Rapidly Progressive Frontotemporal Dementia Associated with MAPT Mutation G389R

Abstract

Frontotemporal dementia includes a large spectrum of neurodegenerative disorders. Here, we report the case of a young patient with MAPT mutation G389R, who was 27 years old when he progressively developed severe behavioral disturbances. Initially, he presented with slowly progressive personality change. After 1 year, he exhibited moderate dementia with extrapyramidal and pyramidal symptoms. MRI showed frontotemporal atrophy. He rapidly progressed to severe dementia 3 years after onset. Genetic testing revealed a heterozygous guanine to cytosine mutation at the first base of codon 389 (c.1165G>A) of MAPT, the tau gene, resulting in a glycine to arginine substitution in the patient and two unaffected relatives. We predicted the model of mutant tau protein through I-TASSER software, and speculated the structural change of tau protein caused by mutant site. We also detected the MAPT gene transcript and methylation of samples from peripheral blood leucocytes in an attempt to explain the possible mechanisms of incomplete penetrance, although there were not positive findings. This case is remarkable because of the early onset and rapid progression of the disease.

Keywords

Early onset dementia frontotemporal dementia G389R mutation MAPT protein structure predicting

INTRODUCTION

Frontotemporal dementia (FTD) is a group of progressive degenerative disorders clinically characterized by behavioral changes and cognitive impairment, and pathologic atrophy of the frontal and/or anterior temporal lobes [1]. It is the second most common cause of dementia in patients under 65 years of age, after Alzheimer’s disease [2]. Three main phenotypes can be distinguished: behavioral variant frontotemporal dementia (bvFTD), progressive nonfluent aphasia (PNFA), and semantic dementia (SD) [3]. Of these, bvFTD is the most common clinical syndrome of FTD, typically characterized by progressive changes in personality,behavior, insight, judgment, reasoning abilities, or language, with relative perseveration of episodic memory [4]. Approximately, 30–50% of FTD patients have a positive family history, and 10% exhibit an autosomal dominant mode of inheritance [5]. Mutations in the genes that encode microtubule associated protein tau (MAPT), progranulin (GRN), and C9orf72 are the most common causes of FTD [6].

MAPT is essential for the development and maintenance of the nervous system. Among its many roles is to regulate the growing and shortening dynamics of microtubules [7]. Here we report an early onset FTD patient with MAPT mutation G389R discovered during a 3-year follow-up. This mutation had previously been described in different patients [8 –10]. Interestingly, there are two relatives who carried the same mutation who did not present any clinical modifications, suggesting incomplete penetrance. It is possible that monoallelic expression of a subset of autosomal genes might contribute to the incomplete penetrance of certain CNS disorders, and differential DNA methylation has been reported to be associated with gene silencing in the cases of random monoallelic expression [11].

CASE REPORT

The patient underwent a clinical evaluation at our institution and was then enrolled in the Foundation of China Alzheimer’s disease and related disorders study. All additional data from his relatives were collected and analyzed.

