450 using an RNeasy Mini Kit (Qiagen) per the manufacturer’s instructions. Reverse transcription (RT) was performed using total RNA primed with oligo-dT in the SuperScript III First-Strand Synthesis System (Invitrogen). Polymerase chain reaction (PCR) was performed using a high-fidelity proofreading polymerase (Accuprime Pfx, Invitrogen) and approximately 10–50 ng of cDNA. PCR cycle settings were 94°C for 30 s, 55°C for 30 s, and 72°C for 30–45 s for a total of 35–40 cycles. For nested PCR, dilutions of the initial PCR reaction (ranging from 0.08% to 6%) were used in a second reaction with primers internal to the first set. All PCR products were confirmed by sequencing as above. All primer sequences are available on request. Gel images were captured using a BioSpectrum AC Imaging System (UVP). Exon numbering is based on the 26 exons known to be expressed by the SETX gene (NM_015046). The SETX coding region is represented by exons 3–26 (with exons 14–26 encoding the DNA/RNA helicase domain). The novel exon reported here is designated exon 25b to match with current exon numbering. Results Sequencing of intron 16 in this patient identified a homozygous insertion of one base pair, thymidine (T), at position +2 of intron 16 (Fig. 1). The patient’s mother was found to be heterozygous for this mutation (data not shown). The father was not available for testing so we cannot determine if the second mutation was inherited (as predicted by the pedigree) or arose de novo. As senataxin is expressed in lymphoblasts, we were able to investigate the functional consequences of this mutation, which we hypothesized would disrupt normal RNA splicing. Total RNA from the proband’s mother was converted to cDNA for use in nested RT-PCR with primers spanning exons 15 and 17 of senataxin. This demonstrated the presence of the expected band representing exons 15–17 as well an additional band corresponding to the skipping of exon 16 (Fig. 2). In the patient, only the misspliced product was detected (Fig. 2). The new protein product generated from this splicing event is predicted to lack a functional DNA/RNA helicase domain. Previously, senataxin has not been thought to undergo significant alternative splicing [9]. However, review of the Alternative Splicing and Transcript Diversity database [18] identifies a total of four additional human senataxin transcripts supported by expressed sequence tag (EST) data which include the DNA/RNA helicase domain (data not shown), and therefore may potentially have functional significance (see “Discussion”). The largest alternate transcript includes an additional 3′ exon (which we term exon 25b) which adds 29 amino acids at the C-terminal Cerebellum (2009) 8:448–453 portion of the DNA/RNA helicase domain after position 2429 (Fig. 3). The remaining isoforms are smaller due to utilization of alternative transcription start sites (in exon 10) and/or alternative splicing involving exon 25b or exon 24 (which removes 33 amino acids in the DNA/RNA helicase domain after position 2366; Fig. 3). We find support for these splicing patterns from multiple unique ESTs (Fig. 3). Furthermore, using RT-PCR, we detected transcripts fea- turing the alternative use of both exon 25b and exon 24 in both human fetal and adult brain (Fig. 3). Discussion Senataxin is a large 2,677 amino acid protein whose function still remains relatively unknown. Although multiple regions of the protein appear to be important for disease, an attractive target is the conserved C-terminal DNA/RNA helicase domain found at amino acids 1931–2456 (encoded by exons 14–26) [1, 8, 12]. Mutations that alter or disrupt this domain cause AOA2, presumably through a loss of function [1, 3, 8, 9, 12]. Recent studies strengthen the relevance of the DNA/RNA helicase domain by providing evidence for a role of senataxin in DNA damage response [17] and potential roles in transcription and RNA process- ing based on work with the yeast ortholog [19]. In this report, we identified a novel intronic mutation resulting in aberrant splicing of the SETX mRNA in a patient with clinical AOA2. Three previous mutations that affect splicing of the senataxin pre-mRNA have been reported [5, 7, 16, 17] (Fig. 2). One of these results from a G>A transition at c.6106, the last nucleotide of exon 15, resulting in skipping of the exon [16], with a frameshift mutation and truncation at amino acid 1989. The other two mutations are intronic—one, IVS25+5G>A, results in skip- ping of exon 25 leading to a frameshift [5, 17] with protein truncation at amino acid 2408, and another, IVS19+5G>T, which skips exon 19, resulting in an in-frame deletion of amino acids 2133–2182 [7]. The mutation reported here, IVS16+2T, results in skipping of exon 16 (Fig. 2). The mechanism of this mutation is novel relative to the other known SETX splicing mutations, as the inserted T at position +2 induces an intronic frameshift in the neighbor- hood of the intron 16 5′ splice site sequence which would be predicted to dramatically reduce splice site utilization [20]. The resultant skipping of exon 16 identified in Fig. 2 would result in the in-frame replacement of amino acids 2036–2070 with a glutamic acid residue. Interestingly, all these RNA processing mutations affect the DNA/RNA helicase domain, further implicating the importance of this region in AOA2 pathogenesis [12]. Additionally, it is also of considerable interest that despite the wide variety of mutations seen in AOA2, only