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Identification of a novel loss-of-function C9orf72 splice site mutation in a patient with amyotrophic lateral sclerosis
Neurobiology of Aging xxx (2016) 1.e1e1.e5
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Identification of a novel loss-of-function C9orf72 splice site mutation in a patient with amyotrophic lateral sclerosis Fang Liu a, b,1, Qing Liu b, c,1, Chao Xia Lu a, b, Bo Cui b, c, Xia Nan Guo a, b, Rong Rong Wang a, b, Ming Sheng Liu b, c, Xiao Guang Li b, c, Li-ying Cui b, c, Xue Zhang a, b, c, * a b c
McKusick-Zhang Center for Genetic Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Beijing, China Neuroscience Center, CAMS & PUMC, Beijing, China Department of Neurology and Laboratory of Clinical Genetics, Peking Union Medical College Hospital, CAMS and PUMC, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2016 Received in revised form 28 June 2016 Accepted 29 July 2016
Abnormal expansion of a hexanucleotide GGGGCC repeat in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasians. However, the underlying pathologic mechanisms remain controversial, and both loss-of-function and gain-of-function models have been proposed. To gain further insight into these mechanisms, we performed mutation analysis of C9orf72 in 276 Han Chinese patients with ALS. We identified GGGGCC expansions in 2 cases of sporadic ALS with 38 and 63 repeats, as well as a novel splice site mutation (c.601-2A>G) in a third case. These genetic alterations were not detected in 332 control patients without neurological disease. Intriguingly, functional analysis revealed that the splice site mutation disrupted the reading frame, creating a premature stop codon (p.I201fsX235). Decreased levels of the mutant messenger RNA were detected in patient cells, suggesting that it may undergo nonsense-mediated messenger RNA decay. Taken together, these results demonstrate that C9orf72 mutation is infrequently associated with ALS in Han Chinese patients and suggest that a splice site mutation in C9orf72 may lead to loss of function due to haploinsufficiency of the resulting protein. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis C9orf72 GGGGCC hexanucleotide repeat expansion Splice site mutation Loss-of-function
1. Introduction Amyotrophic lateral sclerosis (ALS) is a devastating, late-onset neurodegenerative disease that is characterized by progressive loss of motor neurons in the brain and spinal cord, leading to progressive weakness and death within 2e3 years after symptom onset. Approximately, 5%e10% of cases can be classified as familial ALS (FALS), but the vast majority of cases are considered to be sporadic ALS (SALS) (Al-Chalabi and Hardiman, 2013; Kiernan et al., 2011; Mitchell and Borasio, 2007; Robberecht and Philips, 2013; Swinnen and Robberecht, 2014). To date, 26 genes have been identified to be associated with ALS, including several causal genes, such as SOD1, TARDBP, FUS, VAPB, ANG, VCP, OPTN, and UBQLN2. Mutations in these genes account for two-thirds of FALS cases and nearly 11% of SALS cases (Al-Chalabi et al., 2012; Ingre et al., 2015; Pasinelli and Brown, 2006; Renton et al., 2014). Expansion of the hexanucleotide GGGGCC (G4C2) repeat present in the noncoding region of C9orf72 has been identified previously as a * Corresponding author at: McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China. Tel./fax: þ86-10-69155110. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to the study. 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.07.027
major cause of ALS and frontotemporal dementia (FTD) in Caucasian populations (DeJesus-Hernandez et al., 2011; Renton et al., 2011) but is thought to be rare in Asian cohorts with ALS (He et al., 2015; Jiao et al., 2014; Ogaki et al., 2012; Zou et al., 2013). A majority of unaffected individuals harbor C9orf72 alleles with less than 20 G4C2 repeats (DeJesus-Hernandez et al., 2011; Renton et al., 2011), and longer expansions have been reported to result in ALS (Rutherford et al., 2012). However, the pathologic mechanisms underlying development of ALS in patients with expanded C9orf72 G4C2 repeats are not well understood, and both gain-of-function and loss-offunction models have been proposed (Mizielinska and Isaacs, 2014). In this study, we analyzed the frequencies of both G4C2 repeat expansions and rare coding variants of C9orf72 in a Chinese cohort of ALS cases and identified a novel splice site mutation that appears to result in loss of function due to haploinsufficiency. 2. Methods 2.1. Subjects A total of 276 patients of Han Chinese descent diagnosed with probable or definitive ALS-based on El Escorial criteria (Brooks et al., 2000) were recruited from the Department of Neurology of
