Biallelic Mutation of SETX and Additional Likely “In Cis” SETX Sequence Change in Ataxia with Oculomotor Apraxia Type 2

Abstract

Ataxia with oculomotor apraxia type 2 (AOA2) is a slowly progressive, autosomal recessive disease characterized by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy. The genetic basis of AOA2 is biallelic mutation of the SETX gene, resulting in reduced or absent senataxin, a DNA/RNA repair protein essential for genomic stability.

In this case report, we described a case of AOA2 with two clear pathogenic SETX mutations, one of which is novel. We then discussed two further likely “in cis” SETX sequence changes (previously reported in the literature as pathogenic), and presented the case that they are likely benign polymorphisms.

Introduction

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare, slowly progressive, autosomal recessive cerebellar ataxia characterized by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy with a median onset of 12 years of age. ¹ Additional features may include compensatory head thrusting, pyramidal signs, loss of reflexes, strabismus, mild-moderate cognitive impairment, and hyperkinetic movement disorders such as chorea, dystonia, and head tremor. Investigative findings almost invariably show marked cerebellar atrophy, particularly in the vermis, and elevated α-fetoprotein (AFP). ^{1 2} The genetic basis of AOA2 is biallelic mutation of the SETX gene located on chromosome 9q34, resulting in reduced or absent senataxin, a DNA/RNA repair protein essential for genomic stability. ³

In this case report, we described a case of AOA2 with two clear SETX mutations as well as two further SETX changes, likely to be in cis.

Case Presentation

Our patient was a 16-year-old adolescent male of Jamaican parentage who initially presented at 10 years of age with a 2-month history of increasingly unsteady gait and frequent falls. On examination at initial presentation, oculomotor apraxia, difficulty with saccade initiation, and horizontal nystagmus were present bilaterally. There was a sensorimotor neuropathy that featured anesthesia of the feet. The patient was unable to tandem walk, experienced problems negotiating uneven surfaces, and exhibited a positive Romberg's test. Reflexes were absent in both the upper and lower limbs. Sensory examination showed loss of light touch and pinprick sensation below the knees bilaterally. Vibratory sense was impaired distal to the mid-shin, but proprioception was intact. The patient's speech was slow and deliberate. Since his initial presentation, the patient has experienced increasing physical difficulties. On his latest assessment he had lost weight, a result of increasing difficulties with eating. The patient was otherwise healthy with no significant past medical history. His family history was unremarkable with no evidence of consanguinity.

The patient had a background of intellectual difficulties that first manifested with concerns regarding attention and speech when the patient was 7 years old. An assessment of the patient by an educational psychologist at this age using the British Ability Scales II identified low-average to average performance in verbal and nonverbal reasoning as well as memory tasks. Despite modest gains over time in verbal and visual reasoning, memory, attention, and academic function, the patient had a reduced reading speed, most likely a result of oral motor problems, as well as a decline in performance of pencil and paper tasks. This pattern of motor deficits with relatively stable overall cognitive abilities is consistent with intellectual impairment secondary to cerebellar atrophy.

Magnetic resonance imaging of the brain revealed cerebellar atrophy with marked widening of both hemispheres and vermian fissures ( Fig. 1 ). The superior vermis was more affected than the inferior. The supratentorial structures were unaffected. The brain stem, including the pons, appeared to have normal volume. The serum AFP level was elevated at 15 μg/L (normal range: 0.0–7.0 μg/L).

Fig. 1.

Magnetic resonance imaging brain with sagittal T1 volumetric imaging demonstrating marked cerebellar atrophy.

Laboratory Investigations

Western blotting of two independent lymphoblastoid cell lines showed normal levels of ataxia-telangiectasia mutated (ATM) protein and aprataxin ( Fig. 2 ), which are deficient in ataxia-telangiectasia (A-T) and ataxia with oculomotor apraxia type 1 (AOA1), respectively. ^{1 2 3} Expression of the hMre11 protein, a constituent member of the hMre11/hRad50/Nbs1 complex, and which has reduced expression in ataxia-telangiectasia-like disorder (ATLD), was also normal. ^{2 3} The kinase activity of irradiated and unirradiated lysates of the patient's ATM protein was also measured. This showed normal levels of its phosphorylation targets SMC1 ser966, KAP-1 ser824, Nbn ser343, and CREB ser121. Thus, A-T, AOA1, and ATLD were all ruled out.

Fig. 2.

