Neurobiology of Aging xxx (2015) 1e5
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Mutation analysis of C9orf72 in patients with corticobasal syndrome Cassandra J. Anor a, Zhengrui Xi a, Ming Zhang a, Danielle Moreno a, Christine Sato a, Ekaterina Rogaeva a, b, Maria Carmela Tartaglia a, c, * a
Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada c Division of Neurology, University Health Network Memory Clinic, Toronto, Ontario, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2015 Received in revised form 1 May 2015 Accepted 5 June 2015
Corticobasal syndrome (CBS) is a neurodegenerative disease characterized by progressive asymmetrical rigidity and apraxia, cortical sensory loss, myoclonus, dystonia, and cognitive impairment. CBS is usually sporadic and associated with tau pathology but there are reports of TDP-43 pathology. We screened 39 CBS cases to determine if any of the cases could be explained by a G4C2-repeat expansion in a noncoding region of C9orf72 gene, the most common genetic cause of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. One patient with CBS had a large (>50 repeats) expansion in C9orf72. Our case features a 63-year-old right-handed woman who developed mild apathy 9 years before presentation, which progressed to include behavioral symptoms, oral stereotypies, significant language impairment, parkinsonism and apraxia. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric left > right frontotemporal atrophy, including orbitofrontal and parietal areas. Her father developed a behavioral syndrome and died at an early age. This case highlights the importance of genetic screening for C9orf72 in patients with CBS. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: C9orf72 Corticobasal syndrome oral stereotypies
1. Introduction Corticobasal syndrome (CBS) is a clinical condition that belongs to the family of frontotemporal lobar degeneration. CBS is characterized by progressive asymmetrical rigidity and apraxia, alien limb phenomenon, cortical sensory loss, myoclonus, dystonia, tremor, and cognitive impairment (Boeve et al., 2003). CBS is typically associated with tau pathology (Armstrong et al., 2013), but TDP-43 (Huey et al., 2012) and Alzheimer’s disease (Rusina et al., 2013) pathology can also present as CBS. Genetics of CBS is mainly unknown. Few case studies (Galimberti et al., 2013; Lesage et al., 2013; Lindquist et al., 2013) have reported CBS due to C9orf72. In Lesage et al. study, they found 1 CBS patient who carried C9orf72 expansions (CBS n ¼ 21; 4.67% mutation carriers) (Lesage et al., 2013). In Lindquist et al. study on a Danish patient cohort, they found a pathogenic expansion in 1 patient with CBS without autopsy (Lindquist et al., 2013). In contrast to these results, Galimberti et al.
studied an Italian cohort in which no pathogenic repeat expansion was found in any of their 21 genotyped CBS patients (CBS n ¼ 21; 0% mutation carriers) (Galimberti et al., 2013). Typically, the C9orf72 mutation is more commonly known for being the cause of amyotrophic lateral sclerosis and FTD (DeJesus-Hernandez et al., 2011; Xi et al., 2012). Although the exact mechanism of the C9orf72 mutation is unknown, possible mechanisms that have been proposed include the accumulation of toxic RNA (DeJesus-Hernandez et al., 2011); haploinsufficiency of the C9orf72 (DeJesus-Hernandez et al., 2011); and the formation of dipeptide-repeat protein aggregates resulting from non-ATG translation of the repeat (Ash et al., 2013; Mori et al., 2013). Alternatively, all 3 mechanisms have been suggested to contribute to the diverse spectrum of phenotypes to various degrees (Xi et al., 2014). Given that the genetics of CBS is largely unknown, we investigated if some of our CBS cases could be explained by the G4C2-repeat expansion in a noncoding region of C9orf72 gene. 2. Methods
* Corresponding author at: Tanz Centre for Research in Neurodegenerative Disease, Krembil Discovery Tower, 60 Leonard Avenue, 6th Floor, 6KD-407,Toronto, Ontario, M5T 2S8, Canada. Toronto Western Hospital, West Wing 5-449, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416 603 5483; fax: 416 603 5768. E-mail address: [email protected] (M.C. Tartaglia). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.06.008
2.1. Data set We investigated 39 CBS cases with a mean age at onset of 58.9 13.2 years (44% female). Informed consent was obtained from all participants in accordance with the University of Toronto ethical
2
C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
review board. Genomic DNA was isolated from blood using a QIAGEN kit.
