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.

Hereditary spastic paraplegia: Clinical-genetic characteristics and evolving molecular mechanisms

“…The improvement of nucleotide sequencing has recently demonstrated the possibility of accelerating and improving cost-effectiveness of genetic diagnosis using an array-based amplification strategy after generation of amplicon libraries of all currently known HSP genes followed by a pooled next-generation sequencing (NGS) (Chase, 2014;Novarino et al, 2014;Schlipf et al, 2011). Besides, modern methodologies of re-sequencing have been reported as well as a gene chip screening (Luo et al, 2013).…”

Identification of a Novel Homozygous <i>SPG7</i> Mutation in a Japanese Patient with Spastic Ataxia: Making an Efficient Diagnosis Using Exome Sequencing for Autosomal Recessive Cerebellar Ataxia and Spastic Paraplegia

“…The p.His448Profs * 12 mutation is predicted to result in a premature stop codon. Previous reports (7,(9)(10)(11)(12)(13)(14)(15) have revealed that missense and nonsense mutations located closer to the C-terminal of paraplegin than the p.His448Profs * 12 mutation can cause disease ( Figure D). Recently, genotype-phenotype correlations were identified for SPG7: an association between cerebellar ataxia and SPG7 null alleles leading to an absence of protein products or severely truncated protein products and an association between optic nerve atrophy and a missense mutation in exon 10 (pArg470Gln) (9).…”

The applications of massive parallel sequencing (next-generation sequencing) in research and molecular diagnosis of human genetic diseases

“…NGS facilitates researchers with the required capacity to analyze large panels of genes for suspected genetic diseases. These diseases vary from single gene disorders such as Neurofibromatosis Type 1 (NF1), Marfan syndrome (MFS), and spastic paraplegia [50,53,54] to diseases caused by a group of related genes such as hypertrophic cardiomyopathy and congenital disorders of glycosylation (CDG) [19,20,51]. NGS has also been applied to multi-gene disorders including X-linked intellectual disability (XLID) [18] and retinitis pigmentosa [52], as well as defined disorders without identified genetic causes [55][56][57] (Table 4).…”