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Functional characterization of a novel progranulin mutation in a patient with progressive nonfluent aphasia
Neurobiology of Aging xxx (2018) 1.e1e1.e4
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Functional characterization of a novel progranulin mutation in a patient with progressive nonfluent aphasia _ n ska b, Katarzyna Gaweda-Walerych a, *, Emilia J. Sitek b, c, Ewa Naroza Michalina Wezyk a, Bogna Brockhuis d, Cezary Zekanowski a, Jaros1aw S1awek b, c a
Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre, PAS, Warsaw, Poland Neurology Department, St. Adalbert Hospital, Copernicus PL, Gdansk, Poland Neurological and Psychiatric Nursing Department, Medical University of Gdansk, Gdansk, Poland d Department of Nuclear Medicine, Medical University of Gdansk, Gdansk, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2018 Received in revised form 24 May 2018 Accepted 25 June 2018
Loss-of-function mutations in progranulin (PGRN) gene cause frontotemporal lobar degeneration. Here, we report a case of a 63-year-old woman with a 2-year history of speech impairment, diagnosed with a nonfluent variant of primary progressive aphasia, a subtype of frontotemporal lobar degeneration. In this patient, a novel heterozygous frameshift mutation, c.77delG, in exon 2 of PGRN gene, introducing premature stop codon, p.(C26SfsX28), has been identified. Cultured fibroblasts derived from the patient and her asymptomatic firstdegree relative with c.77delG mutation had decreased levels of PGRN messenger RNA (mRNA) and protein. However, PGRN mRNA levels did not recover upon incubation with inhibitors of nonsense-mediated mRNA decay (cycloheximide or puromycin), suggesting involvement of other mRNA degradation pathways. In addition, we observed upregulated wingless-type mouse mammary tumor virus integration site (WNT) signaling pathway gene, WNT3A, in fibroblasts of the patient and her asymptomatic first-degree relative with c.77delG mutation. As reported previously, this is an early hallmark of PGRN deficiency. Ó 2018 Elsevier Inc. All rights reserved.
Keywords: Progranulin (PGRN) mutation PGRN haploinsufficiency Nonfluent variant of primary progressive aphasia (nfvPPA) Fibroblasts WNT3A
1. Introduction
2. Materials and methods
Frontotemporal lobar degeneration manifests clinically as behavioral variant or primary progressive aphasia (PPA), the latter one comprising two subtypes: nonfluent variant PPA (nfvPPA) and semantic variant PPA (Gorno-Tempini et al., 2011). Mutations in the progranulin (PGRN) gene (Baker et al., 2006; Cruts et al., 2006) are among the most common genetic causes of PPA (Rohrer, 2014). To date, nearly 80 PGRN mutations have been reported, in the majority nonsense, frameshift, or splicing ones (Alzheimer Disease & Frontotemporal Dementia Mutation Database; http://www.molgen.ua.ac. be/FTDMutations). They typically introduce premature termination codon (PTC) in the messenger RNA (mRNA), which triggers degradation of the mutated mRNA through nonsense-mediated mRNA decay (NMD) and results in PGRN protein haploinsufficiency (ChenPlotkin et al., 2011; van Swieten and Heutink, 2008). Here, we functionally characterize a novel PGRN mutation detected in a 62-year-old woman (diagnosed with nfvPPA) and her asymptomatic first-degree relative.
