Identification of a FUS splicing mutation in a large family with amyotrophic lateral sclerosis

Article (PDF Available)inJournal of Human Genetics 56(3):247-9 · December 2010with32 Reads
DOI: 10.1038/jhg.2010.162 · Source: PubMed
Abstract
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have led, thus far, to the identification of 12 loci and 9 genes for familial ALS (FALS). Although the distribution and impact of superoxide dismutase 1 mutations has been extensively examined for over a decade, the recently identified FALS-associated FUS gene has been less studied. Therefore, we set out to screen our collection of FALS cases for FUS mutations. All 15 exons of FUS were amplified and sequenced in 154 unrelated FALS cases and 475 ethnically matched healthy individuals. One substitution located in the acceptor splice site of intron 14 was identified in all affected members of a large family, causing the skipping of the last 13 amino acids of the protein and the translation of 7 novel amino acids, resulting from the new translation of a part of the 3' untranslated region. Our study identified a new splicing mutation in the highly conserved C-terminal of the FUS protein. Thus far most FUS mutations are missenses, and our findings, combined with those of others, confirm the importance of the C-terminal portion of the protein, adding additional support for FUS mutations having a critical role in ALS.
2 Figures
SHORT COMMUNICATION
Identification of a FUS splicing mutation in a large
family with amyotrophic lateral sclerosis
Ve
´ronique V Belzil1, Judith St-Onge1, Hussein Daoud1, Anne Desjarlais1, Jean-Pierre Bouchard2,
Nicolas Dupre
´2, William Camu3, Patrick A Dion1,4 and Guy A Rouleau1,5,6
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the degeneration of upper and lower
motor neurons. Genetic studies have led, thus far, to the identification of 12 loci and 9 genes for familial ALS (FALS). Although
the distribution and impact of superoxide dismutase 1 mutations has been extensively examined for over a decade, the recently
identified FALS-associated FUS gene has been less studied. Therefore, we set out to screen our collection of FALS cases for
FUS mutations. All 15 exons of FUS were amplified and sequenced in 154 unrelated FALS cases and 475 ethnically matched
healthy individuals. One substitution located in the acceptor splice site of intron 14 was identified in all affected members of a
large family, causing the skipping of the last 13 amino acids of the protein and the translation of 7 novel amino acids, resulting
from the new translation of a part of the 3¢untranslated region. Our study identified a new splicing mutation in the highly
conserved C-terminal of the FUS protein. Thus far most FUS mutations are missenses, and our findings, combined with those
of others, confirm the importance of the C-terminal portion of the protein, adding additional support for FUS mutations having a
critical role in ALS.
Journal of Human Genetics (2011) 56, 247–249; doi:10.1038/jhg.2010.162; published online 16 December 2010
Keywords: amyotrophic lateral sclerosis; FUS/motor neuron disease; neurodegeneration; RNA-binding protein; splicing mutation
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease
affecting the upper and lower motor neurons. Specifically, neurons of
the motor cortex, brain stem and spinal cord are progressively
involved, causing gradual spasticity and muscle weakness starting
in the limbs in 75% of cases and in the bulbar region in 25% of
cases. In all, 90% of patients are believed to be sporadic (SALS),
without any family history of the disease, while the other 10% of cases
are familial (FALS), primarily segregating in an autosomal dominant
manner.1SALS and FALS patients are clinically indistinguishable,
except for the mean age of onset, which is 56 years for SALS,
compared with an average of 46 years for FALS.2The overall
prevalence is 4–6/100000 and incidence is 1–2/100000,3which
make ALS the most common of motor neuron diseases.
Mutations in the copper superoxide dismutase 1 gene were
first shown to be ALS causative over 15 years ago and they account
for 15–20% of all FALS cases, representing a proportion of 1–2% of all
ALS cases.4In the last 2 years, the identification of ALS causative
mutations in the TAR-DNA-binding protein (TA RDB P encoding
TDP-43) gene in both SALS and FALS cases5and in FUS/TLS
encoding FUS,6,7 thus far mostly but not exclusively in FALS, opened
a new era for the investigation of mechanisms underlying the disease.
Whereas superoxide dismutase 1 mutations have been reported
throughout the full length of the protein, TAR D B P and FUS mutations
are mostly clustered to specific regions. For TAR D BP,mostofthe
mutations identified are in the glycine-rich region encoded by exon 6,
and for FUS/TLS, which also contains a glycine-rich region, mutations
are mostly in the extreme C-terminal part of the protein.8The aim of
this study was to evaluate the proportion of FUS mutations in a
portion of our FALS cohort and to see if any mutations identified
would be clustered to the same region.
