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A dominant vimentin variant causes a rare syndrome with premature aging

Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer’s or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient’s features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.


Introduction
Rare genetics syndromes with an accelerated aging have been classified as progeroid syndromes. At the severe end of the spectrum, the early-onset Hutchinson-Gilford progeria is due to heterozygous variants in the intermediate filament protein LMNA (MIM: 150330), encoding lamin A/C [1]. Lamins are nuclear fibrous proteins belonging to the type V family of intermediate filament (IF) proteins and perform structural and regulatory functions [1]. IFs, together with microtubules and actin filaments, form the cytoskeletal framework in the cytoplasm and nucleus of most eukaryotic cells. IFs are divided into six groups based on their primary sequence and tissue-specific expression [2]. Vimentin, a type III IF protein, is widely expressed in all mesenchymal cells. It shares the typical tripartite domain organization with all IF proteins with a central, highly conserved α-helical rod domain mediating vimentin homo-and hetero-dimerization essential for the early stages of vimentin polymerization, flanked by globular head and tail domains responsible for elongation and compaction of fully formed filaments [3]. Vimentin plays major roles in cell shape, maintenance of the overall integrity of the cytoplasm, and in stabilizing cytoskeletal interactions and cell substrate adhesion [4].
Two missense variants in the human vimentin gene have been identified so far in two unrelated individuals diagnosed with cataract [5,6]. The two variants (c.451 G > A p.(Glu151Lys) and c.623 A > G p.(Gln208Arg) are located in coil1A subdomain of vimentin rod and the variant p.(Glu151Lys) has been shown to act in a dominant manner by inducing aggregation of the vimentin network. Transgenic mice expressing a vimentin p.(Arg113Cys) mutant also developed a cataract without obvious other symptoms [7]. This organ-specific disease was surprising, considering the wide tissue expression of vimentin. In line with this unexpected phenotype, the targeted deletion of vimentin in mice caused widespread but mild phenotypes in unchallenged animals [2,8]. Most recently, the analysis of phosphodeficient vimentin knockin mice indicated a role of vimentin in skin aging, neuronal differentiation, and subcutaneous fat loss [9][10][11].
Here we report a novel vimentin variant (c.1160 T > C; p.(Leu387Pro)) causing a syndromic disorder affecting craniofacial development, peripheral nervous system, and adipose and ectodermal tissues. The pathogenicity of the variant was confirmed by recapitulating the key clinical features in cellulo and in vivo.
Additional procedures are described in the Supplementary Information files.

Clinical features of the affected individual
This study was approved by the review boards of the involved institutions and written informed consents have been obtained from the affected individual and his family. The 39-year-old male proband was born from nonconsanguineous parents. At birth, he presented with congenital frontonasal dysostosis that required surgery at the age of 5 years. Thoracic scoliosis was noted at the age of 15 months and worsened, despite orthopedic treatment, requiring surgery at the age of 16 years (Fig. 1a). At the age of 16 years, severe amyotrophy, thin skin, and lipodystrophy were noted with pes cavus, alopecia, absent eyebrows, micrognathia, and oligodontia (Fig. 1a). Electromyography showed a motor and sensitive peripheral neuropathy. At the age of 17 years, a nerve biopsy of the leg revealed a severe axonal loss associated with demyelination. Progressive bilateral sensorineural deafness began at 10 years and required bilateral hearing aid at 15 years old. At the age of 31 years, ophtalmological examination showed a chronic normal tension glaucoma with progressive bilateral cupping of the optic nerves and visual field loss. At the age of 33 years, two episodes of cerebral ischemic strokes occurred (frontal and cerebellar infarcts) with right hemiparesis. Cerebral magnetic resonance imaging showed diffuse cortical atrophy with bilateral ventricular dilatation. A left patella fracture occurred at the age of 36 years. Osteodensitometry showed osteopenia (T-score -2,4 SD). Given the similarity of these clinical features with a mild form of Hutchinson progeria and Werner syndrome, LMNA and WRN were sequenced but no function-affecting variants have been identified. An array comparative genomic hybridization was performed and did not reveal candidate copy number variations.

Genetic analysis
We performed WES of the DNA from the proband at a depth of 80× and analyzed the data following GATK's best practices (v3.4). We kept variants which were: (1) exonic or at splice sites (±10 bp), (2) with a frequency below 0.5% in gnomAD and our internal database of 1000 exomes, and (3) covered by at least 10 reads and with an allele frequency above 0.2. We identified 245 variants matching those criteria, including 62 variants in known disease-causing genes in OMIM (Supplementary Table S1). For the analysis, in a diagnostic setting, we focused on those disease-causing genes as we suspected it could be a phenotypic expansion of a known disorder. From the 62 variants, none was identified on X-chromosome. One gene, FDXR, carried two rare variants and could fit with an autosomal recessive inheritance. FDXR is known to cause a syndrome associating an auditory neuropathy and an optic atrophy (MIM: 617717), but the phenotype did not match with the patient's clinical presentation. Then, we analyzed the 19 rare variants associated with an autosomal dominant disorder. No variant has been identified in a gene associated with a syndromic phenotype with aging-related symptoms. Then, given the severity of the phenotype, we kept only variants absent from 1000 genome and gnomAD, and with either a predicted loss-of-function effect or a CADD phred score (v1.3) above 20. We ended up with two missense variants: c.293 C > G p.(Pro98Arg) (GenBank: NM_001130698.1) in TRPC3 and c.1160 T > C p.(Leu387Pro) (GenBank: NM_ 003380.4) in VIM. TRPC3 was associated with a spinocerebellar ataxia (MIM: 616410) and VIM to cataract (MIM: 116300). We performed segregation analysis for both genes and we found that the variant in TRPC3 was inherited from the healthy father, and that the VIM variant c.1160 T > C p. (Leu387Pro) had occurred de novo (Fig. 1b). We then decided to explore further the VIM variant, because vimentin is a type III IF protein, a class of structural proteins related to lamins, a well-known gene causing progeroid syndromes.
The Leucine residue at position 387 in vimentin is highly conserved among the IF superfamily and is located in an "e" position along the α-helical wheel of the coil2 subdomain of the rod (Fig. 2a, c) [3]. Leucine to Proline substitutions at the corresponding position of desmin and KRT17 are associated with myofibrillar myopathy and steatocystoma multiplex, respectively (Fig. 2a) [5,12]. Structural homology modeling based on the previously crystalized vimentin coil2 subdomain predicted that c.1160 T > C p.(Leu387Pro) variant should not significantly change vimentin static structure (Fig. 2b). Although it should not involve primary misfolding, it could still affect higher-order interactions among vimentin subunits in filaments or with associated proteins.