2.1. Study subjects
* Corresponding author at: Department of Neurodegenerative Diseases, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, Warsaw 02-106, Poland. Tel.: þ48 226086485; fax: þ48 2266855 32. E-mail address: [email protected] (K. Gaweda-Walerych). 0197-4580/$ e see front matter Ó 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2018.06.033
The patient (age of onset, 61 years), her two first-degree relatives (gender and age are omitted for confidentiality), and three unrelated, neurologically healthy controls (gender and age of skin biopsy donation is provided in Table S1) were enrolled for the study. All the study subjects have given an informed consent, and the study protocol has been approved by the Bioethical Committee of Medical University of Gdansk and the Central Clinical Hospital of Ministry of the Interior and Administration in Warsaw. Experimental procedures are described in the supplementary material (Appendix A). 3. Results 3.1. Clinical assessment Neurological assessment of the 62-year-old proband did not reveal any abnormalities apart from aphasia (1-year history of nonfluent speech). The patient was fully independent in the complex activities of daily living. Follow-up in the consecutive year
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K. Gaweda-Walerych et al. / Neurobiology of Aging xxx (2018) 1.e1e1.e4
revealed significant deterioration of speech and writing, with language and cognitive profile compatible with the diagnosis of nfvPPA. Apraxia of speech was the most prominent feature in spontaneous speech, repetition, and reading. Complex sentence comprehension was mildly impaired, with preserved word comprehension and semantics. Language dysfunction was accompanied by planning deficit (see Table S2). Pegboard task revealed slight bilateral slowing of precise hand movements and involuntary co-movements in the lower limbs. Brain magnetic resonance imaging (62 years 2 months) evidenced left-sided frontotemporal
atrophy (Fig 1F, left panel). Regional cerebral blood flow singlephoton emission computed tomography (rCBF SPECT, 62 years 6 months) showed mild frontal, temporal, and parietal hypoperfusion on the left side (Fig 1F, right panel), which progressed over the period of 10 months (63 years 3 months, not shown). 3.2. Genetic assessment Familial history of the patient suggested early-onset dementia in at least 4 paternal-side family members. The patient and her 2 first-degree
Fig. 1. Decreased PGRN mRNA and protein levels and upregulated WNT3A mRNA levels in nfvPPA patient and her first-degree relative with PGRN c.77delG mutation. (A) Electropherogram showing PGRN WT sequence and (B) heterozygous, 1 nucleotide deletion c.77delG, in PGRN exon 2 of patient’s DNA. (C) Decreased PGRN mRNA and (D) protein levels in cultured fibroblasts of the patient (P) and her asymptomatic first-degree relative (FDR1) with c.77delG mutation, compared to the first-degree relative without the mutation (FDR2-C1) and 3 unrelated control individuals (C2, C3, and C4). (E) Higher WNT3A mRNA levels in carriers of c.77delG mutation versus controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) Brain MRI of the patient evidencing left-sided frontotemporal atrophy (left panel). SPECT demonstrating mild frontal, temporal, and parietal hypoperfusion on the left side (right panel). Abbreviations: MRI, magnetic resonance imaging; mRNA, messenger ribonucleic acid; nfvPPA, nonfluent variant of primary progressive aphasia; PGRN, progranulin.
K. Gaweda-Walerych et al. / Neurobiology of Aging xxx (2018) 1.e1e1.e4
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relatives werescreened for mutations in MAPT, C9orf72, and PGRN genes. The analysis revealed a novel deletion c.77delG in exon 2 of PGRN gene in the patient and one of her first-degree relatives (at a presymptomatic age) (Fig 1A, B). c.77delG mutation was absent in ExAC, 1000gp, and ESP5400 databases. However, there are already known mutations locatedinthesameposition:aframeshiftonec.77_78insC(p.Val28Cysfs, rs1392550887) and 2 missense substitutions c.77 G > T and c.77 G > A (p.Cys26Phe, p.Cys26Tyr, respectively; rs780086363). These mutations are very rare with ExAC MAF/MinorAlleleCount T ¼ 0.000008/1. The latter variant is also annotated in the catalog of somatic mutations in cancer (COSMIC, COSM4382943) and was predicted by SIFT as deleterious and by PolyPhen as possibly damaging. Bioinformatic analysis revealed that c.77delG leads to a frameshift and a premature stop codon, p.(Cys26SerfsX28), implicating degradation of the mutated mRNA by NMD. As expected, cultured fibroblasts with c.77delG mutation derived from the patient and her first-degree relative (FDR1) showed decreased levels of PGRN mRNA (by c.a. 50%) and protein, compared to control fibroblasts, including the patient’s first-degree relative without the mutation (FDR2) (Fig 1C, D). However, PGRN mRNA levels did not recover upon incubation with NMD inhibitors, cycloheximide or puromycin (Fig S1 A, B, C, D). Moreover, sequencing of cDNA derived from the patient’s fibroblasts (cultured under standard conditions or treated with cycloheximide) revealed only wild-type sequence (not shown). This suggests involvement of other mRNA decay pathways in degradation of the mutated mRNA. In addition, fibroblasts with c.77delG mutation exhibited upregulated wingless-type mouse mammary tumor virus integration site (WNT) signaling pathway gene d WTN3A (Fig 1E) d an early hallmark of the PGRN deficiency. Once the genetic testing revealed PGRN mutation, the patient fulfilled level III criteria of nfvPPA (Gorno-Tempini et al., 2011).
