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Abstract
Background Mutations in the tropomyosin receptor kinase fused (TFG) gene are associated with various neurological disorders, including autosomal recessive hereditary spastic paraplegia (HSP), autosomal dominant hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) and autosomal dominant type of Charcot-Marie-Tooth disease type 2.
Methods Whole genome sequencing and whole-exome sequencing were used, followed by Sanger sequencing for validation. Haplotype analysis was performed to confirm the inheritance mode of the novel TFG mutation in a large Chinese family with HSP. Additionally, another family diagnosed with HMSN-P and carrying the reported TFG mutation was studied. Clinical data and muscle pathology comparisons were drawn between patients with HSP and patients with HMSN-P. Furthermore, functional studies using skin fibroblasts derived from patients with HSP and patients with HMSN-P were conducted to investigate the pathomechanisms of TFG mutations.
Results A novel heterozygous TFG variant (NM_006070.6: c.125G>A (p.R42Q)) was identified and caused pure HSP. We further confirmed that the well-documented recessively inherited spastic paraplegia, caused by homozygous TFG mutations, exists in a dominantly inherited form. Although the clinical features and muscle pathology between patients with HSP and patients with HMSN-P were distinct, skin fibroblasts derived from both patient groups exhibited reduced levels of autophagy-related proteins and the presence of TFG-positive puncta.
Conclusions Our findings suggest that autophagy impairment may serve as a common pathomechanism among different clinical phenotypes caused by TFG mutations. Consequently, targeting autophagy may facilitate the development of a uniform treatment for TFG-related neurological disorders.
- genetics
- mutation
- neuromuscular diseases
Data availability statement
Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. Data are available upon reasonable request.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Structural correlations between genotypes and clinical phenotypes have been observed in tropomyosin receptor kinase fused (TFG)-associated neuropathies. Mutations in the carboxyl-terminal domain of the TFG protein lead to late-onset neuropathies inherited in an autosomal dominant manner, including hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P), Charcot-Marie-Tooth disease type 2, amyotrophic lateral sclerosis and Parkinson’s disease. In contrast, mutations in the amino-terminal domain of the TFG protein cause autosomal recessive hereditary spastic paraplegias (HSP).
WHAT THIS STUDY ADDS
We report a novel heterozygous TFG variant causing pure HSP in a Chinese family and provide compelling evidence that the well-documented recessively inherited spastic paraplegia, caused by TFG homozygous mutations, also exists in a dominantly inherited form. Furthermore, in vitro functional studies reveal autophagy impairment as the shared pathogenic mechanism between patients with HSP and patients with HMSN-P with TFG mutations.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
Our findings reveal shared cellular phenotypes across different clinical manifestations caused by TFG mutations and support the notion that targeting autophagy may facilitate the development of a uniform treatment for TFG-related neurological disorders.
Introduction
The tropomyosin receptor kinase fused (TFG) gene is located on chromosome 3q12 and is ubiquitously expressed in various human tissues. TFG mutations were initially identified to contribute to hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P), which is inherited in an autosomal dominant manner.1 HMSN-P is characterised by proximal-predominant muscle weakness and atrophy, distal sensory disturbances, absent deep tendon reflexes, elevated creatine kinase levels and electrophysiological evidence of axonal degeneration in peripheral nerves.2 Unlike classic Charcot-Marie-Tooth disease (CMT), which presents predominantly with distal motor and sensory involvement, HMSN-P shares some characteristics with amyotrophic lateral sclerosis (ALS). Patients with HMSN-P display widespread fasciculations and cramps in the extremities and trunk. Additionally, patients have a higher incidence of hyperlipidaemia. Autosomal recessive spastic paraplegias (SPG57) and autosomal dominant type of CMT2 were later described as the result of TFG mutations.3 4 Recently, it was reported that patients from a Korean family carrying a heterozygous missense mutation of TFG presented with Parkinson’s disease, subclinical parkinsonism or ALS.5
In this study, we describe a novel heterozygous TFG variant causative of pure hereditary spastic paraplegia (HSP) in a Chinese family. Furthermore, in vitro functional studies reveal autophagy impairment as the shared pathogenic mechanism between patients with HSP and patients with HMSN-P with TFG mutations.
