No correlation between amount of aberrant transcript and severity of phenotype in hereditary spastic paraplegia patients with a c.1242A > G splice mutation in the SPG4 gene

Abstract

Sirs: Hereditary spastic paraplegia (HSP, MIM 182601) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterised by progressive spasticity and hyperreflexia of the lower limbs (pure HSP). The most common form (40 %) of autosomal dominant pure HSP is caused by mutations in the SPG4 gene encoding spastin. Spastin belongs to the AAA family of ATPases [5]. So far more than 100 mutations in the SPG4 gene have been described, including missense, nonsense, splice site mutations, deletions and insertions. They are scattered all over the 17 exons of the gene; however, most of them are located in the highly conserved region that encodes the AAA cassette [2, 4–12]. In this study we describe the clinical and genetic findings in a family with the previously described c.1242A > G mutation [11 (nomenclature according to [1, 3])] in exon 9 of the SPG4 gene. We provide experimental evidence that this mutation results in an aberrant splice donor site. The index patient had shown onset of symptoms already in childhood. At the age of 65 years she had spasticity, paresis and hyperreflexia of the lower limbs. A positive Babinski’s sign, ankle clonus, a reduced vibration sense and an urge incontinence were also present. Since the age of 57 years she has needed walking sticks. Her 30-year-old son also developed a gait disorder in childhood. He presented with spasticity and hyperreflexia of the legs and ankle cloni. The 34-year-old daughter of the index patient until recently only had hyperreflexia and a questionable positive Babinski’s sign at the right foot. During recent months she had noticed gait problems. The c.1242A > G mutation in exon 9 of the SPG4 gene, detected in all three patients, is a silent mutation on the DNA level (p.K414K). However, this base exchange could create an alternative splice donor site 4 bp before the end of exon 9 leading to a frameshift with a premature termination codon after eight amino acids. To determine the quantity of the aberrant transcript in relation to the wild type transcript we performed RT-PCR using RNA isolated from whole blood samples. RT-PCR products of each proband were cloned into the pGEM TEasy vector (Promega, Mannheim, Germany). DNA was isolated from approximately 100 colonies per proband. A restriction analysis with the restriction endonuclease MaeII was performed subseLETTER TO THE EDITORS