Demographic features and clinical diagnosis of FTD patients

The demographic features of the 261 FTD patients are shown in Table 1. A total of 32 patients (12.3%) had a positive family history in first-degree or second-degree relatives (Fig. 2A, B). 53.1% (17/32) of individuals with a positive family history and 19.2% (44/229) of sporadic patients could be genetically diagnosed.

Table 1 Demographic data of FTD patientsFig. 2

Genetic analysis of the FTD participants. A Pedigree charts of the 17 familial FTD patients in our cohort. B The proportion of familial and sporadic cases and the phenotype spectrum of the FTD participants in our cohort. C The genetic spectrum of the FTD participants in our cohort

All patients with FTD met the consensus criteria for FTD spectrum [17]: 185 for bv-FTD [18], 14 for nvPPA [2], 40 for svPPA [2], 9 for FTD combined with ALS [19], and 13 for FTD-parkinsonism overlap (FTD-P) [20] (Fig. 2B).

Genetic findings

A total of 261 individuals were screened for dementia-related causing and susceptible genes. We were able to establish a genetic diagnosis in 17 of 32 familial cases (Fig. 2A). Family 1 had a repeat expansion in C9orf72. Family 2, Family 4, and Family 7 had the p.P301L mutation in MAPT. Family 3, Family 5, Family 6, Family 8, Family 9, Family 10, Family 11, and Family 12 had p.N279K, p.V337M, p.N296N, p.R5C, p.G389R, p.E342K, p.Q351R, and p.S285R mutations in MAPT respectively. Family 13 and Family 14 had p.S398F and p.I37T mutations in TBK1, respectively. One family each was found to carry a variant in OPTN, GRN, and UBQLN2, respectively. Of the sporadic cases, repeat-primed PCR revealed one patient with repeat expansion in C9orf72. Exome sequencing led to genetic diagnoses with variants in MAPT in 6 cases, GRN in 5 cases, TBK1 in 5 cases, OPTN in 5 cases, ANXA11 in 4 cases, CHMP2B in 3 cases, SQSTM1 in 2 cases, CYLD in 2 cases, TARDBP in 2 cases, and VCP, CCNF, SIGMAR1, CHCHD10, CHCHD2, FUS, and TMEM106B in 1 case each. One case had a two octarepeats deletion in PRNP, and another case had a V180I mutation in PRNP (Fig. 2C). 29 variants have not been observed so far and can be considered novel, including the MAPT p.D54N, p.E342K, p.R221P, p.T263I, TBK1 p.E696G, p.I37T, p.E232Q, p.S398F, p.T78A, p.Q150P, p.W259fs, OPTN p.R144G, p.F475V, GRN p.V473fs, p.C307fs, p.R101fs, CHMP2B p.K6N, p.R186Q, ANXA11 p.Q155*, CYLD p.T157I, SQSTM1 p.S403A, UBQLN2 p.P509H, CCNF p.S160N, CHCHD10 p.A8T, SIGMAR1 p.S117L, CHCHD2 p.P53fs, FUS p.S235G & p.S236G, and TMEM106B p.L144V variants. No dual diagnoses were made. Identified variants and their predicted pathogenicity are listed in Table 2. There was no statistical difference between the distribution of ApoE alleles in our FTD cohort and the control Chinese population (Table S2) [15].

Table 2 Interpretation of identified variants and their pathogenicityClinical findings of TBK1 and OPTN variants

The clinical characteristics of 61 FTD-gene variant carriers are summarized in Table 3. In this study, we focus on the clinical characteristics of the patients with TBK1 and OPTN variants. Figure 3 presents the neuroimaging results of cranial MRI or 18F-FDG-PET of the patients with TBK1 and OPTN variants.

Table 3 The correlation between genetic features and clinical manifestations of 28 FTD-gene variant carriersFig. 3

Neuroimaging studies of the FTD patients with TBK1 and OPTN variants. The upper lane shows the cranial MRI. The atrophic changes in frontotemporal regions are indicated by white arrows. The lower lane shows the 18F-FDG-PET images. The color bar of the 18F-FDG-PET images indicates the corresponding regional standardized uptake value ratios (SUVR)

TBK1 variants Family 13 TBK1 p.Ser398Phe/c.1193C > T

The proband, a 63-year-old male, began experiencing memory loss, a change in personality, frequent mood swings, and difficulty expressing and understanding speech at the age of 54. Over time, he gradually developed an inability to recognize family members and perform activities of daily living. He was diagnosed with svPPA [2]. The patient's father also exhibited similar symptoms and was diagnosed with "dementia" at the age of 70. He subsequently passed away due to an accident at the age of 73.