mRNA levels in PGRN-deficient fibroblasts from symptomatic and presymptomatic mutation carriers. PGRN-associated PPA usually manifests clinically as a mixed nonfluent and logopenic PPA (lvPPA) phenotype (Rohrer et al., 2010). Although in sporadic cases of nfvPPA speech apraxia is a common core feature, it is not considered typical for PPA patients with PGRN mutations (Rohrer, 2014). In previous studies, PGRN mutation carriers did not show apraxia of speech (Snowden et al., 2015) and PGRN mutations were absent in cases with primary progressive apraxia of speech (Flanagan et al., 2015). However, in our case, not only profound speech apraxia is present, but also other features typical for nfvPPA coexist, such as preserved delayed word recognition and spatial function, and selective executive deficits. Only rapid progression of anomia, observed in our case at follow-up, is typical for PGRN-associated PPA (Rohrer et al., 2010). However, in our patient anomia was not accompanied by word-comprehension deficits, that are frequently associated with PGRN-related PPA but not with the classical nfvPPA phenotype. This highlights the possible heterogeneity in the clinical presentation of PGRN-linked aphasia. Individuals with speech apraxia and PGRN mutations were reported to manifest PPA-unclassified phenotype (Harris et al., 2013). Our patient’s phenotype, with predominant apraxia of speech accompanied by other nvfPPA features both in terms of language impairment (e.g., agrammatism) and cognitive profile (e.g., dysexecutive deficits), suggests that PGRN mutations may be also associated with a rather typical nfvPPA clinical presentation, observed in sporadic cases.
4. Discussion
Acknowledgements
Functional assessment of a novel PGRN mutation c.77delG demonstrated decreased PGRN mRNA and protein levels in fibroblasts derived from the patient and her first-degree relative, which confirms haploinsufficiency as the underlying pathogenic mechanism. To date, this mechanism is considered the most common for PGRN mutations (van Swieten and Heutink, 2008). Lack of mRNA recovery after cycloheximide treatment despite detecting mRNA decay has been reported previously in case of mutations introducing PTC close to the N-terminus of the protein (Montfort et al., 2006; Perrin-Vidoz et al., 2002). Such PTCs have been shown to escape NMD due to reinitiation of transcription from the following ATG sequence (Kozak, 2001; Perrin-Vidoz et al., 2002). In the case of PGRN c.77delG mutation (located at the beginning of the first translated exon, close to the start codon), RNA decay is likely triggered by a mechanism other than NMD, as previously proposed by Montfort et al., 2006. WNT signaling was identified as one of the top defective pathways in genome-wide transcriptome analyses of PGRNdeficient human neural progenitors and neurons derived from induced pluripotent stem cells and postmortem brains obtained from frontotemporal lobar degenerationePGRN patients (Raitano et al., 2015; Rosen et al., 2011). WNT2, WNT3A, WNT5A, WNT6, WNT10a, and CTNNB1 were among the top upregulated transcripts (Raitano