Autosomal recessive cerebellar ataxias

Autosomal recessive cerebellar ataxias (ARCA) are a heterogeneous group of rare neurological disorders involving both central and peripheral nervous system, and in some case other systems and organs, and characterized by degeneration or abnormal development of cerebellum and spinal cord, autosomal recessive inheritance and, in most cases, early onset occurring before the age of 20 years. This group encompasses a large number of rare diseases, the most frequent in Caucasian population being Friedreich ataxia (estimated prevalence 2–4/100,000), ataxia-telangiectasia (1–2.5/100,000) and early onset cerebellar ataxia with retained tendon reflexes (1/100,000). Other forms ARCA are much less common. Based on clinicogenetic criteria, five main types ARCA can be distinguished: congenital ataxias (developmental disorder), ataxias associated with metabolic disorders, ataxias with a DNA repair defect, degenerative ataxias, and ataxia associated with other features. These diseases are due to mutations in specific genes, some of which have been identified, such as frataxin in Friedreich ataxia, α-tocopherol transfer protein in ataxia with vitamin E deficiency (AVED), aprataxin in ataxia with oculomotor apraxia (AOA1), and senataxin in ataxia with oculomotor apraxia (AOA2). Clinical diagnosis is confirmed by ancillary tests such as neuroimaging (magnetic resonance imaging, scanning), electrophysiological examination, and mutation analysis when the causative gene is identified. Correct clinical and genetic diagnosis is important for appropriate genetic counseling and prognosis and, in some instances, pharmacological treatment. Due to autosomal recessive inheritance, previous familial history of affected individuals is unlikely. For most ARCA there is no specific drug treatment except for coenzyme Q10 deficiency and abetalipoproteinemia.     

Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene

Objective

To expand the spectrum of genetic causes of autosomal recessive cerebellar ataxia (ARCA).

Case report

Two brothers are described who developed progressive cerebellar ataxia at 3 1/2 and 18 years, respectively. After ruling out known common genetic causes of ARCA, analysis of blood peroxisomal markers strongly suggested a peroxisomal biogenesis disorder. Sequencing of candidate PEX genes revealed a homozygous c.865_866insA mutation in the PEX2 gene leading to a frameshift 17 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders).

Conclusions

Genetic screening of PEX2 and other PEX genes involved in peroxisomal biogenesis is warranted in children and adults with ARCA.     

The human synaptotagmin IV gene defines an evolutionary break point between syntenic mouse and human chromosome regions but retains ligand inducibility and tissue specificity

Rat synaptotagmin IV (SYT IV) is a depolarization-inducible synaptic vesicle protein. SYT IV homozygous mutant mice are viable and have deficits in fine motor coordination and some forms of memory. In this study, we report the identification of a human SYT IV orthologue. The predicted amino acid sequence of the human SYT IV clone is nearly 90% identical to the rat and mouse SYT IV proteins. In addition, human SYT IV has a characteristic serine for aspartate substitution within the first C2 domain that is conserved among Drosophila, Caenorhabditis elegans, mouse, and rat SYT IV sequences. The human SYT IV gene maps to chromosome band 18q12.3, a region that defines a break point in the synteny with mouse chromosome 18 and has been implicated by associated markers in two human psychiatric disorders. In the human neuroblastoma cell line SK-N-SH, SYT IV is an immediate-early gene inducible by elevated intracellular calcium and by forskolin, an activator of adenylyl cyclase. Expression of human SYT IV mRNA is restricted to brain and is not detectable in non-neuronal tissues. Within brain, human SYT IV mRNA is most highly expressed in hippocampus, with lower levels present in amygdala and thalamus. These results suggest a role for SYT IV in human brain function and in human neurological disease.     

Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight

IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ∼25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.Key words: epigenetic, DNA methylation, genomic imprinting, cerebellum, IGF2, H19, brain, expression, frontal cortex, genetic, single nucleotide polymorphism     

A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia [corrected

Hereditary spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders for which >/=14 different genetic loci have been identified. In some SCA types, expanded tri- or pentanucleotide repeats have been identified, and the length of these expansions correlates with the age at onset and with the severity of the clinical phenotype. In several other SCA types, no genetic defect has yet been identified. We describe a large, three-generation family with early-onset tremor, dyskinesia, and slowly progressive cerebellar ataxia, not associated with any of the known SCA loci, and a mutation in the fibroblast growth factor 14 (FGF14) gene on chromosome 13q34. Our observations are in accordance with the occurrence of ataxia and paroxysmal dyskinesia in Fgf14-knockout mice. As indicated by protein modeling, the amino acid change from phenylalanine to serine at position 145 is predicted to reduce the stability of the protein. The present FGF14 mutation represents a novel gene defect involved in the neurodegeneration of cerebellum and basal ganglia.     

Brain Region–Specific Alterations in the Gene Expression of Cytokines,Immune Cell Markers and Cholinergic System Components during Peripheral Endotoxin–Induced Inflammation

Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune–brain communication, including the impact of peripheral inflammation on brain region–specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region–specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches.     

Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders

Joubert syndrome–related disorders (JSRDs) are a group of clinically and genetically heterogeneous conditions that share a midbrain-hindbrain malformation, the molar tooth sign (MTS) visible on brain imaging, with variable neurological, ocular, and renal manifestations. Mutations in the CEP290 gene were recently identified in families with the MTS-related neurological features, many of which showed oculo-renal involvement typical of Senior-Löken syndrome (JSRD-SLS phenotype). Here, we performed comprehensive CEP290-mutation analysis on two nonoverlapping cohorts of JSRD-affected patients with a proven MTS. We identified mutations in 19 of 44 patients with JSRD-SLS. The second cohort consisted of 84 patients representing the spectrum of other JSRD subtypes, with mutations identified in only two patients. The data suggest that CEP290 mutations are frequently encountered and are largely specific to the JSRD-SLS subtype. One patient with mutation displayed complete situs inversus, confirming the clinical and genetic overlap between JSRDs and other ciliopathies.     

Comparative analysis reveals distinctive epigenetic features of the human cerebellum

Identifying the molecular underpinnings of the neural specializations that underlie human cognitive and behavioral traits has long been of considerable interest. Much research on human-specific changes in gene expression and epigenetic marks has focused on the prefrontal cortex, a brain structure distinguished by its role in executive functions. The cerebellum shows expansion in great apes and is gaining increasing attention for its role in motor skills and cognitive processing, including language. However, relatively few molecular studies of the cerebellum in a comparative evolutionary context have been conducted. Here, we identify human-specific methylation in the lateral cerebellum relative to the dorsolateral prefrontal cortex, in a comparative study with chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta). Specifically, we profiled genome-wide methylation levels in the three species for each of the two brain structures and identified human-specific differentially methylated genomic regions unique to each structure. We further identified which differentially methylated regions (DMRs) overlap likely regulatory elements and determined whether associated genes show corresponding species differences in gene expression. We found greater human-specific methylation in the cerebellum than the dorsolateral prefrontal cortex, with differentially methylated regions overlapping genes involved in several conditions or processes relevant to human neurobiology, including synaptic plasticity, lipid metabolism, neuroinflammation and neurodegeneration, and neurodevelopment, including developmental disorders. Moreover, our results show some overlap with those of previous studies focused on the neocortex, indicating that such results may be common to multiple brain structures. These findings further our understanding of the cerebellum in human brain evolution.     

Identification of a 31-bp Deletion in the RELN Gene Causing Lissencephaly with Cerebellar Hypoplasia in Sheep

Lissencephaly is an inherited developmental disorder in which neuronal migration is impaired. A type of lissencephaly associated with cerebellar hypoplasia (LCH) was diagnosed in a commercial flock of Spanish Churra sheep. The genotyping of 7 affected animals and 33 controls with the OvineSNP50 BeadChip enabled the localization of the causative mutation for ovine LCH to a 4.8-Mb interval on sheep chromosome 4 using genome-wide association and homozygosity mapping. The RELN gene, which is located within this interval, was considered a strong positional and functional candidate because it plays critical roles in neuronal migration and layer formation. By performing a sequencing analysis of this gene’s specific mRNA in a control lamb, we obtained the complete CDS of the ovine RELN gene. The cDNA sequence from an LCH-affected lamb revealed a deletion of 31 bp (c.5410_5440del) in predicted exon 36 of RELN, resulting in a premature termination codon. A functional analysis of this mutation revealed decreased levels of RELN mRNA and a lack of reelin protein in the brain cortex and blood of affected lambs. This mutation showed a complete concordance with the Mendelian recessive pattern of inheritance observed for the disease. The identification of the causal mutation of LCH in Churra sheep will facilitate the implementation of gene-assisted selection to detect heterozygous mutants, which will help breeders avoid at-risk matings in their flocks. Moreover, the identification of this naturally occurring RELN mutation provides an opportunity to use Churra sheep as a genetically characterized large animal model for the study of reelin functions in the developing and mature brain.     

Tissue and Process Specific microRNA–mRNA Co-Expression in Mammalian Development and Malignancy

An association between enrichment and depletion of microRNA (miRNA) binding sites, 3′ UTR length, and mRNA expression has been demonstrated in various developing tissues and tissues from different mature organs; but functional, context-dependent miRNA regulations have yet to be elucidated. Towards that goal, we examined miRNA–mRNA interactions by measuring miRNA and mRNA in the same tissue during development and also in malignant conditions. We identified significant miRNA-mediated biological process categories in developing mouse cerebellum and lung using non-targeted mRNA expression as the negative control. Although miRNAs in general suppress target mRNA messages, many predicted miRNA targets demonstrate a significantly higher level of co-expression than non-target genes in developing cerebellum. This phenomenon is tissue specific since it is not observed in developing lungs. Comparison of mouse cerebellar development and medulloblastoma demonstrates a shared miRNA–mRNA co-expression program for brain-specific neurologic processes such as synaptic transmission and exocytosis, in which miRNA target expression increases with the accumulation of multiple miRNAs in developing cerebellum and decreases with the loss of these miRNAs in brain tumors. These findings demonstrate the context-dependence of miRNA–mRNA co-expression.     