Family 14 TBK1 p.Ile37Thr/c.110 T > C

The proband of this family was a 55-year-old male with a five-year history of anomia and personality changes. He presented with progressive fluent aphasia with impaired naming and loss of understanding of even single words five years ago. On examination, there were also extrapyramidal symptoms of rigidity. The diagnosis of svPPA was confirmed [2]. The patient’s father and older sister all presented similar symptoms.

P7 TBK1 p.Glu696Gly/c.2087A > G

The patient exhibited memory problems and personality changes, with initial short-term memory loss leading to difficulty retaining new information. Subsequently, she struggled with daily practical tasks and experienced behavioral disturbances such as stubbornness and repetitive behaviors. She also showed a decline in personal hygiene and reluctance to engage in self-care. She was diagnosed with bv-FTD [18].

P8 TBK1 p.Glu232Gln/c.694G > C

The patient presented language abnormalities manifesting as deficits in speech production and was initially diagnosed with nvPPA [2]. The patient then exhibited extrapyramidal symptoms of rigidity and bradykinesia in the right limbs, but no personality changes or behavioral abnormalities were noted. The diagnosis was subsequently revised to FTD-P [20].

P9 TBK1 p.Thr78Ala/c.232A > G

The patient presented with a decline in verbal communication accompanied by a decrease in memory function at the age of 67. This decline includes forgetting the names of individuals and places, as well as an inability to recognize written language. She was diagnosed with svPPA [2].

P10 TBK1 p.Gln150Pro/c.449A > C

The patient presented with anxiety accompanied by reduced verbal communication and memory decline. The onset of anxiety occurred at the age of 63, characterized by worries and fears, primarily concerning the possibility of having a serious illness and fear of impending death. Family members gradually noticed changes in the patient's personality and reduced speech output. Additionally, there was evident memory decline, and a notable slowing of response and movement, accompanied by a decline in comprehension and computational abilities. She was diagnosed with bv-FTD [18].

P11 TBK1 p.Trp259GlyfsTer52/c.775del

A male patient, aged 50, has been experiencing unclear speech accompanied by decreased memory for over six months. Six months ago, the patient exhibited unclear speech characterized by indistinct articulation and a hoarse voice. The patient experiences coughing when drinking water and also reports difficulty swallowing. Grip strength in both hands has diminished, evidenced by an inability to operate a lighter and difficulty opening packaged items, while lower limb strength remains unchanged. Memory decline is notable, primarily affecting recent events. The patient has become more introverted, displaying a reduced inclination for communication and social interaction. He was diagnosed with FTD-ALS [19].

OPTN variants Family 15 OPTN p.Glu516Gln/c.1546G > C

The proband exhibited progressive speech and language deficits, with difficulty expressing thoughts and finding words, as well as errors in speech sounds and sentence construction. Additionally, he developed PSP-like symptoms, including mildly elevated axial muscle tone in the trunk and hips, and vertical gaze palsy. He was clinically diagnosed with FTD-P [20]. His older brother was reported to have similar symptoms, but could not be examined. The medical history of their parents, both of whom died at an early age, was unavailable.

P12 OPTN p.Arg144Gly/c.430A > G

The patient exhibited impaired memory and language difficulties, manifesting as short-term memory impairment, difficulty naming objects, comprehending word meanings, and understanding spoken or written language. She was clinically diagnosed with svPPA [2].

P13 OPTN p.Leu494Trp/c.1481 T > G

The patient initially presented with forgetfulness, language difficulties, and personality changes, including short-term memory impairment, difficulty naming familiar items, and struggling to find the right words. With disease progression, she exhibited obsessive behaviors, fear of leaving home, and disinhibition. Her speech became increasingly vague and she experienced difficulty carrying out daily activities. She was clinically diagnosed with svPPA [2].