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Peking Union Medical College Hospital from 2009 to 2015. Routine blood biochemical analysis, neuroimaging, and electromyography were conducted to exclude disorders that mimic ALS. All subjects provided written consent, and this study was approved by the Ethical Review Board of Peking Union Medical College Hospital. Control samples for DNA analysis were obtained from 332 ethnically matched individuals with no diagnosis of a neurological disorder. 2.2. Mutation screening 2.2.1. Analysis of G4C2 hexanucleotide repeat expansion Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s recommended instructions. The repeat region of C9orf72 was amplified by polymerase chain reaction (PCR) using a flourescein-labeled, gene-specific forward primer (6-FAM-50 -AGTCGCTAGAGGCGAAAGC30 ) complementary to a sequence in exon 1; the reverse primer consisted of 4 GGGGCC repeats (50 -TACGCATCCCAGTTTGAGA CGG GGGCCGGGGCCGGGGCCGGGGCC-30 ) and an anchor sequence (50 -TA CGCATCCCAGTTTGAGACG-30 ) (Renton et al., 2011). Reactions contained 0.6-U PrimeSTAR GXL DNA Polymerase (TaKaRa), 1 PrimeSTAR GXL Buffer (TaKaRa), 200-mM dNTPs (TaKaRa), 1-M betaine (SigmaeAldrich), 5% dimethyl sulfoxide (SigmaeAldrich), and 7-deaza-2-deoxy GTP (New England Biolabs) in place of deoxyguanosine triphosphate. Touchdown PCR was carried out under the following conditions: 1 denaturating cycle of 94 C for 3 minutes; 7 cycles of 98 C for 10 seconds, 70 C (decreasing 2 C per cycle) for 30 seconds, and 68 C for 3 minutes; and 23 cycles of 98 C for 10 seconds, 56 C for 30 seconds, and 68 C for 3 minutes (DeJesusHernandez et al., 2011). PCR products were analyzed using an ABI3730 DNA Analyzer (Applied Biosystems) and visualized using GeneMapper software (version 4; Applied Biosystems). 2.2.2. Sequencing of C9orf72-coding variants Targeted sequencing of multiple ALS genes was performed using an Ion PGM Sequencer (Personal Genome Machine, Life Technologies Corporation). Briefly, the National Center for Biotechnology Information (NCBI) consensus coding sequences and untranslated regions of 24 genes previously shown to cause ALS, including C9orf72, were incorporated into a single screening panel. The resulting sequence data were analyzed using Ion Torrent Variant Caller software, version 4.0; annotated using SeattleSeq Annotation 138 (http://snp.gs. washington.edu/SeattleSeqAnnotation138/); and confirmed by Sanger sequencing. The average depth of sequencing was 100, and the uniformity of coverage was 92%. Exome variant data from 60,706 unrelated individuals in the Exome Variant Server (EVS) database served as a control (http://evs.gs.washington.edu/EVS/) and included 4206 individuals of East Asian descent. Single nucleotide variations were confirmed by Sanger sequencing of samples from ALS patients and control unaffected individuals.
2.4. Functional splicing reporter minigene assay The minigene used as a splicing reporter was constructed by PCR amplification of wild-type and mutant genomic DNA sequences as described previously (Cooper, 2005; Gaildrat et al., 2010). The amplified sequences included exons 3 through 5 of the C9orf72 gene, together with approximately 150 basepairs (bp) from the 50 and 30 flanking intronic sequences (illustrated in Fig. S1). C9orf72 minigene products were digested with BamHI and MluI (New England Biolabs) and cloned into the pCAS1 reporter vector. The resulting constructs were transfected into Hela cells. Total cellular RNA was extracted after 24 hours, followed by RT-PCR analysis. 2.5. Statistical analysis The ManneWhitney U test was used to compare differences in expansion frequencies between ALS cases and unaffected controls. p-values less than 0.05 were considered statistically significant. 3. Results A total of 276 ALS patients were enrolled in this study, including 24 with FALS (19.6%) and 252 with SALS (91.3%). The demographic and disease characteristics of the subjects were comparable to those of other center-based cohorts (Harms and Baloh, 2013). The mean ages of onset were 49.7 11.5 (mean standard deviation) and 49.9 11.4 for FALS and SALS, respectively; 22.8% of patients exhibited bulbar onset. Disease-associated expansions (containing more than 30 repeats) were identified in 2 SALS patients (0.79% of SALS patients, 0.72% overall) and consisted of 38 and 63 repeats, respectively (Fig. 1). No rare variants of other known ALS genes were detected in either patient. Both patients with expanded repeats exhibited late-onset disease with typical progression and no obvious documented cognitive decline. Numbers of C9orf72 repeats in other ALS patients were all within normal ranges (1e23 repeats, shown in Fig. 2). None of the control cases (n ¼ 332) exhibited C9orf72 repeat expansion, and the maximum number of repeats
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from blood lymphocytes using TRIzol LS reagent (Life Technologies) according to the manufacturer’s recommended instructions. RT reactions were performed using the GoScript Reverse Transcription System (Promega). Complementary DNA sequences containing the splice site mutation were amplified by PCR using a forward primer complementary to a sequence located in exon 2 of the C9orf72 gene (50 -GAATGGGGATCGCAGCACATA-30 ) and a reverse primer located in exon 8 (50 -GCAGCACAAAGCTTCCAGTTG-30 ). The resulting PCR products were analyzed by Sanger sequencing.
Fig. 1. Electropherograms showing expanded hexanucleotide repeats in C9orf72. The vertical axis represents the fluorescence intensity, and the horizontal axis displays product size. Repeat expansions are associated with a characteristic sawtooth pattern with a 6-basepair periodicity. In comparison to a control individual harboring 2 repeats (top block), abnormal expansions of 38 and 63 repeats were detected in 2 SALS patients (P006 and P331, middle and bottom blocks, respectively). Abbreviation: SALS, sporadic amyotrophic lateral sclerosis.
