Characterization of a novel TFG variant causing autosomal recessive pure hereditary spastic paraplegia

Abstract Objective TFG mutations have previously been implicated in autosomal recessive hereditary spastic paraplegia (HSP), also known as SPG57. This study aimed to investigate the clinical and molecular features of TFG mutations in a Taiwanese HSP cohort. Methods Genetic analysis of TFG was conducted in 242 unrelated Taiwanese HSP patients using a targeted resequencing panel covering the entire coding regions of TFG. Functional assays were performed using an in vitro cell model to assess the impact of TFG variants on protein function. Additionally, other representative TFG mutant proteins were examined to understand the broader implications of TFG mutations in HSP. Results The study identified a novel homozygous TFG c.177A>C (p.(Lys59Asn)) variant in a family with adolescent‐onset, pure form HSP. Functional analysis revealed that the Lys59Asn TFG variant, similar to other HSP‐associated TFG mutants, exhibited a low affinity between TFG monomers and abnormal assembly of TFG homo‐oligomers. These structural alterations led to aberrant intracellular distribution, compromising TFG's protein secretion function and resulting in decreased cellular viability. Interpretation These findings confirm that the homozygous TFG c.177A>C (p.(Lys59Asn)) variant is a novel cause of SPG57. The study expands our understanding of the clinical and mutational spectrum of TFG‐associated diseases, highlighting the functional defects associated with this specific TFG variant. Overall, this research contributes to the broader comprehension of the genetic and molecular mechanisms underlying HSP.


Introduction
Hereditary spastic paraplegia (HSP) represents a heterogeneous group of inherited neurological disorders characterized by progressive degeneration of the motor axons in the corticospinal tracts, resulting in bilateral lower limb spasticity and weakness. 1HSP can be categorized into pure and complex forms, with the former caused by the isolated degeneration of the corticospinal tracts, while the complex form encompassing additional neurological system involvement.In addition to lower limb spasticity and weakness, individuals with complex HSP may present with a wide range of neurological symptoms, including ataxia, seizures, intellectual disability, muscle atrophy, extrapyramidal symptoms, visual abnormalities, and peripheral neuropathy.Up to 90 genes have been identified to be associated with HSP (https://neuromuscular.wustl.edu/spinal/fsp.html), 2 and individual HSP-related genes and mutations can contribute to clinical phenotypes through different mechanisms.Understanding the molecular basis underpinning these disease genes and causal mutations may facilitate the therapeutic development and provide insights into the pathophysiology of HSP.
Mutations in the TFG gene have been linked to various neurodegenerative diseases, including hereditary motor ª 2024 The Author(s).Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.and sensory neuropathy (HMSN) and HSP.In 2012, a Japanese research group identified a heterozygous c.854C>T (p.(Pro285Leu)) variant in TFG as the cause of autosomal dominant hereditary motor and sensory neuropathy with proximal predominance (HMSN-P). 3Our research team later found a heterozygous TFG c.806G>T (p.(Gly269Val)) variant responsible for typical Charcot-Marie-Tooth disease type 2 (CMT2) in a large Taiwanese family, characterized by autosomal dominant axonal polyneuropathy with distal predominant involvement. 4Furthermore, a small subset of TFG variants can result in autosomal recessive HSP, specifically Spastic Paraplegia type 57 (SPG57).In 2013, Beetz et al. reported that the homozygous TFG variant, c.316C>T (p.(Arg106Cys)), in an Indian family led to a complex form of HSP characterized by spastic paraplegia, optic nerve atrophy, and neuropathy, known as SPOAN syndrome. 5Intriguingly, in a consanguineous Pakistani family, another homozygous TFG variant, c.317G>A (p.(Arg106His)), was found to cause a childhood-onset pure form of HSP. 6 Based on the distribution of pathogenic variants in TFG discovered thus far, it can be summarized that TFG variants associated with neuropathy typically occur in a heterozygous pattern at the C-terminal of the protein, while variants linked to SPG57 are the homozygous N-terminus-residing TFG variants.
In this study, we identified a novel homozygous TFG variant, c.177A>C, p.(Lys59Asn), in a consanguineous pedigree with autosomal recessive pure HSP.We assessed the pathogenicity of the TFG variant by utilizing in vitro cell experiments and bioinformatic analysis.Additionally, we compared the influence of TFG protein function between this variant and several previously reported pathogenic TFG variants.

