A Novel Exon Duplication in the SACS Gene in Charlevoix-Saguenay Ataxia and a Summary of Polish Cases

Introduction

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is one of the most prevalent forms of autosomal recessive ataxia caused by biallelic pathogenic variants in the SACS gene (OMIM: 270550).¹ Patients with ARSACS typically exhibit three core features: early-onset cerebellar ataxia, spasticity, and peripheral neuropathy. Additional atypical manifestations may include absence of spasticity, cognitive impairment, epileptic seizures, and hearing loss. Brain magnetic resonance imaging (MRI) often shows distinct cerebellar atrophy and linear T2 hypointensity within the pons, whereas optical coherence tomography (OCT) frequently shows retinal nerve fiber layer (RNFL) hypertrophy. However, some ARSACS cases lack these remarkable findings on brain MRI or retinal OCT.

Sacsin is a large 520kDa protein expressed ubiquitously but is enriched in neurons.² Although it’s exact function is not fully understood it may play a role in recruitment or retention of Drp1 at mitochondrial fission sites, with loss of sacsin function leading to impaired mitochondrial activity.³ Over 500 different pathogenic variants have been identified in the SACS gene,⁴ with three-quarters occurring in exon 10, one of the largest in the human genome. Most mutations are frameshift inserions/deletions, or single nucleotide mostly nonsense variants with only two structural variants within SACS gene reported in Clinvar so far. The same mutations can lead to variable clinical presentations, sometimes even within a single family. This clinical and genetic heterogeneity in ARSACS may complicate the accurate diagnosis. Herein, we report the case of a Polish patient with a novel SACS duplication of the entire exon 6 and a known small deletion, presenting with progressive ataxia and demyelinating peripheral neuropathy.

Case Report

Patient

A 22-year-old woman with a clinical diagnosis of spinocerebellar ataxia was referred to the Genetic Counselling Unit of the Institute of Psychiatry and Neurology in Warsaw. She was born at term via caesarean section due to breech presentation. The birth weight was 3200 g, the length was 57 cm, and the Apgar score was 9. Neonatal examination results were unremarkable. Early motor milestones were within the normal range; she sat independently at seven months and began walking at 13–14 months. However, signs of motor dysfunction, including difficulties in toe walking and balance, appear in early childhood.

By the age of 10, she exhibited high-arched feet (pes cavus), unsteady gait, and tight Achilles tendons. These signs were initially attributed to the perinatal complications. Despite motor issues, cognitive development remained normal. She completed higher education and earned a master’s degree in pedagogy, maintaining employment as an office worker until the age of 31.

At 22 years of age, neurological evaluation revealed subtle horizontal nystagmus in the lateral gaze and incomplete convergence of the left eye. In general, muscle strength was preserved, but tone was significantly decreased. In the upper limbs, tendon and periosteal reflexes were reduced, with slight intention tremor on finger-nose testing, and dysdiadochokinesia. In the lower limbs, the patient showed calf hypertrophy, Achilles tendon contractures, and pes cavus, with limited ankle mobility. Knee reflexes were barely present and Achilles reflexes were absent. Babinski sign was positive. Coordination tests revealed slight dysmetria on heel-to-knee testing, and balance was impaired, as demonstrated by a positive Romberg test and broad-based, uncertain gait. She could walk on tiptoes but not on heels, consistent with the shortened Achilles tendons. Mild dysarthria was noted, but it was non-progressive.

Electrophysiological studies confirmed a diagnosis of sensorimotor peripheral neuropathy. Laboratory testing revealed mildly but persistently elevated creatine kinase levels. Brain and spinal magnetic resonance imaging (MRI) at the age of 12 years showed moderate cerebellar and cervical-thoracic spinal cord atrophy. EMG at 21 years revealed both demyelinating and axonal changes in the motor and sensory fibers of the peripheral nerves.

At the most recent follow-up at 39 years of age, the patient reported mild progression of ataxia and worsening foot deformities. She used elbow crutches for ambulation and underwent orthopedic surgery for Achilles tendon elongation.

