title: Amplicon-based high-throughput pooled sequencing identifies mutations in CYP7B1 and SPG7 in sporadic spastic paraplegia patients.
abstract: Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder defined clinically by progressive lower limb spasticity and weakness. HSP is a genetically highly heterogeneous condition with at least 46 gene loci identified so far, involving X-linked, autosomal recessive (AR) and autosomal dominant inheritance. For correct diagnosis, molecular testing is essential because clinical parameters by themselves are not reliable to differentiate HSP forms. The purpose of this study was to establish amplicon-based high-throughput genotyping for AR-HSP. A sample of 187 index cases with apparently sporadic or recessive spastic paraplegia were analyzed by applying an array-based amplification strategy. Amplicon libraries of the CYP7B1-(SPG5) and SPG7-gene were generated followed by a pooled next-generation sequencing (NGS) approach. We identified three SPG5 and seven SPG7 patients. All had one homozygous or two heterozygous mutations. In total, 20 distinct mutations (CYP7B1,n = 4and SPG7,n = 16) including two novel CYP7B1 mutations (p.G51R and p.E211KfsX3) and eight novel SPG7 mutations (p.Leu8delinsLeuLeu, p.W29X, p.R139X, p.R247X, p.G344D, p.Leu346_Leu347ins11, p.R398X and p.R398Q) were detected by this comprehensive genetic testing. Our study illustrates how amplicon-based NGS can be used as an efficient tool to study genotypes and mutations in large patient cohorts and complex phenotypes.
authors.affiliation: Department of Medical Genetics, Institute of Human Genetics, Tübingen, Germany.
authors.forename: N A
authors.initials: NA
authors.lastname: Schlipf
authors.forename: R
authors.initials: R
authors.lastname: Schüle
authors.forename: S
authors.initials: S
authors.lastname: Klimpe
authors.forename: K N
authors.initials: KN
authors.lastname: Karle
authors.forename: M
authors.initials: M
authors.lastname: Synofzik
authors.forename: J
authors.initials: J
authors.lastname: Schicks
authors.forename: O
authors.initials: O
authors.lastname: Riess
authors.forename: Ludger
authors.initials: L
authors.lastname: Schöls
authors.forename: P
authors.initials: P
authors.lastname: Bauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D001483:Base Sequence; D015331:Cohort Studies; D000072474:Cytochrome P450 Family 7; D004252:DNA Mutational Analysis / Q000379:methods*; D005820:Genetic Testing; D014644:Genetic Variation / Q000235:genetics; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010264:Paraplegia / Q000175:diagnosis* / Q000235:genetics*; D013250:Steroid Hydroxylases / Q000235:genetics*
chemical_list: D013250:Steroid Hydroxylases; D000072474:Cytochrome P450 Family 7; C499571:CYP7B1 protein, human; D008666:Metalloendopeptidases; C112889:SPG7 protein, human; D000074183:ATPases Associated with Diverse Cellular Activities