The 15 coding exons of FUS were amplified and sequenced in 154
unrelated FALS cases, and only one variant was identified. It is an
unreported substitution of an adenine to a cytosine in the acceptor
splice site of intron 14 (c.1542-2A4C). The variant was present in six
samples of a large ALS family (Figure 1) recruited in France: II:5, III:8,
IV:1, IV:3, IV:4, and IV:6. Two of those individuals had an ALS
phenotype (II:5, III:8), while the four unaffected mutation carriers
are in the fourth generation and are younger than the average age of
onset of the first ALS symptoms. By carrying the mutation, they
confirm that their three affected fathers likely had the substitution.
In addition, three mothers married to affected fathers (II:4, III:1 and
III:7) were tested and were negative for the mutation. We prepared
cDNA using total RNA from the immortalized lymphoblast cell
lines of the two affected individuals with the adenine-to-cytosine
Received 30 October 2010; revised 18 November 2010; accepted 26 November 2010; published online 16 December 2010
1Center of Excellence in Neuromics of Universite
´de Montre
´al, CHUM Research Center, Montreal, Quebec, Canada; 2Universite
´Laval, Faculty of Medicine, CHA, Enfant-Je
´sus
Hospital, Quebec, Canada; 3ALS Center, Department of Neurology, CHU Gui de Chauliac, Montpellier, France; 4Department of Pathology and Cellular Biology, Faculty of Medicine,
Universite
´de Montre
´al, Montreal, Quebec, Canada; 5Department of Medicine, Faculty of Medicine, Universite
´de Montreal, Montreal, Quebec, Canada and 6Research Center,
CHU Sainte-Justine, and Department of Pediatrics and Biochemistry, Universite
´de Montreal, Montreal, Quebec, Canada
Correspondence: Dr GA Rouleau, CHUM Research Centre, Notre-Dame Hospital, 1560 Sherbrooke East, Y-3633, Montreal, Quebec, Canada H2L 4M1.
E-mail: guy.rouleau@umontreal.ca
Journal of Human Genetics (2011) 56, 247249
&
2011 The Japan Society of Human Genetics All rights reserved 1434-5161/11 $32.00
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substitution. These cDNAs were PCR amplified, and two products of
425 and 167bp were observed on agarose gel (Figure 2a), demonstrat-
ing that the mutant allele was not degraded by nonsense-mediated
mRNA decay. The heterozygous sequence trace was analyzed, as
well as the separate sequence of the two different alleles after a gel
extraction of the two products (Figure 2b). Sequencing showed that
patients with the c.1542-2A4C change expressed a mutated allele
missing the 40bp coding sequence of exon 15 as well as the first 203bp
of the 3¢untranslated region. More precisely, the sequence of
the mutant mRNA brings together the last 2 bp of amino acid 514
located in exon 14 and the nucleotide at the c.*204 position in the
3¢untranslated region, as an AG alternative acceptor site located at
position c.*202_*203 is encountered (Figure 2c); translation subse-
quently ends with a stop codon at position c.*228. Consequently, the
mutant protein undergoes a silent change at amino acid 514
(p.R514R), followed by seven new amino acids (SMSRSGR) at the
end of the protein. The adenine of the wild-type acceptor site of intron
14 located at base pair 31 110 219 on chromosome 16 is highly
conserved across species and is not listed as a known SNP. The
Alternative Splice Site Predictor bioinformatics program attributed a
score of 15.533 to the wild-type AG acceptor splice site, and a score
of 4.249 for the acceptor splice site with the cytosine substitution.
The alternative AG acceptor splice site located in the 3¢untranslated
region at position c.*202_*203 had a score of 4.620 and is suggested to
Figure 1 Pedigree of the family with the c.1542-2A4C variant. Black
symbols represent affected individuals, while open symbols represent
unaffected family members. Symbols with dots represent unaffected
individuals carrying the substitution. Slashed symbols indicate deceased
individuals. A red square around the individual III:8 symbol indicates the
sample sequenced in the primary screening, and asterisks indicate family
members for whom exon 15 of the FUS gene was sequenced in the
secondary screening. Symbols with two asterisks represent individuals
for whom cDNA was amplified. Genotypes at position c.1542-2 are
indicated in black for the wild-type allele and in red for the mutant allele.
Genotypes in parentheses are inferred.
