Anna Szuto

Exome Sequencing Identifies FUS Mutations as a Cause of Essential Tremor

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Deleterious mutations in the essential mRNA metabolism factor, hGle1, in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.

Replication study of MATR3 in familial and sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by an extensive loss of motor neurons in the primary motor cortex, brainstem, and spinal cord. Genetic studies report a high heritability of ALS. Recently, whole-exome sequencing analysis of familial ALS (FALS) patients allowed the identification of missense variations within the MATR3 gene. MATR3 was previously associated to distal myopathy 2 and encodes for a nuclear matrix and DNA/RNA binding protein that has been shown to interact with TDP43 in an RNA-dependent manner. Here, we assessed the MATR3 mutation frequency in French-Canadian ALS and control individuals (nFALS = 83, sporadic ALS [nSALS] = 164, and ncontrols = 162) and showed that MATR3 mutations were found in 0%, 1.8%, and 0% of FALS, SALS, and controls, respectively. Interestingly, among the mutations identified in SALS, the splicing mutation c.48+1G>T was found to result in the insertion of 24 amino acids in MATR3 protein. These findings further support the role of MATR3 in ALS, and more studies are needed to shed more light on MATR3 proteinopathy.

UBQLN2 mutations are rare in French and French–Canadian amyotrophic lateral sclerosis

Mutations in the UBQLN2 gene, which encodes a member of the ubiquitin-like protein family (ubiquilin-2), have been recently identified in patients with dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS with dementia. We report here the sequencing of the UBQLN2 gene in 590 ALS patients of French and French-Canadian ancestry. We identified two novel missense mutations (p.S155N and p.P189T) in two individuals with sporadic ALS. Bioinformatic analysis predicts that these missense mutations affect the normal proteins function. Importantly, these findings further highlight the importance of the proline residues located in the conserved domains of the ubiquilin-2 protein, suggesting that mutations affecting these residues are particularly relevant to the development of ALS. Our findings further support a causative role of the UBQLN2 gene in the pathogenesis of ALS and suggest that UBQLN2 mutations are rare in the French and French-Canadian population.

C9orf72 Hexanucleotide Repeat Expansions as the Causative Mutation for Chromosome 9p21–Linked Amyotrophic Lateral Sclerosis and Frontotemporal Dementia

OBJECTIVE To further assess the presence of a large hexanucleotide repeat expansion in the first intron of the C9orf72 gene identified as the genetic cause of chromosome 9p21-linked amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) in 4 unrelated families with a conclusive linkage to c9ALS/FTD. DESIGN A repeat-primed polymerase chain reaction assay. SETTING Academic research. PARTICIPANTS Affected and unaffected individuals from 4 ALS/FTD families. MAIN OUTCOME MEASURE The amplified C9orf72 repeat expansion. RESULTS We show that the repeat is expanded in and segregated perfectly with the disease in these 4 pedigrees. CONCLUSION Our findings further confirm the C9orf72 hexanucleotide repeat expansion as the causative mutation for c9ALS/FTD and strengthen the hypothesis that ALS and FTD belong to the same disease spectrum.

Exome sequencing identifies recessive CDK5RAP2 variants in patients with isolated agenesis of corpus callosum.

Agenesis of the corpus callosum (ACC) is a common brain malformation which can be observed either as an isolated condition or as part of numerous congenital syndromes. Therefore, cognitive and neurological involvements in patients with ACC are variable, from mild linguistic and behavioral impairments to more severe neurological deficits. To date, the underlying genetic causes of isolated ACC remains elusive and causative genes have yet to be identified. We performed exome sequencing on three acallosal siblings from the same non-consanguineous family and identified compound heterozygous variants, p.[Gly94Arg];[Asn1232Ser], in the protein encoded by the CDK5RAP2 gene, also known as MCPH3, a gene previously reported to cause autosomal recessive primary microcephaly. Our findings suggest a novel role for this gene in the pathogenesis of isolated ACC.