In late 2012, five years after graduating from university, a right-handed 27-year-old patient appeared gradually more inert and stopped working and taking care of personal hygiene. Instead, he spent his time sitting in front of the computer playing games. Progressively, his movements became slow, stiff, and his ability to play the same games declined. Emotional responses became odd and unusual. When he witnessed his sister suffer a fall, the patient merely looked at his sister and giggled. On admission to the local hospital (June 2013), his brain MRI, including T1 and T2 weighted images, revealed mild atrophy of frontal lobe (Fig. 1Aa at age 29), which led to a suspicion of FTD. Subsequently, therapy with Aricept, piracetam, and meclofenoxate were initiated. In September 2013, the patient suffered an accidental head injury that caused external bleeding. He left the wound untreated, holding his head in his hands. In the aftermath of this injury, he became severely apathetic and exhibited emotional blunting. He was referred to our department in October 2013. He had no complaints and displayed a mood-incongruent smile. He seemed indifferent and unconcerned about his health. His Mini-Mental State Examination (MMSE) score was 17/30, including deficits in orientation (4/10), attention and calculation (1/5), recall (6/6), language (6/8), and visual construction (0/1) (Table 1). On Montreal Cognitive Assessment (MoCA) testing, the patient scored 11/30, including deficits in visual construction (0/4), alternate line (0/1), recall (0/5), attention and calculation (2/6), language (3/6), abstraction (1/2), and orientation (5/6) (Table 2). The neurological evaluation showed postural tremor of bilateral upper extremities, positive left Babinski, and incoordination in movements. His second MR imaging revealed moderate atrophy of the frontal and temporal lobes (Fig. 1Ab at age 29). Brain DAT PET showed normal functioning of the dopamine transporter (Fig. 1D). Electroencephalogram was normal. Cerebrospinal fluid biomarkers measurements showed a moderate increase in total proteins (0.492 g/L, N < 0.45 g/L), and protein qualitative test was negative. Standard blood investigations were normal. These findings were consistent with the diagnosis of probable FTD [4] and, subsequently, therapy with Aricept and memantine were initiated. In October 2014, one year after the previous evaluation, cognitive decline progressed rapidly. MMSE score was 11/30, including deficits in orientation (4/10), attention and calculation (0/5), recall (4/6), language (3/8), and visual construction (0/1) (Table 1). On MoCA testing, the patient scored 8/30, with deficits in visual-construction (0/4), alternate line (0/1), recall (0/5), attention and calculation (1/6), language (3/6), abstraction (0/2), and orientation (4/6) (Table 2). The clinical examination revealed muscle weakness of right lower extremity (IV level), hypermyotonia and tendon hyperreflexia of bilateral lower extremities, positive left Babinski, and decline in movement coordination. His third MR imaging revealed increased atrophy of frontal and temporal lobes when compared to the previous MR imaging (Fig. 1Ac at age 30). At 3 years post-onset of symptoms, his cognitive functions and behavior worsened significantly. The patient was increasingly apathetic and exhibited severe language dysfunction, including reduced spontaneous speech and comprehension difficulties, leading to complete inability to communicate with others. MMSE and MoCA scores were all 0/30 at this point (Tables 1 and 2). He developed pyramidal features such as increased muscle weakness (III level) and bilateral positive Babinski signs, and extrapyramidal features such as distal tremor, rigidity, and akinesia. Because of the patient’s difficulty with cooperation, the brain MRI at 3 years post-onset of symptoms was not conducted.

The family history was as follows (Fig. 1B). There are no similar presentations in relatives of the patient. We tested the gene mutation c.1165G>A SNP site (rs63750512), causing guanidine to cytosine substitution at codon 389 (exon 13) in the MAPT sequence in the patient (III-2). The tau gene was assessed by evaluating the 12 members in the family including I-2, II-1, II-2, II-4, II-5, II1-0, III-1, III-3, III-4, III-6, III-9, and IV-1. The same mutation was identified in the patient’s unaffected father (II-1) and sister (III-1) (Fig. 1C).

GENETIC/BIOCHEMICAL PROCEDURES

Genetic procedures

Total genomic DNA was prepared and amplified from peripheral blood according to standard procedures. All coding exons and intron-exon junctions of MAPT gene were sequenced in both directions on an ABI3730 automated sequencer (ABI, USA). One hundred nanograms of the extracted DNA were amplified by polymerase chain reaction (PCR) using specific MAPT primers. The primers of exon 13 in MAPT gene are listed below. Sequences were analyzed using SeqMan software Package.

Forward primer A: 5^′-GAGCAAGACCCTGTCTCAAA-3^′

Reverse primer A: 5^′-ATTACTAGCCCACCCATCAA-3^′

Forward primer B: 5^′-GCAGGGCTGGTCTTTCT-3^′

Reverse primer B: 5^′-TTTTTTTCCACACTCTCTCAT-3^′

MAPT structure model prediction

As the crystal structure of wild type MAPT has not yet been solved, the complete sequence of MAPT (441 aa) was submitted to the NCBI BLAST [12] tool for detecting the homologues sequences against the PDB database. However, there was no homologues were identified with the identity percentage of 30% for homology modeling. The homolog-based structural prediction service I-TASSER [13] was used to generate the 3D models of wild type and mutant type MAPT protein. I-TASSER (as “Zhang-Server”) was ranked as the No. 1 server for protein structure prediction in the 7th, 8th, 9th, 10th, and 11th Critical Assessment of protein Structure Prediction (CASP) experiments from years 2006 to 2014. Multiple templates (PDB Hits: 1w0sA, 2qzvA, 1w0rA, 4durA, 3ov0A, 1s58A) were used to create the 3D model. The predicted model for wild and mutant type protein were evaluated using different validation tools including Procheck [14], Verify_3D [15], and ERRAT [16] at NIH SAVES (http://nihserver.mbi.ucla.edu/SAVES/). Molecular graphics were performed using Swiss-Pdbviewer 4.1.0 software [17].

