A novel c.1468 G > A GRN mutation causes frontotemporal dementia in a Chinese Han family

Abstract

Background/purpose

GRN mutations act as causative factors in patients with FTD clinical phenotype or FTD pathology and exhibit high clinical heterogeneity. The discovery of these mutations and the analysis of their associations with resembling Alzheimer’s disease should be critical to understand the pathogenesis of FTD.

Methods

Clinical analysis, neuroimaging, target region capture and high-throughput sequencing were performed in a family of 3 generations. The underlying Alzheimer’s pathology was evaluated by using biomarker evidence obtained from cerebrospinal fluid (CSF) amyloid testing, 18F-florbetapir (AV-45) PET imaging and FDG18-positron emission tomography imaging.

Results

Through target region capture and high-throughput sequencing, a three-generation family was able to identify a heterozygous G to A point mutation at position 490 (c.1468)G > A, which led to a valine to methionine substitution (V490M) at exon 12. This unique missense mutation was found at codon 1468. Eight members of the proband's family—two sisters and the proband himself—had the mutation found by Sanger sequencing. Interestingly, biomarker tests for amyloid in the proband's cerebrospinal fluid (CSF) indicated pathology consistent with Alzheimer's disease (AD). The mutation was expected to have a high likelihood of being pathogenic.

Conclusions

We firstly reported a novel mutation in the GRN gene at codon 490 (V490M) in exon 12 in a China FTD family. The CSF biomarker alterations of the proband revealed a reduction in Aβ42 and the Aβ42/Aβ40 ratio. The analysis of mutation might support the role of GRN in patients with FTD and contribute to the discovery of a new pathological mechanism underlying the disease.

Introduction

Frontotemporal dementia (FTD) encompasses a range of neurodegenerative illnesses that are genetically intricate and marked by behavioral disruptions, language impairments, and various cognitive abnormalities. FTD is primarily categorized into three sub-types based on clinical phenotype: semantic dementia (SD), progressive non-fluent aphasia (PNFA), and behavioral variant FTD (bvFTD) [1]. Additionally, there is a phenotype that shares the characteristics of both FTD and other dementias and motor diseases, including motor neuron disease, atypical parkinsonism, corticobasal degeneration, progressive supranuclear palsy (PSP), and Alzheimer's disease (AD) [2,3,4,5]. The genes associated with FTD, such as Chromosome 9 open reading frame 72 (C9orf72), granulin (GRN), and, microtubule-associated protein tau (MAPT), are widely recognized as the primary genetic factors contributing to FTD, particularly in cases of early-onset familial FTD (FFTD) [6]. GRN mutations are responsible for around 5–10% of all occurrences of FTD globally [7, 8]. Recent research indicates that GRN mutation carriers may be diagnosed with AD [9]. In addition, GRN contributes to the development of AD by various mechanisms, such as the preservation of neuronal function, the buildup of phosphorylated tau within neurons, neuroinflammation, and the accumulation and removal of Aβ [10, 11]. GRN mutations can potentially increase the chance of developing AD in persons who exhibit either the clinical symptoms or the pathological characteristics of AD [12, 13]. FTD induced by GRN mutations is less prevalent in China compared to Western countries [4, 14]. Most pathogenic GRN gene variants are frameshift, stop codon, or splice site mutations. However, the contribution of missense mutations to disease progression cannot be overlooked [15]. In this investigation, we identified a new missense mutation in the GRN gene at codon 1468 (V490M), which is associated with familial FTD with a clinical phenotype manifested as AD and MCI in China. The examination of this mutation revealed the involvement of GRN in patients with FTD and AD and helped with the identification of a novel pathogenic mechanism that underlies the disease.

Materials and methods

Participants

The proband (II:3) and his younger sister (II:5) received a clinical diagnosis at the Department of Neurology of Zhengzhou University People's Hospital. At the Shenzhen Hospital’s Department of Neurology, the elder sister (II:2) was assessed and given a clinical diagnosis. The family hailed from the central-southern region of Henan province in China and comprised three successive generations. The diagnosis of AD prior to undergoing gene analysis was established based on the criteria set out by the National Institute on Aging-Alzheimer's Association (NIA-AA) [16]. In the present investigation, 150 control participants, the proband, and seven more family members were enrolled.