Patient evaluations and ethics
A consecutive series of 242 unrelated patients with HSP were enrolled at Neurology Services of Taipei Veterans General Hospital from January 1998 to January 2022.The diagnosis of HSP is based on the manifestation of either (1) pure spastic paraplegia, (2) spastic quadriparesis with more severe and earlier involvement of lower limbs, or (3) spastic paraplegia as an early and prominent sign with involvement of multiple parts of the nervous system.8][9] This study was approved by the Institutional Review Board of Taipei Veterans General Hospital.Written informed consent was obtained from all participants prior to their involvement.A comprehensive neurological examination was conducted, including the assessment of the Spastic Paraplegia Rating Scale, 9 to characterize the patients' clinical features.Nerve conduction studies (NCS) and electromyography (EMG) were carried out to assess the involvement of the peripheral nervous systems.Neuroimaging studies using magnetic resonance imaging (MRI) were conducted to evaluate any structural abnormalities in the brain and spinal cord.

Genetic analyses
Genomic DNA was extracted from peripheral white blood cells.Mutational analysis of TFG was conducted by utilizing a targeted sequencing panel covering 76 HSP genes and the 57 other genes implicated in diseases with an HSP-like phenotype on an Illumina HiSeq2500 (Table S1). 10 Sequence variants within the targeted regions were called by aligning the sequenced reads to the reference Human Genome version 38 (hg38/GRCh38).The TFG variants were further confirmed through Sanger sequencing and named according to the reference TFG sequence (NM_006070.6).In silico prediction of the variant's pathogenicity utilized multiple bioinformatics tools, including MutationTaster, 11 SIFT, 12 PolyPhen-2, 13 PredictSNP, 14 and CADD. 15Conservation scores from ConSurf 16 and LIST-S2 17 were employed to assess conservation.The pathogenicity of the novel TFG variant was evaluated according to the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP). 18
CRISPR/Cas9 knockout of TFG in HEK293T, neuro-2a, and NSC-34 cells To deplete endogenous TFG in cells using CRISPR/Cas9 technology, TFG gRNA (target sequence in Exon 2: ATCCT GGAGTCCACCATGAA for human TFG; ATCCTGGACT CCACAATGAA for mouse Tfg) in pCas-Guide-EF1a-GFP vector (OriGene, Rockville, MD, USA) was transfected into cells (Fig. S1A).After 3 days, cells were seeded as single colonies in 96-well plates.Once individual colonies had formed, they were isolated and cultured in 6-well plates until cell numbers were sufficient for western blot analyses (Fig. S1B).Genomic DNA was isolated from cells using QuickExtract solution (Epicentre, Madison, WI, USA), following the manufacturer's instructions.The modified region was amplified using designed validation primers and sequenced to determine the clonal genotype (Fig. S1A).

Cross-linking of oligomeric TFG proteins
To examine the oligomeric state of TFG, lysates from TFG-overexpressing cells were cross-linked by incubating them with various concentrations of Bis (sulfosuccinimidyl) suberate sodium salt (BS3) in conjugation buffer (100 mM sodium phosphate, 0.15 M NaCl) for 30 min.The cross-linking reactions were then quenched by incubating with 50 mM Tris HCl, pH 7.5.Subsequently, the reaction mixtures were analyzed using western blotting.

Co-immunoprecipitation (co-IP) analysis
To investigate the self-assembly ability of TFG proteins, co-IP analysis was performed to assess the binding affinity between TFG molecules.In brief, HEK293T cells were cotransfected with flag-and myc-tagged TFG constructs.Cell extracts were prepared using Pierce IP Lysis Buffer (Thermo Fisher Scientific).With the exception of 10% of the sample volume used for input, the lysates and antiflag antibody-conjugated Dynabeads (Invitrogen) were incubated overnight at 4°C.After washing, the target proteins were eluted by boiling in 1X SDS loading buffer at 95°C for 5 min, and these eluates were subsequently analyzed by western blotting.