3. Results 3.1. ID #9660
2.2. Genetic analysis The C9orf72 G4C2-repeat was genotyped by a 2-step strategy as previously described (Xi et al., 2012). Briefly, the first step was the fluorescent fragment length genotyping to obtain the number of repeats of the small alleles (50 repeats). Pathological expansions were defined using the previously suggested 30-repeat cutoff (DeJesus-Hernandez et al., 2011; Renton et al., 2011).
2.3. Epigenetic studies The methylation of the G4C2-repeats itself was studied using our (G4C2)n-methylation assay as previously reported (Xi et al., 2015). In brief, this qualitative assay combines rp-PCR (detect expansion in C9orf72) with methylation-specific PCR, where the sample was amplified by rp-PCR using primers specific for methylated versus unmethylated DNA after bisulfite conversion. The primers were labeled by FAM for methylated DNA amplification (blue channel) and HEX for unmethylated DNA amplification (green channel). Data were visualized by Genotyper software (version 3.6, Applied Biosystems). If the G4C2-repeat is methylated in all DNA copies, only the blue channel is expected to have products; whereas if the G4C2repeat is unmethylated in all DNA copies, only the green channel is expected to have products. If the G4C2-repeat is methylated in some DNA copies, both channels are expected to have products.
3.1.1. Genetic and epigenetic analysis One of our CBS samples (ID #9660) was detected to carry an expansion allele (Fig. 1). The first-step genotyping detected a 10-repeat allele and the second-step genotyping detected an expansion allele. The (G4C2)n-methylation assay demonstrated CpG methylation at the expanded G4C2-repeat itself (Fig. 2), indicating that ID #9660 has a typical large expansion (Xi et al., 2015). 3.1.2. Medical history This was a 63-year-old Italian right-handed woman who developed mild apathy 9 years before presentation. Four and a half years later, this apathetic behavior progressed into notable personality changes including disinhibition, loss of social graces, aggression, impulsivity, sexual inappropriateness, and perseverative behaviors. She also appeared to be anxious at times. There were prominent executive deficits as she demonstrated poor judgment and trouble problem solving. Her language problems included anomia, substitution of words in speech, trouble following a conversation with multiple participants, and an inability to follow oral instructions. In addition, she developed prosopagnosia. Her memory at the time was not of concern. On presentation, 7 years after symptom onset, she had hallucinations and delusions. She wandered out of her home. She had difficulty using utensils due to her apraxia and she developed significant hyperphagia. Swallowing assessment revealed mild oral preparatory dysphagia and possible laryngeal penetration of thin liquids. Furthermore, she became incontinent of bladder and occasionally bowel. In terms of her memory, there were notable
Fig. 1. Genotyping results are shown for sample #9660 and a representative sample of nonexpansion carriers.
C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
3
Fig. 2. Chromatograms of the (G4C2)n-methylation assay for ID #9660. Methylated and unmethylated G4C2-repeat alleles are detected by methylation channel and unmethylation channel, respectively. Detectable repeat size was marked along with the corresponding peak.
changes as she became repetitive and would forget to turn off the stove and tap. There was marked difficulty in concentrating and severe restlessness. She nearly became mute except for the echolalia and occasional singing. Her minimal speech output was apraxic. She was bradykinetic in both her upper and lower extremities, bilaterally rigid, and she had a wide-based, shuffling gait with increasing apraxia in her right leg, but with no falls. She also had 4 episodes of what appeared to be extreme fear. During these episodes, her arms would stiffen and she became incontinent but with no loss of awareness; she could only state that something was happening without explaining further. Unfortunately, at this time she was also diagnosed with breast cancer. Eight years after symptom onset she developed new orolingual involuntary movements that were present all the time but worsened with stress and nervousness. There had been no excoriation of her lips, which might have been expected with this much tongue movement. Her swallowing difficulties were managed, and her weight was stable. She had deteriorated to complete dependence for all activities of daily living and was wheelchair-bound because of gait apraxia. A proper neurological examination was impossible, as she would try to bite you if your hand got too close to her mouth. She introduced organic and inorganic objects into her mouth
including clothes, bingo chips, and so forth. Her memory at this time could not be assessed because of severe aphasia and her prosopagnosia had worsened as she recognized her family less and less. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric frontotemporal atrophy, worse on the left. She also had significant orbitofrontal and biparietal atrophy (See Fig. 3). There were no microangiopathic changes noted. Her past medical history was unremarkable except for the recent diagnosis of breast cancer and she had no previous head injury. 3.1.3. Social and family history She never smoked nor tried any recreational drugs. Her mother died at the age of 77 years of cervical cancer; but before this was wheelchair-bound for many years because of intrauterine radiation. The patient also has 2 healthy siblings aged 57 and 62 years. She lives with her husband who is the primary caregiver; they have 2 healthy daughters aged 42 and 43 years. Her father had presented with behavioral changes at the age of 59 years when he developed apathy and became less motivated for household chores and interactions with his family. He became
Fig. 3. Axial and L-hemisphere sagittal fluid-attenuated inversion recovery image at age 60 years, 6 years after symptom onset.
