ARCA: autosomal recessive cerebellar ataxias

NGS: next-generation sequencing

SARA: scale for the assessment and rating of ataxia

SCAR: spinocerebellar ataxias

VUS: variants of uncertain significance

Autosomal recessive cerebellar ataxias (ARCA)/ Spinocerebellar ataxias (SCAR) are a heterogeneous group of rare conditions, most frequent of which are Friedreich’s ataxia and SPG7-associated ataxia [1]. However, of patients with early-onset of symptoms, ataxia telangiectasia (ATX-ATM) is the second most common type [1]. This is a neurodegenerative and immunodeficiency multisystem disorder, characterised by cerebellar ataxia, ocular telangiectasia, oculomotor apraxia, predisposition to recurrent sinopulmonary infections, radiosensitivity. Both patients and heterozygous carriers have increased cancer risk [2]. Pathogenic variants in ATM affecting the level of ATM and activity/signalling capacity of the ATM kinase are responsible for the phenotype [2]. Multiple causative variants have been described; functional studies assessing the presence of retained enzyme activity predict age of onset, clinical symptoms and progression better than the variant nomenclature alone [3]. Patients with retained ATM kinase activity due to leaky splice site or missense variants have a milder phenotype, variant ATX-ATM [3].

ATX-SETX is another early-onset ARCA, caused by pathogenic SETX variants, which shares many similar features with ATX-ATM including oculomotor dysfunction, cerebellar atrophy, sensorimotor axonal neuropathy, chorea and/or dystonia. However, it is not associated with immunodeficiency or predisposition to cancer and telangiectasia is rare. Variants in both SETX and ATM affect DNA repair and contribute to cell death [4].

Here, we report a patient initially suspected to have ATX-ATM due to progressive ataxia with a history of malignancy in association with elevated levels of CA-125 and AFP and homozygous VUS in ATM. However, functional analysis reclassified the ATM VUS to likely benign and identified a reduced level of senataxin, leading to a diagnosis of ATX-SETX.

A 42-year-old Slovakian lady presented with progressive unsteadiness since age 17 years. She had a history of strabismus surgery at 4 years, left temporal haemorrhage at 21 years secondary to an angioma, splenomegaly, prior breast cancer and ovarian mass under investigation by gynaecology at time of referral. There was no family history of ataxia, breast cancer, or consanguinity. On examination she had tortuous conjunctival vessels, gaze-evoked horizontal nystagmus and jerky pursuit movements (Suppl. Video 1). She had cerebellar dysarthria, myoclonic jerks and 4 limb incoordination. Vibration sensation was impaired to the anterior superior iliac spine and reflexes were absent in the lower limbs. She was wheelchair dependent. Scale for the assessment and rating of ataxia (SARA) score was 19.5/40 [5].

MRI brain demonstrated diffuse cerebellar atrophy along with vermian atrophy resulting in enlargement of the 4th ventricule (Figure 1). Neurophysiology confirmed a moderately severe length-dependent sensorimotor axonal neuropathy. Investigations showed elevated AFP at 65.7 IU/mL (normal value <5 IU/mL) and CA-125 at 76 U/mL (normal value <35 U/mL). It was decided to proceed with sequencing of the ATM gene, given the clinical suspicion for ATX-ATM. This demonstrated a homozygous missense variant c.1010G>A p.(Arg337His) in ATM affecting a highly conserved nucleotide and amino acid. This variant is classified by American College of Medical Genetics (ACMG) criteria as a variant of uncertain significance (VUS), although listed on ClinVar as benign, likely benign and VUS [6]. This variant had a minor allele frequency of 0.000079. Both parents were heterozygous. This ATM variant has been suggested as a risk for cancer development [7], raising the initial possibility that both her breast cancer and ataxic syndrome occurred as a consequence of this ATM variant. However, subsequent analysis did not demonstrate cytogenetic chromosomal instability. Furthermore, western blotting revealed a normal level of ATM protein and functional studies assessing differential phosphorylation of proteins demonstrated normal ATM kinase activity/signalling, suggesting that this ATM variant was not pathogenic.