Immunofluorescence studies
Cells were fixed in 4% paraformaldehyde, followed by permeabilization with 0.2% Tween-20 and blocking with 1% BSA.The cells were then incubated overnight at 4°C with primary antibodies.The bound primary antibodies were detected using Alexa-conjugated secondary antibodies.Imaging was conducted using an Olympus FluoView FV10i confocal laser scanning fluorescence microscopy system with a 60X oil immersion objective (Olympus, Tokyo, Japan).

Investigating protein secretory efficiency through the Gaussia luciferase reporter assay
Gaussia luciferase (Gluc) is commonly used to monitor the protein secretory pathway. 19,20Secreted Gluc activity in the culture medium from co-transfected cells expressing both Gluc and Firefly luciferase (Fluc) serves as an indicator of protein secretory efficiency.To evaluate this, cells were co-transfected with plasmids expressing Gluc (pCMV-Gaussia Luc; Thermo Fisher Scientific), Fluc (pCMV-Red Firefly Luc; Thermo Fisher Scientific), and either TFG wild-type, variants, or an empty vector.After 48 h of transfection, conditioned medium was collected to measure Gluc activity, which was then normalized to the corresponding Fluc activity in the cell lysate for each sample set.

Cell viability assay
Cells were seeded onto 96-well culture plates and transfected with the specified TFG constructs.After 48 h, cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich) following the manufacturer's instructions.

Neurite outgrowth assays
Neurite outgrowth assays were performed using the Neurite Outgrowth Staining Kit (Thermo Fisher Scientific).N2a cells in a 96-well plate were transfected with the specified TFG constructs.After 16 h of transfection, cells were induced to differentiate for 48 h and fixed with 4% paraformaldehyde.Neurite outgrowth and cell viability were assessed by staining with fluorescent dyes for the cell membrane and viability.The samples were then analyzed using a fluorescence microplate reader.

Identification of the TFG variant
Among the 242 unrelated HSP patients, only one patient was identified to carry a TFG variant, which was the homozygous TFG c.177A>C (p.(Lys59Asn)) (Fig. 1A,B).
The patient manifested pure HSP and originated from a consanguineous family.Further analysis revealed the same homozygous variant in one of his brothers, who also presented with pure HSP.Four other individuals in the family carrying a heterozygous TFG c.177A>C (p.(Lys59Asn)) variant did not exhibit any neurological deficits.This TFG variant had not been previously reported and was absent in the Genome Aggregation Database (gnomAD v4.0.0; http://gnomad.broadinstitute.org/) and the Taiwan Biobank database (http://www.twbiobank.org.tw/), which contains 1517 genomes from healthy Taiwanese individuals.The pathogenicity of TFG p.(Lys59Asn) was also supported by multiple bioinformatics prediction tools (Table 1).According to the ACMG-AMP guidelines, 18 TFG c.177A>C (p.(Lys59Asn)) meets several criteria, including cosegregation with HSP in a recessively inherited pattern within the family (PP1 criterion), absence in the population databases (PM2), and its deleterious effect supported by multiple predicting programs (PP3) and functional studies (PS3).Collectively, TFG c.177A>C (p.(Lys59Asn)) is classified as a likely pathogenic variant.