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C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
lethargic and would spend much of his time in bed. He would wander around the neighborhood to smoke. He changed his eating habits and insisted on eating only pasta and bread. There was also loss of hygiene. Over time he lost empathy and only wanted his wife around, completely disinterested in his children although he recognized them until the end. He enjoyed playing cards and did so until his death. He developed hepatocellular carcinoma with metastatic disease to the spine with spinal cord involvement. He died at the age of 63 years from complications of decompression surgery. Language function was preserved until the end and he never developed any stereotypies. He was never formally diagnosed but his signs and symptoms are most consistent with possible behavioral variant frontotemporal dementia. 4. Discussion We present 1 clinically diagnosed CBS case due to a C9orf72 mutation. To our knowledge, only 2 CBS cases (Lesage et al., 2013; Lindquist et al., 2013) have been reported to have C9orf72 repeat expansions. Our review of all published CBS cases investigated for C9orf72 repeat expansion [n ¼ 81 (Galimberti et al., 2013; Lesage et al., 2013; Lindquist et al., 2013), including current report with an unknown number of CBS cases in the article by Lindquist et al.] revealed approximately 3.7% mutation carriers (n ¼ 3). In our previous study, we demonstrated that noncarriers (
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Mutation analysis of C9orf72 in patients with corticobasal syndrome Cassandra J. Anor a, Zhengrui Xi a, Ming Zhang a, Danielle Moreno a, Christine Sato a, Ekaterina Rogaeva a, b, Maria Carmela Tartaglia a, c, * a
Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada c Division of Neurology, University Health Network Memory Clinic, Toronto, Ontario, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2015 Received in revised form 1 May 2015 Accepted 5 June 2015
Corticobasal syndrome (CBS) is a neurodegenerative disease characterized by progressive asymmetrical rigidity and apraxia, cortical sensory loss, myoclonus, dystonia, and cognitive impairment. CBS is usually sporadic and associated with tau pathology but there are reports of TDP-43 pathology. We screened 39 CBS cases to determine if any of the cases could be explained by a G4C2-repeat expansion in a noncoding region of C9orf72 gene, the most common genetic cause of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. One patient with CBS had a large (>50 repeats) expansion in C9orf72. Our case features a 63-year-old right-handed woman who developed mild apathy 9 years before presentation, which progressed to include behavioral symptoms, oral stereotypies, significant language impairment, parkinsonism and apraxia. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric left > right frontotemporal atrophy, including orbitofrontal and parietal areas. Her father developed a behavioral syndrome and died at an early age. This case highlights the importance of genetic screening for C9orf72 in patients with CBS. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: C9orf72 Corticobasal syndrome oral stereotypies
1. Introduction Corticobasal syndrome (CBS) is a clinical condition that belongs to the family of frontotemporal lobar degeneration. CBS is characterized by progressive asymmetrical rigidity and apraxia, alien limb phenomenon, cortical sensory loss, myoclonus, dystonia, tremor, and cognitive impairment (Boeve et al., 2003). CBS is typically associated with tau pathology (Armstrong et al., 2013), but TDP-43 (Huey et al., 2012) and Alzheimer’s disease (Rusina et al., 2013) pathology can also present as CBS. Genetics of CBS is mainly unknown. Few case studies (Galimberti et al., 2013; Lesage et al., 2013; Lindquist et al., 2013) have reported CBS due to C9orf72. In Lesage et al. study, they found 1 CBS patient who carried C9orf72 expansions (CBS n ¼ 21; 4.67% mutation carriers) (Lesage et al., 2013). In Lindquist et al. study on a Danish patient cohort, they found a pathogenic expansion in 1 patient with CBS without autopsy (Lindquist et al., 2013). In contrast to these results, Galimberti et al.