et al., 2015). Increased expression of various WNT pathway genes has been further confirmed in cultured keratinocytes (with siRNA-silenced PGRN) and lymphoblastoid cell lines derived from carriers of PGRN mutations (Alquezar et al., 2016; Tian et al., 2016). In addition, aberrant WNT signaling has emerged as an early hallmark of PGRN deficiency as it was observed also in asymptomatic PGRN mutation carriers (Alquezar et al., 2016). Here, for the first time, we report increased WNT3A
This work was supported by National Science Center, Poland (DEC-2017/01/X/NZ3/00266), the funding obtained by Dr GawedaWalerych. The authors thank the patient, her relatives, and other subjects who participated in this study and Dr Dawid Walerych and ski for critical reading of the article. Dr Mariusz Berdyn
Disclosure statement The authors declare no conflict of interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.neurobiolaging.2018. 06.033. References Alquezar, C., de la Encarnacion, A., Moreno, F., Lopez de Munain, A., MartinRequero, A., 2016. Progranulin deficiency induces overactivation of WNT5A expression via TNF-alpha/NF-kappaB pathway in peripheral cells from frontotemporal dementia-linked granulin mutation carriers. J. Psychiatry Neurosci. 41, 225e239. Baker, M., Mackenzie, I.R., Pickering-Brown, S.M., Gass, J., Rademakers, R., Lindholm, C., Snowden, J., Adamson, J., Sadovnick, A.D., Rollinson, S., Cannon, A., Dwosh, E., Neary, D., Melquist, S., Richardson, A., Dickson, D., Berger, Z., Eriksen, J., Robinson, T., Zehr, C., Dickey, C.A., Crook, R., McGowan, E., Mann, D., Boeve, B., Feldman, H., Hutton, M., 2006. Mutations in progranulin cause taunegative frontotemporal dementia linked to chromosome 17. Nature 442, 916e919. Chen-Plotkin, A.S., Martinez-Lage, M., Sleiman, P.M., Hu, W., Greene, R., Wood, E.M., Bing, S., Grossman, M., Schellenberg, G.D., Hatanpaa, K.J., Weiner, M.F., White, C.L., 3rd, Brooks, W.S., Halliday, G.M., Kril, J.J., Gearing, M., Beach, T.G., Graff-Radford, N.R., Dickson, D.W., Rademakers, R., Boeve, B.F., PickeringBrown, S.M., Snowden, J., van Swieten, J.C., Heutink, P., Seelaar, H., Murrell, J.R., Ghetti, B., Spina, S., Grafman, J., Kaye, J.A., Woltjer, R.L., Mesulam, M., Bigio, E., Llado, A., Miller, B.L., Alzualde, A., Moreno, F., Rohrer, J.D., Mackenzie, I.R., Feldman, H.H., Hamilton, R.L., Cruts, M., Engelborghs, S., De Deyn, P.P., Van Broeckhoven, C., Bird, T.D., Cairns, N.J., Goate, A., Frosch, M.P., Riederer, P.F., Bogdanovic, N., Lee, V.M., Trojanowski, J.Q., Van Deerlin, V.M., 2011. Genetic and
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clinical features of progranulin-associated frontotemporal lobar degeneration. Arch. Neurol. 68, 488e497. Cruts, M., Gijselinck, I., van der Zee, J., Engelborghs, S., Wils, H., Pirici, D., Rademakers, R., Vandenberghe, R., Dermaut, B., Martin, J.J., van Duijn, C., Peeters, K., Sciot, R., Santens, P., De Pooter, T., Mattheijssens, M., Van den Broeck, M., Cuijt, I., Vennekens, K., De Deyn, P.P., Kumar-Singh, S., Van Broeckhoven, C., 2006. