Objective: The aim of this study is to reveal molecular pathogenicity and genotype-phenotype correlations in spastic paraplegia type 4 (SPG4).

Background: Mutations in SPG4/SPAST represent the most frequent molecular etiology in an autosomal dominant form of Hereditary Spastic Paraplegia (HSP). Previous studies demonstrated various molecular pathogenesis in SPG, including loss-of-function, haploinsufficiency, dominant-negative effect, and dysregulation of microtubule-severing activity.

Methods: A cohort of patients with spastic paraplegia recruited in Italy and Japan was investigated clinically and genetically. Initial diagnostic approach included targeted resequencing of currently-known HSP genes or resequencing of whole exomes. Biological effects by nucleotide variation were predicted using bioinformatic tools and confirmed by reverse transcription polymerase chain reaction (RT-PCR) experiment. Measurement of SPG4 transcripts were conducted in cultured T cells treated with nonsense pre-mRNA mediated decay (NMD) inhibitor.

Results: A novel SPAST intronic variation was found in two families, one from Italy and the other from Japan. The variant is located at the third position at 5′-splice-site of intron 6, c.1004+3A>C. RNA secondary structure seems to remain unchanged by the variant; however, skipping of exon 6 was shown by RT-PCR experiment, presumably leading to generation of frame shifts and premature stop codons (p.Gly290Trpfs*5). Measurement of SPAST transcripts in lymphocytes demonstrated a reduction through NMD. Intra- and inter-familial phenotypic variations were observed, including age-at-onset, severity of spasticity, as well as scoliosis.

Conclusions: Our study demonstrated further evidence of allelic heterogeneity in SPG4, dosage effects through NMD, and broad clinical features of SPAST mutation.

References: 1. Lo Giudice T, Lombardi F, Santorelli FM, Kawarai T, Orlacchio A. 2014. Hereditary spastic paraplegia: clinical-genetic characteristics and evolving molecular mechanisms. Exp Neurol. 261: 518-539.

2. Orlacchio A, Kawarai T, Totaro A, Errico A, St George-Hyslop PH, Rugarli EI, Bernardi G. 2004. Hereditary spastic paraplegia: clinical genetic study of 15 families. Arch Neurol. 61(6): 849-855.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/spastic-paraplegia-type-4-a-novel-spast-splice-site-donor-mutation-and-expansion-of-the-phenotype-variability/