Case presentation
The proband (V-1) harboring the homozygous c.177A>C (p.(Lys59Asn)) TFG variant was born to the consanguineous parents who were neurologically healthy and were genotyped for the heterozygous TFG c.177A>C (p.(Lys59Asn)) variant (Fig. 1A,B).Patient V-1 had an unremarkable birth history and achieved normal developmental milestones during childhood.He began to experience gradual worsening of gait characterized by intermittent stiffness of the legs and frequent falls since age 15 years.These symptoms persisted and worsened after age 31 years when he suffered from fatigue after walking long distances and had difficulty walking downstairs.Despite these symptoms, he did not require walking aids and retained normal function of daily activities.Neurological examinations at age 36 revealed normal muscle strength but lower limbs spasticity, resulting in limited dorsi-and plantar-flexion of the ankles and mild spasticity in the hips and knees.The deep tendon reflex (DTR) examination revealed bilateral brisk knee jerks and ankle clonus, with normal tendon reflex at the upper limbs.Although he was able to walk without assistance, a scissor gait was evident during the evaluation (Video S1).No sensory abnormalities or sphincter dysfunction were reported.The spastic paraplegia rating scale (SPRS) yielded a score of 13/60.The MRI studies excluded structural abnormalities at the cervical and thoracic levels.Electrophysiological evaluations, including NCS and EMG, revealed normal sensory and motor conduction, as well as normal electromyographic activities.
The proband's younger brother (V-2), who harbored homozygous TFG c.177A>C (p.(Lys59Asn)) variant, had suffered from progressive gait abnormalities and frequent falls since age 15 years.He reported that the leg stiffness became more prominent in cold weather, leading to difficulty going downstairs and mild fatigue while walking upstairs.Neurological examinations at age 34 years revealed lower limbs spasticity, bilateral brisk knee and ankle jerks, and a spastic gait (Video S2).The SPRS score was 9/60 at age 34 years.His spine MRI demonstrated unremarkable findings.Electrophysiological examinations further revealed normal results.In contrast, neurological examinations of the family members carrying the heterozygous TFG c.177A>C (p.(Lys59Asn)) variant were all normal, including the proband's parents (III-8 and IV-1) at age 60 and 55 and his youngest brother (V-3) at age 25 (Fig. 1A).

Impact of TFG variants on protein oligomerization
To assess the functional alterations induced by the TFG p.(Lys59Asn) variant, our investigation began by examining the effects associated with Lys59Asn TFG in the absence of wild-type TFG.CRISPR-Cas9 was initially employed to knock out TFG expression in HEK293T, N2a, and NSC-34 cells for two primary reasons: First, TFG c.177A>C (p.(Lys59Asn)) causes autosomal recessive HSP in its homozygous status, and second, TFG protein is ubiquitously expressed in neuronal and non-neuronal cells.This approach aimed to establish a cellular model for analyzing homozygous variants linked to recessive disorders.To comprehensively understand the effects of mutant proteins and elucidate potential pathological mechanisms of TFG p.(Lys59Asn), our functional studies included other representative pathogenic TFG variants: p.(Arg106Cys) and p.(Arg106His) linked to SPG57, and p.(Gly269Val) and p.(Pro285Leu) associated with CMT2 and HSMN-P (Fig. 1C,D).
Previous study has shown that TFG forms an octameric structure capable of self-association to generate larger polymers. 21TFG p.(Lys59Asn) induces alterations in the Phox and Bem1p (PB1) domain of TFG, known to govern self-association and homo-oligomer formation, as evidenced by yeast two-hybrid experiments. 22To investigate whether p.(Lys59Asn) affects TFG protein homooligomerization, we conducted an in vitro cross-linking assay with the BS3 chemical.In Fig. 2A, untreated wildtype TFG proteins showed a single band at approximately 57 kDa, representing the monomeric form.In the presence of BS3, the intensity of this band decreased, and at 1 mM BS3, five new bands with molecular masses exceeding 100 kDa emerged.With a gradual increase in BS3 The LIST-S2 score, the closer it is to 1, indicates a higher level of conservation. c The SIFT score ranges from 0.0 (deleterious) to 1.0 (tolerated).concentration (1-5 mM), the intensity of these new bands increased, indicating the formation of homo-oligomers of TFG proteins in vitro.However, the assembly states of Lys59Asn, Arg106Cys, and Arg106His TFG mutant proteins lacked the highest band (>315 kDa), whereas the wild-type TFG protein and the two neuropathy-associated Gly269Val and Pro285Leu TFG variants displayed five distinct bands representing the complete oligomeric form of TFG proteins.
To further validate the impact of the SPG57-associated TFG variants on protein assembly, we conducted coimmunoprecipitation experiments to examine the selfinteraction abilities of these disease-related TFG proteins within the TFG-knockout HEK293T cells.We cotransfected cells with myc-tagged and flag-tagged TFG proteins and found that the three TFG mutants associated with SPG57, including Lys59Asn, Arg106Cys, and Arg106His TFG, all exhibited weaker self-interaction abilities than the self-interaction abilities between wild-type TFG molecules (Fig. 2B).Notably, among these TFG mutants, Arg106Cys TFG displayed the weakest selfinteraction ability.In contrast, the Gly269Val TFG, which is associated with CMT2, exhibited an enhanced protein self-interaction ability.The protein self-interaction ability of the Pro285Leu TFG, which is associated with HMSN-P, was similar to that of wild-type TFG.
Next, we utilized immunofluorescence techniques to observe the intracellular distribution of wild-type or mutant TFG proteins overexpressed in the Tfg-knockout N2a cells.As illustrated in Fig. 3A, the wild-type TFG protein exhibited a granular aggregation pattern within the cytoplasm, highlighting its oligomeric nature.In contrast, the protein expression of Lys59Asn, Arg106Cys, and Arg106His TFG appeared to be less concentrated and distributed extensively throughout the cytoplasm with significantly reduced granular aggregation.Among them, Arg106Cys TFG exhibited the most dispersed pattern with the smallest protein foci, followed by Arg106His and Lys59Asn TFG.Moreover, the Gly269Val TFG protein shows larger protein foci accumulations, consistent with our prior observations. 4On the other hand, the Pro285-Leu TFG displayed a protein appearance similar to the wild-type TFG.

