Cerebellum (2009) 8:448–453 Fig. 2. Aberrant splicing of senataxin in patients with AOA2. a Total peripheral blood RNA from the proband (P) and her mother (M) as well as an unrelated normal control individual (C) was used for RT-PCR with primers amplifying across exons 15 and 17 (black arrows) followed by internal nested primers (gray arrows). Exons are shown in genomic context with intronic sequences indicated by dashed lines. The detected spliced products (shown) either include or exclude exon 16 (291 or 189 base pairs, respectively). HPRT was amplified as a positive control (489 base pair product). Size markers are indicated in base pairs. The predicted effect on senataxin protein is shown graphically with the DNA/RNA helicase domain (aa 1931–2456) indicated by a black line and an in-frame deletion shown by the dashed segment. b Previously published senataxin mutations which affect RNA splicing are shown with the corresponding mutation (arrow) in the genomic context. The abnormal splicing event is indicated by boxes, and the resulting effect on the protein is shown graphically as in part a. Protein truncation is shown by a dashed line 451 a few distinct heterozygous missense mutations have thus far been identified in motor neuron disease [8–10]. This observation would be consistent with the proposed gain-of- function hypothesis in ALS4. It is worth noting that one of the SETX splicing mutations seen in AOA2, IVS19+5 G>T [7], removes residue 2136 which has been detected as mutated in cases of ALS4 (R2136H) [8, 9]. As the patients clinically have AOA2 [7], this demonstrates that deletion of this residue is insufficient to produce a motor neuron phenotype even when homozygous, suggesting that an intact DNA/RNA helicase domain is required for the development of ALS4. In addition to AOA2, the patient reported here also carries a diagnosis of polycystic ovarian syndrome. This may be relevant, as early menopause and premature ovarian failure have been reported in other cases of AOA2 [2, 5, 13], suggesting a germ line effect, at least in affected women. While the genetic risk for polycystic ovarian syndrome is complex [21], given the above findings, it is interesting to speculate that senataxin mutations (or perhaps a specific subset thereof) may alter the risk of this associated condition in affected women. Finally, we have demonstrated the presence of alterna- tively spliced senataxin transcripts in human fetal and adult brain. It is of considerable interest that the variability in these transcripts resides in the C-terminal portion of DNA/ RNA helicase domain. The role of these (and potentially other) isoforms of senataxin in endogenous function and in the pathogenesis of AOA2 and ALS4 is an important new area of study. In conjunction with the known patient data,