All three cases (II:2, II:3, II:5) had standard brain magnetic resonance imaging (MRI) to establish the diagnosis and rule out any other possible explanations. FDG¹⁸-positron emission tomography (FDG¹⁸-PET) and brain 18F-florbetapir (AV-45) PET imaging were performed on a single patient (II:5).

Analysis and capture of target regions

The subjects who volunteered to have their blood drawn included the proband (II:3), members of their immediate relatives (II:2, II:4, II:5, III:4, III:5, III:6, and III:9), and even unrelated normal controls who gave their informed consent. The study protocol received approval from the Institutional Review Board of Zhengzhou University People's Hospital.

A set of 60-mer biotin-labeled probes was developed herein to bind to the target region's exon. Initially, we readied the library for sequencing on the Illumina platform. Mygenostics Co., Ltd.'s GenCap Kit^® was employed to capture the target gene areas. After that, we ran bioinformatics analyses and high-throughput sequencing on the Illumina NextSeq 500, a next-generation sequencer. Using the Mygenostics Library Preparation Kit, the library was set up following the Illumina platform specifications. An enzymatic cleavage was performed on a DNA sample of 1–5 μg. The genomic DNA fragments obtained were subjected to end-repair, followed by the addition of adapters. The libraries were created to have a length of around 350–400 bp. They were amplified using thermal cyclers and then analyzed using an Agilent 2100 Bioanalyzer. The target gene was captured by covalently binding the biotin-labeled probe to streptavidin-modified magnetic beads following its hybridization with library DNA under particular conditions. After washing, eluting, and amplifying the magnetic rack adsorption beads containing the target gene, the libraries were prepared for parallel sequencing. Illumina employs a distinctive "bridge" amplification response. To image the NextSeq 500 sequencer, the library was fed into the sequencing Flowcell. The sequencing-by-synthesis technique was used to sequence two types of nucleotides that were labeled with fluorescence and could be reversed. Every cycle reaction is capable of only elongating a precise complimentary basis. The identification of the base species was validated using four distinct fluorescence signals, which served as indicators of the accuracy of the nucleic acid sequence. The entire sequence of nucleic acids was read after a few cycles. After filtering, separating, and annotating the data, the variant was identified and its biological relevance was assessed. The single nucleotide polymorphism was detected by referencing the NCBI dbSNP137, HapMap, the Genome Aggregation Database (GnomAD), ExAC Browser Beta (http://exac.broadinstitute.org/), and 1000 human genome dataset (20110521 release, http://www.1000genomes.org/). The single nucleotide mutation's pathogenicity was predicted using the conservation score, an internet program called MutationTaster, and web-based programs SIFT and PolyPhen-2. Moreover, we used UniProt (https://www.uniprot.org/) to get the amino acid sequences that were similar to this mutation and other species. The DNAMAN software and WebLogo tool (https://weblogo.berkeley.edu/logo.cgi) were employed to assess the evolutionary conservation of amino acids. This was done by aligning the sequences of GRN orthologs from various species. In addition, the SWISS-MODEL tool (https://swissmodel.expasy.org/) was utilized to generate protein models before and after mutation. The PyMol software was used to simulate both the wild type (WT) and mutant models.

Analyzing cerebrospinal fluid (CSF) biomarkers

Proband (II:3) CSF was extracted in the morning via lumbar puncture, and the fluid was centrifuged at 2000 × g for 10 min. The supernatants were then kept at – 80 °C until examination. The following proteins were measured via a solid-phase enzyme-linked immune absorbent assay (Fujirebio, Belgium): phosphorylated tau at T181 (p-tau), total tau (t-tau), amyloid-beta 1–40 (Aβ1–40), and amyloid-beta 1–42 (Aβ1–42), all per the instructions provided by the manufacturer. In summary, the 96-well plate was coated with antigen-specific monoclonal antibodies. Human CSF was then added to the plate, and the plate was incubated with a biotinylated secondary antibody. Streptavidin labeled with horseradish peroxidase was employed to identify the antigen–antibody complex. The color intensity after adding the substrate is a quantitative indicator of the protein concentration in the CSF sample.