Methods
Genetics
Blood samples for genomic DNA were obtained from two families. Whole exome sequencing (WES) and Sanger sequencing were performed on relevant family members. To further rule out potential pathogenic variants, whole genome sequencing (WGS) was performed on the proband III-4 in Family 1. The pathogenicity of the identified mutation was assessed using the American College of Medical Genetics (ACMG) guidelines.6 To determine whether the disease is linked to the TFG locus, linkage analysis was conducted for Family 1. Four highly polymorphic short tandem repeat markers, D3S2419, D3S1271, D3S3655 and D3S3652, spanning the TFG gene on chromosome 3q12, were genotyped for all individuals. Primer sequences for these markers were obtained from the Genome database (http://www.gdb.org). Each marker was individually amplified by PCR, and the PCR products were subjected to direct bidirectional sequencing.
Immunohistochemical analysis of biopsied muscle tissue
Muscle specimens, including the right tibialis anterior muscle of patient III-4 from Family 1 and the right biceps brachii muscle of patient II-4 from Family 2, were obtained and analysed to assess muscle pathology. Histological and immunohistochemical analyses were conducted on 8 µm transverse sections of each muscle specimen. For routine histochemical stains, H&E staining, reduced nicotinamide adenine dinucleotide-tetrazolium reductase staining and adenosine triphosphatase staining at pH 4.3 and 10.4 were performed, according to standard protocols.7
Immunofluorescence analysis of biopsied muscle and nerve tissue
In our current study, we examined muscle specimens from patients diagnosed with ALS, spinal muscular atrophy, spinal and bulbar muscular atrophy (SBMA), CMT and limb-girdle muscular dystrophy (LGMD) type 2A, which were confirmed by genetic testing. These patients were part of our cohort who previously underwent open muscle biopsies for diagnostic purposes at Qilu Hospital of Shandong University. Additionally, muscle specimens from patients with sporadic inclusion body myositis (sIBM) were used as positive controls based on prior researches,8 9 and muscle specimens from non-muscular medical conditions presenting no recognisable muscle histological abnormalities served as negative controls. The majority of these muscle specimens were derived from the biceps brachii, and the remaining samples were obtained from the tibialis anterior and quadriceps femoris. In addition, a sural nerve biopsy was performed on patient II-4 from Family 2. Frozen sections were fixed with acetone at 4°C and blocked with 10% normal goat serum (Solarbio) in phosphate-buffered saline (PBS) at room temperature.
The primary antibodies used included rabbit anti-TRK fused gene antibody (ab156866, 1:100 (Abcam)), TFG polyclonal antibody (11 571–1-AP, 1:50 (ProteinTech Group)), rabbit anti-TDP43 antibody (ab41972, 1:100 (Abcam)), rabbit phospho-TDP43 (Ser409/410) polyclonal antibody (22309–1-AP, 1:100 (ProteinTech Group)), rabbit anti-ubiquitin antibody (ab7780, 1:100 (Abcam)) and rabbit p62/SQSTM1 polyclonal antibody (18420–1-AP, 1:100 (ProteinTech Group)). Additionally, mouse dystrophin (Rod Domain) monoclonal antibody (NCL-DYS1, 1:100 (Leica)), mouse dystrophin (C-terminus) monoclonal antibody (NCL-DYS2, 1:100 (Leica)), or mouse dystrophin (N-terminus) monoclonal antibody (NCL-DYS3, 1:100 (Leica)) were used to label the sarcolemma. Immunolabelled proteins were detected using an anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (A11012, 1:1000 (Life Technologies)) and an anti-mouse secondary antibody conjugated with Alexa Fluor 488 (A11001, 1:1000 (Life Technologies)). Sections were photographed using an LSM880 confocal microscope (ZEISS).
Skin fibroblast culture
Punch biopsies were obtained from the skin of patients II-4 and III-4 from Family 1, and II-3 and II-4 from Family 2. Primary skin fibroblasts from healthy volunteers were obtained from discarded skin tissue after medical cosmetic surgery, with informed consent. Dermal skin fibroblasts were cultured in Dulbecco’s modified Eagle medium (M&C GENE TECHNOLOGY) supplemented with 10% foetal bovine serum (Gibco) and antibiotics and antimycotics.
Quantitative real-time PCR
Total RNA was isolated using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme) and subsequently reverse transcribed to cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) according to the manufacturer’s protocols. Quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on QuantStudio 3 and 5 Real-Time PCR Systems (Thermo Fisher Scientific). Primer sequences are listed in the online supplemental material.