Expression and localization of TFG variants in neuronal cells
We evaluated the expression of TFG variants in differentiated neuronal cell lines by transfecting various TFG variants into Tfg-knockout N2a cells.Following differentiation, immunofluorescence staining was utilized to visualize TFG protein expression and localization in differentiated neurons.As shown in Fig. 3B, the wild-type TFG exhibited a granular aggregation pattern within the cytoplasm and extended into the neurites.However, the protein expression of p.(Lys59Asn), p.(Arg106Cys), and p.(Arg106His) variants predominantly distributed within the cytoplasm of neurons, with minimal observation in the neurites.Variants associated with neuropathy, including p.(Gly269Val) and p.(Pro285Leu), exhibit a distribution pattern similar to that of the wild type.Overall, these results indicate that the three SPG57-associated variants disrupt TFG protein assembly, leading to impaired oligomerization and altered distribution within both the cytoplasm and neurites.

Effects of TFG variants on protein transport and cell viability
Previous studies have emphasized TFG's role in the COPII-mediated vesicle transport pathway. 21,23,24In this study, we evaluated the impact of these TFG variants on protein transport by co-transfecting TFG variants with two reporter plasmids in TFG-knockout HEK293T or N2a cells.In TFG-knockout cells transfected with an empty vector, we observed significantly reduced protein secretion efficiency compared to the original cell control without TFG-knockout (Fig. 4A).Introducing the wildtype TFG into TFG-knockout cells significantly improved protein secretion efficiency.However, cells expressing other external TFG variants were unable to rescue protein secretion efficiency, indicating that all five TFG variants disrupt cellular protein transport.We also performed a CCK-8 cell viability assay to assess the effect of TFG variants on cell viability.This revealed significantly reduced cell viability in TFG-knockout vector control cells compared to the original cells without TFG-knockout (Fig. 4B).The introduction of external wild-type TFG into TFG-knockout cells notably increased cell viability, but the expression of other external TFG mutant variants failed to rescue cell viability, demonstrating that all five disease-associated TFG variants can influence cell viability.

