High throughput genotyping: microarray-based resequencing for autosomal-dominant hereditary spastic paraplegia

Background: The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive spasticity of the lower limbs. Forty-two different loci have been mapped so far, and 17 responsible genes have been identified. Autosomal-dominant (AD-HSP), autosomal-recessive (AR-HSP), and X-linked (XL-HSP) modes of inheritance have been described. Clinically, pure forms are limited to affection of the pyramidal tracts whereas complicated forms present additional neurological signs. For correct diagnosis moleculargenetic testing is essential because clinical parameters alone are not reliable to differentiate HSP forms.

Objective: We wanted to establish high throughput genotyping for AD-HSP and XL-HSP by designing a resequencing microarray in order to determine the relative frequencies of mutations in index patients of AD-HSP families for whom SPG4 mutations had been excluded.

Methods: After multiplex PCR amplification of coding sequences we hybridized labeled amplicons to a CustomSeq HSP resequencing microarray (Affymetrix platform) covering the coding exons and flanking intronic sequences of 6 autosomal-dominant and 2 X-linked genes. Data analysis was performed using the Sequence Pilot Software module C (JSI medical systems GmbH). In order to validate the HSP-array, amplified exons of 10 patients with known mutations in L1CAM (SPG1) were hybridized to the array. Furthermore, 32 DNA samples with known AD-HSP mutations were included as positive controls. All known mutations have been automatically detected by the SeqC module. Sequencing variants in our patient group have been validated by Sanger sequencing.

Results: A sample of 53 index patients with a family history compatible with AD-HSP was resequenced for nine HSP genes. Several new polymorphisms and non pathogenic sequence variants but no disease-causing mutations have been identified. Therefore, the frequencies of Atlastin (SPG3A), NIPA1 (SPG6), Paraplegin (SPG7), KIF5A (SPG10), HSP60 (SPG13) and BSCL2 (SPG17) in SPG4 mutation negative patients is rather low.

Conclusion: Our findings demonstrated that the AD-HSP resequencing array is a reliable and effective tool to detect sequence alterations. The AD-HSP diagnostic microarray will enable us to diagnose a larger genetic spectrum of dominant HSPs and therefore rapidly select families with so far undefined HSP subtypes which are of special interest for mapping and cloning projects.