An Intronic SINE Insertion in FAM161A that Causes Exon-Skipping Is Associated with Progressive Retinal Atrophy in Tibetan Spaniels and Tibetan Terriers

Progressive retinal atrophy (PRA) in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 19 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s) responsible have yet to be identified. Using genome-wide association with 22 Tibetan Spaniel PRA cases and 10 controls, we identified a novel PRA locus, PRA3, on CFA10 (p_raw = 2.01×10⁻⁵, p_genome = 0.014), where a 3.8 Mb region was homozygous within 12 cases. Using targeted next generation sequencing, a short interspersed nuclear element insertion was identified near a splice acceptor site in an intron of a provocative gene, FAM161A. Analysis of mRNA from an affected dog revealed that the SINE causes exon skipping, resulting in a frame shift, leading to a downstream premature termination codon and possibly a truncated protein product. This mutation segregates with the disease in 22 out of 35 cases tested (63%). Of the PRA controls, none are homozygous for the mutation, 15% carry the mutation and 85% are homozygous wildtype. This mutation was also identified in Tibetan Terriers, although our results indicate that PRA is genetically heterogeneous in both Tibetan Spaniels and Tibetan Terriers.     

Mutations in SNX14 Cause a Distinctive Autosomal-Recessive Cerebellar Ataxia and Intellectual Disability Syndrome

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.     

Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12

Wilson’s disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 ^del/del), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 ^del/del).A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 ^del/del Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene.The study has identified a significant disease association with a region on chromosome 37 containing identified SNP’s which are highly significantly associated with non-COMMD1 ^del/del Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilson’s disease in man.     

A novel homozygous mutation in <Emphasis Type="Italic">SZT2</Emphasis> gene in Saudi family with developmental delay,macrocephaly and epilepsy

Epileptic encephalopathies are genetically heterogeneous disorders which leads to epilepsy and cause neurological disorders. Seizure threshold 2 (SZT2) gene located on chromosome 1p34.2 encodes protein mainly expressed predominantly in the parietal and frontal cortex and dorsal root ganglia in the brain. Previous studies in mice showed that mutation in this gene can confers low seizure threshold, enhance epileptogenesis and in human may leads to facial dysmorphism, intellectual disability, seizure and macrocephaly. Objective of this study was to find out novel gene or novel mutation related to the gene phenotype. We have identified a large consanguineous Saudi family segregating developmental delay, intellectual disability, epilepsy, high forehead and macrocephaly. Exome sequencing was performed in affected siblings of the family to study the novel mutation. Whole exome sequencing data analysis, confirmed by subsequent Sanger sequencing validation study. Our results showed a novel homozygous mutation (c.9368G>A) in a substitution of a conserved glycine residue into a glutamic acid in the exon 67 of SZT2 gene. The mutation was ruled out in 100 unrelated healthy controls. The missense variant has not yet been reported as pathogenic in literature or variant databases. In conclusion, the here detected homozygous SZT2 variant might be the causative mutation that further explain epilepsy and developmental delay in this Saudi family.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

Absence of an orphan mitochondrial protein, c19orf12, causes a distinct clinical subtype of neurodegeneration with brain iron accumulation

The disease classification neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive neurodegenerative disorders characterized by brain iron deposits in the basal ganglia. For about half of the cases, the molecular basis is currently unknown. We used homozygosity mapping followed by candidate gene sequencing to identify a homozygous 11 bp deletion in the orphan gene C19orf12. Mutation screening of 23 ideopathic NBIA index cases revealed two mutated alleles in 18 of them, and one loss-of-function mutation is the most prevalent. We also identified compound heterozygous missense mutations in a case initially diagnosed with Parkinson disease at age 49. Psychiatric signs, optic atrophy, and motor axonal neuropathy were common findings. Compared to the most prevalent NBIA subtype, pantothenate kinase associated neurodegeneration (PKAN), individuals with two C19orf12 mutations were older at age of onset and the disease progressed more slowly. A polyclonal antibody against the predicted membrane spanning protein showed a mitochondrial localization. A histopathological examination in a single autopsy case detected Lewy bodies, tangles, spheroids, and tau pathology. The mitochondrial localization together with the immunohistopathological findings suggests a pathomechanistic overlap with common forms of neurodegenerative disorders.     

Rat and Drosophila Synaptotagmin 4 Have Opposite Effects during SNARE-catalyzed Membrane Fusion

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca²⁺. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca²⁺. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca²⁺ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca²⁺ when the conserved Asp-to-Ser Ca²⁺ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca²⁺ sensor capable of coupling Ca²⁺ to membrane fusion.