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observed in the control group was 15. No significant difference was observed in the distributions of repeat lengths between patients and control cases (p ¼ 0.805). Targeted resequencing of C9orf72 in the patient cohort revealed a heterozygous splice site mutation (c.601-2A>G) in a single patient that was subsequently confirmed by Sanger sequencing (Fig. S2). No other rare C9orf72 variants or known variants of other ALS genes were detected. The patient was female who exhibited bulbar onset at the age of 51 and committed suicide 4 years later. Her family refused autopsy. The patient’s history was consistent with SALS, as there was no reported family history of muscle weakness and dementia, and both of her parents died at ages greater than sixty years old due to unrelated causes. Her mother died of a cerebral vascular attack, and her father died of cancer. The patient’s initial neuropsychological evaluation was not documented. According to reports by her family during follow up, her disease manifested with rapidly progressive muscle weakness, dysarthria and dysphagia, pathologic laughter and crying, stereotyped speech, and loss of sympathy. She exhibited no obvious dyspnea before her death. The mutation, which altered a highly conserved nucleotide, was not found in 4206 individuals of East Asian descent in the EVS database or in 332 ethnically matched controls. Sequence analysis indicated that the A-to-G transition (c.6012A>G) abolished the invariable consensus AG splice acceptor of intron 4 and may have activated a cryptic splice site in the third and fourth nucleotides of exon 5 (c.601_604 del ATAG; shown in Fig. 3A). We also observed that levels of the mutant C9orf72 messenger RNA (mRNA) were decreased relative to the wild type in leukocytes from the patient (Fig. 3B). Further analysis using a functional splicing reporter minigene assay confirmed that the mutation disrupted the reading frame of the resulting mRNA by deleting 4 bases (ATAG), creating a premature stop codon (p.I201fsX235). 4. Discussion The precise pathologic cutoff for expansion of the hexanucleotide G4C2 repeat in C9orf72 is currently poorly defined (DeJesusHernandez et al., 2011; Hardy and Rogaeva, 2014; Mackenzie et al., 2014; Renton et al., 2011). Normal G4C2 expansions are considered to range from 2 to 23 units, and pathogenic expansions generally exceed 30 repeats and may even contain hundreds or thousands of repeats (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012; Renton et al., 2011). However, intermediate repeat
Fig. 2. Distributions of C9orf72 repeat expansions in patients and unaffected controls. No significant difference was observed between the distributions of repeat lengths between patients (n ¼ 276) and controls (n ¼ 332) using ManneWhitney U test (p ¼ 0.805). Abbreviation: ALS, amyotrophic lateral sclerosis.
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lengths (15e30 units) have been proposed to be associated with ALS, and expansions of 20e22 repeats have been reported in familial FTD (Byrne et al., 2014). In our cohort of patients of Chinese Han descent, with the exception of 2 affected individuals with highly expanded repeats (38 and 63 units), the number of G4C2 repeats was no more than 15 in either control cases or ALS patients. Intermediate length expansions were not observed in our ALS cohort or healthy controls, and the distributions of G4C2 repeat lengths were also not significantly different between the control and ALS groups (p ¼ 0.805). It is possible that the patients with a small expansion in blood (e.g., 38 and 63 repeats) could have a large pathologic expansion in the central nervous system as a result of somatic instability (Fratta et al., 2015). The frequencies of C9orf72 repeat expansions have been shown to vary substantially in ALS cohorts from different geographic regions worldwide (Alavi et al., 2014; Debray et al., 2013; Fahey et al., 2014; Ratti et al., 2012). Among Caucasian populations, high frequencies of expanded repeats have been reported, ranging from 25% to 50% for FALS and 4% to 7% for SALS (Corrado et al., 2011; Ratti et al., 2012), although several studies have excluded a founder effect (Fratta et al., 2015; Xi et al., 2015). In contrast, low frequencies of expanded repeats have been observed in Asian populations, including 18% (4/22) of FALS and 2% (2/102) of SALS cases in Taiwanese patients (Tsai et al., 2012), 0% (0/11) of FALS and 0.4% (2/552) of SALS in Japanese patients (Ogaki et al., 2012), 10% (1/10) of FALS and 0.8% (1/128) of SALS in Chinese patients (Jiao et al., 2014), and 0% of ALS cases in Korean patients (Jang et al., 2013). In accordance with reports from other Asian cohorts, we identified only 2 SALS cases (<1%) harboring pathogenic expansions of >30 repeats, suggesting that abnormal expansion of C9orf72 is infrequent in Chinese patients with ALS. The pathologic mechanisms underlying development of ALS in patients with G4C2 repeat expansions are