A novel c.5308_5311delGAGA mutation in Senataxin in a Cypriot family with an autosomal recessive cerebellar ataxia

Autosomal recessive cerebellar ataxias (ARCA) form a group of heterogeneous disorders characterised by autosomal recessive inheritance, cerebellar ataxia and an early onset of disease, usually before the age of 20 [1]. The so called Ataxia with Oculomotor Apraxia type 2 was recently found as the second most frequent ARCA, after Freidreich's Ataxia in a group of 77 families, most of them (69%) of French origin [2]. AOA2 has been mapped to chromosome 9q34 [3, 4] and more recently mutations in the senataxin (SETX) gene were identified in AOA2 patients [5]. Senataxin contains a domain found in superfamily 1 of helicases and is the human ortholog of yeast Sen1p gene [5]. Autosomal dominant mutations in the same gene occur in a form of juvenile amyotrophic lateral sclerosis (ALS4) [6].

In the late 1980s a cluster of 7 Friedreich's Ataxia families with 13 affected members was identified in the Cypriot population and the highest estimated carrier frequency of 1-in-6 to 1-in-7 was reported for two neighbouring villages in the western part of the island [7]. Since then 16 additional FRDA patients, 13 non-FRDA ARCA families, 1 ADSCA family and 37 sporadic cerebellar ataxia patients were ascertained in the Cypriot population.

Initial linkage studies of the non-FRDA ARCA families showed linkage of family 909 to the senataxin locus on chromosome 9q34. We hereby report the identification of the second ARCA mutation in the Cypriot population, a novel SETX deletion. This mutation has not been identified in 204 normal chromosomes and it has not been detected in any of the remaining non-FRDA ARCA families or sporadic cerebellar ataxia patients in our population.

We then performed sequence analysis of the proband (II-2) DNA sample for the 26 SETX exons and detected a novel homozygous SETX gene deletion c.5308-5311delGAGA (Figure 3). This 4 bp deletion causes a frame shift mutation that results in a Glu-to-Ile change at amino acid 1770 of the SETX gene and also introduces a stop codon 15 amino acids further down the new reading frame (p.Glu1770IlefsX15). We designed a PCR and agarose gel electrophoresis based assay of this mutation in order to further investigate the remaining affected and non-affected members of the family and other additional Cypriot samples. The c.5308-5311delGAGA mutation co-segregates with the disease haplotype (5-2-2-1-1) of the family and is found in a homozygous state in all four affected individuals (Figure 1). It was not detected in any of 204 normal Cypriot population chromosomes analysed, thus further supporting the hypothesis that this is the disease causing mutation in this Cypriot ARCA family.

The above novel SETX mutation is the second ARCA mutation, after the FRDA GAA repeat expansion, thus far identified in the Cypriot population. Therefore, we used the PCR based assay to investigate whether any of the remaining molecularly undiagnosed Cypriot cerebellar ataxia patients have this newly identified Cypriot ARCA mutation. The proband of 12 ARCA families and 37 sporadic SCA patients, previously excluded for the FRDA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17 and DRPLA mutations, were analysed for this novel SETX mutation. The mutation was not detected in any of the above patients, thus indicating that this is a rare ARCA mutation in our population.

We report a novel SETX gene mutation in a Cypriot ARCA family. The clinical phenotype of the reported patients is similar to other reported SETX mutation patients. Overall the clinical presentation of the affected family members included an age at onset of 8 to 14 years old, slow progressive ataxia of limb and trunk and eye movements with multidirectional nystagmus, peripheral neuropathy (clinically), spasticity, Babinski sign and areflexia. No significant intrafamilial clinical variability was observed although one patient (II-3) had a slightly milder form of the disease. AFP levels were measured in 3 of the 5 patients (Table 1) and all were highly elevated, as characteristically found in AOA2 patients.

Senataxin encodes a ubiquitously expressed protein of 2,677 amino acids that shares similarities with the yeast Sen1 proteins mainly at the carboxy-terminus conserved superfamily I DNA/RNA helicase domain spanning amino acids 1,931 to 2,456 [5, 6]. Although the function of senataxin is poorly understood, it is speculated that, like the DNA/RNA helicases, it is involved in DNA repair, replication, recombination, transcription, RNA processing, transcript stability and translation initiation. Evidence that SETX plays a role in the defence against oxidative DNA damage has recently been reported [11]. In human cultured cells SETX was found as expected in the nucleus, but also diffused in the cytoplasm, thus indicating additional functions of the protein beyond those of the yeast Sen1p [12]. SETX gene mutations were identified in autosomal recessive AOA2 [5, 13–17], autosomal dominant ALS4 [6] and more recently in autosomal dominant tremor/ataxia syndrome [18]. Missense mutations have been identified both in the C-terminus conserved helicase domain and the N-terminal part of the SETX protein, thus indicating that other than the helicase regions of the protein are also important for the pathogenesis of AOA2.

The homozygous c.5308-5311delGAGA mutation is a frame shift deletion that results in a Glu-to-Ile change at amino acid 1770 of the SETX gene and also introduces a stop codon 15 amino acids further down the new reading frame (p.Glu1770IlefsX15). It is likely that this mutation most probably results in a truncated protein lacking the c-terminal helicase domain of the SETX protein. This novel mutation adds to 30 already known SETX gene mutations causing AOA2, most of them reported by Fogel et al [16]. Identification of additional AOA2 mutations may contribute towards conclusive genotype/phenotype correlations.

We hereby describe the second ARCA mutation thus far identified in the Cypriot population after the FRDA GAA repeat expansion. Absence of this novel mutation from 12 Cypriot ARCA families and 37 sporadic cerebellar ataxia patients pending molecular diagnosis indicates that further genetic heterogeneity of cerebellar ataxia exists in the Cypriot population. The Cypriot population under study was 766,400 at the 2005 census; therefore, the frequency of patients with this novel SETX mutation is estimated to be 1 in approximately 150,000 of the population. Although the family excluded any known relation between the parents, a common ancestor or founder effect is more likely.

We identified the second ARCA mutation in the Cypriot population, after the FRDA GAA repeat expansion, a novel SETX homozygous c.5308_5311delGAGA mutation that co-segregates with the ataxia phenotype in a Cypriot ARCA family with 5 affected individuals.