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 442, 920e924. Flanagan, E.P., Baker, M.C., Perkerson, R.B., Duffy, J.R., Strand, E.A., Whitwell, J.L., Machulda, M.M., Rademakers, R., Josephs, K.A., 2015. Dominant frontotemporal dementia mutations in 140 cases of primary progressive aphasia and speech apraxia. Dement Geriatr. Cogn. Disord. 39, 281e286. Gorno-Tempini, M.L., Hillis, A.E., Weintraub, S., Kertesz, A., Mendez, M., Cappa, S.F., Ogar, J.M., Rohrer, J.D., Black, S., Boeve, B.F., Manes, F., Dronkers, N.F., Vandenberghe, R., Rascovsky, K., Patterson, K., Miller, B.L., Knopman, D.S., Hodges, J.R., Mesulam, M.M., Grossman, M., 2011. Classification of primary progressive aphasia and its variants. Neurology 76, 1006e1014. Harris, J.M., Gall, C., Thompson, J.C., Richardson, A.M., Neary, D., du Plessis, D., Pal, P., Mann, D.M., Snowden, J.S., Jones, M., 2013. Classification and pathology of primary progressive aphasia. Neurology 81, 1832e1839. Kozak, M., 2001. Constraints on reinitiation of translation in mammals. Nucleic Acids Res. 29, 5226e5232. Montfort, M., Chabas, A., Vilageliu, L., Grinberg, D., 2006. Analysis of nonsensemediated mRNA decay in mutant alleles identified in Spanish Gaucher disease patients. Blood Cells Mol. Dis. 36, 46e52. Perrin-Vidoz, L., Sinilnikova, O.M., Stoppa-Lyonnet, D., Lenoir, G.M., Mazoyer, S., 2002. The nonsense-mediated mRNA decay pathway triggers degradation of
most BRCA1 mRNAs bearing premature termination codons. Hum. Mol. Genet. 11, 2805e2814. Raitano, S., Ordovas, L., De Muynck, L., Guo, W., Espuny-Camacho, I., Geraerts, M., Khurana, S., Vanuytsel, K., Toth, B.I., Voets, T., Vandenberghe, R., Cathomen, T., Van Den Bosch, L., Vanderhaeghen, P., Van Damme, P., Verfaillie, C.M., 2015. Restoration of progranulin expression rescues cortical neuron generation in an induced pluripotent stem cell model of frontotemporal dementia. Stem Cell Rep. 4, 16e24. Rohrer, J.D., 2014. The genetics of primary progressive aphasia. Aphasiology 28, 941e947. Rohrer, J.D., Crutch, S.J., Warrington, E.K., Warren, J.D., 2010. Progranulin-associated primary progressive aphasia: a distinct phenotype? Neuropsychologia 48, 288e297. Rosen, E.Y., Wexler, E.M., Versano, R., Coppola, G., Gao, F., Winden, K.D., Oldham, M.C., Martens, L.H., Zhou, P., Farese Jr., R.V., Geschwind, D.H., 2011. Functional genomic analyses identify pathways dysregulated by progranulin deficiency, implicating Wnt signaling. Neuron 71, 1030e1042. Snowden, J.S., Adams, J., Harris, J., Thompson, J.C., Rollinson, S., Richardson, A., Jones, M., Neary, D., Mann, D.M., Pickering-Brown, S., 2015. Distinct clinical and pathological phenotypes in frontotemporal dementia associated with MAPT, PGRN and C9orf72 mutations. Amyotroph. Lateral Scler. Frontotemporal Degener. 16, 497e505. Tian, R., Li, Y., Yao, X., 2016. PGRN suppresses inflammation and promotes autophagy in keratinocytes through the wnt/beta-catenin signaling pathway. Inflammation 39, 1387e1394. van Swieten, J.C., Heutink, P., 2008. Mutations in progranulin (GRN) within the spectrum of clinical and pathological phenotypes of frontotemporal dementia. Lancet Neurol. 7, 965e974.