controversial, as both loss-of-function and gain-of-function mechanisms have been proposed (Lewis, 2015; Mizielinska and Isaacs, 2014; Zhang et al., 2015). Haploinsufficiency of C9orf72 was originally suggested as a causative factor because reduced transcript abundance was detected in expansion carriers (DeJesus-Hernandez et al., 2011; Renton et al., 2011). However, other studies failed to find differences in C9orf72 transcript levels (Al-Chalabi and Hardiman, 2013; Al-Chalabi et al., 2012; Mitchell and Borasio, 2007; Mizielinska and Isaacs, 2014; Ogaki et al., 2012; Pasinelli and Brown, 2006; Ratti et al., 2012; Renton et al., 2014; Robberecht and Philips, 2013). Similarly, sequencing of the coding region of C9orf72 failed to detect any rare causative variants (Koppers et al., 2013). Our study identified a C9orf72 splice site mutation in a Chinese ALS patient, and subsequent functional analyses revealed that the mutation created a premature translation stop codon. Decreased levels of the mutant C9orf72 mRNA, potentially resulting from nonsense-mediated mRNA decay, were also observed, suggesting that haploinsufficiency may be a possible pathologic mechanism in this case. The patient exhibited characteristics of radical disease progression and would have probably been diagnosed as ALS-FTD if she had survived, since she developed prominent changes in personality and social behavior. Because pathogenicity of C9orf72 in ALS has been shown to be associated with accumulation of TDP43 aggregates in the cytoplasm (Chew et al., 2015; Mori et al., 2013), an autopsy would have been highly valuable for verifying whether the mutation was associated with TDP43 pathology typical of cases harboring C9orf72 mutations. Although autopsy was declined by the family of the patient in this case, it may prove useful in the future if additional pathologic variants are identified in the coding regions of C9orf72. In conclusion, in this study, we have identified a novel and potentially pathogenic splice site mutation in C9orf72. Analysis of
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Fig. 3. A splice site loss-of-function mutation identified in C9orf72. (A) The splice site mutation (c.601-2A>G) was located in the acceptor site of intron 4. It disrupted the reading frame and created a premature termination codon (p.I201fsX235). (B) Levels of the mutant C9orf72 mRNA were decreased in patient leukocytes, potentially resulting from nonsense-mediated mRNA decay. (C) A functional splicing reporter minigene assay confirmed deletion of 4 bases (ATAG) in the mutant C9orf72 messenger RNA (mRNA) compared with the wild type mRNA.
this variant provides evidence supporting a loss-of-function mechanism in the pathogenesis of C9orf72-associated cases of ALS. Disclosure statement The authors disclose no conflicts of interest. Acknowledgements The authors thank all of the patients for their collaboration. This study was supported by the Beijing Municipal Science and Technology Commission (grant number Z151100003915078), the National Natural Science Foundation of China (NSFC; grant number 81230015), and a grant from Neuroscience Center of Chinese Academy of Medical Sciences. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neurobiolaging.2016.07.027.
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Mizielinska, S., Isaacs, A.M., 2014. C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia: gain or loss of function? Curr. Opin. Neurol. 27, 515e523. Mori, K., Weng, S.-M., Arzberger, T., May, S., Rentzsch, K., Kremmer, E., Schmid, B., Kretzschmar, H.A., Cruts, M., Van Broeckhoven, C., 2013. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335e1338. Ogaki, K., Li, Y., Atsuta, N., Tomiyama, H., Funayama, M., Watanabe, H., Nakamura, R., Yoshino, H., Yato, S., Tamura, A., Naito, Y., Taniguchi, A., Fujita, K., Izumi, Y., Kaji, R., Hattori, N., Sobue, G. Japanese Consortium for Amyotrophic Lateral Sclerosis Research, 2012. Analysis of C9orf72 repeat expansion in 563 Japanese patients with amyotrophic lateral sclerosis. Neurobiol. Aging 33, 2527.e11e2527.e16. Pasinelli, P., Brown, R.H., 2006. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat. Rev. Neurosci. 7, 710e723. Ratti, A., Corrado, L., Castellotti, B., Del Bo, R., Fogh, I., Cereda, C., Tiloca, C., D’Ascenzo, C., Bagarotti, A., Pensato, V., Ranieri, M., Gagliardi, S., Calini, D., Mazzini, L., Taroni, F., Corti, S., Ceroni, M., Oggioni, G.D., Lin, K., Powell, J.F., Soraru, G., Ticozzi, N., Comi, G.P., D’Alfonso, S., Gellera, C., Silani, V. SLAGEN Consortium, 2012. C9ORF72 repeat expansion in a large Italian ALS cohort: evidence of a founder effect. Neurobiol. Aging 33, 2528.e7e2528.e14. Renton, A.E., Chiò, A., Traynor, B.J., 2014. State of play in amyotrophic lateral sclerosis genetics. Nat. Neurosci. 