Sanger sequencing

To confirm the DNA sequence variations identified using Sanger sequencing, we developed primers specifically for the locations in the GRN gene that contain the variant. The primer sequences used were as follows: the forward primer sequence was 5′-AGAGACATCGGCTGTGACCAG-3′ while the reverse primer sequence was 5′-CGAGAGGGTTGGACGAGG-3′. The Multigene OptiMix (LABNET, USA) was utilized for conducting the polymerase chain reaction (PCR). Using an ABI3100 automated sequencer (Applied Biosystems, Foster City, CA, USA), the PCR amplification products of the proband and the remaining seven family members were purified and sequenced. The NCBI BLAST tool and the Chromas program were used to compare the sequencing reads.

Results

Case reports

At the age of 69, the proband (II:3), a 72-year-old man, began to lose his memory, particularly regarding recent events. This was followed by forgetting how to go home in a familiar setting. The clinical signs showed a gradual deterioration. At 71 years old, he had a decline in cognitive function characterized by delayed responses and difficulty finding the right words, known as cataphasia. At 72 years old, the person's personality changed, becoming more obstinate, irritable, and uninterested. Over time, he developed a reluctance to leave his home and experienced memory loss and a deterioration in cognitive abilities. He was dependent on his son and daughter for everyday tasks since he was unable to take care of himself. In addition, he exhibited infrequent verbal communication and displayed childlike behavior. The neurological evaluation identified memory and cognitive impairments, and disorientation. At 72 years of age, the Mini-Mental State Examination performance was 16/30. An MRI performed at 72 years of age showed modest hippocampal shrinkage and temporal lobe cortical atrophy (Fig. 1A). Over the next years, the proband's severity needed further evaluation. Nevertheless, his kid rejected every test, including the FDG-PET and MRI. In 2020, the proband had a lumbar puncture and cerebral computed tomography (CT) after being admitted to the hospital with acute pneumonia. A brain CT scan showed considerable hippocampal shrinkage and temporal lobe cortical atrophy (Fig. 1B). The proband's CSF amyloid test revealed a rise in total-tau and phospho-tau levels together with a reduction in Aβ42 (Table 1).

Fig. 1

A Hippocampus atrophy and mild cortical atrophy were revealed by A MRI, and B brain CT compared to A

Table 1 Cerebrospinal fluid biomarker analysis of the proband (II:3)

Although his father (I1) had dementia for 7 years before his death at the age of 84, no specific information on his cognitive state was accessible. The sister of the index patient (II:2) had a gradual decline in memory function and experienced challenges in comprehending spoken language and doing everyday tasks. At the age of 76, she had an MRI of her brain in 2016 after battling dementia for 6 years. The results showed moderate to severe overall shrinkage in the hippocampus, subcortical temporal area, and cortical and subcortical regions. Her lateral ventricular capacity was increased, with the exception of the periventricular and subcortical white matter abnormalities (Fig. 2). Accordingly, a clinical diagnosis of likely AD for her was established at the Shenzhen Hospital. The family declined to do further testing such as lumbar puncture, cranial MRI, and FDG-PET/18F-florbetapir (AV-45) PET, and we only received limited data from the first hospitalization. Presently, she is in the late stage of the illness, rendering her unable to conduct neuropsychological evaluations or other examinations, and confined to bed as reported during the telephone follow-up.

Fig. 2

MRI shows moderate general atrophy in cortical and subcortical temporal and hippocampus regions. Furthermore, subcortical and periventricular white matter lesions are observed, and lateral ventricular volume is enlarged

At the age of 67 years, the proband's other sister (II:5) had modest memory impairments, but was unaffected in her employment or day-to-day activities. At 70 years old in 2022, she still had trouble remembering to switch off her stove while cooking and never knew what to look for on her phone. Interestingly, her overall test findings were within the normal range, and her MMSE score was 26 out of 30, particularly in terms of delayed memory. The MRI scan showed a little decrease in the cortex without any shrinkage in the hippocampus (Fig. 3A). The FDG-PET scan indicated reduced metabolism in the right temporoparietal region and the left local parietal lobe (Fig. 3B). Additionally, the 18F-florbetapir (AV-45) PET scan showed no abnormal accumulation of Aβ in the cerebral cortex (Fig. 3C). Consequently, she received a clinical diagnosis of mild cognitive impairment prior to undergoing gene analysis. Characteristics of the family affected members are summarized in Table 2.

