Diagnostic rates of whole exome sequencing in Hereditary Spastic Paraplegia

Background and Objective: Hereditary Spastic Paraplegia (HSP) is a group of inherited neurodegenerative disorders encompassing pure and complicated forms, characterized by progressive spasticity and weakness of lower limbs. Complicated HSP often clinically overlap with a broad number of neurodegenerative disorders and their correct diagnosis can be challenging. HSP can be inherited by autosomal-dominant (AD-HSPs), autosomal-recessive (AR-HSPs), X-linked, or mitochondrial patterns. To date, more than 70 genes have been discovered for HSP and identification of causative genes by direct sequencing is time consuming and expensive in these patients. So, a rapid, high-throughput and cost-effective approach; whole exome sequencing (WES) can be useful for identifying the genetic causes of HSP. Here we carried out WES to assessment the effective diagnostic power of this approach in HSP patients. Materials and Methods: 20 unrelated Iranian HSP probands were recruited. DNA was isolated from peripheral blood leukocytes of all probands and their family members using salting out method. WES (Illumina HiSeq 2500 system) was performed on all probands. Preliminary filtering of sequence variations was done to identify all nonsynonymous, stopgain, stoploss, deletion, insertion, and splice sites variants present in the probands. Subsequently, variations with a MAF> 0.01 in public databases (1000Genomes, ExAc, ESP, GnomAD, Iranome, HEX, SISu, GME-Variome and 100 in-house exomes) were removed to find the probable disease-causing variations. In silico tools and ACMG criteria were used in order to estimate the pathogenicity or deleterious impact of genetic variants on protein function. Candidate variants were PCR-amplified and sequenced by Sanger method subsequently checked in family members in order to co-segregation analysis, based on mode of inheritance in the families. Findings: This approach led us to identify the causing genes in 11 out of 20 patients. The patients carried mutations in 10 known HSP-causing genes: SPG11, SPG7, ERLIN1, ERLIN2, CAPN1, CYP7B1, ZFYVE26, GJC2, SPAST (two cases), and ENTPD1. Respectively, variations of SPAST and SPG11 were heterozygous and compound heterozygous, whereas the other variations presented in homozygous state. These variations are corresponding with SPG11, SPG7, SGP62, SPG18, SPG76, SPG5A, SPG15, SPG44, SPG4 and SPG64, respectively. Conclusion: The power of WES as a first-tier diagnostic method in HSP has been reported ~40%-70%. Despite the use of WES, genetic analysis has failed in finding of causative genes in ~45% of the AD-HSPs and > 60% of the AR-HSPs, indicating that, some of HSP-genes especially AR-HSPs have remained unknown. In present study, the potentials of WES were explored for diagnosis of disease-causing genes among 20 Iranian HSP families. The results showed a WES diagnostic rate 55% for this cohort. Now, we can search and re-analyze WES data in order to identify novel HSP-genes in remaining 45% of patients. Identification of novel genes and molecular pathways will enable a deeper understanding of the underlying molecular bases and our knowledge about pathogenesis of the disease.

Keywords:

Hereditary Spastic Paraplegia , HSP , Whole exome sequencing , WES , Diagnostic rate