Figure 2 Splicing mutation in FUS.(a) Agarose gel electrophoresis of FUS cDNA amplified in one unrelated control, and two affected family members
(II:5, III:8), showing the wild-type allele (425 bp) and the mutant allele (167bp) for the fragment amplified using foward primer 5¢-GAGGGGG
ACCAGGTGGCTCTCAC in exon 14 and reverse primer 5¢-TCATTTGGCCTTCTCCCCGAACAC in the 3¢untranslated region (UTR). (b) cDNA sequence
chromatogram of the wild-type and mutant alleles in one patient, confirming the skipping of exon 15 and the first 203bp of the 3¢UTR. (c) Schematic
(not to scale) representation of exons 14, 15 and the 3¢UTR of the FUS gene for the wild-type and mutant alleles. In the mutant allele, a part of the 3¢UTR
is translated, which adds seven new amino acids to the protein (SMSRSGR).
FUS splicing mutation in an ALS family
VV Belzil et al
248
Journal of Human Genetics
be responsible for this new isoform. As this FUS mutation segregates
in all affected individuals of the family as well as in the children of
deceased affected members, and was not present in 475 control
participants, we believe that it is likely responsible for the ALS
phenotype observed in this family.
The substitution is located in the RGG domain enriched in
arginine–glycine–glycine motifs, where most other FUS mutations
were reported. Importantly, truncated proteins were previously
observed with the two other major ALS causative genes.9–11 But,
more interestingly, this is the second splicing mutation reported
in FUS, as another one was recently shown to lead to the skipping
of exon 14 after the substitution of the adenine located in the acceptor
splice site of intron 13.12 Unfortunately, no clinical information could
be retrieved for the family presented in our study, so we were unable
to compare the disease evolution caused by our splicing mutation in
intron 14 with the splicing mutation in intron 13 reported by Dejesus-
Hernandez et al.12 The splicing mutation identified here removes
the last 13 amino acids that partly encode the C-terminal portion
of FUS, in which a number of mutations have been associated with
ALS. Our finding reinforces the notion that this portion has a key role
in the pathology of ALS, most likely by way of affecting the RNA-
binding domain it encodes for. In the future, such splicing defect
causative mutations may become treatable through drugs or gene
therapies aimed at correcting splicing, as explored for a number
of disorders.13–15
ACKNOWLEDGEMENTS
VVB, HD and GAR are supported by the Canadian Institutes of Health Research.
We would like to thank the patients involved in this study; Me
´lanie Benard,
Isabelle Thibault and Pierre Provencher for sample collection and organization;
Anne Noreau, Cynthia Bourassa, Sophie Massart and Bertrand Boutie
´for
technical support; and to acknowledge support from the Association pour la
Recherche sur la Scle
´rose Late
´rale Amyotrophique (ARS), the Association
Franc¸aisecontrelesMyopathies(AFM)andtheFrenchGrouponMND.
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lateral sclerosis. Nature 362, 59–62 (1993).
5 Sreedharan, J., Blair, I. P., Tripathi, V. B., Hu, X., Vance, C., Rogelj, B. et al. TDP-43
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FUS splicing mutation in an ALS family
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    • Flexibility attained by mutant complex protein structures is many times greater than the wild FUS-Kapβ2, resulting in decreased Cα atom contacts in mutant FUS (R521C)-Kapβ2 and mutant FUS (R521H)-Kapβ2 structures. interrupt the binding of FUS with Kapβ2 and thereby increase the severity of ALS (Belzil et al., 2009Belzil et al., , 2011aBelzil et al., , 2011b Corrado et al., 2010; Chio et al., 2011; Damme et al., 2010; Hewitt et al., 2010; Kwiatkowski et al., 2009; Lai et al., 2011; Nagayama et al., 2012; Ticozzi et al., 2009; Tsai et al., 2011; Vance et al., 2009; Yamashita et al., 2012; Yan et al., 2010). In present study, we investigated the binding behaviour of mutant FUS (R521C) and mutant FUS (R521H) with Kapβ2 and compared with wild FUS-Kapβ2.
    [Show abstract] [Hide abstract] ABSTRACT: FUS (fused in sarcoma) gene encodes the RNA binding protein FUS. This gene is mapped to chromosome 16p11.2. The FUS protein binds with Karyopherineβ2 (Kapβ2) through its proline/tyrosine-nuclear localization signal (PY-NLS) that helps in the localization of FUS protein within the nucleus. Arginine residue in 521 position (R521) of PY-NLS plays a vital role in the binding of FUS protein with Kapβ2. Mutations in this position (R521C and R521H) are the most predominant mutations associated with Amyotrophic lateral sclerosis (ALS). However, the mechanism by which these mutations lead to ALS is poorly understood. We examined the binding behaviour of the mutants FUS (R521C) and FUS (R521H) with Kapβ2 through protein-protein docking and molecular dynamics (MD) simulation. The binding patterns of mutants were compared with the binding behaviour of wild FUS-Kapβ2. Our results suggest that these mutants have relatively weak binding affinity with Kapβ2 when compared with wild FUS-Kapβ2 as indicated by the lesser number of interactions found between the mutant FUS and Kapβ2. Hence, these mutations weakens the binding and this results in the cytoplasmic mislocalization of mutant FUS; and thereby it increases the severity of ALS.