Binding site prediction

To determine the binding site of MAPT, the active sites of modeled MAPT protein were predicted using MetaPocket 2.0 [18]. The MetaPocket 2.0 is a meta server used to identify ligand binding sites on protein surface, in which the predicted binding sites from eight methods: LIGSITEcs, PASS, Q-SiteFinder, SURFNET, Fpocket, GHECOM, ConCavity, and POCASA are combined together to improve the prediction success rate. The prediction results can be visualized by using the MetaPocket web visualization system built based on Jmol.

The characterization of MAPT mutation G389R transcript by pyrosequencing

The mutation G389R (c.1165G>A, rs63750512) of MAPT cDNA 176-bp amplicons were obtained from the patient, the unaffected carriers, and control person mRNA samples (III-2, II-1, III-1, and III-3) by RT-PCR reaction. The cycling parameters of RT-PCR were 3 min at 95°C, followed by 30 s at 94°C, followed by 35 cycles of 60°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 5 min. The target sequence was gaccacRgggcgga (the base with box was variant site). The pair of PCR primer sequence included the forward primer 5^′-CCCTGGACAATATCACCCAC-3^′ and reverse primer 5^′-GGTGGAGGAGACATTGCTGA-3^′(5^′ marked by biotin), and the sequencing primer sequence was 5^′-AACGCCAAAGCCAAGACA-3^′. The pyrosequencing was performed following the manufacturer’s protocols (Sangon, China). The analysis was used through PyroMark Software 1.0.11 software environment (Sangon, China).

MAPT methylation by bisulfite sequencing

For detecting the methylation of MAPT gene including the region of promoter and the region containing variant site c.1165G>A, we performed bisulfite sequencing. Unmethylated cytosine was converted to uracil by sodium bisulfite. Three microliters of the converted samples were used as PCR template for amplification. PCR products were ligated into pUC18-T Vector (Sangon, China). Ligations were transformed into Escherichia coli JM109 and plasmid extracted from cultures of recombinant colonies using SanPrep Column Endotoxin-Free Plasmid Mini-Preps Kit (Sangon, China). At least ten clones per sample were sequenced using the ABI 3730 automated sequencer (ABI, USA).

The first target detection sequence included 54 CpG sites within the promoter and the first exon of MAPT gene. The forward primer was 5^′-AAGATTTTAATTATAGGAGGTGGAG-3^′ and the reverse primer was 5^′-CTACTATTAATACCRAAACTAATAAATAAC-3^′. The second target detection sequence included 14 CpG sites around the variant site c.1165G>A. The forward primer was 5^′- AGGGTAGTTGGTAGGGTTGG-3^′ and the reverse primer was 5^′-TTAACCAAAAAAACAAACACCTC-3^′.

Ethics and patient consents

We received approval from the regional ethical standards committee on human experimentation for our experiments using human materials. We also received written informed consent for research from all patients and guardians.

