Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs covalently bound topoisomerase I-DNA complexes and is essential for preventing the formation of double-strand breaks that result when stalled topoisomerase I complexes interfere with DNA replication in yeast. Here we show that a deficiency of this DNA repair pathway in humans does not predispose to neoplasia or dysfunctions in rapidly replicating tissues, but instead causes spinocerebellar ataxia with axonal neuropathy (SCAN1) by affecting large, terminally differentiated, non-dividing neuronal cells. Using genome-wide linkage mapping and a positional candidate approach in a Saudi Arabian family affected with autosomal recessive SCAN1, we identified a homozygous mutation in TDP1 (A1478G) that results in the substitution of histidine 493 with an arginine residue. The His493 residue is conserved in TDP1 across species and is located in the active site of the enzyme. Protein modeling predicts that mutation of this amino acid to arginine will disrupt the symmetric structure of the active site. We propose that loss-of-function mutations in TDP1 may cause SCAN1 either by interfering with DNA transcription or by inducing apoptosis in postmitotic neurons.

Mutations in mitofusin 2 (MFN2) have been reported in Charcot-Marie-Tooth type 2 (CMT2) families. To study the distribution of mutations in MFN2 we screened 323 families and isolated patients with distinct CMT phenotypes. In 29 probands, we identified 22 distinct MFN2 mutations, and 14 of these mutations have not been reported before. All mutations were located in the cytoplasmic domains of the MFN2 protein. Patients presented with a classical but rather severe CMT phenotype, since 28% of them were wheelchair-dependent. Some had additional features as optic atrophy. Most patients had an early onset and severe disease status, whereas a smaller group experienced a later onset and milder disease course. Electrophysiological data showed in the majority of patients normal to slightly reduced nerve conduction velocities with often severely reduced amplitudes of the compound motor and sensory nerve action potentials. Examination of sural nerve specimens showed loss of large myelinated fibres and degenerative mitochondrial changes. In patients with a documented family history of CMT2 the frequency of MFN2 mutations was 33% indicating that MFN2 mutations are a major cause in this population.

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We describe here the results of a search of Mendelian inheritance in man, GENDIAG and other sources which suggest that, in comparison with autosomes 1, 2, 3, 4 and 11, the X chromosome may contain a significantly higher number of sex- and reproduction-related (SRR) genes. A similar comparison between X-linked entries and a subset of randomly chosen entries from the remaining autosomes also indicates an excess of genes on the X chromosome with one or more mutations affecting sex determination (e.g. DAX1), sexual differentiation (e.g. androgen receptor) or reproduction (e.g. POF1). A possible reason for disproportionate occurrence of such genes on the X chromosome could be that, during evolution, the 'choice' of a particular pair of homomorphic chromosomes for specialization as sex chromosomes may be related to the number of such genes initially present in it or, since sex determination and sexual dimorphism are often gene dose-dependent processes, the number of such genes necessary to be regulated in a dose-dependent manner. Further analysis of these data shows that XAR, the region which has been added on to the short arm of the X chromosome subsequent to eutherian-marsupial divergence, has nearly as high a proportion of SRR genes as XCR, the conserved region of the X chromosome. These observations are consistent with current hypotheses on the evolution of sexually antagonistic traits on sex chromosomes and suggest that both XCR and XAR may have accumulated SRR traits relatively rapidly because of X linkage.