17, 17e23. Renton, A.E., Majounie, E., Waite, A., Simon-Sanchez, J., Rollinson, S., Gibbs, J.R., Schymick, J.C., Laaksovirta, H., van Swieten, J.C., Myllykangas, L., Kalimo, H., Paetau, A., Abramzon, Y., Remes, A.M., Kaganovich, A., Scholz, S.W., Duckworth, J., Ding, J., Harmer, D.W., Hernandez, D.G., Johnson, J.O., Mok, K., Ryten, M., Trabzuni, D., Guerreiro, R.J., Orrell, R.W., Neal, J., Murray, A., Pearson, J., Jansen, I.E., Sondervan, D., Seelaar, H., Blake, D., Young, K., Halliwell, N., Callister, J.B., Toulson, G., Richardson, A., Gerhard, A., Snowden, J., Mann, D., Neary, D., Nalls, M.A., Peuralinna, T., Jansson, L., Isoviita, V.M., Kaivorinne, A.L., Holtta-Vuori, M., Ikonen, E., Sulkava, R., Benatar, M., Wuu, J., Chio, A., Restagno, G., Borghero, G., Sabatelli, M., Consortium, I., Heckerman, D., Rogaeva, E., Zinman, L., Rothstein, J.D., Sendtner, M., Drepper, C., Eichler, E.E., Alkan, C., Abdullaev, Z., Pack, S.D., Dutra, A., Pak, E., Hardy, J., Singleton, A., Williams, N.M., Heutink, P., Pickering-Brown, S., Morris, H.R., Tienari, P.J., Traynor, B.J., 2011. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257e268. Robberecht, W., Philips, T., 2013. The changing scene of amyotrophic lateral sclerosis. Nat. Rev. Neurosci. 14, 248e264. Rutherford, N.J., Heckman, M.G., Dejesus-Hernandez, M., Baker, M.C., Soto-Ortolaza, A.I., Rayaprolu, S., Stewart, H., Finger, E., Volkening, K., Seeley, W.W., Hatanpaa, K.J., Lomen-Hoerth, C., Kertesz, A., Bigio, E.H., Lippa, C., Knopman, D.S., Kretzschmar, H.A., Neumann, M., Caselli, R.J., White 3rd, C.L., Mackenzie, I.R., Petersen, R.C., Strong, M.J., Miller, B.L., Boeve, B.F., Uitti, R.J., Boylan, K.B., Wszolek, Z.K., Graff-Radford, N.R., Dickson, D.W., Ross, O.A., Rademakers, R., 2012. Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype. Neurobiol. Aging 33, 2950.e5e2950.e7. Swinnen, B., Robberecht, W., 2014. The phenotypic variability of amyotrophic lateral sclerosis. Nat. Rev. Neurol. 10, 661e670. Tsai, C.P., Soong, B.W., Tu, P.H., Lin, K.P., Fuh, J.L., Tsai, P.C., Lu, Y.C., Lee, I.H., Lee, Y.C., 2012. A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan. Neurobiol. Aging 33, 2232.e11e2232.e18. Xi, Z., van Blitterswijk, M., Zhang, M., McGoldrick, P., McLean, J.R., Yunusova, Y., Knock, E., Moreno, D., Sato, C., McKeever, P.M., 2015. Jump from pre-mutation to pathologic expansion in C9orf72. Am. J. Hum. Genet. 96, 962e970. Zhang, K., Donnelly, C.J., Haeusler, A.R., Grima, J.C., Machamer, J.B., Steinwald, P., Daley, E.L., Miller, S.J., Cunningham, K.M., Vidensky, S., 2015. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56e61. Zou, Z.Y., Li, X.G., Liu, M.S., Cui, L.Y., 2013. Screening for C9orf72 repeat expansions in Chinese amyotrophic lateral sclerosis patients. Neurobiol. Aging 34, 1710.e5e1710.e6.
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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Identification of a novel loss-of-function C9orf72 splice site mutation in a patient with amyotrophic lateral sclerosis Fang Liu a, b,1, Qing Liu b, c,1, Chao Xia Lu a, b, Bo Cui b, c, Xia Nan Guo a, b, Rong Rong Wang a, b, Ming Sheng Liu b, c, Xiao Guang Li b, c, Li-ying Cui b, c, Xue Zhang a, b, c, * a b c
McKusick-Zhang Center for Genetic Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Beijing, China Neuroscience Center, CAMS & PUMC, Beijing, China Department of Neurology and Laboratory of Clinical Genetics, Peking Union Medical College Hospital, CAMS and PUMC, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2016 Received in revised form 28 June 2016 Accepted 29 July 2016
Abnormal expansion of a hexanucleotide GGGGCC repeat in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasians. However, the underlying pathologic mechanisms remain controversial, and both loss-of-function and gain-of-function models have been proposed. To gain further insight into these mechanisms, we performed mutation analysis of C9orf72 in 276 Han Chinese patients with ALS. We identified GGGGCC expansions in 2 cases of sporadic ALS with 38 and 63 repeats, as well as a novel splice site mutation (c.601-2A>G) in a third case. These genetic alterations were not detected in 332 control patients without neurological disease. Intriguingly, functional analysis revealed that the splice site mutation disrupted the reading frame, creating a premature stop codon (p.I201fsX235). Decreased levels of the mutant messenger RNA were detected in patient cells, suggesting that it may undergo nonsense-mediated messenger RNA decay. Taken together, these results demonstrate that C9orf72 mutation is infrequently associated with ALS in Han Chinese patients and suggest that a splice site mutation in C9orf72 may lead to loss of function due to haploinsufficiency of the resulting protein. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis C9orf72 GGGGCC hexanucleotide repeat expansion Splice site mutation Loss-of-function