Fig. 3

A MRI shows little thinning of the outer layer of the brain without any shrinkage of the hippocampus. B 18F-FDG-PET reveals decreased metabolic activity in the right temporoparietal region and the left local parietal lobe. C The 18F-florbetapir (AV-45) PET scan revealed no presence of Aβ deposition in the cerebral cortex. PET: positron emission tomography; MRI: Magnetic resonance image

Table 2 Clinical features of GRN p.V490M family patients and other pathogenic GRN missense mutations

Mutation analysis

The individual being studied has a confirmed family history that aligns with the inheritance pattern of autosomal dominant dementia (Fig. 4A). A new missense mutation (c.1468G > T, p.Val490Met) was identified in the proband with AD-related symptoms by target region capture sequencing. No other genes have any other mutations that caused the condition. The GRN gene, which has been linked to FTD, has the mutation at exon 12. According to Cruts et al. (2012) and Stenson et al. (2014), neither the AD&FTD Mutation Database (www.molgen.ua.ac.be/admutations/) nor The Human Genetics Mutation Database (HGMD) and the Genome Aggregation Database(GnomAD) recorded this missense mutation. The Exome Variant Server (http://evs.gs.washington.edu/EVS/) and dbSNP were also used to forecast the mutation's pathogenicity. An examination of evolutionary conservation showed that the codon 490 side chain was changed by the extremely stable amino acid change that occurred as a consequence of the missense mutation p.V490M (Fig. 4B, C). Also, the mutation was predicted to have damaging, likely damaging, conserved, and disease-causing consequences by SIFT, Polyphen2, Phylop, and Mutation Taster, respectively. These findings showed that the new missense mutation is likely the main reason why certain families get FTD.

Fig. 4

A An analysis of the pedigree of FTD related to the GRN Val490Met mutation. The arrow designates the proband, the circle represents a female, the square represents a male, a black symbol represents an impacted family member, and a slashed symbol represents a dead family member. Proband (III: 3) and family members II: 2, II: 4, II: 5, III: 4, III: 5, III: 6, III: 9 provided blood samples. B Evolutionary conservation analysis. Val at position 490 in GRN (red square) is conserved among Homo sapiens (NP_002078.1), horse (XP_001489791.1), chimpanzee (XP_016787144.1), mouse (NP_032201.2), Bos taurus (XP_010814707.2), Mus musculus (NP_032201.3), and Canis lupus (XP_038341266.1). The protein sequences were retrieved from GenBank. C 3D model of GRN was constructed from Val to Met at position 490 in GRN. The mutation p.V490M resulted in a modification of the side chain of the residue located at position 490. WT: wild type, MUT: p. V490Mmutation. D Chromatograph of exon 12 of the GRN gene obtained from DNA sequencing

A heterozygous missense mutation was found in the GRN gene during direct DNA sequencing analysis. This mutation occurred in the first position of codon 490 in exon 12 (c.1468G > A), causing a substitution of Val-to-Met (p.Val490Met) in the proband (II:3) and two other family members (II:2, II:5) (Fig. 4D). However, this mutation was not present in the other five family members or the 150 normal controls. The aforementioned results suggested that this mutation did not exhibit a prevalent polymorphic nature. The Apolipoprotein E (APOE) genotypes of the proband (II:3) and his younger sister (II:5) were ε3/ε3.