Influence of TFG variant expression on neurite outgrowth
We assessed the influence of TFG variant expression on neurite outgrowth in Tfg-knockout N2a cells using the Neurite Outgrowth Staining Kit.As illustrated in Fig. 4C, when compared to wild-type TFG expression, the introduction of neuropathy-associated TFG variants (i.e., Gly269Val and Pro285Leu) led to a substantial reduction in neurite outgrowth.In the contrast, the three variants linked to SPG57 showed no significant difference from the wild-type.

Discussion
The significance of TFG-associated HSP has been increasingly recognized due to the growing number of TFG variants identified in HSP patients.These patients predominantly exhibit an autosomal recessive inheritance pattern, denoted as SPG57, 5,6,[25][26][27] or less frequently as an autosomal dominant subtype. 28In this study, we identified a novel homozygous TFG c.177A>C (p.(Lys59Asn)) variant in a pair of siblings presenting with adolescentonset pure spastic paraplegia within a consanguineous family in Taiwan.This finding introduces TFG p.(Lys59Asn) as the seventh variant associated with HSP.
Our study revealed that the p.(Lys59Asn) variant in the PB1 domain of the TFG protein, as well as the p.(Arg106Cys) and p.(Arg106His) variants in the coiled-coil domain, all resulted in reduced intermolecular affinity, leading to abnormal protein assembly and disruption of TFG's high-ordered oligomeric structure.Previous studies have also noted the impact of the p.(Arg106Cys) variant on TFG intermolecular assembly, 5,29,30 and another SPG57-associated variant, TFG p.(Arg22Trp), in the PB1 domain, compromised TFG oligomerization. 252][33][34] Therefore, the proper folding and homo-oligomerization of these two N-terminal domains are essential for maintaining TFG's normal structure.Altering certain conserved amino acids within the PB1 or coiled-coil domain may hinder the self-assembly function of TFG through different mechanisms, thereby affecting TFG's structure and function.Furthermore, in this study, we observed that the p.(Lys59Asn) variant, along with two other SPG57associated variants, exhibited altered cellular localization and distribution.Wild-type TFG typically accumulates at the ER exit site (ERES) within the cytoplasm, where its oligomeric structure acts as a scaffold protein facilitating the transport of COPII-coated vesicles. 21,23,24Additionally, TFG is known to aggregate and distribute in the axons and dendrites of nerve cells, supporting the cycling transport of endosome cargoes. 35However, our study revealed that SPG57-associated TFG mutants exhibited reduced aggregation at the ERES, ultimately affecting the intracellular protein transport efficiency.In differentiated neuronal cells, while SPG57-associated TFG mutant proteins did not significantly affect differentiation and neurite growth, the granule structure of TFG was visibly smaller, and these proteins were sparsely aggregated in neurites.A previous study has also noted that the p.(Arg106Cys) variant resulted in fewer TFG foci in neurites of primary rat cortical neurons and disrupted the normal endosomal trafficking of gephyrin to synapses. 35Therefore, it is suggested that TFG p.(Lys59Asn) may also influence TFG's role in endosome cargo transport.
Moreover, remarkably, there is a positive correlation between the severity of alterations in molecular affinity, structural size, and intracellular distribution among TFG molecules and the clinical severity of these variants.The Lys59Asn TFG protein showed the mildest impact on molecular affinity, oligomeric structure size, and intracellular distribution, corresponding to the mildest clinical symptoms.In contrast, the Arg106Cys TFG showed the smallest oligomeric structure and the most diffuse distribution, and was nearly absent in neurites, aligning with the most severe clinical symptoms.We employed the machine learning-based software MutPred2 36 to predict the effects of these three SPG57-associated variants.The results indicated that the p.(Arg106Cys) variation, located in the coiled-coil domain, may potentially induce an "altered coiled coil" effect (p value = 0.002), while p.(Arg106His) did not exhibit this effect, suggesting that p.(Arg106Cys) has a more significant impact on the protein structure.On the other hand, the p.(Lys59Asn) variant might lead to a "loss of ubiquitylation at K59" (p value = 0.01).In the results presented in Fig. S2, we also observed that the protein steady-state expression level of Lys59Asn TFG is indeed higher than that of the wild type, possibly due to the variant affecting its protein degradation regulation.Furthermore, the steady-state expression levels of Arg106Cys TFG are the lowest, followed by Arg106His.The TFG levels inside the cells may also play a role in determining the clinical phenotype's severity.However, these speculations require further experimental validation.
Additionally, SPG57 mutations at different sites yield distinct clinical manifestations.The TFG p.(Lys59Asn) variant identified in this study is the sole PB1 domain variant known to cause pure HSP.Patients with this variant typically experience symptom onset in their late adolescence and exhibit milder clinical symptoms than other SPG57-associated variants.][27] Notably, p.(Ile66Thr) shares similarities with TFG p.(Arg106Cys) and is associated with optic nerve atrophy.Therefore, mutations at different sites may induce varying degrees of structural and functional impairments, potentially affecting interactions with distinct proteins, disrupting diverse downstream signaling pathways, and leading to a wide array of clinical symptoms.
In addition to SPG57-associated TFG variants, we also examined two variants, p.(Gly269Val) and p.(Pro285Leu), linked to CMT2 and HMSN-P, for functional comparisons.Interestingly, these two variants, responsible for peripheral neuropathy, do not disrupt TFG molecular assembly and oligomerization.Instead, the TFG p.(Gly269Val) variant significantly increases the molecular affinity among TFG molecules, leading to larger TFG protein aggregates within cells.Our previous research indicated that the TFG p.(Gly269Val) variant increases the tendency for protein deposition, exhibiting a dominant negative effect, resulting in the accumulation of insoluble protein aggregates and a reduction in functional TFG levels within cells. 4Furthermore, the TFG p.(Gly269Val) and p.(Pro285Leu) variants do not alter the distribution of TFG within the cytoplasm or neurites; however, they have a more pronounced impact on neurite outgrowth.These findings highlight significant differences in the mechanisms responsible for structural or functional abnormalities induced by TFG variants associated with peripheral neuropathies compared to those related to SPG57.
Combining the functional results of this study, the pathogenic mechanisms underlying the three SPG57associated variants, TFG Lys59Asn, Arg106Cys, and Arg106His proteins, appear to lean toward a loss of function.TFG plays a critical role in various membrane system-mediated protein transport processes, 21,23,24,35,37 which are particularly crucial for maintaining the normal physiology of neurons compared to other cell types.Many pathogenic genes in HSP are involved in membrane modeling, including proteins such as Spastin M1, REEP1, Atlastin, Spastizin, Spatacsin, and Strumpellin, which are associated with the ER, endosomes, autophagosomes, and lysosomes.Any functional defects in these proteins can easily lead to neuronal and axonal pathological conditions, subsequently causing the disease.
In conclusion, this study identifies a novel SPG57associated TFG variant, c.177A>C (p.(Lys59Asn)) and delineates the relevant functional disturbance of TFG proteins.These findings expand the clinical knowledge and mutational spectrum of SPG57 and provide insights into the molecular pathogenic mechanisms of HSP.