    Full-text · Article · Jul 2016
    • None USA [Kwiatkowski et al., 2009] 48 c.1554 1557delACAG p.R518del Exon 15/RGG rich 1 Spinal None United Kingdom [Bäumer et al., 2010] 49 c.1555 C>T p.Q519X Exon 15/RGG rich 1 n.a. None France [Belzil et al., 2011]
    [Show abstract] [Hide abstract] ABSTRACT: Mutations in the TAR DNA Binding Protein gene (TARDBP), encoding the protein TDP-43, were identified in amyotrophic lateral sclerosis (ALS) patients. Interestingly, TDP-43 positive inclusion bodies were first discovered in ubiquitin-positive, tau negative ALS and frontotemporal dementia (FTD) inclusion bodies, and subsequently observed in the majority of neurodegenerative disorders. To date, 47 missense and one truncating mutations have been described in a large number of familial (FALS) and sporadic (SALS) patients. Fused in Sarcoma (FUS) was found to be responsible for a previously identified ALS6 locus, being mutated in both FALS and SALS patients. TARDBP and FUS have a structural and functional similarity and most of mutations in both genes are also clustered in the C-terminus of the proteins. The molecular mechanisms through which mutant TDP-43 and FUS may cause motor neuron degeneration are not well understood. Both proteins play an important role in mRNA transport, axonal maintenance and motor neuron development. Functional characterization of these mutations in in vitro and in vivo systems is helping to better understand how motor neuron degeneration occurs. This report summarizes the biological and clinical relevance of TARDBP and FUS mutations in ALS. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database(LOVD) and is accessible online at www.lovd.nl/TARDBP, www.lovd.nl/FUS.
    Full-text · Article · Jun 2013
    • The p.P525L mutation has been identified in 8 FALS (Chio et al., 2009; Fecto and Siddique, 2011; Ito et al., 2011b; Kwiatkowski et al., 2009; Yan et al., 2010) and 9 SALS patients (including the 1 in our study) (Baumer et al., 2010; Brown et al., 2012; Conte et al., 2012; Huang et al., 2010; Sproviero et al., 2012), 12 of which had a juvenile onset (Table 1) (Baumer et al., 2010; Brown et al., 2012; Chio et al., 2009; Conte et al., 2012; Huang et al., 2010; Ito et al., 2011b; Kwiatkowski et al., 2009; Sproviero et al., 2012). Twelve frameshift mutations have been identified in 7 FALS probands and 7 SALS cases (including the 1 in this study) (Supplementary Table 1) (Baumer et al., 2010; Belzil et al., 2011a Belzil et al., , 2011b Belzil et al., , 2012 Corrado et al., 2010; DeJesus-Hernandez et al., 2010; Fecto and Siddique, 2011; Kwon et al., 2012; Lai et al., 2011; Yamashita et al., 2011; Yan et al., 2010). Among them, 6 FALS probands and 4 SALS patients had a juvenile onset (Table 1) (Baumer et al., 2010; Belzil et al., 2012; DeJesus-Hernandez et al., 2010; Fecto and Siddique, 2011; Yamashita et al., 2011; Yan et al., 2010).
    [Show abstract] [Hide abstract] ABSTRACT: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of motor neuron disease and occurs before 25 years of age. Only very few sporadic cases of juvenile-onset ALS have been reported. Rare SOD1 mutations and several FUS mutations have been identified in juvenile-onset ALS patients. To define the genetics of juvenile-onset sporadic ALS (SALS) of Chinese origin, we sequenced all 5 exons of SOD1, exons 3-6 and 12-15 of FUS in 11 juvenile-onset SALS patients, 105 adult-onset ALS patients (including 6 familial ALS [FALS] pedigrees), and 245 healthy controls. For the 11 juvenile-onset SALS and 6 FALS cases, the other 7 exons of FUS were also screened. A heterozygous de novo missense mutation c.1574C>T (p.P525L), a heterozygous de novo 2-base pair deletion c.1509_1510delAG (p.G504Wfs*12), and a nonsense mutation c.1483C>T (p.R495X) was each identified in 1 juvenile SALS patient. A heterozygous missense mutation c.1561C>G (p.R521G) was identified in a FALS proband. In the Chinese population, the frequency of FUS mutation in FALS is 11.4% (95% confidence interval [CI], 0.9%-22.0%), higher than the Japanese (10%; 95% CI, 0.7%-19.3%), and Caucasians (4.9%; 95% CI, 3.9%-6.0%). The frequency of FUS mutation in SALS patients is 1.5% (95% CI, 0.2%-2.9%), which is similar to Koreans (1.6%; 95% CI, 0%-3.2%), but higher than in Caucasians (0.6%; 95% CI, 0.4%-0.8%). Our findings suggest that de novo FUS mutations are associated with juvenile-onset SALS of Chinese origin and that this gene should be screened in ALS patients with a young age of onset, aggressive progression, and sporadic occurrence.