1. Introduction Amyotrophic lateral sclerosis (ALS) is a devastating, late-onset neurodegenerative disease that is characterized by progressive loss of motor neurons in the brain and spinal cord, leading to progressive weakness and death within 2e3 years after symptom onset. Approximately, 5%e10% of cases can be classified as familial ALS (FALS), but the vast majority of cases are considered to be sporadic ALS (SALS) (Al-Chalabi and Hardiman, 2013; Kiernan et al., 2011; Mitchell and Borasio, 2007; Robberecht and Philips, 2013; Swinnen and Robberecht, 2014). To date, 26 genes have been identified to be associated with ALS, including several causal genes, such as SOD1, TARDBP, FUS, VAPB, ANG, VCP, OPTN, and UBQLN2. Mutations in these genes account for two-thirds of FALS cases and nearly 11% of SALS cases (Al-Chalabi et al., 2012; Ingre et al., 2015; Pasinelli and Brown, 2006; Renton et al., 2014). Expansion of the hexanucleotide GGGGCC (G4C2) repeat present in the noncoding region of C9orf72 has been identified previously as a * Corresponding author at: McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China. Tel./fax: þ86-10-69155110. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to the study. 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.07.027
major cause of ALS and frontotemporal dementia (FTD) in Caucasian populations (DeJesus-Hernandez et al., 2011; Renton et al., 2011) but is thought to be rare in Asian cohorts with ALS (He et al., 2015; Jiao et al., 2014; Ogaki et al., 2012; Zou et al., 2013). A majority of unaffected individuals harbor C9orf72 alleles with less than 20 G4C2 repeats (DeJesus-Hernandez et al., 2011; Renton et al., 2011), and longer expansions have been reported to result in ALS (Rutherford et al., 2012). However, the pathologic mechanisms underlying development of ALS in patients with expanded C9orf72 G4C2 repeats are not well understood, and both gain-of-function and loss-offunction models have been proposed (Mizielinska and Isaacs, 2014). In this study, we analyzed the frequencies of both G4C2 repeat expansions and rare coding variants of C9orf72 in a Chinese cohort of ALS cases and identified a novel splice site mutation that appears to result in loss of function due to haploinsufficiency. 2. Methods 2.1. Subjects A total of 276 patients of Han Chinese descent diagnosed with probable or definitive ALS-based on El Escorial criteria (Brooks et al., 2000) were recruited from the Department of Neurology of
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Peking Union Medical College Hospital from 2009 to 2015. Routine blood biochemical analysis, neuroimaging, and electromyography were conducted to exclude disorders that mimic ALS. All subjects provided written consent, and this study was approved by the Ethical Review Board of Peking Union Medical College Hospital. Control samples for DNA analysis were obtained from 332 ethnically matched individuals with no diagnosis of a neurological disorder. 2.2. Mutation screening 2.2.1. Analysis of G4C2 hexanucleotide repeat expansion Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s recommended instructions. The repeat region of C9orf72 was amplified by polymerase chain reaction (PCR) using a flourescein-labeled, gene-specific forward primer (6-FAM-50 -AGTCGCTAGAGGCGAAAGC30 ) complementary to a sequence in exon 1; the reverse primer consisted of 4 GGGGCC repeats (50 -TACGCATCCCAGTTTGAGA CGG GGGCCGGGGCCGGGGCCGGGGCC-30 ) and an anchor sequence (50 -TA CGCATCCCAGTTTGAGACG-30 ) (Renton et al., 2011). Reactions contained 0.6-U PrimeSTAR GXL DNA Polymerase (TaKaRa), 1 PrimeSTAR GXL Buffer (TaKaRa), 200-mM dNTPs (TaKaRa), 1-M betaine (SigmaeAldrich), 5% dimethyl sulfoxide (SigmaeAldrich), and 7-deaza-2-deoxy GTP (New England Biolabs) in place of deoxyguanosine triphosphate. Touchdown PCR was carried out under the following conditions: 1 denaturating cycle of 94 C for 3 minutes; 7 cycles of 98 C for 10 seconds, 70 C (decreasing 2 C per cycle) for 30 seconds, and 68 C for 3 minutes; and 23 cycles of 98 C for 10 seconds, 56 C for 30 seconds, and 68 C for 3 minutes (DeJesusHernandez et al., 2011). PCR products were analyzed using an ABI3730 DNA Analyzer (Applied Biosystems) and visualized using GeneMapper software (version 4; Applied Biosystems). 2.2.2. Sequencing of C9orf72-coding variants Targeted sequencing of multiple ALS genes was performed using an Ion PGM Sequencer (Personal Genome Machine, Life Technologies Corporation). Briefly, the National Center for Biotechnology Information (NCBI) consensus coding sequences and untranslated regions of 24 genes previously shown to cause ALS, including C9orf72, were incorporated into a single screening panel. The resulting sequence data were analyzed using Ion Torrent Variant Caller software, version 4.0; annotated using SeattleSeq Annotation 138 (http://snp.gs. washington.edu/SeattleSeqAnnotation138/); and confirmed by Sanger sequencing. The average depth of sequencing was 100, and the uniformity of coverage was 92%. Exome variant data from 60,706 unrelated individuals in the Exome Variant Server (EVS) database served as a control (http://evs.gs.washington.edu/EVS/) and included 4206 individuals of East Asian descent. Single nucleotide variations were confirmed by Sanger sequencing of samples from ALS patients and control unaffected individuals.
