Application of the designed panel in ataxia patients revealed that the overall mutation detection rate amounted for 55.2% and varied from 33.3% in those with a familial history and autosomal dominant mode of inheritance (3/9 cases) to 53.3% in those with sporadic individuals (8/15 cases). Among the latter cases, we identified variants in genes associated with disorders that are inherited in both autosomal recessive and autosomal dominant manners. The obtained results present significant molecular effectiveness in comparison to other NGS-based studies. For example, Németh et al. reported definite molecular diagnosis by gene panel sequencing in 18.0% of 50 probands with ataxia, although the detection rate varied from 8.3–40.0% depending on the age of symptoms onset (adult-, childhood- or adolescent-onset disease) [Németh et al., 2013]. Other studies using NGS exome sequencing revealed a diagnostic yield of 21.0% in a cohort of 76 patients with chronic progressive cerebellar ataxias and potential additional diagnoses in 40.0% [Fogel et al., 2014]. A study of 319 cerebellar ataxia cases showed a yield of 22.6%, with possible additional diagnoses in 5.9% [Coutelier et al., 2018].
Due to comprehensive diagnostic investigations that involve clinical assessment and differentiate ataxia from acquired, primary cause, or secondary cause by examining clinical findings, such as blood tests, neuroimaging tests, and genetic tests, a definitive molecular diagnosis of patients with suspected clinically hereditary ataxia often takes years and is highly complex.
In our study, the most frequently pathogenic mutations were located in the POLG, CACNA1A, SACS and SLC33A1 genes. Other variants were detected in the following genes: STUB1, SPTBN2, TGM6, SETX, ANO10 and SPAST. The most common type of mutation was missense mutation.
In the POLG gene, we identified two known pathogenic variants most frequently present in Caucasians: c.1399G>A (p.Ala467Thr) and c.2243G>C (p.Trp748Ser). These variants usually occur in the homozygous state and have high variability in their clinical presentation [Neeve et al., 2012, Van Goethem et al., 2004], which may be caused by genetic, epigenetic and environmental factors [Neeve et al., 2012]. Notably, our tested individual with a heterozygous mutation of c.1399G>A in the POLG gene clinically presented with gait and limb ataxia, motor and sensory neuropathy, positive Romberg sign, decreased sensory nerve conduction velocity, impaired vibration sense in the right foot, cardiomyopathy and an age of onset of 70 years (patient RM). However, it was previously assumed that heterozygous carriers were unaffected cases [Neeve et al., 2012, Van Goethem et al., 2004]. The patient KW had the same variant in the POLG gene and also had the c.1506delA (p.Lys502AsnfsTer28) mutation in the SPAST gene. However, in this case, the mutation in the SPAST gene should be considered the more likely cause of the patient’s disease.
The gold standard in clinical practice should be using both a focused approach for NGS and Sanger sequencing. As an example, the homozygous splice-site pathogenic mutation c.1218+1G>C in ANO10 was confirmed by Sanger sequencing in the proband and both parents in a consanguineous family. In addition, the analysis of cosegregation of the variants with the disease in families should always be performed when possible.
Interestingly, genetic analysis of two unrelated patients with suspected hereditary ataxia identified the heterozygous mutation c.1559T>C (p.Ile520Thr) in the SLC33A1 gene, which is related to autosomal dominant spastic paraplegia type 42 [Lin et al., 2008] or recessive congenital cataracts, hearing loss, and neurodegeneration [Huppke et al., 2012]. To the best of our knowledge, the heterozygous missense mutation c.339T>G (p.Ser113Arg) in the SLC33A1 gene was identified in one Chinese family with autosomal dominant pure HSP [Lin et al., 2008]. In addition, no mutations in SLC33A1 were detected among 220 Caucasian patients with autosomal dominant hereditary spastic paraplegias and negative for mutations in the SPAST gene [Schlipf et al., 2010]. Our results may suggest that mutations in the SLC33A1 gene can be associated with spinocerebellar ataxia, hereditary spastic paraplegia or both. However, further segregation analysis of gene variants in SLC33A1, functional assays or analysis of a large group of ataxia patients should be performed.