Supplemental material
Western blotting
Total protein was extracted from patient-derived skin fibroblasts and biopsied muscles using RIPA lysis buffer (Solarbio) supplemented with 1× protein phosphatase inhibitor and 1× protease inhibitor mixture (Solarbio). The resulting cell extracts were centrifuged at 12 000×g for 15 min, and the supernatants were collected. Cell lysates were subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis, and immunoblot analysis was performed as previously described. The primary antibodies used were anti-TRK fused gene antibody (ab156866, 1:1000 (Abcam)), anti-LAMP1 antibody (ab24170,1:1000 (Abcam)), p62/SQSTM1 Polyclonal antibody (18420–1-AP, 1:1000 (ProteinTech Group)), BECN1/Beclin-1 antibody (E-8) (sc-48341, 1:500 (Santa Cruz Biotechnology)), LC3B antibody (2775, 1:1000 (Cell Signaling Technology)), GAPDH monoclonal antibody (60004–1-Ig, 1:1000 (ProteinTech Group)) and anti-beta actin mouse monoclonal loading control antibody (A2-F6, 1:10000 (HUABIO)). Goat anti-rabbit IgG -HRP (HA1001, 1:10000 (HUABIO)) or goat anti-mouse IgG -HRP (HA1006, 1:10000 (HUABIO)) was used as the secondary antibody.
Immunofluorescence analysis of human fibroblasts
Cells were cultured on glass coverslips prior to fixation using 4% paraformaldehyde for 15 min. Next, the cells were permeabilized with 0.5% Triton X-100 (Solarbio) for 10 min and blocked in a solution containing 3% bovine serum albumin (BSA, Solarbio) and 0.1% Triton X-100 in PBS for 1 hour at room temperature. Coverslips were then incubated with the anti-TRK fused gene antibody (ab156866, 1:100 (Abcam)) diluted in a solution of 0.1% BSA and 0.1% Triton X-100 in PBS overnight at 4°C After washing, the slides were incubated for 1 hour at 4°C in the dark with an Alexa Fluor 594-conjugated anti-rabbit secondary antibody (A11012, 1:1000 (Life Technologies)). Finally, coverslips were mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) and observed under an Olympus IX73 inverted microscope.
Statistical analysis
Statistical analyses were conducted using SPSS V.26.0. Data normality was assessed using the Shapiro-Wilk test. One-way analysis of variance followed by post-hoc Tukey’s test was applied to normally distributed data. For non-normally distributed data, the Kruskal-Wallis H test was used. Statistical significance was accepted as *p<0.05, **p<0.01, and ***p<0.001. All charts were generated using GraphPad Prism V.8, and data are presented as the means±SEMs.
Results
Clinical phenotypes of two Han Chinese families
Family 1, with a clinical diagnosis of HSP, was recruited for the current study (figure 1A). The family had a medical profile spanning four generations, and all affected members exhibited an onset of symptoms in childhood. All four patients manifested progressive spasticity and weakness of the lower limbs. There were no reported cases of vision problems or cognitive impairment among any family members. Electrodiagnostic studies, including nerve conduction studies and needle electromyography (EMG), were unremarkable in all four patients. Nerve conduction and needle EMG data are summarised in table 1 and detailed in online supplemental table 1. Additionally, the brain and muscle MRI of patient III-4 were normal (figure 2A).
Pedigree charts and linkage analysis. (A) The pedigree of Family 1 with HSP and the haplotypes of markers spanning the tropomyosin receptor kinase fused (TFG) gene. The haplotypes co-segregating with HSP are boxed. (B) The clinical spectrum of TFG mutations and their relevant locations in protein domains. The novel variant of TFG (NM_006070.6: c.125G>A (p.R42Q)) causing autosomal-dominant spastic paraplegia in the current study is indicated in red. ALS, amyotrophic lateral sclerosis; CMT, Charcot-Marie-Tooth; HMSN-P, hereditary motor and sensory neuropathy with proximal dominant involvement; HSP, hereditary spastic paraplegia; PD, Parkinson’s disease.