2.4. Functional splicing reporter minigene assay The minigene used as a splicing reporter was constructed by PCR amplification of wild-type and mutant genomic DNA sequences as described previously (Cooper, 2005; Gaildrat et al., 2010). The amplified sequences included exons 3 through 5 of the C9orf72 gene, together with approximately 150 basepairs (bp) from the 50 and 30 flanking intronic sequences (illustrated in Fig. S1). C9orf72 minigene products were digested with BamHI and MluI (New England Biolabs) and cloned into the pCAS1 reporter vector. The resulting constructs were transfected into Hela cells. Total cellular RNA was extracted after 24 hours, followed by RT-PCR analysis. 2.5. Statistical analysis The ManneWhitney U test was used to compare differences in expansion frequencies between ALS cases and unaffected controls. p-values less than 0.05 were considered statistically significant. 3. Results A total of 276 ALS patients were enrolled in this study, including 24 with FALS (19.6%) and 252 with SALS (91.3%). The demographic and disease characteristics of the subjects were comparable to those of other center-based cohorts (Harms and Baloh, 2013). The mean ages of onset were 49.7 11.5 (mean standard deviation) and 49.9 11.4 for FALS and SALS, respectively; 22.8% of patients exhibited bulbar onset. Disease-associated expansions (containing more than 30 repeats) were identified in 2 SALS patients (0.79% of SALS patients, 0.72% overall) and consisted of 38 and 63 repeats, respectively (Fig. 1). No rare variants of other known ALS genes were detected in either patient. Both patients with expanded repeats exhibited late-onset disease with typical progression and no obvious documented cognitive decline. Numbers of C9orf72 repeats in other ALS patients were all within normal ranges (1e23 repeats, shown in Fig. 2). None of the control cases (n ¼ 332) exhibited C9orf72 repeat expansion, and the maximum number of repeats
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from blood lymphocytes using TRIzol LS reagent (Life Technologies) according to the manufacturer’s recommended instructions. RT reactions were performed using the GoScript Reverse Transcription System (Promega). Complementary DNA sequences containing the splice site mutation were amplified by PCR using a forward primer complementary to a sequence located in exon 2 of the C9orf72 gene (50 -GAATGGGGATCGCAGCACATA-30 ) and a reverse primer located in exon 8 (50 -GCAGCACAAAGCTTCCAGTTG-30 ). The resulting PCR products were analyzed by Sanger sequencing.
Fig. 1. Electropherograms showing expanded hexanucleotide repeats in C9orf72. The vertical axis represents the fluorescence intensity, and the horizontal axis displays product size. Repeat expansions are associated with a characteristic sawtooth pattern with a 6-basepair periodicity. In comparison to a control individual harboring 2 repeats (top block), abnormal expansions of 38 and 63 repeats were detected in 2 SALS patients (P006 and P331, middle and bottom blocks, respectively). Abbreviation: SALS, sporadic amyotrophic lateral sclerosis.
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observed in the control group was 15. No significant difference was observed in the distributions of repeat lengths between patients and control cases (p ¼ 0.805). Targeted resequencing of C9orf72 in the patient cohort revealed a heterozygous splice site mutation (c.601-2A>G) in a single patient that was subsequently confirmed by Sanger sequencing (Fig. S2). No other rare C9orf72 variants or known variants of other ALS genes were detected. The patient was female who exhibited bulbar onset at the age of 51 and committed suicide 4 years later. Her family refused autopsy. The patient’s history was consistent with SALS, as there was no reported family history of muscle weakness and dementia, and both of her parents died at ages greater than sixty years old due to unrelated causes. Her mother died of a cerebral vascular attack, and her father died of cancer. The patient’s initial neuropsychological evaluation was not documented. According to reports by her family during follow up, her disease manifested with rapidly progressive muscle weakness, dysarthria and dysphagia, pathologic laughter and crying, stereotyped speech, and loss of sympathy. She exhibited no obvious dyspnea before her death. The mutation, which altered a highly conserved nucleotide, was not found in 4206 individuals of East Asian descent in the EVS database or in 332 ethnically matched controls. Sequence analysis indicated that the A-to-G transition (c.6012A>G) abolished the invariable consensus AG splice acceptor of intron 4 and may have activated a cryptic splice site in the third and fourth nucleotides of exon 5 (c.601_604 del ATAG; shown in Fig. 3A). We also observed that levels of the mutant C9orf72 messenger RNA (mRNA) were decreased relative to the wild type in leukocytes from the patient (Fig. 3B). Further analysis using a functional splicing reporter minigene assay confirmed that the mutation disrupted the reading frame of the resulting mRNA by deleting 4 bases (ATAG), creating a premature stop codon (p.I201fsX235). 4. Discussion The precise pathologic cutoff for expansion of the hexanucleotide G4C2 repeat in C9orf72 is currently poorly defined (DeJesusHernandez et al., 2011; Hardy and Rogaeva, 2014; Mackenzie et al., 2014; Renton et al., 2011). Normal G4C2 expansions are considered to range from 2 to 23 units, and pathogenic expansions generally exceed 30 repeats and may even contain hundreds or thousands of repeats (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012; Renton et al., 2011). However, intermediate repeat
Fig. 2. Distributions of C9orf72 repeat expansions in patients and unaffected controls. No significant difference was observed between the distributions of repeat lengths between patients (n ¼ 276) and controls (n ¼ 332) using ManneWhitney U test (p ¼ 0.805). Abbreviation: ALS, amyotrophic lateral sclerosis.
