Vitamin D3 deficiency and osteopenia in spastic paraplegia type 5 indicate impaired bone homeostasis.

Hereditary spastic paraplegia type 5 (SPG5) is an autosomal recessively inherited movement disorder characterized by progressive spastic gait disturbance and afferent ataxia. SPG5 is caused by bi-allelic loss of function mutations in CYP7B1 resulting in accumulation of the oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol in serum and cerebrospinal fluid of SPG5 patients. An effect of 27- hydroxycholesterol via the estrogen and liver X receptors was previously shown on bone homeostasis. This study analyzed bone homeostasis and osteopenia in 14 SPG5 patients as a non-motor feature leading to a potential increased risk for bone fractures. T-Scores in CT bone density measurements were reduced, indicating osteopenia in SPG5 patients. Further, we analyzed various metabolites of bone homeostasis by ELISA in serum samples of these patients. We identified a lack of vitamin D3 metabolites (Calcidiol and Calcitriol), an increase in Sclerostin as a bone formation/mineralization inhibiting factor, and a decrease in cross-linked N-telopeptide of type I collagen (NTX), a marker indicating reduced bone resorption. As statin treatment has been found to lower oxysterol levels, we evaluated its effect in samples of the STOP-SPG5 trial and found atorvastatin to normalize the increased sclerostin levels. In summary, our study identified osteopenia as a non-motor feature in SPG5 and suggests the need for vitamin D3 substitution in SPG5 patients. Sclerostin may be considered a therapeutic target and biomarker in upcoming therapeutical trials in SPG5.

BiP inactivation due to loss of the deAMPylation function of FICD causes a motor neuron disease.

PURPOSE: The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. METHODS: Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. RESULTS: We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. CONCLUSION: Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.

Clinical and molecular characterization of adult patients with late-onset MTHFR deficiency.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

Confirmation of TACO1 as a Leigh Syndrome Disease Gene in Two Additional Families.

BACKGROUND: In 2009, we identified TACO1 as a novel mitochondrial disease gene in a single family, however no second family has been described to confirm the role of TACO1 in mitochondrial disease. OBJECTIVE: In this report, we describe two independent consanguineous families carrying pathogenic variants in TACO1, confirming the phenotype. METHODS: Detailed clinical investigations and whole exome sequencing with haplotype analysis have been performed in several members of the two reported families. RESULTS: Clinical phenotype of the patients confirms the originally reported phenotype of a childhood-onset progressive cerebellar and pyramidal syndrome with optic atrophy and learning difficulties. Brain MRI showed periventricular white matter lesions with multiple cystic defects, suggesting leukoencephalopathy in both patients. One patient carried the previously described homozygous TACO1 variant (p.His158ProfsTer8) and haplotype analysis suggested that this variant is a rare founder mutation. The second patient from another family carried a homozygous novel frame shift variant (p.Cys85PhefsTer15). CONCLUSIONS: The identification of two Turkish families with similar characteristic clinical presentation and an additional homozygous nonsense mutation confirms that TACO1 is a human mitochondrial disease gene. Although most patients with this clinical presentation undergo next generation sequencing analysis, screening for selected founder mutations in the Turkish population based on the precise clinical presentation may reduce time and cost of finding the genetic diagnosis even in the era of massively parallel sequencing.

ATPase Domain AFG3L2 Mutations Alter OPA1 Processing and Cause Optic Neuropathy.

OBJECTIVE: Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed. METHODS: We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts. RESULTS: Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells. INTERPRETATION: This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.

Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein.

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.

Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions.

CNTNAP1 mutations cause CNS hypomyelination and neuropathy with or without arthrogryposis.