Next-Generation Sequencing

DNA was extracted from the peripheral blood of the proband by using standard methods. Whole exome sequencing (WES) was performed commercially at the BGI Tech Solutions (Hong Kong) using the SureSelect Human All Exon v5 + UTR enrichment kit and paired-end 100 nt sequencing on the Illumina HiSeq2000 platform, as described previously.⁵

The SNVs and indels were initially filtered for a Phred quality score of at least 30. Only variants with an impact on the coding regions were retained for further consideration: missense, nonsense, frameshift, and essential splice site mutations. Further filtering was based on allele frequency in the ExAC (Exome Aggregation Consortium) database (< 1%), association with HPO (Human Phenotype Ontology) terms, and predicted pathogenicity. The HPO terms used were: “ataxia” and “neuropathy”. Variants predicted to be pathogenic by at least one of the following programs were considered: MutationTaster, PolyPhen2, and SIFT. The remaining variants were confirmed by Sanger sequencing.

In total, 20 variants SNVs and indels passed filtering and prioritization (Supplementary Table 1). All of them were absent in 306 exomes and genomes from individuals of Polish origin deposited in our laboratory database. The SACS c.12923_12927del and VCP c.1460G>A variants were confirmed using Sanger sequencing in the proband (Table 1). The SACS variant was found in the proband’s mother, and the VCP variant in her father.

Table 1 Putative Causative or Phenotype-Modifying Genetic Variants Identified in the Proband

A targeted ataxia panel analysis was additionally performed. The DNA library was prepared using the KAPA HyperPlus Kit and MiSeq 2×75 bp paired-end sequencing was performed using the MiSeq Reagent Kit v3 (150 cycles) according to the manufacturer’s procedure as described previously.⁶ The targeted gene panel included the coding sequence of 152 known genes associated with hereditary ataxias, hereditary spastic paraplegias, and rare disorders presenting with ataxia symptoms. The results confirmed the Whole Exome Sequencing results, including SACS c.12923_12927del and VCP c.1460G>A variants, indicating no additional putative pathogenic variants.

Multiplex Ligation-Dependent Probe Amplification

SALSA MLPA Probemix P441 SACS (MRC Holland, Netherlands) was used to determine the presence of microrearrangements (exon deletion/duplication) in the SACS gene in the patient and her relatives. MLPA was performed according to the manufacturer’s protocol and analyzed using a Coffalyser.net (MRC Holland, Netherlands). The results revealed the presence of the NM_014363.6:c.(345+1_346-1)_(457+1_458-1)dup covering exon 6 of the SACS gene in the patient and her father.

RNA Sequencing

Total RNA was isolated from the peripheral blood mononuclear cells of the proband, her parents, and controls (without neurodegenerative disorders nor SACS rare variants) using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Total RNA (500 ng) was converted to cDNA libraries using TruSeq Stranded Total RNA with a Ribo-Zero kit (Illumina, Analityk Genetyka, Warsaw, Poland) according to the manufacturer’s protocol. Libraries were assessed qualitatively on a Bioanalyzer 2100 using a High-Sensitivity DNA Kit, quantitatively on a CFX96 Real-Time PCR system (Bio-Rad) using the KAPA Library Quantification Kit (Kapa Biosystems), and then sequenced at 2×76 bp on a HiSeq2500 Illumina platform. Sequencing data were analyzed according to previously described protocols.⁷

Although the observed decrease in SACS mRNA expression in carriers of SACS variants was not statistically significant, the change was greatest in the proband with variants on both alleles. (Table 2). RNA sequencing also confirmed a heterozygous five-nucleotide deletion within the SACS exon 10 in the proband and her mother.

Table 2 SACS RNAseq Fold Change, with p-values in Brackets

Discussion

We present the fourth case and seventh Polish patient with a clinical and morphological diagnosis of ARSACS carrying a compound heterozygous mutation in the SACS gene.

Twenty rare genetic variants that might be associated with the disease were identified in the proband (Supplementary Table 1). The interpretation of identified variants was made according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) Standards and Guideline.⁸ To assess the clinical significance of DNA variants ClinVar and ClinGen databases were used, whereas the population frequency of the variants was determined by gnomAD v3.1.2 (Genome Aggregation Database).

