Evaluating the effect of spastin splice mutations by quantitative allele-specific expression assay.

BACKGROUND: mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP. METHODS: a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed. RESULTS: we have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414-1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%). CONCLUSIONS: our finding supports a "threshold-effect-model" for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.

Compound heterozygosity in the SPG4 gene causes hereditary spastic paraplegia.

The SPG4 gene is frequently mutated in autosomal dominant form of hereditary spastic paraplegia (HSP). We report that the compound heterozygous sequence variants S44L, a known polymorphism, and c.1687G>A, a novel mutation in SPG4 cause a severe form of HSP in a patient. The family members carrying solely c.1687G>A mutation are asymptomatic for HSP. The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the c.1687G>A mutation is a splice site mutation and causes skipping of the exon 15 of spastin. Furthermore, quantification of RT-PCR products by sequencing and quantification of allele-specific expression by pyrosequencing assay revealed that c.1687G>A is a leaky or hypomorphic splice site mutation. At the protein level, c.1687G>A mutation in SPG4 leads to E563K substitution. In ex vivo study, about 10% of cells expressing E563K mutant spastin showed filamentous expression pattern, suggesting a hypomorphic effect at the protein level. Collectively, our results suggest that S44L in association with c.1687G>A (E563K) drops the functional level of spastin below a threshold limit sufficient to manifest HSP.

Novel mutations in the Atlastin gene (SPG3A) in families with autosomal dominant hereditary spastic paraplegia and evidence for late onset forms of HSP linked to the SPG3A locus.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterised by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia linked to the SPG3A locus on chromosome 14q11-21 accounts for approximately 10% of autosomal dominant hereditary spastic paraplegia (ADHSP). It is caused by mutations in the SPG3A gene encoding the protein atlastin. To date, only five disease-causing mutations in the SPG3A gene have been described. We analysed 13 SPG4-negative families for mutations in the SPG3A gene and identified a mutation in 38% (5/13). Two of the mutations are novel, c.481G>C (p.A161P) and c.740A>C (p.H247P). One of the novel mutations was found both in a family with early onset of symptoms and in a late onset family. Furthermore, we report on numerous polymorphisms detected in the SPG3A gene.

Partial trisomy of distal 19q detected by quantitative real-time PCR and FISH in a girl with mild facial dysmorphism, hypotonia and developmental delay.

We report on a 2 7/12-year-old girl who was referred to us because of psychomotor developmental delay. She is the second child of healthy, non-consanguineous parents. Pregnancy and birth were uneventful. Milestones of motor development were delayed: grasping at 6 months, sitting without support at 16 months, crawling at 16 months and walking at 2 4/12 years of age. She spoke about five words and followed simple instructions. Banding cytogenetics revealed a numerically and structurally normal female karyotype of 46,XX. By quantitative real-time PCR analysis of all subtelomeric regions, a partial trisomy of the subtelomeric region of 19q could be detected. This result was confirmed by FISH-analysis with a subtelomeric probe for 19q. The additional material of chromosome 19q was localized on chromosome 6q. However, a deletion of the subtelomeric region of 6q could not be detected with a subtelomeric FISH probe for 6q. Conventional cytogenetic analysis as well as FISH with subtelomeric probes for 19q and 6q showed normal results in the parents. The detected chromosomal aberration probably occurred de novo. The clinical features are very likely to be caused solely by the partial trisomy 19q.

Molecular cytogenetic characterization of a de novo supernumerary ring chromosome 7 resulting in partial trisomy, tetrasomy, and hexasomy in a child with dysmorphic signs, congenital heart defect, and developmental delay.

We report on a girl with mosaicism (65%) of a de novo supernumerary ring chromosome 7. The main clinical features were delayed psychomotor development, congenital heart defect, facial dysmorphisms, and long hands, fingers, feet and toes. Molecular cytogenetic analysis revealed that the ring chromosome was duplicated in 20% of the analyzed metaphases with marker chromosome and quadruplicated in 5% thereof. Uniparental disomy (UPD) of the two normal sister chromosomes 7 was excluded. This is, to our knowledge, the first report of a partial tetrasomy to hexasomy due to a ring chromosome 7. Additionally, the ring evolution could be reconstructed according to the FISH-results.