OBJECTIVE: To explore the phenotypic spectrum and pathophysiology of human disease deriving from mutations in the CNTNAP1 gene. METHODS: In a field study on consanguineous Palestinian families, we identified 3 patients carrying homozygous mutations in the CNTNAP1 gene using whole-exome sequencing. An unrelated Irish family was detected by screening the GENESIS database for further CNTNAP1 mutations. Neurophysiology, MRI, and nerve biopsy including electron microscopy were performed for deep phenotyping. RESULTS: We identified 3 novel CNTNAP1 mutations in 5 patients from 2 families: c.2015G>A:p.(Trp672*) in a homozygous state in family 1 and c.2011C>T:p.(Gln671*) in a compound heterozygous state with c.2290C>T:p.(Arg764Cys) in family 2. Affected patients suffered from a severe CNS disorder with hypomyelinating leukodystrophy and peripheral neuropathy of sensory-motor type. Arthrogryposis was present in 2 patients but absent in 3 patients. Brain MRI demonstrated severe hypomyelination and secondary cerebral and cerebellar atrophy as well as a mega cisterna magna and corpus callosum hypoplasia. Nerve biopsy revealed very distinct features with lack of transverse bands at the paranodes and widened paranodal junctional gaps. CONCLUSIONS: CNTNAP1 mutations have recently been linked to patients with arthrogryposis multiplex congenita. However, we show that arthrogryposis is not an obligate feature. CNTNAP1-related disorders are foremost severe hypomyelinating disorders of the CNS and the peripheral nervous system. The pathology is partly explained by the involvement of CNTNAP1 in the proper formation and preservation of paranodal junctions and partly by the assumed role of CNTNAP1 as a key regulator in the development of the cerebral cortex.

STUB1/CHIP mutations cause Gordon Holmes syndrome as part of a widespread multisystemic neurodegeneration: evidence from four novel mutations.

BACKGROUND: CHIP, the protein encoded by STUB1, is a central component of cellular protein homeostasis and interacts with several key proteins involved in the pathogenesis of manifold neurodegenerative diseases. This gives rise to the hypothesis that mutations in STUB1 might cause a far more multisystemic neurodegenerative phenotype than the previously reported cerebellar ataxia syndrome. METHODS: Whole exome sequencing data-sets from n = 87 index subjects of two ataxia cohorts were screened for individuals with STUB1 mutations. In-depth phenotyping by clinical evaluation and neuroimaging was performed in mutation carriers. RESULTS: We identified four novel STUB1 mutations in three affected subjects from two index families (frequency 2/87 = 2.3%). All three subjects presented with a severe multisystemic phenotype including severe dementia, spastic tetraparesis, epilepsy, and autonomic dysfunction in addition to cerebellar ataxia, plus hypogonadism in one index patient. Diffusion tensor imaging revealed degeneration of manifold supra- and infratentorial tracts. CONCLUSIONS: Our findings provide clinical and imaging support for the notion that CHIP is a crucial converging point of manifold neurodegenerative processes, corresponding with its universal biological function in neurodegeneration. Further, our data reveal the second STUB1 family with ataxia plus hypogonadism reported so far, demonstrating that Gordon Holmes syndrome is indeed a recurrent manifestation of STUB1. However, it does not present in isolation, but as part of a broad multisystemic neurodegenerative process. This supports the notion that STUB1 disease should be conceptualized not by historical or clinical syndromic names, but as a variable multisystemic disease defined by disturbed function of the underlying STUB1 gene, which translates into a multidimensional gradual spectrum of variably associated clinical signs and symptoms.

Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78).

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

Generation of induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia patient carrying a homozygous R486C mutation in CYP7B1 (SPG5).

Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation R486C in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-R486C was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-R486C line may be a useful resource for disease modelling of SPG5.

SYNE1 ataxia is a common recessive ataxia with major non-cerebellar features: a large multi-centre study.

Mutations in the synaptic nuclear envelope protein 1 (SYNE1) gene have been reported to cause a relatively pure, slowly progressive cerebellar recessive ataxia mostly identified in Quebec, Canada. Combining next-generation sequencing techniques and deep-phenotyping (clinics, magnetic resonance imaging, positron emission tomography, muscle histology), we here established the frequency, phenotypic spectrum and genetic spectrum of SYNE1 in a screening of 434 non-Canadian index patients from seven centres across Europe. Patients were screened by whole-exome sequencing or targeted panel sequencing, yielding 23 unrelated families with recessive truncating SYNE1 mutations (23/434 = 5.3%). In these families, 35 different mutations were identified, 34 of them not previously linked to human disease. While only 5/26 patients (19%) showed the classical SYNE1 phenotype of mildly progressive pure cerebellar ataxia, 21/26 (81%) exhibited additional complicating features, including motor neuron features in 15/26 (58%). In three patients, respiratory dysfunction was part of an early-onset multisystemic neuromuscular phenotype with mental retardation, leading to premature death at age 36 years in one of them. Positron emission tomography imaging confirmed hypometabolism in extra-cerebellar regions such as the brainstem. Muscle biopsy reliably showed severely reduced or absent SYNE1 staining, indicating its potential use as a non-genetic indicator for underlying SYNE1 mutations. Our findings, which present the largest systematic series of SYNE1 patients and mutations outside Canada, revise the view that SYNE1 ataxia causes mainly a relatively pure cerebellar recessive ataxia and that it is largely limited to Quebec. Instead, complex phenotypes with a wide range of extra-cerebellar neurological and non-neurological dysfunctions are frequent, including in particular motor neuron and brainstem dysfunction. The disease course in this multisystemic neurodegenerative disease can be fatal, including premature death due to respiratory dysfunction. With a relative frequency of ∼5%, SYNE1 is one of the more common recessive ataxias worldwide.

