An inherited large-scale rearrangement in SACS associated with spastic ataxia and hearing loss

Abstract

Autosomal recessive spastic ataxia of Charlevoix–Saguenay is a neurodegenerative disorder characterized by early-onset, spastic ataxia and peripheral neuropathy, with or without mental retardation. The array of mutations in SACS has expanded worldwide after the first description in Quebec. We herein report the identification of an unconventional SACS mutation, a large-scale deletion sized ∼1.5 Mb encompassing the whole gene, in two unrelated patients. The clinical phenotype of the patients was similar to more canonical ARSACS cases, though it is was complicated by the unusual presence of hearing loss. Our findings suggest that a “microdeletion” on chromosome 13q12 represents a novel allelic variant associated with ARSACS, stressing the need for an expanded testing in molecular diagnostic laboratories.

E-Methods

Haplotyping

Markers flanking the SACS locus (D13S1275, D13S232, D13S1034, D13S292, D13S1243, and D13S221) were genotyped by PCR using fluorescence labeled primers, 3130xl Genetic Analyzer, and Genemapper software (Applied Biosystems). Haplotypes were reconstructed manually assuming the minimal number of recombinations. SNPs surrounding the SACS gene (rs725600, rs1008814, rs7998705, rs1630, rs751853, rs977655) were analyzed by PCR and direct sequencing (primers listed in E-Table 1) using BigDye 3.1 chemistry on an ABI 3130xl (Applied Biosystems).

Quantitative real-time PCR (qPCR)

To test gene copy number, we designed four amplicons located in the exons 1, 8 and 10 of SACS using Primer Express 3.0 software (Applied Biosystems) (primers listed in E-Table 1) and tested their specificity using BLAST software (http://blast.ncbi.nlm.nih.gov). We also analyzed the exon 10 of the SACS gene employing a predesigned, commercially available TaqMan-MGB assay (Hs00274210_m1) (Applied Biosystems). As a reference gene, we used RNase P as recommended by the manufacturers. PCR reactions were carried out using an ABI Prism 7500 (Applied Biosystems) in a 96-well optical plate with a final reaction volume of 50 μl. About 10 ng of DNA was subjected to thermal cycling conditions with a prerun of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 s and 60°C for 1 min according to the TaqMan Universal PCR Protocol. Using the comparative Ct method, outlined in reference [6], the starting copy number of the unknown samples was determined in comparison with the known copy number of the calibrator sample, using the following formula: ΔΔCt = [ΔCt RNAaseP (calibrator sample) − ΔCt SACS (calibrator sample)] − [ΔCt RNAaseP (unknown sample) − ΔCt SACS (unknown sample)]. The relative gene copy number was calculated by the expression 2-(ΔΔCt). Using this calculation, a ΔΔCt ratio of about 1 for a diploid sample and about 0.5 for a haploid sample are expected. Values in patients were compared to results obtained in ten normal individuals, deemed free of neurological disorders.

Array-CGH

CGH was performed in patient 1-LF using the Agilent Technologies Array CGH Kits (Agilent, Santa Clara, CA, USA). These platforms are 60-mer oligonucleotide-based microarrays that allow genome-wide survey and molecular profiling of genomic aberrations with a resolution of 75-Kb (kit 4x44K). Aliquots of 1 μg of DNA from patient and same-sex reference samples were double-digested with the endonucleases RsaI and AluI for 2 h at 37°C. After heat inactivation of the enzymes at 65°C for 20 min, each digested sample was labeled by random priming (Agilent Technologies) for 2 h using Cy5-dUTP for patient DNAs and Cy3-dUTP for reference DNA. Labeled products were purified using Microcon Y-30 filters (Millipore, Billerica, MA, USA). After probe denaturation and preannealing with 5 μg of Cot-1 DNA, hybridization was performed at 65°C with maximum speed rotation for 24 h. After two washing steps, the array was analyzed using GenePix 4000B scanner (Axon, Sunnyvale, CA, USA) and Feature Extraction V.9.1.5 software (Agilent). A graphical overview of the results was obtained using CGH Analytics V.3.5.14 software (Agilent).

Fluorescent in situ hybridization (FISH)

FISH analysis was performed on metaphase chromosomes, with the BAC probe RP11-72P19 spanning the SGCG gene in the 13q12.12 region. The probe was directly labeled with Spectrum Orange (Vysis) and hybridized with DAPI counterstain according to standard procedures. Metaphases were visualized using a Zeiss Axioplan 2 imaging fluorescent microscope equipped with single-band pass filters (Zeiss, Germany). Digital images were captured and analyzed with the ISIS program (Meta Systems, Germany). At least 20 metaphases were analyzed by direct microscopic observation and digital-imaging techniques. As internal control, we also used the 13q-subtelomeric probe.