Considering the similarities between ATX-ATM and ATX-SETX, Western blotting was performed to assess levels of senataxin, which was confirmed to be reduced (Figure 2). Sequencing of SETX revealed two variants: a previously reported c.5825T>C, p.(Ile1942Thr), and another variant listed in ClinVar c.1484T>C, p.(Leu495Pro) [8-10]. The first variant is located inside the helicase domain and allows some expression of mutant senataxin. It has a minor allele frequency of 0.00001195. Pathogenicity prediction tools, Mutation Taster and Polyphen-2 suggest that the second variant is damaging. The variant was not present in gnomAD. Her father was a heterozygous carrier of p.(Ile1942Thr), and her mother a heterozygous carrier of p.(Leu495Pro), confirming compound heterozygosity in the proband. Both ACMG and ACGS (Association for Clinical Genomic Science) criteria classify both of these variants as pathogenic (Table 1).

Subsequently, her ovarian mass was revealed to be an ovarian endometriotic cyst and benign paratubal serous cystadenoma following investigation through a total abdominal hysterectomy and bilateral salpingo-oophorectomy.

In the present study, we have reported a patient with an initial suspected diagnosis of ATX-ATM and homozygous variants in ATM in whom subsequent functional analysis was critical in establishing the diagnosis of ATX-SETX, highlighting that ATX-SETX should be considered in the differential diagnosis of ATX-ATM.

Although sequential single gene sequencing was performed in this patient, first seen some years ago, it is widely accepted that NGS with either whole exome or genome sequencing is a more efficient diagnostic approach [1].

Advances in genetic technology have significantly improved diagnostic yield in rare diseases; however, these improvements have led to an increase in VUS [1]. VUS represent major challenges for diagnosis, management, and genetic counselling. A large study assessing utility of multigene testing identified VUS in 53% of individuals; only 0.7% of these variants were reclassified as clinically significant [11]. However, as the literature and our understanding of variants expand, it is expected that more variants are likely to be reclassified. With the increase in genetic tests being sent from general neurology clinics, access to a neurogenetics multidisciplinary meeting or discussion with local genetics laboratories is important. About 40% of ATM variants in breast cancer are VUS [12]. When ATM sequencing in an ataxia patient demonstrates VUS, functional studies to measure radiosensitivity, ATM protein level and ATM kinase activity/signalling should be performed. Functional and cytogenetic studies and an alternate molecular diagnosis in our patient support that ATM c.1010G>A is not pathogenic, also indicated by other authors, and should be reclassified as likely benign [6]. This has important clinical implications for the patient and her family, as her breast cancer is likely sporadic. Furthermore, the reclassification of this variant has implications for carriers of this variant, as they are not at increased cancer risk.

Pathogenic recessive variants in SETX, encoding an enzyme thought to function as a helicase in DNA transcription and RNA processing, cause ATX-SETX [12]. Our patient had a known pathogenic variant c.5825T>C but a younger age at symptom onset than reported previously with this variant. She also had peripheral neuropathy, not previously documented with this variant, but a common finding in patients with ATX-SETX [8]. This may be due to the effect of the second variant c.1484T>C. Functional studies demonstrated reduced level of senataxin, supporting pathogenicity.

In summary, this case highlights the challenges with interpretation of variants of uncertain significance. The ease of access and widespread use of genomic sequencing will increase the return of VUS; interpretation should be cautious based on careful clinical phenotyping, easily accessible pre-existing databases (e.g. ClinVar) [6] and consideration of additional diagnostic steps, including familial segregation and functional studies. There are limitations to in silico algorithms and where possible, in vitro functional analysis can help discriminate between pathogenic and non-pathogenic genetic variants.

Author contribution statement. PJM, PBM, AG, MT and SMM were involved in the conception, acquisition, analysis, and interpretation of data, drafting and revising of the manuscript. KS, LYS, PB, HD and RAW were involved in the acquisition of the data and analysis, interpretation and revising of the manuscript.

Ethics statement. The authors confirm that this work complies to the journal's guidelines on issues involved in ethical publication, which state that written informed consent was obtained from individual participants involved in case studies or through a surrogate where appropriate.

Declaration of interest. The authors declare that they have no conflict of interest.

Funding. None.

Supplementary Video 1. Eye movements. This video demonstrates tortuous conjunctival vessels and telangiectasia, interrupted pursuit movements, gaze-evoked nystagmus and hypometric saccades. The supplementary material is available at https://doi.org/10.5281/zenodo.8308340.

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