Lack of enzyme activity in GBA2 mutants associated with hereditary spastic paraplegia/cerebellar ataxia (SPG46).

Glucosylceramide is a membrane glycolipid made up of the sphingolipid ceramide and glucose, and has a wide intracellular distribution. Glucosylceramide is degraded to ceramide and glucose by distinct, non-homologous enzymes, including glucocerebrosidase (GBA), localized in the endolysosomal pathway, and ß-glucosidase 2 (GBA2), which is associated with the plasma membrane and/or the endoplasmic reticulum. It is well established that mutations in the GBA gene result in endolysosomal glucosylceramide accumulation, which triggers Gaucher disease. In contrast, the biological significance of GBA2 is less well understood. GBA2-deficient mice present with male infertility, but humans carrying mutations in the GBA2 gene are affected with a combination of cerebellar ataxia and spastic paraplegia, as well as with thin corpus callosum and cognitive impairment (SPastic Gait locus #46, SPG46). To improve our understanding of the biochemical consequences of the GBA2 mutations, we have evaluated five nonsense and five missense GBA2 mutants for their enzyme activity. In transfected cells, the mutant forms of GBA2 were present in widely different amounts, ranging from overabundant to very minor, compared to the wild type enzyme. Nevertheless, none of the GBA2 mutant cDNAs raised the enzyme activity in transfected cells, in contrast to the wild-type enzyme. These results suggest that SPG46 patients have a severe deficit in GBA2 activity, because the GBA2 mutants are intrinsically inactive and/or reduced in amount. This assessment of the expression levels and enzyme activities of mutant forms of GBA2 offers a first insight in the biochemical basis of the complex pathologies seen in SPG46.

First de novo KCND3 mutation causes severe Kv4.3 channel dysfunction leading to early onset cerebellar ataxia, intellectual disability, oral apraxia and epilepsy.

BACKGROUND: Identification of the first de novo mutation in potassium voltage-gated channel, shal-related subfamily, member 3 (KCND3) in a patient with complex early onset cerebellar ataxia in order to expand the genetic and phenotypic spectrum. METHODS: Whole exome sequencing in a cerebellar ataxia patient and subsequent immunocytochemistry, immunoblotting and patch clamp assays of the channel were performed. RESULTS: A de novo KCND3 mutation (c.877_885dupCGCGTCTTC; p.Arg293_Phe295dup) was found duplicating the RVF motif and thereby adding an extra positive charge to voltage-gated potassium 4.3 (Kv4.3) in the voltage-sensor domain causing a severe shift of the voltage-dependence gating to more depolarized voltages. The patient displayed a severe phenotype with early onset cerebellar ataxia complicated by intellectual disability, epilepsy, attention deficit hyperactivity disorder, strabismus, oral apraxia and joint hyperlaxity. CONCLUSIONS: We identified a de novo KCND3 mutation causing the most marked change in Kv4.3's channel properties reported so far, which correlated with a severe and unique spinocerebellar ataxia (SCA) type 19/22 disease phenotype.

Cholestenoic acids regulate motor neuron survival via liver X receptors.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3ß,7α-dihydroxycholest-5-en-26-oic acid (3ß,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3ß-hydroxy-7-oxocholest-5-en-26-oic acid (3ßH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3ß,7α-diHCA and 3ßH,7O-CA, 3ß-hydroxycholest-5-en-26-oic acid (3ß-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3ß-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3ß,7α-diHCA. Moreover, 3ß,7α-diHCA prevented the loss of motor neurons induced by 3ß-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Motor protein mutations cause a new form of hereditary spastic paraplegia.