P14 OPTN p.Phe475Val/c.1423 T > G

The patient first developed a memory deficit and depression, with rapid progression of behavioral symptoms, including irritability, anxiety, and apathy. She then exhibited impaired word-finding, deteriorating naming abilities, and progressive difficulty comprehending spoken or written language, with unreasonable responses. Disorientation in time and space also manifested. She was clinically diagnosed with svPPA [2].

P15 OPTN p.Thr282Pro/c.844A > C

The patient, currently 54 years old, progressively displayed changes in personality and behavior, including stubbornness, irritability, diminished concern for family members, disregard for others' feelings, and a lack of social etiquette since the age of 51. Furthermore, symptoms comprised declining recent memory, reduced executive function, impaired language abilities, and challenges with naming and writing. He was clinically diagnosed with svPPA [2].

P16 OPTN p. Ala136Val/c.407C > T

The patient experienced memory decline at the age of 51, predominantly affecting recent memory, characterized by an inability to recall recent events and difficulty remembering familiar names. The patient also exhibited personality changes. She experienced behavioral disturbances such as stubbornness and repetitive behaviors. She was diagnosed with bv-FTD [18].

Functional analysis of TBK1 variants

Serine/threonine-protein kinase TBK1 regulates selective autophagy pathways, specifically mitophagy [21] and xenophagy [22]. TBK1 phosphorylates a selective autophagy receptor, OPTN, at Ser177, that enhances interaction with ubiquitinated cargoes and is an autophagy modifier of the LC3 family [23]. The three mutations of TBK1 p.I37T, p.T77I, and p.E232Q were located in the kinase catalytic domain, and p.E696G was located in the coiled-coil 2 (CCD2) domain involved in binding with OPTN (Fig. 4A). We examined whether TBK1 and its mutants actively phosphorylated and associated with OPTN in the HEK293T cells transfected with TBK1 and OPTN expression plasmids. The T77I variant found in a normal elderly person without symptoms was used as a control. Overexpression of wild-type TBK1 robustly phosphorylated OPTN. However, OPTN phosphorylation by the TBK1 I37T mutant was significantly reduced compared to the wild type (Fig. 4B, C). Generally, autophosphorylation of kinases is defined as the phosphorylation of kinase itself, which normally regulates the catalytic activity. We next examined the autophosphorylation (pS172) of wild-type and mutants TBK1. Autophosphorylation of the mutant I37T was significantly reduced (Fig. 4D). This was consistent with the result of the OPTN-phosphorylation. Autophosphorylation of the E232Q was also reduced (Fig. 4D), but it did not affect the OPTN-phosphorylation. It is known that dimerization of TBK1 is required for kinase activation and autophosphorylation [24]. Therefore, we next examined TBK1-dimerization using non-reducing SDS-PAGE. Although the autophosphorylation and kinase activity for OPTN were reduced, the dimerization ability of the I37T mutant and the other mutants were not changed compared to wild-type TBK1 (Fig. 4B).

Fig. 4

Biochemical characterization of TBK1 variants. A Domain structure and OPTN-binding site of TBK1 protein. The variants identified in this study are indicated. B Top five lanes: HEK293T cells expressing Flag-TBK1 wild-type and indicated mutants were co-transfected with mCherry-OPTN for 48 h before lysis. The binding of TBK1 variants was analyzed via immunoblot (IB). The asterisk points to OPTN pS177 bands. Bottom two lanes: Coimmunoprecipitation (co-IP) of Flag-TBK1 (wild-type and indicated mutants) and mCherry-OPTN wild-type from HEK293T cell lysates. C Phosphorylation of OPTN S177 was confirmed by IB using the pS177 OPTN antibody generated by immunoGlobe GmbH. EV: empty vector. D Cell lysates were subjected to immunoblot analysis and active TBK1 (pS172) was detected with a phosphospecific antibody (pS172; no. 5483; Cell Signaling Technology). E Co-immunoprecipitation (Co-IP) of Flag-TBK1 wild-type and the indicated mutants with mCherry-OPTN wild-type from HEK293T cells using an antibody to Flag. Co-immunoprecipitated proteins were analyzed by immunoblotting (IB) with the Flag and mCherry antibodies

The mutation E696G is located in the CCD2 domain, which involves interaction with OPTN (Fig. 4A) [25]. We next examined TBK1-OPTN interaction using immunoprecipitation. It was found that OPTN-TBK1 complex formation is significantly enhanced in cells transfected with human TBK1 E696G mutant (Fig. 4B, E). The TBK1 E232Q mutant also slightly increased the OPTN-TBK1 complex formation, and the TBK1 I37T mutant marginally decreased the OPTN-TBK1 binding, but these effects did not reach statistical significance (Fig. 4B, E).