Discussion

Currently, the absence of large sample epidemiological studies in China has restricted an accurate understanding of the prevalence and incidence of FTD and its pathogenic mutations [6, 17]. As a result, GRN mutations are infrequent in China [6]. A total of 14 Chinese patients with 13 distinct mutations—six missense, four frameshifts, one nonsense, one deletion, and one splice site mutation—have been reported in ten papers regarding GRN mutations thus far [18]. Previous literature reports suggest that most missense mutations in the GRN gene are considered non-pathogenic [19]. However, some studies or case reports indicate that certain GRN missense mutations have clear pathogenicity, and the role of missense mutations in disease progression cannot be excluded [15, 20,21,22]. The clinical phenotypes, imaging manifestations, and pathological characteristics of selected pathogenic GRN missense mutations are summarized in Table 2. In this study, we have discovered a new type of genetic mutation called a missense mutation. This mutation occurs in a specific part of the GRN gene known as exon 12. The affected family in our research has been diagnosed with probable AD based on clinical observations prior to undergoing gene analysis. In other words, the clinical phenotype of this FTD family is that of Alzheimer's phenotype. We are unaware of any previous reports involving a Chinese Han household. The residue's phylogenetic conservation suggests that the Val490Met mutation may be harmful. The GRN was aligned with numerous sequences from several organisms. The examination of evolutionary conservation revealed that the newly identified mutation p.V490M caused a significant and consistent alteration in the amino acid sequence. Relative to the algorithm approaches used to forecast mutation pathogenicity, the p.V490M mutation is positioned within the conserved domain housing the likely pathogenic variants of the gene, although no mutation has been recorded at the GRN gene residue 490 [12]. Contrarily, PolyPhen-2 HumVar and PolyPhen-2 HumDiv both predicted that V490M would be harmful, with a score of 1.00 (specificity: 1.00; sensitivity: 0.00). The combined annotation-dependent depletion (CADD) score of the V490M mutation is 26.2 (Reference score > 15), also suggesting it is a deleterious variant. The data from PS4, PM1, PM2, PM5, PM6, PP3, and PP4 support the classification of this unique GRN p.V490M variation as pathogenic, following the recommendations made via the American College of Medical Genetics and Genomics (ACMG) [23]. According to our prediction results, the new missense mutation in this family was most likely the cause of the dementia.

Individuals with GRN mutations display significant variability in the age at which symptoms first appear. As a result, they may have difficulty identifying patterns of inheritance in households with only a few affected members, particularly when the inheritance follows an autosomal dominant pattern [1]. Three individuals (II:2, II:3, and II:5) from this family were found to have the Val490Met genotype. Conversely, both the proband (II:3) and his sister (II:2) exhibited the clinical phenotype of Alzheimer's disease (AD), while another sister (II:5) showed the clinical phenotype of mild cognitive impairment (MCI) in the present investigation. The individuals’ ages at the disease onset were 70, 69, and 67 years, respectively. A prior investigation of the GRN in the Belgian population revealed that the manifestation of GRN mutation in both sporadic and FAD phenotypes may occur as late as 89 years of age [12, 24]. A broad range of 66 to 89 years was observed in certain studies as the age of onset linked to GRN mutations associated with AD [12, 24, 25]. However, the age at which symptoms first appeared in the current family was between 67 and 77 years. And then, the onset age of the GRN-mutated family in this study matches that in previous studies. This heterogeneity suggested the possibility of modifying the age of start and clinical phenotype by additional genetic or environmental factors. There will be a follow-up on the family's pathogenic state to back up the genetic test results.

The proband (II:3) underwent a lumbar puncture to obtain CSF biomarkers. The analysis showed total-tau, phospho-tau, and pathogenic Aβ42 (Table 1). The V490M mutation's impaired expression of these pathological biomarkers was consistent with an AD profile according to the ATN biological diagnostic marker categorization established by the Alzheimer's Disease Association [26]. First and foremost, our study found a CSF biomarker characteristic of the p.V490M mutation, which is typical for AD. This suggests that there is AD pathology in the proband’s brain, and it also supports the idea that GRN plays a role in the pathogenic mechanism of AD in FTD individuals who express clinical symptoms of AD. It also partially represented the heterogeneity of pathological features across patients with the same genetic mutation. At the same time, it is of significant note that both the proband (II:3) and his younger sister (II:5) in our research exhibited an APOE genotype of ε3/ε3. Consequently, the ApoE4 risk factor, which is closely associated with the development of sporadic AD, was not present in either of these two individuals. This observation is in exact accordance with the findings reported by Redaelli et al. in their investigation of an AD family harboring the GRN C139R mutation [15], thereby reinforcing the consistency and reliability of our research outcomes within the context of genetic factors influencing AD.