Muscle imaging and muscle biopsy pathology. (A) Axial view of T1-weighted muscle MRI of lower extremities. Substantial muscle atrophy with fatty degeneration was observed in the gluteus medius, gluteus minimus and lateral posterior thigh muscles of the patients with HMSN-P. Muscle imaging of the patient with HSP was nearly normal. (B) The right tibialis anterior muscle of patient III-4 from Family 1 with HSP showed replacement of muscle fibres by fat (H&E), deep staining under the sarcolemma (NADH-TR) and group of type 1 and type 2 fibres (ATPase pH 10.4). The right biceps brachii muscle of patient II-4 from Family 2 with HMSN-P showed large group atrophy (H&E), target fibres (NADH-TR) and type 1 fibre predominance (ATPase pH 10.4). ATPase, adenosine triphosphatase; HMSN-P, hereditary motor and sensory neuropathy with proximal dominant involvement; HSP, hereditary spastic paraplegia; NADH-TR, nicotinamide adenine dinucleotide-tetrazolium reductase.
Comparison of clinical characteristics of patients with TFG mutations from two Chinese families
Two patients from Family 2 were diagnosed with HMSN-P (online supplemental figure 1A). In their 50s, they developed predominantly proximal muscle weakness and atrophy followed by distal sensory disturbances. Fasciculations and cramps were also observed, and both patients had hyperlipidaemia. Notably, patient II-3 exhibited extensive muscle atrophy, including the pectoralis major, shoulder and hip girdle and limb muscles (online supplemental figure 1B). Muscle MRI of patient II-4 showed fat replacement in the gluteus minimus muscles and muscle atrophy of the lateral posterior thighs (figure 2A). Nerve conduction studies of both individuals indicated abnormal sensory nerve conduction but completely normal motor nerve conduction. Needle EMG revealed a neurogenic pattern in four separate body part regions: craniobulbar, cervical, thoracic and lumbosacral. Sural nerve biopsy in patient II-4 showed a marked reduction in the myelinated fibre population (online supplemental figure 1C).
The clinical presentation of the two families did not share common characteristics. The results of the detailed comparison are presented in table 1, with extended case reports provided in the online supplemental material.
Novel heterozygous TFG mutation identified as the cause of HSP
Family 1 had a four-generation family history consistent with an autosomal dominant disease. WES revealed that patients III-4, IV-2 and IV-3 from Family 1 had a novel heterozygous variant in the TFG gene (NM_006070.6: c.125G>A (p.R42Q)). Sanger sequencing of relevant family members confirmed that the mutation co-segregated with the disease. All other variants associated with neuromuscular diseases, identified through WGS and WES, have been excluded through co-segregation analysis and pathogenicity prediction using bioinformatics tools. Moreover, four polymorphic markers flanking the TFG gene used for family linkage studies confirmed the disease linkage to the TFG locus. Haplotype analysis further demonstrated that the four markers flanking the TFG gene co-segregated with the disease (figure 1A). The results of DNA agarose gel electrophoresis and the sequence data of the cDNA sample of the proband III-4 in Family 1 showed normal splicing of the TFG mRNA (online supplemental figure 2). The novel TFG variant was classified as pathogenic variants (PS3+PM1+PM2+PP1+PP4), according to the criteria of ACMG. In addition, two patients from Family 2 with HMSN-P carried the reported TFG mutation (NM_006070.6: c.854C>T (p.P285L)) (online supplemental figure 1A).
Neurogenic features of muscle pathology in patients carrying TFG mutations
The comparison of the histological staining of muscle samples obtained from patient III-4 from Family 1 and patient II-4 from Family 2 revealed neurogenic changes in both patients (figure 2B). Immunofluorescence analysis of TAR DNA-binding protein of 43 kDa (TDP-43) indicated the presence of sarcoplasmic positive staining in atrophic fibres of patient II-4 from Family 2 (figure 3). However, such signals were not observed in patient III-4 from Family 1 due to the lack of atrophic fibres (figure 3). Moreover, we did not detect any pTDP-43, ubiquitin, or p62 signals in either of the muscle samples (figure 3). Immunofluorescence analysis of TFG revealed positive signals localised in the perinuclear regions of biopsied muscle and nerve tissues in both healthy controls and patients harbouring TFG mutations. However, no TFG aggregates were observed in the intramuscular nerve bundles from patient III-4 with HSP and the sural nerve from patient II-4 with HMSN-P (online supplemental figure 3). The absence of aggregates was also noted in the muscle specimens from the two patients (online supplemental figure 3).