studied an Italian cohort in which no pathogenic repeat expansion was found in any of their 21 genotyped CBS patients (CBS n ¼ 21; 0% mutation carriers) (Galimberti et al., 2013). Typically, the C9orf72 mutation is more commonly known for being the cause of amyotrophic lateral sclerosis and FTD (DeJesus-Hernandez et al., 2011; Xi et al., 2012). Although the exact mechanism of the C9orf72 mutation is unknown, possible mechanisms that have been proposed include the accumulation of toxic RNA (DeJesus-Hernandez et al., 2011); haploinsufficiency of the C9orf72 (DeJesus-Hernandez et al., 2011); and the formation of dipeptide-repeat protein aggregates resulting from non-ATG translation of the repeat (Ash et al., 2013; Mori et al., 2013). Alternatively, all 3 mechanisms have been suggested to contribute to the diverse spectrum of phenotypes to various degrees (Xi et al., 2014). Given that the genetics of CBS is largely unknown, we investigated if some of our CBS cases could be explained by the G4C2-repeat expansion in a noncoding region of C9orf72 gene. 2. Methods
* Corresponding author at: Tanz Centre for Research in Neurodegenerative Disease, Krembil Discovery Tower, 60 Leonard Avenue, 6th Floor, 6KD-407,Toronto, Ontario, M5T 2S8, Canada. Toronto Western Hospital, West Wing 5-449, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416 603 5483; fax: 416 603 5768. E-mail address: [email protected] (M.C. Tartaglia). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.06.008
2.1. Data set We investigated 39 CBS cases with a mean age at onset of 58.9 13.2 years (44% female). Informed consent was obtained from all participants in accordance with the University of Toronto ethical
2
C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
review board. Genomic DNA was isolated from blood using a QIAGEN kit.
3. Results 3.1. ID #9660
2.2. Genetic analysis The C9orf72 G4C2-repeat was genotyped by a 2-step strategy as previously described (Xi et al., 2012). Briefly, the first step was the fluorescent fragment length genotyping to obtain the number of repeats of the small alleles (50 repeats). Pathological expansions were defined using the previously suggested 30-repeat cutoff (DeJesus-Hernandez et al., 2011; Renton et al., 2011).
2.3. Epigenetic studies The methylation of the G4C2-repeats itself was studied using our (G4C2)n-methylation assay as previously reported (Xi et al., 2015). In brief, this qualitative assay combines rp-PCR (detect expansion in C9orf72) with methylation-specific PCR, where the sample was amplified by rp-PCR using primers specific for methylated versus unmethylated DNA after bisulfite conversion. The primers were labeled by FAM for methylated DNA amplification (blue channel) and HEX for unmethylated DNA amplification (green channel). Data were visualized by Genotyper software (version 3.6, Applied Biosystems). If the G4C2-repeat is methylated in all DNA copies, only the blue channel is expected to have products; whereas if the G4C2repeat is unmethylated in all DNA copies, only the green channel is expected to have products. If the G4C2-repeat is methylated in some DNA copies, both channels are expected to have products.
3.1.1. Genetic and epigenetic analysis One of our CBS samples (ID #9660) was detected to carry an expansion allele (Fig. 1). The first-step genotyping detected a 10-repeat allele and the second-step genotyping detected an expansion allele. The (G4C2)n-methylation assay demonstrated CpG methylation at the expanded G4C2-repeat itself (Fig. 2), indicating that ID #9660 has a typical large expansion (Xi et al., 2015). 3.1.2. Medical history This was a 63-year-old Italian right-handed woman who developed mild apathy 9 years before presentation. Four and a half years later, this apathetic behavior progressed into notable personality changes including disinhibition, loss of social graces, aggression, impulsivity, sexual inappropriateness, and perseverative behaviors. She also appeared to be anxious at times. There were prominent executive deficits as she demonstrated poor judgment and trouble problem solving. Her language problems included anomia, substitution of words in speech, trouble following a conversation with multiple participants, and an inability to follow oral instructions. In addition, she developed prosopagnosia. Her memory at the time was not of concern. On presentation, 7 years after symptom onset, she had hallucinations and delusions. She wandered out of her home. She had difficulty using utensils due to her apraxia and she developed significant hyperphagia. Swallowing assessment revealed mild oral preparatory dysphagia and possible laryngeal penetration of thin liquids. Furthermore, she became incontinent of bladder and occasionally bowel. In terms of her memory, there were notable
Fig. 1. Genotyping results are shown for sample #9660 and a representative sample of nonexpansion carriers.