An 18 alanine repeat in a severe form of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant adult-onset neuromuscular disease that usually develops in the fifth or sixth decade of life and which is characterized by progressive ptosis, dysphagia and proximal limb weakness as well as a positive family history1. The disease is usually inherited with complete penetrance and without gender preference1. It is caused by trinucleotide repeat expansions that result in a lengthened polyalanine tract in the polyadenylate binding protein nuclear-1 gene (PABPN1), located on chromosome 14q11.2-q13. These polyalanine expansions referred to as (GCN)n are believed to arise from unequal allelic homologous recombination during meiosis and/or mitosis2. The wild type allele of the PABPN1 gene harbors 10 alanine codons that are located immediately downstream to the start codon in exon 1 where they code for an homopolymeric stretch of 10 alanine residues, also referred as (GCN)10. Mutant dominant PABPN1 alleles underlying OPMD range between 12 and 17 (GCN) repeats. A (GCN)11 polymorphism has been reported in North America, Europe and Japan but it does not lead to OPMD or another specific phenotype when in an heterozygous state2. However, rare cases of (GCN)11 homozygous carriers have been reported and, by comparison to individuals with (GCN)12-17 alleles, they present with relatively mild OPMD symptoms and at a later age of onset (sixties). To date, PABPN1 is the only known gene to be associated with OPMD. Interestingly, in our study based on 72 French-Canadian families, we have shown that 5% of familial cases had a (GCN)12, 40% a (GCN)13, 26% a (GCN)14, 21% a (GCN)15, 7% a (GCN)16 and finally 1% a (GCN)17 PABPN1 allele3. A relationship between the length of the (GCN) repeat and symptom severity was found because compound heterozygote carriers of (GCN)11 (GCN)13 alleles displayed a more severe phenotype than siblings who were compound heterozygous carriers of (GCN)10 (GCN)13 alleles or homozygous siblings with (GCN)11 alleles3,4. In that vein, Hill et al observed an earlier age of onset for individuals with longer repeat expansions5. However, Tondo and colleagues reported a range of symptom severity among carriers of a mutated (GCN)n of the same size, while Semmler and colleagues reported autosomal recessive OPMD cases harboring (GCN)11 alleles with severe phenotypes6,7. Therefore, it has been suggested that additional genetic and environmental factors could modulate disease expression and consequently, the correlation between severity and the polyalanine expansion size is still a subject of debate. Here, we report a family with two affected sisters who are both carriers of an unusual expansion of 18 polyalanines in PABPN1, the longest (GCN)n expansion reported to date. We believe this An 18 Alanine Repeat in a Severe Form of Oculopharyngeal Muscular Dystrophy

Clinical and genetic study of hereditary spastic paraplegia in Canada

Objective: To describe the clinical, genetic, and epidemiologic features of hereditary spastic paraplegia (HSP) in Canada and to determine which clinical, radiologic, and genetic factors determine functional outcomes for patients with HSP. Methods: We conducted a multicenter observational study of patients who met clinical criteria for the diagnosis of HSP in the provinces of Alberta, Ontario, and Quebec from 2012 to 2015. Characteristics of the participants were analyzed using descriptive statistics. The main outcome measure for a subset of the cohort (n = 48) was the Spastic Paraplegia Rating Scale. We also used the SPATAX-EUROSPA disability stage (disability score) to assess disability (n = 65). Results: A total of 526 patients were identified with HSP across the country, and 150 patients had a confirmed genetic diagnosis. Mutations were identified in 15 different genes; the most common were SPAST (SPG4, 48%), ATL1 (SPG3A, 16%), SPG11 (8%), SPG7 (7%), and KIAA0196 (SPG8, 5%). The diagnosis of SPG4 was associated with older age at symptom onset (p = 0.0017). SPG4 and SPG3A were less associated with learning disabilities compared to other subtypes of HSP, and SPG11 was strongly associated with progressive cognitive deficits (odds ratio 87.75, 95% confidence interval 14.04–548.24, p < 0.0001). SPG3A was associated with better functional outcomes compared to other HSP subtypes (p = 0.04) on multivariate analysis. The strongest predictor of significant disability was abnormal brain MRI (p = 0.014). Conclusions: The most important predictors of disability in our HSP cohort were SPG11 mutations and abnormal brain MRI. Accurate molecular characterization of well-phenotyped cohorts and international collaboration are essential to establish the natural history of these rare neurodegenerative disorders.

CYP7B1 mutations in French-Canadian hereditary spastic paraplegia subjects.

Lorrain disease, refers to progressive motor neurodegenerative disorders characterized by lower limb spasticity and weakness. This group of diseases has been shown to include very heterogeneous clinical manifestation and various modes of inheritance (dominant, recessive and X-linked) have been reported. Hereditary spastic paraplegia are classified as pure or complicated forms. Cases with pure forms only present lower limb spasticity but complicated forms have additional neurological features such as cerebellar ataxia, intellectual disabilities, dementia, thin corpus callosum, and/or visual dysfunction. To date, over 48 spastic paraplegia loci including 20 genes have been identified. In 1994, one of these loci, SPG5A, was mapped to chromosome 8q21.3 and deemed to contain a gene underlying a pure-autosomal recessive form of HSP (MIM#270800)1. Many HSP families have been mapped to this particular locus and the causative gene was finally identified in 2008. Mutations in Cytochrome P450, family 7, subfamily B polypeptide 1 (CYP7B1) gene were then found to underlie this particular disorder; homozygous mutations were originally identified in a consanguineous English family2. CYP7B1 extends over ~220 Kb gene and contain six exons that encode for an evolutionarily conserved steroid metabolizing enzyme, cytochrome P450. This particular enzyme is widely expressed in many tissues, with the highest mRNA levels found in the liver and the brain. Containing 506 amino-acid, this enzyme catalyzes the 6α-7α hydroxylation of several endogenous substrates such as: dehydroepiandrosterone, pregnenolone, oxysterols, 25-and 27-hydroxycholestrol. In the liver, CYP7B1 is implicated in the synthesis of bile salt that regulate bile flow and indirectly the excretion of metabolites (e.g. porphyrins arising from heme breakdown). Interestingly, mutations in the CYP7B1 gene are also known to cause liver failure in children. Additional roles for CYP7B1 were described in the metabolism of oestrogen receptor ligands and in immunoglobulin production3. The exact role of CYP7B1 in the brain is unknown, other than its link to HSP. The main goal of our study was to determine if CYP7B1 mutations could explain disease in some of our HSP patients.