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Functional characterization of a novel progranulin mutation in a patient with progressive nonfluent aphasia _ n ska b, Katarzyna Gaweda-Walerych a, *, Emilia J. Sitek b, c, Ewa Naroza Michalina Wezyk a, Bogna Brockhuis d, Cezary Zekanowski a, Jaros1aw S1awek b, c a
Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre, PAS, Warsaw, Poland Neurology Department, St. Adalbert Hospital, Copernicus PL, Gdansk, Poland Neurological and Psychiatric Nursing Department, Medical University of Gdansk, Gdansk, Poland d Department of Nuclear Medicine, Medical University of Gdansk, Gdansk, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2018 Received in revised form 24 May 2018 Accepted 25 June 2018
Loss-of-function mutations in progranulin (PGRN) gene cause frontotemporal lobar degeneration. Here, we report a case of a 63-year-old woman with a 2-year history of speech impairment, diagnosed with a nonfluent variant of primary progressive aphasia, a subtype of frontotemporal lobar degeneration. In this patient, a novel heterozygous frameshift mutation, c.77delG, in exon 2 of PGRN gene, introducing premature stop codon, p.(C26SfsX28), has been identified. Cultured fibroblasts derived from the patient and her asymptomatic firstdegree relative with c.77delG mutation had decreased levels of PGRN messenger RNA (mRNA) and protein. However, PGRN mRNA levels did not recover upon incubation with inhibitors of nonsense-mediated mRNA decay (cycloheximide or puromycin), suggesting involvement of other mRNA degradation pathways. In addition, we observed upregulated wingless-type mouse mammary tumor virus integration site (WNT) signaling pathway gene, WNT3A, in fibroblasts of the patient and her asymptomatic first-degree relative with c.77delG mutation. As reported previously, this is an early hallmark of PGRN deficiency. Ó 2018 Elsevier Inc. All rights reserved.
Keywords: Progranulin (PGRN) mutation PGRN haploinsufficiency Nonfluent variant of primary progressive aphasia (nfvPPA) Fibroblasts WNT3A
1. Introduction
2. Materials and methods
Frontotemporal lobar degeneration manifests clinically as behavioral variant or primary progressive aphasia (PPA), the latter one comprising two subtypes: nonfluent variant PPA (nfvPPA) and semantic variant PPA (Gorno-Tempini et al., 2011). Mutations in the progranulin (PGRN) gene (Baker et al., 2006; Cruts et al., 2006) are among the most common genetic causes of PPA (Rohrer, 2014). To date, nearly 80 PGRN mutations have been reported, in the majority nonsense, frameshift, or splicing ones (Alzheimer Disease & Frontotemporal Dementia Mutation Database; http://www.molgen.ua.ac. be/FTDMutations). They typically introduce premature termination codon (PTC) in the messenger RNA (mRNA), which triggers degradation of the mutated mRNA through nonsense-mediated mRNA decay (NMD) and results in PGRN protein haploinsufficiency (ChenPlotkin et al., 2011; van Swieten and Heutink, 2008). Here, we functionally characterize a novel PGRN mutation detected in a 62-year-old woman (diagnosed with nfvPPA) and her asymptomatic first-degree relative.
2.1. Study subjects
* Corresponding author at: Department of Neurodegenerative Diseases, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, Warsaw 02-106, Poland. Tel.: þ48 226086485; fax: þ48 2266855 32. E-mail address: [email protected] (K. Gaweda-Walerych). 0197-4580/$ e see front matter Ó 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2018.06.033
The patient (age of onset, 61 years), her two first-degree relatives (gender and age are omitted for confidentiality), and three unrelated, neurologically healthy controls (gender and age of skin biopsy donation is provided in Table S1) were enrolled for the study. All the study subjects have given an informed consent, and the study protocol has been approved by the Bioethical Committee of Medical University of Gdansk and the Central Clinical Hospital of Ministry of the Interior and Administration in Warsaw. Experimental procedures are described in the supplementary material (Appendix A). 3. Results 3.1. Clinical assessment Neurological assessment of the 62-year-old proband did not reveal any abnormalities apart from aphasia (1-year history of nonfluent speech). The patient was fully independent in the complex activities of daily living. Follow-up in the consecutive year
