Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia. We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP). Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.We previously identified a 16-cM interval on chromosome 9q34 associated with an autosomal recessive adolescent-onset cerebellar ataxia segregating in two families 1,2 , one with additional oculomotor apraxia 1 and the second with associated elevated serum AFP, immunoglobulins and creatine kinase levels but no oculomotor apraxia 2,3 . We identified nine additional families with ataxia linked to 9q34 by homozygosity mapping (Supplementary Methods online). As most affected individuals had both oculomotor apraxia and elevated AFP levels we assumed that they were affected by the same disorder, which we named AOA2 (OMIM 606002). We identified distal and proximal recombinations in families with two affected individuals (Fig. 1a), localizing the defective gene underlying AOA2 to a 1.1-Mb interval containing 13 genes ( Fig. 1b) and three groups of overlapping spliced expressed-sequence tags, which we analyzed for nucleotide changes but found no mutations. We also found that the unspliced mRNA AK024331 overlaps with the KIAA0625 cDNA and is part of a larger transcript overlapping with additional exons on the 5′ side. We obtained an open reading frame of 8,031 nucleotides and 24 exons (Fig. 1c), of which exon 8 was 4,177 nucleotides long. We confirmed the prediction and size of the transcript by long-range RT-PCR experiments spanning the putative exon 1 and 3′ untranslated region in human fibroblast and lymphoblastoid cell lines (data not shown) and by hybridization of a human northern blot with a probe spanning putative exons 8-24 (Fig. 1d). We also identified an alternative transcript that is 2.4 kb longer, resulting from a second polyadenylation site (human mRNAs AB014525 and AK022902; Fig. 1d).We sequenced exons 1-18 and flanking intronic sequences in families with ataxia linked to this region and in additional individuals with either AOA or ataxia with elevated AFP levels and found 15 different disease-associated mutations in 15 families ( Table 1). Ten of these mutations, including mutations in the two families in whom we first identified AOA2, cause truncation of the protein, indicating that this is the gene underlying AOA2. We found the nonsense mutation R1363X in three unrelated families originating from Portugal, Cabo Verde (once a Portuguese colony) and Spain, suggestive of an Iberian founder event, although recurrent C→T changes on this CpG dinucleotide cannot be formally excluded. Absence of the five missense mutations in 150 unrelated and unaffected individuals sharing the same ethnic origin as the affected in...

Autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) is a common and clinically distinct form of familial spastic paraplegia that is linked to the SPG11 locus on chromosome 15 in most affected families. We analyzed 12 ARHSP-TCC families, refined the SPG11 candidate interval and identified ten mutations in a previously unidentified gene expressed ubiquitously in the nervous system but most prominently in the cerebellum, cerebral cortex, hippocampus and pineal gland. The mutations were either nonsense or insertions and deletions leading to a frameshift, suggesting a loss-of-function mechanism. The identification of the function of the gene will provide insight into the mechanisms leading to the degeneration of the corticospinal tract and other brain structures in this frequent form of ARHSP.

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.

Background: Autosomal dominant cerebellar ataxias comprise a clinically, neuropathologically, and genetically heterogeneous group of neurodegenerative disorders. The vast majority of cases are caused by trinucleotide or pentanucleotide repeat expansions in 9 different genes. Spinocerebellar ataxia type 14 (SCA14) is a relatively pure form of autosomal dominant cerebellar ataxia mapped to chromosome 19q and caused by missense mutations in the gene encoding protein kinase C ␥ (PRKCG), which are all located in the regulatory domain.Objectives: To identify new SCA14 families and to describe the associated phenotype. Methods:We describe a new SCA14 family of French ancestry with 14 patients and 4 probably affected individuals. Linkage to the SCA14 locus was evaluated according to standard procedures using 5 markers covering the SCA14 candidate interval. All 18 exons of the PRKCG gene and splice junctions were screened with direct sequencing in the index patient.Results: Linkage to the SCA14 locus was established with lod scores greater than 3 in the interval between DNA segments D19S571 and D19S926. Direct sequencing of the PRKCG gene revealed a T-to-C transition in exon 18 respon-

Autosomal dominant cerebellar ataxias (ADCA) are a heterogeneous group of neurological disorders. Point mutations in the gene encoding protein kinase Cgamma (PRKCG) are responsible for spinocerebellar ataxia 14 (SCA14). We screened for mutations in the PRKCG gene, in a large series of 284 ADCA index cases, mostly French (n=204) and German (n=48), in whom CAG repeat expansions in the known SCA genes were previously excluded. Six mutations were found that segregated with the disease and were not detected on 560 control chromosomes, including F643L (exon 18), already reported in another French kindred. Five new missense mutations were identified in exons 4 (C114Y/G123R/G123E), 10 (G360S) and 18 (V692G). All but one (V692G) were located in highly conserved regions of the regulatory or catalytic domains of the protein. All six SCA14 families were French and there was no evidence of reduced penetrance. The phenotype consisted in a very slowly progressive cerebellar ataxia with a mean age at onset of 33.5+/-14.2 years (range 15 to 60 years), occasionally associated with executive dysfunction, myoclonus, myorythmia, tremor or decreased vibration sense. SCA14 represented only 1.5% (7/454) of French ADCA families but none of the German families. It should, however, be considered in patients with slowly progressive ADCA, particularly when myoclonus and cognitive impairment are present.

Kinesin genes encode a family of cargo/motor proteins and are known to cause HSP if mutated. Here we identified nonsense and missense mutations in a further member of this protein family. The KIF1C mutation is associated with a HSP subtype (SPAX2/SAX2) that combines spastic paraplegia and weakness with cerebellar dysfunction.

Autosomal dominant cerebellar ataxias constitute one of the most clinically, neuropathologically, and genetically heterogeneous groups of neurodegenerative disorders. Approximately 50 to 80% of the families carry mutations in genes known to be implicated in spinocerebellar ataxias (SCAs). Numerous loci (SCAn) also have been mapped, often in single families, but the responsible genes have not yet been identified. This suggests further genetic heterogeneity. We have ascertained 18 subjects from a large French family in which cerebellar ataxia and prominent sensory neuropathy segregated as a dominant trait. Intrafamilial variability was high regarding age at onset (17 months to 39 years), severity, and the clinical picture that ranged from pure sensory neuropathy with little cerebellar involvement to a Friedreich's ataxia-like phenotype. After excluding known genes/loci responsible for SCA and hereditary sensory neuropathies, we detected linkage with chromosome 2p markers in a genomewide screen. We designated this new locus SCA25 after testing of 16 additional markers. Maximum two-point logarithm of odds scores of 3.15 and 3.10 were obtained at D2S2378 and D2S2734, respectively. Haplotype analysis defined a critical 12.6cM region of 15Mb between D2S2174 and D2S2736. No linkage to this locus was found in four other families. This interval contains several genes that could be responsible for the disease. One of these genes, CRIPT, encodes a postsynaptic protein, but no mutations were found by direct sequencing, excluding its responsibility in the disease. CAG repeat expansions often are involved in SCA pathogenesis, but no pathological expansions were found at the protein or at the DNA level using the 1C2 antibody and the repeat expansion detection method, respectively. The gene responsible for SCA25 remains to be identified.