Additionally, we assume that further reanalysis of the most likely variants, e.g., c.2062C>G (p.Pro688Ala) in the AFG3L2 gene, which range from unknown significance to pathogenic, may increase the diagnostic yield. Therefore, eventually, it may be extremely important to check for updates of the databases, i.e., HGMD, OMIM, ClinVar, and LOVD, related to gene variants that were previously classified as VUS. At present, these diagnoses cannot be unequivocally settled due to the lack of samples from parents.
The analysis of co-segregation of the variant c.146A>G in the STUB1 gene for the affected mother and cousin of the proband (Figure 2) showed the presence of a heterozygous mutation in both, confirming that this variant co-segregates with SCA48. These genetic findings enable the renaming of this variant from VUS to pathogenic. Moreover, 128 affected patients tested for NGS did not reveal the presence of this mutation, which could be strong evidence for the presumed pathogenic variant. These findings together account for the pathogenicity of this variant in the STUB1 gene. Therefore, we propose that the most suspect VUS should be reported in the scientific literature because with the growing knowledge in online databases, some gene variants can be reclassified from VUS to pathogenic.
The gene-specific NGS approach is subject to some limitations. The first limitation is the overlapping of neurological phenotypes and the presence of pathogenic mutations in genes encompassing other neurodegenerative disorders. The second limitation is the identification of more than one VUS per single case. The third limitation is the increasing information about novel variants in databases and renaming of VUS as pathogenic variants. Thus, we do not exclude the possibility of a genetic cause of the disorder in patients with no known pathogenic variants detected in the SCA-SPG genes to date. In one such case, we were able to detect a deleterious heterozygous missense mutation of c.305C>T (p.Pro102Leu) in the PRNP gene that was associated with autosomal dominant Gerstmann-Straussler disease. A 32-year-old man (patient BW) presented with gait ataxia, progressive lower limb weakness, imbalance and incoordination, clumsiness, cerebellar dysarthria, scanning speech, rigidity, postural tremor, dysmetria, bilateral extensor plantar responses, hypertonia, dysdiadochokinesia, excessive reflexes of lower limbs, axonal polyneuropathy and an age at onset of 27 years old. MRI neuroimaging showed cerebellar and brainstem atrophy and thinning of the corpus callosum. The mutation was found through the analysis of different NGS panels that included 118 genes related to neurodegenerative and dementia disorders, which included the PRNP gene. However, we believe that the high detection rate confirmed that our strategy of using a targeted NGS approach that focuses on genes associated with both hereditary ataxias and hereditary spastic paraplegias was appropriate. This establishment is associated with clinical and genetic overlapping of these two diseases, which is termed ataxia-spasticity spectrum [Synofzik and Schüle, 2017, Elert-Dobkowska et al., 2019].
Furthermore, together with single nucleotide variants, rare cerebellar ataxias are also caused by different types of mutations undetectable by NGS. Copy number variants (CNVs) in GRID2 are a main cause of SCAR18 [Ceylan et al., 2020]. In addition, Friedreich’s ataxia is caused by noncoding GAA repeat expansion or ultrarare point mutations in the FXN gene. Recently, Cortese A. et al. (2019) identified a biallelic intronic pentanucleotide AAGGG repeat expansion in the RFC1 gene that is associated with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) [Cortese et al., 2019]. This finding expands the known molecular genetic basis of autosomal recessive cerebellar ataxia. All these examples can explain the likelihood of a lack of a definite molecular diagnosis in some ataxic patients after the application of only select genetic tests.
In conclusion, we demonstrated that an approach based on a targeted NGS panel can be a highly effective and useful tool in the final molecular genetic diagnosis of ataxia patients. Furthermore, we highlight that a sequencing panel that targets ataxias together with hereditary spastic paraplegia genes increases diagnostic success. Due to the complexity of the clinical picture and overlapping phenotypes between distinct neurological disorders, NGS testing is more common and is the gold standard in neurodegenerative disorder diagnostics.