1.e2
K. Gaweda-Walerych et al. / Neurobiology of Aging xxx (2018) 1.e1e1.e4
revealed significant deterioration of speech and writing, with language and cognitive profile compatible with the diagnosis of nfvPPA. Apraxia of speech was the most prominent feature in spontaneous speech, repetition, and reading. Complex sentence comprehension was mildly impaired, with preserved word comprehension and semantics. Language dysfunction was accompanied by planning deficit (see Table S2). Pegboard task revealed slight bilateral slowing of precise hand movements and involuntary co-movements in the lower limbs. Brain magnetic resonance imaging (62 years 2 months) evidenced left-sided frontotemporal
atrophy (Fig 1F, left panel). Regional cerebral blood flow singlephoton emission computed tomography (rCBF SPECT, 62 years 6 months) showed mild frontal, temporal, and parietal hypoperfusion on the left side (Fig 1F, right panel), which progressed over the period of 10 months (63 years 3 months, not shown). 3.2. Genetic assessment Familial history of the patient suggested early-onset dementia in at least 4 paternal-side family members. The patient and her 2 first-degree
Fig. 1. Decreased PGRN mRNA and protein levels and upregulated WNT3A mRNA levels in nfvPPA patient and her first-degree relative with PGRN c.77delG mutation. (A) Electropherogram showing PGRN WT sequence and (B) heterozygous, 1 nucleotide deletion c.77delG, in PGRN exon 2 of patient’s DNA. (C) Decreased PGRN mRNA and (D) protein levels in cultured fibroblasts of the patient (P) and her asymptomatic first-degree relative (FDR1) with c.77delG mutation, compared to the first-degree relative without the mutation (FDR2-C1) and 3 unrelated control individuals (C2, C3, and C4). (E) Higher WNT3A mRNA levels in carriers of c.77delG mutation versus controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) Brain MRI of the patient evidencing left-sided frontotemporal atrophy (left panel). SPECT demonstrating mild frontal, temporal, and parietal hypoperfusion on the left side (right panel). Abbreviations: MRI, magnetic resonance imaging; mRNA, messenger ribonucleic acid; nfvPPA, nonfluent variant of primary progressive aphasia; PGRN, progranulin.
K. Gaweda-Walerych et al. / Neurobiology of Aging xxx (2018) 1.e1e1.e4
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relatives werescreened for mutations in MAPT, C9orf72, and PGRN genes. The analysis revealed a novel deletion c.77delG in exon 2 of PGRN gene in the patient and one of her first-degree relatives (at a presymptomatic age) (Fig 1A, B). c.77delG mutation was absent in ExAC, 1000gp, and ESP5400 databases. However, there are already known mutations locatedinthesameposition:aframeshiftonec.77_78insC(p.Val28Cysfs, rs1392550887) and 2 missense substitutions c.77 G > T and c.77 G > A (p.Cys26Phe, p.Cys26Tyr, respectively; rs780086363). These mutations are very rare with ExAC MAF/MinorAlleleCount T ¼ 0.000008/1. The latter variant is also annotated in the catalog of somatic mutations in cancer (COSMIC, COSM4382943) and was predicted by SIFT as deleterious and by PolyPhen as possibly damaging. Bioinformatic analysis revealed that c.77delG leads to a frameshift and a premature stop codon, p.(Cys26SerfsX28), implicating degradation of the mutated mRNA by NMD. As expected, cultured fibroblasts with c.77delG mutation derived from the patient and her first-degree relative (FDR1) showed decreased levels of PGRN mRNA (by c.a. 50%) and protein, compared to control fibroblasts, including the patient’s first-degree relative without the mutation (FDR2) (Fig 1C, D). However, PGRN mRNA levels did not recover upon incubation with NMD inhibitors, cycloheximide or puromycin (Fig S1 A, B, C, D). Moreover, sequencing of cDNA derived from the patient’s fibroblasts (cultured under standard conditions or treated with cycloheximide) revealed only wild-type sequence (not shown). This suggests involvement of other mRNA decay pathways in degradation of the mutated mRNA. In addition, fibroblasts with c.77delG mutation exhibited upregulated wingless-type mouse mammary tumor virus integration site (WNT) signaling pathway gene d WTN3A (Fig 1E) d an early hallmark of the PGRN deficiency. Once the genetic testing revealed PGRN mutation, the patient fulfilled level III criteria of nfvPPA (Gorno-Tempini et al., 2011).
