haplotype analysis could help us to identify among the affected members haplotype pairs not present among other healthy members older enough to have developed the disease. DNA was available from four affected members with complete clinical information and from 16 healthy family members. Microsatellite genotyping was subsequently performed for three markers (D15S118-D15S659-D15S117) located in a 18.63 cM interval on 15q13-15 which covers the region linked to ARHSP-TCC.1 For this purpose, 5’-6FAM fluorescently tagged primers were used to amplify the DNA with the standard conditions. PCR reactions included 10 ng of DNA in a 10 microliter (l) reaction mixture containing 10 pmol of each primer, 25 mM deoxyribonucleotide triphosphates, 2U of Taq polymerase (Biolase, Bioline), 10X PCR buffer, and 10% DMSO. PCR fragments were separated using a capillary-based system (ABI 3130xl). Alleles were scored for fragment length (base pairs) using the GeneMapper software version 4.0 for all the markers (Applied Biosystems, Co). 1331-01 and 1331-02 CEPH individuals were used as controls to standardize the scoring of alleles.

Information on marker order and inter-marker distances was obtained from the Center for Medical Genetics ( The Pedcheck program was used to identify any Mendelian inconsistencies and genotyping errors.2 Afterwards genotyping data were formatted for haplotype analysis using Mega2 version 2.33 and Simwalk2 version 2.82.4

Results:Haplotype reconstruction on chromosome 15q13-15 did not show any common disease haplotypes shared by any of the kindreds suggesting the presence of different mutational events across the families (figure 1A). The two affected individuals (HSP1 II-2 and HSP1 II-4) of the HSP1 family shared the paternal and maternal haplotypes across the sibship. The other affected individuals from HSP2 (HSP2 II-1) and HSP3 (HSP3 II-3) families showed haplotype pairs which were absent in healthy family members.

SPG11 gene copy-number analysis

Methods:The presence of deletions or duplications of the SPG11 gene was analyzed by quantitative real-time PCR (qPCR). Fluorogenic allele-specific TaqManprobes (TMP) for SPG11 intron 1, exon 25 and exon 40 were designed in order to cover most of the SPG11 gene region (primer sequences and experimental conditions available on request). A pre-designed TMP for the RNAseP gene (Part#: 4316831) was used for copy-number normalization. qPCR runs included within-plate triplicates and the experiment was performed three times for each DNA sample from the HPS 2 family. Comparative standard curve method and Ct method 5 was used to calculate the fold change in relation to DNA control samples.

Results: qRT-PCR of the SPG11 did not show any copy-number variation in HSPs families nor in 40 independent controls indicating the absence of large sequence rearrangements at the SPG11 gene locus.

References

1.Lossos A, Stevanin G, Meiner V, Argov Z, Bouslam N, Newman JP, et al. Hereditary spastic paraplegia with thin corpus callosum: reduction of the SPG11 interval and evidence for further genetic heterogeneity. Arch Neurol 2006;63(5):756-760.

2.O'Connell JR, Weeks DE. PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 1998;63(1):259-266.

3.Mukhopadhyay N, Almasy L, Schroeder M, Mulvihill WP, Weeks DE. Mega2, a data-handling program for facilitating genetic linkage and association analyses. Am J Hum Genet 1999;65:A436.

4.Sobel E, Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics. Am J Hum Genet 1996;58(6):1323-1337.

5.Bookout A.L., Cummins C.L., Mangelsdorf D.J. High-Throughput Real-Time Quantitative Reverse Transcription PCR. In Current Protocols In Molecular Biology. John Wiley & Sons Inc. 2005:15.18.11-15.18.21.