Immunofluorescence staining of muscle specimens with TFG mutations. TDP-43-positive sarcoplasmic staining was detected in angulated atrophic fibres of the patient with HMSN-P (right top panel). Due to the lack of atrophic fibres, the signals were not detected in the patient with HSP (middle right top panel). Additionally, phospho-TDP43, ubiquitin and p62 signals were not observed in either patient with TFG mutations. sIBM was used as a positive control (left panel). HCs, healthy controls; HMSN-P, hereditary motor and sensory neuropathy with proximal dominant involvement; HSP, hereditary spastic paraplegia; sIBM, sporadic inclusion body myositis.
Fibroblasts of patients with TFG mutations reveal autophagy impairment
At the mRNA level, the expression of TFG was reduced in fibroblasts derived from both patients with HSP and patients with HMSN-P (figure 4A). However, there was no significant change at the TFG protein level (figure 4B). Furthermore, TFG aggregates were observed in fibroblasts of patients carrying TFG mutations (figure 4C). Moreover, western blot analysis revealed that the protein expression levels of p62, Beclin 1 and LC3B-II, were significantly reduced (figure 4D).
Autophagy impairment may act as a common pathogenic mechanism among different clinical phenotypes caused by TFG mutations. (A) RT-qPCR and (B) western blotting results for TFG expression levels in fibroblasts of patients with TFG mutations and healthy control specimens. (C) Immunofluorescence of human fibroblasts using anti-TRK fused gene antibody (red). TFG aggregates (white arrowheads) were present in both patients with HSP and patients with HMSN-P. (D) The protein expression levels of p62, Beclin 1 and LC3B-II were reduced to varying degrees in patients with HSP and patients with HMSN-P as compared with control specimens. Error bars indicate SEM. Comparisons were made via one-way analysis of variance followed by post-hoc Tukey’s test for normally distributed data or the Kruskal-Wallis H test for non-normally distributed data. Statistical significance was accepted as *p<0.05, **p<0.01, and ***p<0.001, and non-significant data are indicated as ns. HCs, healthy controls; HMSN-P, hereditary motor and sensory neuropathy with proximal dominant involvement; HSP, hereditary spastic paraplegia.
Discussion
Structural correlations between genotype and clinical phenotype have been observed in TFG-associated neuropathies. Mutations in the carboxyl-terminal proline and glutamate (P/Q)-rich domain and Sec23-binding domain of the TFG protein lead to late-onset neuropathies inherited in an autosomal dominant manner, including HMSN-P, CMT2, ALS and Parkinson’s disease (figure 1B). Conversely, mutations in the amino-terminal Phox and Bem1p (PB1) and coiled-coil domains cause autosomal recessive HSP, which commonly occurs in families with consanguineous marriages and presents as early-onset complicated forms of HSP (online supplemental table 2). In this study, we report for the first time a non-consanguineous family carrying a novel heterozygous variant in TFG resulting in pure HSP. The pedigree has a positive family history spanning over four generations, and haplotype analysis further confirms autosomal dominant inheritance.
We retrospectively analysed patients with TFG mutations in our hospital and identified an HMSN-P pedigree harbour a previously reported TFG mutation (p.P285L). The clinical data and muscle pathology characteristics of patients with HSP and patients with HMSN-P were compared. Strikingly, no remarkable common clinical features were found. Additionally, TDP-43-positive aggregates were present in atrophic fibres in the biopsied muscle from the patient with HMSN-P, but not in the patient with HSP (figure 3). Recent studies have been increasingly indicating a primary and pathogenic role of TDP-43 in muscle degeneration. However, it remains unclear whether muscle pathology of abnormal protein aggregates contributes to the pathophysiology of HMSN-P. Previous pathological studies have revealed the presence of cytoplasmic inclusions containing TFG and TDP-43 in spinal and cortical motor neurons, frontotemporal lobes and biopsied muscles from patients with the p.P285L variant.1 10–12 Indeed, the presence of TDP-43 aggregates has been previously reported in the skeletal muscles of patients with various neuromuscular disorders such as sIBM, ALS, SMA, LGMD and oculopharyngeal muscular dystrophy.13 Analogously, pTDP-43, TDP-43 and ubiquitin aggregates were observed in our cohort of muscle biopsy patients diagnosed with sIBM (online supplemental figures 5 and 6A). Conversely, the deposition of these proteins was not found in muscle specimens of patients with ALS (online supplemental figures 5 and 6A), partly because these patients carry fused-in-sarcoma or optineurin mutations rather