Figure 1 .
Figure 1.Identification of the homozygous TFG missense variant in the pedigree with pure hereditary spastic paraplegia.(A) Pedigree of the HSP patient harboring the TFG p.(Lys59Asn) variant.The proband is indicated by an arrow."M" denotes the mutant TFG allele, while "W" represents the wild-type allele.Squares and circles correspond to males and females, respectively.Filled and open symbols indicate affected and unaffected individuals, respectively.Half-filled symbols represent carriers of the heterozygous mutation without displaying the disease phenotype.A slash indicates deceased individuals.(B) Electropherograms of TFG Exon 2 in a wild-type control, the HSP-manifested siblingship, and their unaffected carrier family members.The altered nucleotides are highlighted with red star symbols.(C) Schematic representation of the functional domains of the TFG protein and the comparison of the amino acid sequences of TFG orthologous proteins from various species.Several known pathogenic TFG variants are labeled, with the TFG p.(Lys59Asn) variant identified in this study highlighted in red.Additional variants examined in this study are marked in blue.Alignment of multiple TFG orthologs demonstrates conservation of the K59 residue from human to Caenorhabditis elegans.(D) Visualization of the AlphaFold-predicted TFG protein (PDB: AF-Q92734-F1) structure on the UCSF Chimera software (V1.14).The loci of TFG variants characterized in this study are labeled in red.