    Full-text · Article · Oct 2012
    • The identification of the R521L and R521H substitutions in our patients with ALS confirms that codon 521 is the most commonly mutated codon in the FUS gene. In fact, mutations in codon 521 account for more than 30% of all FUS-mutated ALS cases [6][7][8][9][10][11][12][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32], suggesting the functional importance of this codon in triggering motor neuron degeneration. The FUS protein continuously shuttles between the nucleus and the cytoplasm.
    [Show abstract] [Hide abstract] ABSTRACT:   According to studies in European, North American, Australian, and Asian populations, FUS gene mutations occur in 0.6-20.2% of the patients with familial amyotrophic lateral sclerosis (ALS) and 0.4-2.0% of sporadic ALS cases. In China, FUS mutations have been reported in several familial ALS pedigrees but not in sporadic ALS cases. Here, we screened for FUS mutations in Chinese patients with ALS.   We sequenced all of the 15 exons of FUS in 10 familial ALS pedigrees, exons 5, 6, 14, and 15 in 210 patients with sporadic ALS and 151 healthy controls. All patients were negative for SOD1, TARDBP, and ANG mutations.   A c.1562G>T (p.R521L) missense mutation was identified in one familial ALS proband and her asymptomatic daughter. A c.1562G>A (p.R521H) missense mutation was identified in two patients with sporadic ALS. Three synonymous mutations (c.453C>T, c.648C>T, and c.1464C>T) were detected among four patients with sporadic ALS, and a untranslated region variant (*14C>T) was identified in one familial ALS proband and one patient with sporadic ALS.   The frequency of FUS mutations is approximately 1.0% in our SOD1-, ANG-, TARDBP-mutation-negative sporadic ALS cohort and similar to that reported in previous studies from Asia in our familial ALS cohort. [Correction added on 31 May 2012, after first online publication: the gene FUS- was changed to ANG-]. Our findings provide an overview of the occurrence of FUS mutations in Chinese sporadic and familial ALS cases and highlight the importance of screening for FUS mutations in ALS patients of Chinese origin.
    Full-text · Article · Feb 2012
    • These transgenes included: (i) full-length (FL) WT human FUS (WT-FUS); (ii) four different missense mutations associated with varying clinical severity (as defined by age at onset and by disease duration) of human ALS (R514G and R521G = mild; R522G = moderate and P525L = severe) (1–3) (Supplementary Material, Table S1) and (iii) two different C-terminal-truncated FUS constructs (FUS513 and FUS501—lacking the C-terminal 13 and 25 amino acids of FUS, respectively). While the C-terminal-truncated constructs are artificial, they are very similar to several human C-terminal splicing/frame-shifting truncation mutations that are also associated with severe ALS phenotypes (15,16). Each construct was cloned into a vector containing a C. elegans pan-neuronal promoter Prgef-1and an in-frame green fluorescent protein (GFP S65T) or red fluorescent protein (TagRFP) at the N-terminus.
    [Show abstract] [Hide abstract] ABSTRACT: It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.
    Full-text · Article · Sep 2011
  • [Show abstract] [Hide abstract] ABSTRACT: The recent identification of ALS-linked mutations in FUS and TDP-43 has led to a major shift in our thinking in regard to the potential molecular mechanisms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA-mediated proteinopathy is increasingly being recognized as a potential cause of neurodegenerative disorders. FUS and TDP-43 are structurally and functionally similar proteins. FUS is a DNA/RNA binding protein that may regulate aspects of RNA metabolism, including splicing, mRNA processing, and micro RNA biogenesis. It is unclear how ALS-linked mutations perturb the functions of FUS. This review highlights recent advances in understanding the functions of FUS and discusses findings from FUS animal models that provide several key insights into understanding the molecular mechanisms that might contribute to ALS pathogenesis.
    Full-text · Article · Jan 2012
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