than TAR DNA binding protein 43 mutations. Moreover, TDP-43 aggregates are more commonly visualised in axial muscles than appendicular muscles of patients with ALS,14 15 and most of our muscle specimens were derived from the biceps brachii. Abnormal protein deposition of pTDP-43, TDP-43 and ubiquitin was also not found in patients with SMA, SBMA, CMT or LGMD2A in our cohort (online supplemental figures 5 and 6A). Additionally, p62-immunopositive signals were present in muscle specimens of patients with sIBM, ALS and SBMA (online supplemental figure 6B). Furthermore, muscle sections from patients with LGMD2A showed only sporadic dot-like p62 signals (online supplemental figure 6B). Notably, the present study found no evidence of TFG-positive aggregates in muscle specimens from either patients with HMSN-P or patients with HSP (online supplemental figure 3). Previous research has shown that motor neuron-specific TFG knockout, as opposed to muscle-specific TFG knockout, results in a decline in motor ability, indicating that muscle TFG does not appear essential for motor functions.16 Overall, despite the ubiquitous expression of TFG,3 17 18 its role in muscle cells remains obscure, and there is a lack of evidence regarding whether TFG mutations are associated with TDP-43 pathological deposition in muscle.
TFG localises to endoplasmic reticulum (ER) exit sites and functions in COPII-mediated export of secretory cargoes from the ER to the ER-Golgi intermediate compartments.19–22 Several pathogenic mechanisms have been described in terms of a single clinical phenotype associated with TFG mutations. For example, the TFG p.P285L variant for HMSN-P was reported to cause cytoplasmic aggregation of TDP-43 and impairment of the ubiquitin‒proteasome system.1 23 24 The p.G269V variant of CMT2 exhibits a propensity to generate insoluble TFG aggregates and induces haploinsufficiency.4 25 Furthermore, the p.R106C mutant for HSP is associated with defects in secretory and endosomal protein sorting, mitochondrial function and axon fasciculation.3 26–29 Recently, it was reported that TFG regulates UNC-51-like kinase 1 localisation and autophagosome formation by interacting with LC3C.30 This finding was further confirmed in patient-derived fibroblasts homozygous for the TFG p.R106C variant. Consistent with these observations, our data demonstrated that the levels of autophagy-related proteins (p62, Beclin 1 and LC3B-II) were decreased in skin fibroblasts from both patients with HSP and patients with HMSN-P (figure 4D), suggesting that autophagy defects may be a common pathogenic mechanism in TFG-related neurological diseases. Additionally, TFG-positive puncta were present in patient fibroblasts (figure 4C), while cytoplasmic TDP-43-positive and ubiquitin-positive inclusions were not detected (data not shown). Interestingly, we observed that TFG expression in patient-derived skin fibroblasts and biopsied muscle was reduced at the mRNA level but remained normal at the protein level (figure 4A,B, and online supplemental figure 4). Consequently, it appears that the mutant TFG protein is difficult to degrade. The underlying cause of this condition remains unclear; it may involve a loss of functional TFG, which is postulated to participate in autophagosome formation. Notably, further studies are necessary to determine whether TFG aggregates exacerbate disease progression and thereby create a vicious cycle. In summary, our findings reveal shared cellular phenotypes across different clinical manifestations caused by TFG mutations and support the notion that targeting autophagy may facilitate the development of a uniform treatment for TFG-related neurological disorders.
Data availability statement
Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
The Institutional Review Board at Qilu Hospital of Shandong University approved all studies (KYLL-2023(ZM)-017), and all participants provided written informed consent.
Acknowledgments
We express our utmost gratitude to the families who participated in this study.
References
Supplementary materials
Supplementary Data
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Footnotes
YZ and PL contributed equally.
Contributors CY, PL and LX contributed to study conception and design. LX drafted the manuscript text and prepared the figures. All authors contributed to patient clinical data and sequencing data acquisition and analysis, and manuscript review and revision. PL acts as a guarantor.
Funding This study was funded by the National Natural Science Foundation of China (Grant No. 82271436), Shandong Provincial Natural Science Foundation (Grant No. ZR2022MH190) and Qingdao Science and Technology Benefit People Demonstration Guide Special Project (Grant/Award Number: 22-8-7-smjk-1-nsh).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.