dCADD,Figure 2 .
Figure 2. Defective oligomerization and self-assembly of HSP-associated TFG mutants.(A) Oligomeric structure of TFG proteins in vitro.TFG variant-expressing cells underwent cross-linking reactions with varying concentrations of BS3.The resulting products were analyzed through western blotting.The HSP-related TFG Lys59Asn, Arg106Cys, and Arg106His mutants exhibited the absence of the highest-tier oligomeric products.Arrowheads indicate the positions of bands where higher-order oligomers appear.(B) Evaluation of the self-assembly capacity of TFG proteins using co-immunoprecipitation assays.Cells were co-transfected with myc-tagged and flag-tagged TFG expression plasmids, followed by lysis 48 h posttransfection.The interaction among TFG protein molecules was demonstrated by co-immunoprecipitation pull-downs of myc-tagged TFG using an anti-flag antibody against flag-tagged TFG.The degree of binding between TFG protein molecules is graphically presented in a quantitative statistical chart on the right-hand panel.Values are shown as means AE SEM of three independent experiments (*p < 0.05; **p < 0.01)."WT" denotes wild-type.

Figure 3 .
Figure 3. Expression and subcellular localization of the TFG variants.(A) Confocal fluorescence images of transfected N2a cells (Tfg knockout) labeled with an Alexa 488-conjugated anti-flag antibody to visualize TFG (shown in green).Cell nuclei were counterstained with DAPI (blue).The quantitative graph in the bottom right panel shows the analysis of the size of TFG foci formed by various TFG mutants in N2a cells (Tfg knockout).Error bars represent mean AE SEM.*p < 0.05, **p < 0.01.(B) Confocal fluorescence images of differentiated N2a cells (Tfg knockout) transfected with individual TFG constructs (labeled with a flag antibody, green) and a neurite membrane marker (highlighted by the NRP1 antibody, red).Cell nuclei were stained with DAPI (blue).Arrowheads indicate the positions of distinct high-intensity TFG oligomer structures in the neurite.Scale bar: 10 lm.

Figure 4 .
Figure 4.In vitro functional analysis of the TFG variants (A) Quantitative analysis of secreted Gaussia Luciferase (Gluc) activities in the culture medium of co-transfected HEK293T or N2a cells expressing Gluc, Firefly Luciferase (Fluc), and TFG plasmids.The measured Gluc activities were normalized to the corresponding Fluc activities in cell lysates to evaluate protein secretory efficiency.The Gluc activity percentage of TFG or Tfg knockout cells transfected with an empty vector was set as 100% for comparison.Results are presented as means AE SEM from 9 (HEK293T) or 12 (N2a) independent transfections (*p < 0.05; **p < 0.01)."KO" indicates knockout.(B) Assessment of cell viability in HEK293T or NSC-34 cells expressing TFG using the Cell Counting Kit-8.The viability percentage of TFG or Tfg knockout cells transfected with an empty vector was set as 100% for comparison.The presented values represent means AE SEM from 6 (HEK293T) or 18 (NSC-34) independent transfections.(C) Neurite outgrowth analysis in N2a cells expressing TFG mutants.N2a cells (Tfg knockout) were transfected with the TFG construct or an empty vector, cultured under differentiation conditions, and stained with a cell membrane fluorescent dye and a cell viability indicator fluorescent dye.Normalized neurite outgrowth indicates the relative fluorescence units of the cell membrane dye normalized to the corresponding cell viability indicator dye.The presented values represent means AE SEM from 16 independent transfections.

Table 1 .
The TFG coding variant identified in the study. b