RESULTS

Protein modeling

The structure for mutant MAPT protein containing 441 amino acids has a substitution of glycine residue to arginine at the 389th amino acid. The predicted models of wild type and mutant proteins were selected on the basis of C-scores of –1.03 and –0.73 respectively by I-TASSER software. Then, the models were evaluated using different validation tools. In regards to the wild type MAPT protein structure, Ramachandran plot via Procheck sever showed a favorable quality plot which displayed 78.1% of the residues in the core regions, 15.6% in allowable regions, 4.6% in general regions, and just 1.7% in disallowable regions. Essentially, Ramachandran plot showed 98.3% of the residues in the acceptable regions. In regards to the mutant MAPT protein structure, Ramachandran plot also showed 77.3% of the residues in the core regions, 15.8% in allowable regions, 5.2% in general regions, and just 1.7% in disallowable regions, which, in summary, also displayed 98.3% of the residues in the acceptable regions. Verify_3D sever and ERRAT sever had values of 48.19% and 6.989 in the wild type MAPT structure, and 56.56% and 10.109 in the mutant MAPT structure. Almost all regions were predicted as random coil from the secondary structure prediction, which caused the values from Verify_3D and ERRAT to be low. Values above these ensure that the quality of the generated model is good. In the mutant structure, substitution of glycine to arginine located at the 389th amino acid resulted in obvious change of protein structure around the 389th amino acid, when compared to the wild type structure. The substitution led to an extension of half a spiral in the α-helix, and formation of a stable H-bound between Arg389 and Ile360 in the predicted mutant protein structure (Fig. 2A–C). The binding sites of the developed model were predicted using MetaPocket 2.0 [18]. There were in total 6 ligand binding site pockets in the MAPT protein. All the binding pockets are shown in Fig. 2, and the 6th binding site pocket close to the 389th amino acid is marked with color (Fig. 2D).

The analysis of MATP mutation G389R cDNA by pyrosequencing

By pyrosequencing, MATP mutation G389R (c.1165G>A) cDNA was amplified by RT-PCR from mRNA extracted from the peripheral blood leucocyte of the patient (III-2), the unaffected carriers (II-1 and III-1), and a control person (III-3). We found that there was no mutation in MAPT cDNA from all samples (Fig. 3A), which was inconsistent with the sequencing results of DNA.

The DNA methylation diversity of MAPT gene CpG islands

To determine the DNA methylation status of MAPT gene between the patient and the unaffected carriers, we performed bisulfite allelic sequencing using DNA from III-2, II-1, and III-1. Additionally, III-3 was as tested as a normal phenotype. First, we tested the DNA methylation of a 351-bp region containing 54 CpGs within the promoter and the first exon of MAPT gene (Fig. 3C). Second, we tested the DNA methylation of a 272-bp region containing 14 CpGs which includes the variant site c.1165G>A (Fig. 3D). Therein, we tested the methylation of two parental allele strands (G-strand and A-strand, respectively) (Fig. 3D). We found there were not significant differences in methylation between the patient and other family members on the CpG enrichmentregions.

DISCUSSION

The patient exhibited early-onset cognitive disorder combined with the gradual appearance of personality change and behavioral disturbances. Despite the very young age of onset, the typical clinical manifestations and MR imaging still revealed findings related to FTD. The patient lacked a family history, but two unaffected subjects (II-1 and III-1) carried the same mutation (MAPT G389R, c.1165G>A) as the patient. To date, more than 40 mutations have been found in the MAPT gene, and they account for 40% of autosomal-dominant FTD (http://www.molgen.ua.ac.be/FTDmutations) [19]. In 2008, a similar MAPT G389R mutation was found in a 21-year-old woman that presented with postpartum depression as her initial symptom [8]. In 2013, another similar mutation was found in a 17-year-old girl who initially suffered from atypical depression and emotional blunting [9]. The mean age at onset of FTD is 55 years and usually shows a small intrafamilial variation between 45 and 65 years. In some cases, age of onset as early as the third decade can occur, when tau mutations such as P301S, S305N, L351R, or G335V are present [9]. It seems that early onset cases of FTD are more likely to be found in tau G389R carriers. During three-year-follow-up, we observed a rapid progression of cognitive dysfunction in our patient, as his MMSE score decreased from 17 points to 0. The mean life expectancy with FTD is approximately 9 years, but varies between 5 and 20 years [19]. The present case showed the proband had earlier onset age and more rapid progression.

G389R tau reduces the affinity of tau for microtubles, as indicated by a reduction in the ability of mutant tau to promote microtubule assembly [20]. These defects induce tau hyperphosphorylation and the formation of filaments resulting in the aggregation of these abnormal proteins into unusual Pick bodies [21]. This mechanism applies to coding region mutations in exons 9, 11, 12, and 13. Van Swieten isolated tau filaments from the cerebral cortex of an FTD patient with the MAPT G389R mutation, which showed two distinct morphologies, including a major species consisting of a straight filament, and a minor species consisting of a twisted filament [20]. However, in Alzheimer’s disease pathology, straight filaments account for only about 10% of isolated filaments, with paired helical filaments constituting the major species [22].