Functional analysis of OPTN variants

OPTN is an autophagy adaptor protein that plays a critical role in multiple stages of the autophagic pathway. In addition, it is associated with several human disorders that are closely linked to autophagy. It contains two coiled-coil domains, a leucine zipper (LZ) domain, a microtubule-associated protein 1 light chain (LC3)-interacting region (LIR), a ubiquitin-binding domain (UBD), and a zinc finger (ZnF) domain [26] (Fig. 5A). Previous studies have demonstrated that ALS-linked OPTN mutants (E478G and Q398X) prevented vesicle formation and induced non-vesicular localization of optineurin in cells [27]. We investigated whether OPTN mutants identified in our FTD cohort could be recruited to autophagosomes in Neuro2a cells. Exogenous expression of OPTN wild-type, L494W, and E516Q resulted in the formation of vesicular structures that colocalized with LC3 puncta. In contrast, OPTN R144G and F475V mutants were significantly diffused throughout the cytoplasm ((Fig. 5B, C). Our image analysis revealed a significant reduction in the recruitment to LC3 puncta compared to OPTN wild-type (Fig. 5B, C) indicating functional defects as autophagy adaptor proteins.

Fig. 5

Recruitment of OPTN variants to the autophagosomes. A Schematic representation of domain structure in human optineurin protein and the localization of these domains relative to its amino acid sequence. CC, coiled-coil; LZ, leucine zipper domain; LIR, LC3-interacting region; UBD, ubiquitin-binding domain; ZnF, zinc finger. Rab8-interacting region (amino acids 1–209) and myosin VI-interacting region (amino acids 417–512) were indicated. The mutations identified in our study are indicated by red arrows. B Representative confocal images in Neuro2a cells showing co-localization of endogenous LC3 puncta (green) with overexpressed OPTN (magenta) wild-type and mutants. In Neuro2A cells transfected with OPTN wild-type, L494W, and E516Q mutants, optineurin-positive vesicles co-localized with LC3. In Neuro2A cells expressing the OPTN R144G and F475V mutants, optineurin is not vesicular and displayed decreased colocalization with LC3. Insets show higher magnification of the areas outlined in the merged images. Scale bar: 10 μm. C The co-localization of OPTN wild-type or mutants with LC3-positive autophagosome. WT = wild-type. Pearson's R values between LC3 puncta (autophagosomes) and mCherry-OPTN proteins were shown. n = 30 cells. * p < 0.05, ** p < 0.01

The frequencies of genes implicated in FTD in China

In the literature review, rare variants in genes such as MAPT, GRN, C9orf72, CHCHD10, VCP, TBK1, OPTN, SQSTM1, SIGMAR1, TARDBP, UBQLN2, FUS, CCNF, and CYLD were identified in Chinese FTD populations [8, 28,29,30,31,32,33,34,35,36]. The genetic spectrum of the major FTD cohorts previously reported in China is shown in (Fig. 6A). Including the patients identified in our cohort, the top six genes with the highest frequency implicated in FTD in China are shown in Table 4. The pooled frequency of TBK1 and OPTN in Chinese FTD patients was 2.0% (CI: 1.0%-3.1%) and 0.3% (CI: 0.0%-0.9%), respectively (Fig. 6B). No substantial heterogeneity was found in the pooled frequency meta-analysis for both TBK1 and OPTN across all datasets (I2: 0%). Notably, the frequency of TBK1 mutations was the second highest among Chinese FTD patients, surpassing the mutation frequencies of GRN (1.7%) and C9orf72 (0.5%).

Fig. 6

The genetic spectrum and the variant frequency of Chinese FTD patients. A The genetic spectrum of the major FTD cohorts in China. B The overall variant frequency of TBK1 and OPTN in Chinese FTD patients

Table 4 The top six genes with the highest frequency implicated in FTD in China