C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
3
Fig. 2. Chromatograms of the (G4C2)n-methylation assay for ID #9660. Methylated and unmethylated G4C2-repeat alleles are detected by methylation channel and unmethylation channel, respectively. Detectable repeat size was marked along with the corresponding peak.
changes as she became repetitive and would forget to turn off the stove and tap. There was marked difficulty in concentrating and severe restlessness. She nearly became mute except for the echolalia and occasional singing. Her minimal speech output was apraxic. She was bradykinetic in both her upper and lower extremities, bilaterally rigid, and she had a wide-based, shuffling gait with increasing apraxia in her right leg, but with no falls. She also had 4 episodes of what appeared to be extreme fear. During these episodes, her arms would stiffen and she became incontinent but with no loss of awareness; she could only state that something was happening without explaining further. Unfortunately, at this time she was also diagnosed with breast cancer. Eight years after symptom onset she developed new orolingual involuntary movements that were present all the time but worsened with stress and nervousness. There had been no excoriation of her lips, which might have been expected with this much tongue movement. Her swallowing difficulties were managed, and her weight was stable. She had deteriorated to complete dependence for all activities of daily living and was wheelchair-bound because of gait apraxia. A proper neurological examination was impossible, as she would try to bite you if your hand got too close to her mouth. She introduced organic and inorganic objects into her mouth
including clothes, bingo chips, and so forth. Her memory at this time could not be assessed because of severe aphasia and her prosopagnosia had worsened as she recognized her family less and less. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric frontotemporal atrophy, worse on the left. She also had significant orbitofrontal and biparietal atrophy (See Fig. 3). There were no microangiopathic changes noted. Her past medical history was unremarkable except for the recent diagnosis of breast cancer and she had no previous head injury. 3.1.3. Social and family history She never smoked nor tried any recreational drugs. Her mother died at the age of 77 years of cervical cancer; but before this was wheelchair-bound for many years because of intrauterine radiation. The patient also has 2 healthy siblings aged 57 and 62 years. She lives with her husband who is the primary caregiver; they have 2 healthy daughters aged 42 and 43 years. Her father had presented with behavioral changes at the age of 59 years when he developed apathy and became less motivated for household chores and interactions with his family. He became
Fig. 3. Axial and L-hemisphere sagittal fluid-attenuated inversion recovery image at age 60 years, 6 years after symptom onset.
4
C.J. Anor et al. / Neurobiology of Aging xxx (2015) 1e5
lethargic and would spend much of his time in bed. He would wander around the neighborhood to smoke. He changed his eating habits and insisted on eating only pasta and bread. There was also loss of hygiene. Over time he lost empathy and only wanted his wife around, completely disinterested in his children although he recognized them until the end. He enjoyed playing cards and did so until his death. He developed hepatocellular carcinoma with metastatic disease to the spine with spinal cord involvement. He died at the age of 63 years from complications of decompression surgery. Language function was preserved until the end and he never developed any stereotypies. He was never formally diagnosed but his signs and symptoms are most consistent with possible behavioral variant frontotemporal dementia. 4. Discussion We present 1 clinically diagnosed CBS case due to a C9orf72 mutation. To our knowledge, only 2 CBS cases (Lesage et al., 2013; Lindquist et al., 2013) have been reported to have C9orf72 repeat expansions. Our review of all published CBS cases investigated for C9orf72 repeat expansion [n ¼ 81 (Galimberti et al., 2013; Lesage et al., 2013; Lindquist et al., 2013), including current report with an unknown number of CBS cases in the article by Lindquist et al.] revealed approximately 3.7% mutation carriers (n ¼ 3). In our previous study, we demonstrated that noncarriers (