The VCP variant, although predicted by most programs as possibly damaging, does not match the symptoms well. This ultra-rare variant has been found in Japanese patients with amyotrophic lateral sclerosis (ALS),⁹ but there are conflicting reports in Clinvar database. Pathogenic VCP mutations are known to cause inclusion body myopathy (IBM) with Paget’s disease (PDB) frontotemporal dementia (FTD), FTD with amyotrophic lateral sclerosis (FTDALS6), and Charcot-Marie-Tooth Disease type 2Y (CMT2Y). Of these, only CMT2Y has peripheral neuropathy; however, the clinical presentation of the patient is not limited to polyneuropathy. Furthermore, a healthy father with no neurodegenerative disorders was found to be a carrier of the VCP variant. Although so called incomplete penetration of some VCP variants has been reported, this is uncommon and unlikely. The SACS variant was the only variant that fully matched the proband’s clinical and morphological phenotypes. As SACS variants are responsible for autosomal recessive ataxia, identification of a single nucleotide variant is not sufficient for genetic diagnosis. Therefore, ataxia-targeted panel and subsequently MLPA analyses were performed. The duplication of exon 6 found both in the patient and her father is assumed to be a tandem duplication, thereby disrupting the open reading frame and resulting in p.(Pro154IlefsTer14) protein sequence change. Together with the frameshift variant c.12923_12927del, p. Lys4308SerfsTer21, found in both the patient and her mother, it plausibly explains the cause of the disease, however this was not confirmed by any functional studies.

The SACS mRNA level in the proband is two-thirds of that in healthy controls without rare SACS variants. Both parents also have reduced mRNA levels, although less significantly, to (on average) three quarters of the controls. A nonsense-mediated decay is the most plausible explanation as both variants introduce stop codons more than 50 nucleotides upstream of the last exon-exon junction. Lower expression in the father carrying exon duplication than in the mother with a frameshift variant (Table 2). Thus, our observations are consistent with previous studies of SACS expression in both ARSASC patients and carriers of a single heterozygous pathogenic SACS variant.¹⁰

This is the fourth Polish ARSACS case and the seventh patient described (Table 3).^6,11 Among them, were four women and three men. The age of first symptoms in all described cases was in early childhood, with slow disease progression. All patients had ataxia, five of them had spastic gait, one patient showed an absence of spasticity, all patients showed pes cavus, and five patients presented with peripheral neuropathy. All of them revealed cerebellar atrophy on the brain MRI. In five patients, MRI displayed signal hypointensities within the pons, and four showed thickening of the RNFL.

Table 3 Polish SACS (NM_014363.6) Putative Pathogenic Variants Detected in Clinically Diagnosed ARSACS Patients

All patients carried compound heterozygous mutations in the SACS gene. A total of nine SACS gene mutations were identified, including three missense mutations, one nonsense mutation, three small deletions, one small insertion, and one gross duplication. The ACMG criteria resulted in mostly pathogenic of likely pathogenic classification, with only one missense classified as of uncertain significance. In the latter variant more pathogenic than benign criteria were met. Except for a gross duplication, all other mutations were identified in exon 10 of the SACS gene, which is a hotspot for pathogenic SACS mutations.¹² This localization of variants might not be surprising, as exon 10 is by far the largest one spanning almost 13kb - 80% of the coding sequence. However, we did not identify that many rare or uncommon non-pathogenic variants located in this exon in our in-house database. Of the 16 such variants, only nine were found in exon 10, while five were found in the second-largest exon 8, one in exon 6, and one in the intronic region adjacent to exon 3 (Table 4).^5,6,13,14 Many rare SACS variants were found in the Polish population, and some were more common than in the GnomAD non-Finnish European cohort (eg rs147099630 MAF was 0,8% in GnomAD NFE, and 4,7% in our in-house database), albeit usually with no phenotypic consequences.

Table 4 Polish SACS (NM_014363.6) Variants of Unknown Significance (VUS)

Conclusion

The development and widespread use over the past two decades of next-generation sequencing (NGS) methods and algorithmic methods for prioritizing genomic variants identified by NGS allowed quicker genetic diagnosis but also changed the way that inherited diseases are viewed. It has become clear that often more than one gene may be associated with a given disease, different variants in a given gene can cause different phenotypes, and some variants are associated with multiple clinical phenotypes. The presented results indicate that also testing for rearrangements should be performed in patients in whom only one heterozygous mutation was detected. Only the combined use of these two approaches has enabled the establishment of genetic diagnosis.