A deep intronic splice variant advises reexamination of presumably dominant SPG7 Cases

Abstract

Objective: To identify causative mutations in a patient affected by ataxia and spastic paraplegia. Methods: Whole-exome sequencing (WES) and whole-genome sequencing (WGS) were performed using patient's DNA sample. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from patient's fibroblasts, as well as western blot. Results: A novel missense variant in SPG7 (c.2195T> C; p.Leu732Pro) was first found by whole-exome sequencing (WES), while the second, also unreported, deep intronic variant (c.286 + 853A>G) was identified by whole-genome sequencing (WGS). RT-PCR confirmed the in silico predictions showing that this variant activated a cryptic splice site, inducing the inclusion of a pseudoexon into the mRNA sequence, which encoded a premature stop codon. Western blot showed decreased SPG7 levels in patient's fibroblasts. Interpretation: Identification of a deep intronic variant in SPG7, which could only have been detected by performing WGS, led to a diagnosis in this HSP patient. This case challenges the notion of an autosomal dominant inheritance for SPG7, and illustrates the importance of performing WGS subsequently or alternatively to WES to find additional mutations, especially in patients carrying one variant in a gene causing a predominantly autosomal recessive disease.

Challenges and Controversies in the Genetic Diagnosis of Hereditary Spastic Paraplegia

“…In a recent study, a patient with spastic paraplegia and ataxia was investigated with WES, revealing a novel missense variant in SPG7 (c.2195T>C; p.Leu732Pro) [ 94 •]. To seek a second variant, WGS was performed, revealing an unreported, deep intronic variant (c.286 + 853A>G), shown to activate a cryptic splice site [ 94 •]. The deep intronic variant would not have been identified with WES alone, highlighting the usefulness of WGS to increase diagnostic yield [ 94 •].…”

“…To seek a second variant, WGS was performed, revealing an unreported, deep intronic variant (c.286 + 853A>G), shown to activate a cryptic splice site [ 94 •]. The deep intronic variant would not have been identified with WES alone, highlighting the usefulness of WGS to increase diagnostic yield [ 94 •]. Furthermore, it sheds light on the apparent dominant pattern of inheritance of SPG7 [ 95 ], which may be due to the mutation on the other allele being missed [ 94 •].…”

“…The deep intronic variant would not have been identified with WES alone, highlighting the usefulness of WGS to increase diagnostic yield [ 94 •]. Furthermore, it sheds light on the apparent dominant pattern of inheritance of SPG7 [ 95 ], which may be due to the mutation on the other allele being missed [ 94 •]. Another report highlights the importance of an intronic variant in POLR3A , a gene previously associated with hypomyelinating leukodystrophy type 7 (Table 1 ), as a frequent cause of HSP and cerebellar ataxia [ 82 ].…”

Ocular genetics in the genomics age

“…However, there are multiple examples of well characterized intronic variants in retinal diseases, identified through various strategies (den Hollander et al, 2006; Mayer & Aguilera, 1990; Mayer et al, 2016; van den Hurk et al, 2003). Now, with access to whole genome sequencing in research and clinical laboratories (Turnbull et al, 2018; Turro et al, 2020) and public availability of large population genome datasets such as gnomAD, researchers are beginning to apply similar variant rarity filtering strategies regularly performed in exome filtering pipelines to noncoding variants in order to identify candidate‐disease variants in rare diseases (Carss et al, 2017; Cassini et al, 2019; Khan et al, 2017; Verdura et al, 2020). To date, the reports have broadly identified noncoding alleles in recessive retinal diseases (either homozygous noncoding alleles or the presence of second noncoding allele in an individual carrying a coding mutation) and noncoding variants that cause activation of a deep intronic splice site leading to pseudoexon incorporation in the transcript.…”

Novel Compound Missense and Intronic Splicing Mutation in ALDH18A1 Causes Autosomal Recessive Spastic Paraplegia

“…HSP exhibits considerable genetic heterogeneity with more than 80 different HSP (or SPG) loci reported, including ALDH18A1 (2, 7). Massively parallel high throughput sequencing strategies, such as targeted gene-panel sequencing, whole exome sequencing (WES), and whole genome sequencing (WGS), have facilitated genetic diagnosis of HSP (8)(9)(10)(11). As a first-line test, WES can significantly reduce the time and cost of the diagnostic process through accelerated identification of the causative variants associated with HSP.…”