Smith-Magenis syndrome (SMS) is a mental retardation/multiple congenital anomalies disorder associated with a heterozygous approximately 4-Mb deletion in 17p11.2. Patients with SMS show variability in clinical phenotype despite a common deletion found in>75-80% of patients. Recently, point mutations in the retinoic acid induced 1 (RAI1) gene, which lies within the SMS critical interval, were identified in three patients with many SMS features in whom no deletion was detected. It is not clear if the entire SMS phenotype can be accounted for by RAI1 haploinsufficiency, nor has the precise function of RAI1 been delineated. We report two novel RAI1 mutations, one frameshift and one nonsense allele, in nondeletion SMS patients. Comparisons of the clinical features in these two patients, three of the previously reported RAI1 point mutation cases, and the patients with a common deletion suggest that the majority of the clinical features in SMS result from RAI1 mutation, although phenotypic variability exists even among the individuals with RAI1 point mutations. Bioinformatics analyses of RAI1 and comparative genomics between human and mouse orthologues revealed a zinc finger-like plant homeo domain (PHD) at the carboxyl terminus that is conserved in the trithorax group of chromatin-based transcription regulators. These findings suggest RAI1 is involved in transcriptional control through a multi-protein complex whose function may be altered in individuals with SMS.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited peripheral neuropathies characterized by progressive weakness and atrophy of distal limb muscles. Recently, SIMPLE/LITAF was shown to be responsible for an autosomal dominant demyelinating form of CMT linked to 16p (CMT1C). Although two transcripts encoding different proteins (SIMPLE and LITAF) have been reported from the same gene, we could not confirm the existence of LITAF. Here we show that the LITAF transcript appears to result from a DNA sequencing error. We screened the SIMPLE gene for mutations in a cohort of 192 patients with CMT or related neuropathies, each of whom tested negative for other known genetic causes of CMT. In 16 unrelated CMT families we identified nine different nucleotide variations in SIMPLE that were not detected in control chromosomes. SIMPLE mutations can occur de novo, associated with sporadic CMT1 and may convey both demyelinating and axonal forms. Bioinformatics analyses and other observations of SIMPLE suggest that 1) it could be a member of the RING finger motif-containing subfamily of E3 ubiquitin ligases that are associated with the ubiquitin-mediated proteasome processing pathway, 2) it could interact through its PPXY motifs with a WW domain containing protein, for instance with NEDD4, an E3 ubiquitin ligase, and 3) it could interact through the PSAP motif with TSG10, a protein associated with endosomal multivesicular protein sorting. Since both SIMPLE and Hrs are endosomal proteins and have both PPXY and P(S/T)AP motifs, we hypothesize that SIMPLE, like Hrs, is potentially a clathrin adaptor aiding in the retention of ubiquitinated proteins on to the endosomes. Thus the potential E3 ubiquitin ligase activity of SIMPLE, alteration in its interactions with NEDD4 or TSG101, or changes in its properties as a clathrin coat adaptor may underlie the pathogenesis of Charcot-Marie-Tooth disease.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation disorder characterized by distinct craniofacial features and neurobehavioral abnormalities usually associated with an interstitial deletion in 17p11.2. Heterozygous point mutations in the retinoic acid induced 1 gene (RAI1) have been reported in nine SMS patients without a deletion detectable by fluorescent in situ hybridization (FISH), implicating RAI1 haploinsufficiency as the cause of the major clinical features in SMS. All of the reported point mutations are unique and de novo. RAI1 contains a polymorphic CAG repeat and encodes a plant homeo domain (PHD) zinc finger-containing transcriptional regulator. We report a novel RAI1 frameshift mutation, c.3103delC, in a non-deletion patient with many SMS features. The deletion of a single cytosine occurs in a heptameric C-tract (CCCCCCC), the longest mononucleotide repeat in the RAI1 coding region. Interestingly, we had previously reported a frameshift mutation, c.3103insC, in the same mononucleotide repeat. Furthermore, all five single base frameshift mutations preferentially occurred in polyC but not polyG tracts. We also investigated the distribution of the polymorphic CAG repeats in both the normal population and the SMS patients as one potential molecular mechanism for variability of clinical expression. In this limited data set, there was no significant association between the length of CAG repeats and the SMS phenotype. However, we identified a 5-year-old girl with an apparent SMS phenotype who was a compound heterozygote for an RAI1 missense mutation inherited from her father and a polyglutamine repeat of 18 copies, representing the largest known CAG repeat in this gene, inherited from her mother.