Neither the crystal structure nor template of the MAPT protein is available for homology modeling. So, protein threading method was used to construct the 3D structure of MAPT using service I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [13]. Although this represents a prediction of the protein 3D structure, it is still able to provide clues for differences between the wild type and the G389R mutant type tau. In the mutant tau structure, substitution of glycine residue to arginine residue located at the position of the 389th amino acid, led to the formation of stable hydrogen bond between Arg389 and Ile360, and extension of half a spiral in the α-helix. In order to identify the effects of the tau structural change on protein function, we predicted the ligand binding site pockets. We found that there are six predicted binding site pockets in MAPT protein, and the sixth binding site pocket is just adjacent to the 389th amino acid. The structural change of mutant tau, including formation of the stable hydrogen bound and extension of half a spiral in the α-helix caused by G389R mutation, can alter the binding activity of the sixth binding site. As we known, MAPT is coined to be abnormally hyperphosphorylated by kinases like cyclin dependent kinase5 (CDK5) and glycogen synthase kinase 3β (GSK3β) in Alzheimer’s disease [23]. Numerous studies suggested that abnormal phosphorylation impedes tau binding to microtubules, leading on the one hand to the depolymerization and loss of the latter, and on the other hand to the formation of toxic aggregated tau species. We speculate that the structural change of the binding site caused by G389R mutation in MAPT protein leads its hyperphosphorylation caused by kinases to occur, and impedes MAPT binding to microtubules. The verification research needs to be carried out in further experiments.

In this family, the proband had a young onset of FTD, and there were two clinically normal mutation carriers, his father and elder sister (II-1 and III-1), suggesting autosomal dominant inheritance with incomplete penetrance. Chaunu et al. reported the same G389R tau mutation in a 17-year-old girl, and the mutation was identified in her unaffected father [9]. In this context of autosomal dominant transmission, but incomplete penetrance, several classical genetic diseases were screened, such as Huntington’s disease and hereditary Creutzfeldt-Jakob disease, but not confirmed [9]. Why did the disease present with incomplete penetrance in this case? Methylation of cytosine residues in DNA is a highly conserved epigenetic marker in most eukaryotic organisms [24]. DNA methylation at the promoter is an important epigenetic mechanism that causes gene silencing, and DNA methylation at the exon will reduce transcriptional elongation rate and promote the exon inclusion that causes monoallelic expression [25]. To better understand the methylation of the MAPT gene, we detected CpG islands, including promoter and variant sites, by bisulfite sequencing. We did not find a significant difference in methylation status between the patient and other family members. We quantitatively analyzed the variant site of the MAPT cDNA from peripheral blood leucocytes of the patient and three family members (II-1, III-1, and III-3) by pyrosequencing. We did not detect the mutation in any of the cDNA samples. Tissue-specific factors play a role in gene expression aside from the methylation status, based on the variation in methylation among different cell lines [26]. This is a possible reason that there are different patterns of methylation and transcription between peripheral blood leucocytes and CNS neurons. Beyond DNA methylation, dynamic control of gene expression could be exerted by other epigenetic mechanisms, including histone post-translational modifications, nucleosome positioning, and occupancy [27]. Our experiment is only a first attempt to research the mechanism of incomplete penetrance of autosomal dominant inheritance, and clarifying the etiology requires more samples and further in-depth study.

CONCLUSION

The case presented here was an example of an early-onset, rapidly progressive frontotemporal dementia. Genetic testing revealed a heterozygous mutation in G389R of the MAPT protein in the patient and two unaffected relatives, which suggested an autosomal dominant inheritance pattern of FTD, with incomplete penetrance. We predicted the model of the mutant tau protein through I-TASSER software, and speculated the structural change to the tau protein caused by the mutant site. We also detected the MAPT transcript and methylation of samples from peripheral blood leucocytes in hopes of explaining the possible mechanisms of incomplete penetrance, although there were no positive findings using this particular strategy.

Footnotes

ACKNOWLEDGMENTS

This study was supported by a grant of Natural Science Foundation of China (81301139), National Key Clinical Disciplines at Shanghai Mental Health Center (Office of Medical Affairs, Ministry of Health, 2011–873; OMAMH, 2011–873).

Authors’ disclosures available online (.

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