OBJECTIVE: To identify a novel disease gene in 2 families with autosomal recessive hereditary spastic paraplegia (HSP). METHODS: We used whole-exome sequencing to identify the underlying genetic disease cause in 2 families with apparently autosomal recessive spastic paraplegia. Endogenous expression as well as subcellular localization of wild-type and mutant protein were studied to support the pathogenicity of the identified mutations. RESULTS: In 2 families, we identified compound heterozygous or homozygous mutations in the kinesin gene KIF1C to cause hereditary spastic paraplegia type 58 (SPG58). SPG58 can be complicated by cervical dystonia and cerebellar ataxia. The same mutations in a heterozygous state result in a mild or subclinical phenotype. KIF1C mutations in SPG58 affect the domains involved in adenosine triphosphate hydrolysis and microtubule binding, key functions for this microtubule-based motor protein. CONCLUSIONS: KIF1C is the third kinesin gene involved in the pathogenesis of HSPs and is characterized by a mild dominant and a more severe recessive disease phenotype. The identification of KIF1C as an HSP disease gene further supports the key role of intracellular trafficking processes in the pathogenesis of hereditary axonopathies.

The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation.

We previously localized a new form of recessive ataxia with generalized tonic-clonic epilepsy and mental retardation to a 19 Mb interval in 16q21-q23 by homozygosity mapping of a large consanguineous Saudi Arabian family. We now report the identification by whole exome sequencing of the missense mutation changing proline 47 into threonine in the first WW domain of the WW domain containing oxidoreductase gene, WWOX, located in the linkage interval. Proline 47 is a highly conserved residue that is part of the WW motif consensus sequence and is part of the hydrophobic core that stabilizes the WW fold. We demonstrate that proline 47 is a key amino acid essential for maintaining the WWOX protein fully functional, with its mutation into a threonine resulting in a loss of peptide interaction for the first WW domain. We also identified another highly conserved homozygous WWOX mutation changing glycine 372 to arginine in a second consanguineous family. The phenotype closely resembled the index family, presenting with generalized tonic-clonic epilepsy, mental retardation and ataxia, but also included prominent upper motor neuron disease. Moreover, we observed that the short-lived Wwox knock-out mouse display spontaneous and audiogenic seizures, a phenotype previously observed in the spontaneous Wwox mutant rat presenting with ataxia and epilepsy, indicating that homozygous WWOX mutations in different species causes cerebellar ataxia associated with epilepsy.

PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.

Boucher-Neuhäuser and Gordon Holmes syndromes are clinical syndromes defined by early-onset ataxia and hypogonadism plus chorioretinal dystrophy (Boucher-Neuhäuser syndrome) or brisk reflexes (Gordon Holmes syndrome). Here we uncover the genetic basis of these two syndromes, demonstrating that both clinically distinct entities are allelic for recessive mutations in the gene PNPLA6. In five of seven Boucher-Neuhäuser syndrome/Gordon Holmes syndrome families, we identified nine rare conserved and damaging mutations by applying whole exome sequencing. Further, by dissecting the complex clinical presentation of Boucher-Neuhäuser syndrome and Gordon Holmes syndrome into its neurological system components, we set out to analyse an additional 538 exomes from families with ataxia (with and without hypogonadism), pure and complex hereditary spastic paraplegia, and Charcot-Marie-Tooth disease type 2. We identified four additional PNPLA6 mutations in spastic ataxia and hereditary spastic paraplegia families, revealing that Boucher-Neuhäuser and Gordon Holmes syndromes in fact represent phenotypic clusters on a spectrum of neurodegenerative diseases caused by mutations in PNPLA6. Structural analysis indicates that the majority of mutations falls in the C-terminal phospholipid esterase domain and likely inhibits the catalytic activity of PNPLA6, which provides the precursor for biosynthesis of the neurotransmitter acetylcholine. Our findings show that PNPLA6 influences a manifold of neuronal systems, from the retina to the cerebellum, upper and lower motor neurons and the neuroendocrine system, with damage of this protein causing an extraordinarily broad continuous spectrum of associated neurodegenerative disease.