mRNA levels in PGRN-deficient fibroblasts from symptomatic and presymptomatic mutation carriers. PGRN-associated PPA usually manifests clinically as a mixed nonfluent and logopenic PPA (lvPPA) phenotype (Rohrer et al., 2010). Although in sporadic cases of nfvPPA speech apraxia is a common core feature, it is not considered typical for PPA patients with PGRN mutations (Rohrer, 2014). In previous studies, PGRN mutation carriers did not show apraxia of speech (Snowden et al., 2015) and PGRN mutations were absent in cases with primary progressive apraxia of speech (Flanagan et al., 2015). However, in our case, not only profound speech apraxia is present, but also other features typical for nfvPPA coexist, such as preserved delayed word recognition and spatial function, and selective executive deficits. Only rapid progression of anomia, observed in our case at follow-up, is typical for PGRN-associated PPA (Rohrer et al., 2010). However, in our patient anomia was not accompanied by word-comprehension deficits, that are frequently associated with PGRN-related PPA but not with the classical nfvPPA phenotype. This highlights the possible heterogeneity in the clinical presentation of PGRN-linked aphasia. Individuals with speech apraxia and PGRN mutations were reported to manifest PPA-unclassified phenotype (Harris et al., 2013). Our patient’s phenotype, with predominant apraxia of speech accompanied by other nvfPPA features both in terms of language impairment (e.g., agrammatism) and cognitive profile (e.g., dysexecutive deficits), suggests that PGRN mutations may be also associated with a rather typical nfvPPA clinical presentation, observed in sporadic cases.
4. Discussion
Acknowledgements
Functional assessment of a novel PGRN mutation c.77delG demonstrated decreased PGRN mRNA and protein levels in fibroblasts derived from the patient and her first-degree relative, which confirms haploinsufficiency as the underlying pathogenic mechanism. To date, this mechanism is considered the most common for PGRN mutations (van Swieten and Heutink, 2008). Lack of mRNA recovery after cycloheximide treatment despite detecting mRNA decay has been reported previously in case of mutations introducing PTC close to the N-terminus of the protein (Montfort et al., 2006; Perrin-Vidoz et al., 2002). Such PTCs have been shown to escape NMD due to reinitiation of transcription from the following ATG sequence (Kozak, 2001; Perrin-Vidoz et al., 2002). In the case of PGRN c.77delG mutation (located at the beginning of the first translated exon, close to the start codon), RNA decay is likely triggered by a mechanism other than NMD, as previously proposed by Montfort et al., 2006. WNT signaling was identified as one of the top defective pathways in genome-wide transcriptome analyses of PGRNdeficient human neural progenitors and neurons derived from induced pluripotent stem cells and postmortem brains obtained from frontotemporal lobar degenerationePGRN patients (Raitano et al., 2015; Rosen et al., 2011). WNT2, WNT3A, WNT5A, WNT6, WNT10a, and CTNNB1 were among the top upregulated transcripts (Raitano et al., 2015). Increased expression of various WNT pathway genes has been further confirmed in cultured keratinocytes (with siRNA-silenced PGRN) and lymphoblastoid cell lines derived from carriers of PGRN mutations (Alquezar et al., 2016; Tian et al., 2016). In addition, aberrant WNT signaling has emerged as an early hallmark of PGRN deficiency as it was observed also in asymptomatic PGRN mutation carriers (Alquezar et al., 2016). Here, for the first time, we report increased WNT3A
This work was supported by National Science Center, Poland (DEC-2017/01/X/NZ3/00266), the funding obtained by Dr GawedaWalerych. The authors thank the patient, her relatives, and other subjects who participated in this study and Dr Dawid Walerych and ski for critical reading of the article. Dr Mariusz Berdyn
Disclosure statement The authors declare no conflict of interest.
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