Western blot showing a reduced level of senataxin in two cell lines derived from the case patient (lanes 4 and 5). Lane 1 is a normal positive control; lane 2 is a known classical ataxia-telangiectasia (A-T) patient with absence of ataxia-telangiectasia mutated (ATM) (negative control for ATM); lane 3 is a known ataxia with oculomotor apraxia type 2 (AOA2) patient with a SETX missense mutation. ATLD, ataxia-telangiectasia-like disorder; EDTA, ethylenediaminetetraacetic acid.

However, the levels of senataxin were reduced ( Fig. 2 ) and SETX was therefore sequenced. This revealed two clear SETX mutations ( Fig. 3 ). One of these was a novel mutation, c.2990delG; p.(Cys997PhefsTer32), a deletion causing a frameshift and predicted to result in truncation and loss of expression of senataxin protein from this allele; indeed no smaller protein was observed on western blot. The other, c.6638C > T; p.(Pro2213Leu), was a missense mutation within the helicase domain that has previously been reported in the homozygous state in a Japanese AOA2 patient. ⁴ Our patient is the first case of this mutation documented outside of Japan. The reduced level of senataxin was consistent with the presence of this SETX missense mutation. Together, the clinical presentation, reduced senataxin, and presence of two SETX mutations strongly suggested a diagnosis of AOA2.

Fig. 3.

Electropherograms demonstrating SETX mutations present in the proband. Parts A and B show the two pathogenic SETX mutations; the c.2990delG; p.(Cys997PhefsTer32) single base deletion and consequent frameshift seen to its right ( A ) and the c.6638C > T; p.(Pro2213Leu) missense mutation within the helicase domain ( B ). ( C ) and ( D ) are the two additional SETX sequence changes, c.1807A > G; p.(Asn603Asp) ( C ) and c.1957C > A; p.(Gln653Lys) ( D ) in this patient and likely to be in cis.

Two further sequence changes, c.1807A > G; p.(Asn603Asp) and c.1957C > A; p.(Gln653Lys), were also identified in our patient ( Fig. 3 ). Interestingly, these were described previously in cis in a mother and daughter who both had symptoms of cerebellar atrophy with oculomotor defects and tremor, although not unambiguous AOA2. ⁵

DNA sequencing in one parent showed the presence of the c.2990delG; p.(Cys997PhefsTer32) mutation and not the c.6638C > T; p.(Pro2213Leu), nor the c.1807A > G; p.(Asn603Asp) or c.1957C > A; p.(Gln653Lys). This absence of both additional sequence changes would be consistent with them being in cis in the second parent whose DNA was not available.

Discussion

A variety of mutations of the SETX gene were reported to cause AOA2. The protein encoded by this gene is senataxin, a 2,677 amino acid DNA/RNA repair protein. The majority of these mutations are in the C-terminal DNA/RNA helicase domain from amino acids 1931–2456 (encoded by exons 14–26). It is thought that mutations in this helicase domain result in loss of function of senataxin. Of the remaining mutations known to cause AOA2, most are found in the 466 amino acid N-terminal domain, the other major functional domain of the SETX gene. Only a small number of mutations outside of these two domains have been shown to cause AOA2. ^{1 4}

In this patient, we found two pathogenic mutations of the SETX gene. One is a novel frameshift mutation resulting from a deletion located outside of the N-terminal and C-terminal helicase domains. This mutation is predicted to cause truncation and loss of expression of senataxin from the affected allele. The other is a missense mutation in the helicase domain, previously described in a Japanese AOA2 patient. The reduced level of full length senataxin protein seen on western blot, therefore, likely arises from this mutation.

We do not know with certainty what contribution, if any, the presence of the c.1807A > G; p.(Asn603Asp) and c.1957C > A; p.(Gln653Lys) sequence changes might make to the clinical phenotype. In 2007, these changes were proposed by Bassuk et al as possibly pathogenic in cis in a symptomatic mother and daughter. ^{5 6} In our patient's family, the father was asymptomatic and was shown not to have the two cis changes and the patient's mother's estrangement meant her genetic profile and phenotype are not known.

Subsequent examination of the gnomAD database has since revealed that both the Asn603Asp and Gln653Lys alleles are quite common at 691/143320 alleles (and four homozygotes) and 916/143216 alleles (and seven homozygotes), respectively, compared with 1/251,440 with Pro2213Leu and no reported cases with Cys997PhefsTer32. ⁶ Furthermore, the phenotypes of the family described by Bassuk et al do not resemble typical descriptions of AOA2; the patients lacked peripheral neuropathy, true oculomotor ataxia, and elevated AFP.

Together, this raises the possibility that these alleles may in fact be benign polymorphisms and lead us to conclude that the two bona-fide SETX mutations we have identified, alone, would be a sufficient cause of the typical AOA2 described in our patient.