Significant memory impairment in the early stage of FTD patients may lead to misdiagnosis as AD. The reason for the occasional reporting and identification of GRN mutations linked to AD remains unclear. Our research indicates that the proband (II:3) and his sister (II:2) were clinically diagnosed with AD prior to undergoing gene analysis because, although they did not exhibit obvious language dysfunction over the course of the disease, brain imaging revealed moderate cortical atrophy in the hippocampus and temporal lobe, without typical imaging manifestations of FTD. The proband also initially presented with memory loss and abnormal mental behavior. On the other hand, the second sister (II:5) of the proband simply had memory loss, without any other cognitive decline, and received a clinical diagnosis of MCI. Our hypothesis suggests that GRN is among the top three genetic contributors to FTD or FTLD, which are characterized by a wide range of clinical variations. These mutations often go unnoticed due to the similarities in symptoms with AD [2]. Prior research has shown a possible genetic connection between AD, FTD, and DLB. Specifically, individuals with a hereditary type of dementia that exhibits clinical symptoms characteristic of FTD and occurs at an early age have been found to possess mutations associated with FAD [2, 27]. The patients who were first identified with AD had inherited pathogenic mutations associated with FTD. Additionally, mutations in C9orf72, GRN, and MAPT have also been found in AD patients, although at lesser frequencies [12, 24]. Hence, diagnosing and differentiating the primary types of dementia is a challenging task.

PGRN (epithelial-transforming growth factor) is a glycoprotein that is released and composed of several copies of granulin, a module with 12 cysteine residues, often referred to as an epithelin domain [10]. It has been shown that mutations in the GRN gene elevate the likelihood of developing AD, and the presence of PGRN in the blood or plasma serves as a biomarker for AD [13]. A growing body of research has shown that PGRN plays many roles in neuroinflammation, tau phosphorylation, Aβ clearance, and neuronal survival in AD and functions as a neuroprotective agent [10, 11]. The current investigation shows that the majority of GRN mutations, particularly functional deletion mutations, cause the loss of an allele. As a consequence, the amounts of PGRN protein and GRN mRNA that can be detected in blood and CSF are reduced by about 50% [28, 29].

The quantities of PGRN mRNA are decreased as a result of these mutations, which disrupt the mutant transcript and cause nonsense-mediated mRNA attenuation. This, in turn, leads to neurodegenerative diseases, such as AD [30]. Furthermore, the position of the valine at GRN protein position 490 indicated strong cross-species phylogenetic conservation. Hence, almost all GRN mutations diminish the amounts or production of the protein, likely via diverse molecular mechanisms [31, 32]. The subject (II:3) in the present investigation, who was clinically diagnosed with likely AD, had a CSF biomarker profile that is characteristic of AD pathology. His sister (II:5) received a clinical diagnosis of MCI without the presence of Aβ accumulation in the cerebral cortex. Furthermore, we postulated that the depletion of functional proteins might be caused by the GRN mutation V490M, and further research is needed to determine if AD, FTD, or a mix of the two is the underlying disease in our study. In general, mutations in the GRN gene may go undetected in households with few or no afflicted ancestors owing to the wide range of ages at which symptoms appear. Additionally, cases with FTD with a beginning beyond the age of 70 might be mistakenly identified as AD [1].

This research has several limitations. Firstly, it lacked a functional evaluation of the pathogenic variations, which may have aided in identifying the underlying pathological process. It would be advantageous to verify the role of this mutation, particularly in the pathological phase of AD-associated disease. Furthermore, it is necessary to do further verification via follow-up studies including bigger cohorts that are recruited prospectively, as well as animal models.

Conclusions

We reported a new mutation at codon 490 (V490M) in exon 12 of the GRN gene in a case of familial FTD with a clinical phenotype manifested as AD and MCI. This discovery expands the range of clinical characteristics and visual representations linked to GRN mutations. The CSF biomarker alterations of the proband revealed a reduction in Aβ42 and the Aβ42/Aβ40 ratio. Further research is necessary to elucidate the impact of the p.V490M mutation in the GRN gene on β-amyloid formation, therefore uncovering novel pathogenic processes of FTD and AD.