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lengths (15e30 units) have been proposed to be associated with ALS, and expansions of 20e22 repeats have been reported in familial FTD (Byrne et al., 2014). In our cohort of patients of Chinese Han descent, with the exception of 2 affected individuals with highly expanded repeats (38 and 63 units), the number of G4C2 repeats was no more than 15 in either control cases or ALS patients. Intermediate length expansions were not observed in our ALS cohort or healthy controls, and the distributions of G4C2 repeat lengths were also not significantly different between the control and ALS groups (p ¼ 0.805). It is possible that the patients with a small expansion in blood (e.g., 38 and 63 repeats) could have a large pathologic expansion in the central nervous system as a result of somatic instability (Fratta et al., 2015). The frequencies of C9orf72 repeat expansions have been shown to vary substantially in ALS cohorts from different geographic regions worldwide (Alavi et al., 2014; Debray et al., 2013; Fahey et al., 2014; Ratti et al., 2012). Among Caucasian populations, high frequencies of expanded repeats have been reported, ranging from 25% to 50% for FALS and 4% to 7% for SALS (Corrado et al., 2011; Ratti et al., 2012), although several studies have excluded a founder effect (Fratta et al., 2015; Xi et al., 2015). In contrast, low frequencies of expanded repeats have been observed in Asian populations, including 18% (4/22) of FALS and 2% (2/102) of SALS cases in Taiwanese patients (Tsai et al., 2012), 0% (0/11) of FALS and 0.4% (2/552) of SALS in Japanese patients (Ogaki et al., 2012), 10% (1/10) of FALS and 0.8% (1/128) of SALS in Chinese patients (Jiao et al., 2014), and 0% of ALS cases in Korean patients (Jang et al., 2013). In accordance with reports from other Asian cohorts, we identified only 2 SALS cases (<1%) harboring pathogenic expansions of >30 repeats, suggesting that abnormal expansion of C9orf72 is infrequent in Chinese patients with ALS. The pathologic mechanisms underlying development of ALS in patients with G4C2 repeat expansions are controversial, as both loss-of-function and gain-of-function mechanisms have been proposed (Lewis, 2015; Mizielinska and Isaacs, 2014; Zhang et al., 2015). Haploinsufficiency of C9orf72 was originally suggested as a causative factor because reduced transcript abundance was detected in expansion carriers (DeJesus-Hernandez et al., 2011; Renton et al., 2011). However, other studies failed to find differences in C9orf72 transcript levels (Al-Chalabi and Hardiman, 2013; Al-Chalabi et al., 2012; Mitchell and Borasio, 2007; Mizielinska and Isaacs, 2014; Ogaki et al., 2012; Pasinelli and Brown, 2006; Ratti et al., 2012; Renton et al., 2014; Robberecht and Philips, 2013). Similarly, sequencing of the coding region of C9orf72 failed to detect any rare causative variants (Koppers et al., 2013). Our study identified a C9orf72 splice site mutation in a Chinese ALS patient, and subsequent functional analyses revealed that the mutation created a premature translation stop codon. Decreased levels of the mutant C9orf72 mRNA, potentially resulting from nonsense-mediated mRNA decay, were also observed, suggesting that haploinsufficiency may be a possible pathologic mechanism in this case. The patient exhibited characteristics of radical disease progression and would have probably been diagnosed as ALS-FTD if she had survived, since she developed prominent changes in personality and social behavior. Because pathogenicity of C9orf72 in ALS has been shown to be associated with accumulation of TDP43 aggregates in the cytoplasm (Chew et al., 2015; Mori et al., 2013), an autopsy would have been highly valuable for verifying whether the mutation was associated with TDP43 pathology typical of cases harboring C9orf72 mutations. Although autopsy was declined by the family of the patient in this case, it may prove useful in the future if additional pathologic variants are identified in the coding regions of C9orf72. In conclusion, in this study, we have identified a novel and potentially pathogenic splice site mutation in C9orf72. Analysis of
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Fig. 3. A splice site loss-of-function mutation identified in C9orf72. (A) The splice site mutation (c.601-2A>G) was located in the acceptor site of intron 4. It disrupted the reading frame and created a premature termination codon (p.I201fsX235). (B) Levels of the mutant C9orf72 mRNA were decreased in patient leukocytes, potentially resulting from nonsense-mediated mRNA decay. (C) A functional splicing reporter minigene assay confirmed deletion of 4 bases (ATAG) in the mutant C9orf72 messenger RNA (mRNA) compared with the wild type mRNA.
this variant provides evidence supporting a loss-of-function mechanism in the pathogenesis of C9orf72-associated cases of ALS. Disclosure statement The authors disclose no conflicts of interest. Acknowledgements The authors thank all of the patients for their collaboration. This study was supported by the Beijing Municipal Science and Technology Commission (grant number Z151100003915078), the National Natural Science Foundation of China (NSFC; grant number 81230015), and a grant from Neuroscience Center of Chinese Academy of Medical Sciences. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neurobiolaging.2016.07.027.
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