Characterization of c.1160 T > C p.(Leu387Pro) variant in cultured cells
To examine whether the c.1160 T > C p.(Leu387Pro) variant affected vimentin expression or cytoskeletal organization, induced pluripotent stem (iPS) cells were reprogrammed from mononuclear cells enriched from peripheral blood of the patient and were analyzed by immunofluorescence microscopy. The overall filamentous organization of the vimentin cytoskeleton in patient iPS cells appeared highly similar to that in controls ( Fig. 3a and Supplementary Fig. S1A). However, the overall staining intensity and the number of vimentin-positive cells were reduced in the patient's iPS cell population ( Fig. 3a and Supplementary Fig. S1A). This prompted us to investigate vimentin protein levels by western blotting and fluorescence-activated cell sorting analysis. This revealed reduced vimentin protein levels ( Fig. 3b and Supplementary  Fig. S1B). Notably, in the patient's iPS cells, vimentin was processed into distinct fragments of 48 and 25 kDa with little full-length vimentin left ( Fig. 3b and Supplementary  Fig. S1B). Given that the vimentin antiserum used recognizes a carboxy-terminal vimentin epitope, the fragments detected by western blotting result from cleavage of vimentin at its head domain [13,14]. This is consistent with a reduced vimentin protein stability caused by the variant. In this setting, the vimentin variant's mRNA levels remained unaffected (Fig. 3c). To examine whether the destabilizing effect of the novel vimentin mutant was more general, vimentin wild-type (wt) and the p.(Leu387Pro) mutant were transiently transfected as hemagglutinin (HA)tagged variants in 3T3-L1 preadipocytes. In analogy to iPS cells, immunofluorescence analysis showed that HA-tagged vimentin p.(Leu387Pro) was co-distributed with the endogenous cytoplasmic vimentin network of 3T3-L1 cells, highly similar to wt vimentin (Fig. 4a). Strikingly, in a minority (<1%) of vimentin p.(Leu387Pro) cells, cytoplasmic aggregatelike structures reminiscent of those seen in other IF disorders were observed [2].
We then investigated the impact of vimentin p.(Leu387Pro) in the absence of endogenous vimentin, following transient expression of wt and/or mutant vimentin in vimentin knockout (KO) mouse embryonic fibroblasts (MEFs) and vimentin-negative MCF-7 cells. In both cell types, vimentin p.(Leu387Pro) was unable to form an extensive filamentous network on its own and accumulated in cytoplasmic aggregate-like structures as depicted by immunofluorescence microscopy (Figs. 4b, c). When co-expressed with wt vimentin, either native or as HA-tagged variant, vimentin p.(Leu387Pro) formed extensive filament networks in the majority of MCF-7 transfectants, although large aggregates were observed in a subset of cells (Fig. 4b).
HA-tagged and non-tagged vimentin behaved similarly, showing that the fusion did not affect vimentin behavior.
Thus, vimentin p.(Leu387Pro) forms normal IF in the heterozygous state and negatively influences vimentin protein stability by promoting vimentin proteolysis in a highly selective manner. In analogy to other dominant IF variants, it fails to assemble into filaments in the absence of wt vimentin or other type III/IV IF proteins.

Proteasomal degradation of vimentin p.(Leu387Pro) is accelerated compared with wt vimentin
The decreased abundance of vimentin in the patient's iPS cells (  these vimentin species (Supplementary Fig. S3C). Upon inhibition of total protein biosynthesis by cycloheximide in 3T3-L1/Vim-HA and 3T3-L1/Vim p.(Leu387Pro) -HA cells, the mutant decreased to lower levels than the wt protein, confirming the higher turnover of vimentin p.(Leu387Pro) -HA (Fig. 5c).
Overexpression of mutant vimentin RNA in zebrafish larvae leads to craniofacial, axonal guidance, and lipid distribution defects To date, missense variants in IF genes have been described to cause postnatal, tissue-specific diseases [2]. The multi-organ defects, such as malformations of facial bones and of peripheral nerves, identified in our patient were highly suggestive of early-onset defects, possibly with a developmental origin. To challenge this possibility, we chose to utilize the developing zebrafish as an in vivo model. Purified mRNAs encoding human wt vimentin or vimentin p.(Leu387Pro) were injected into zebrafish one-cell-stage eggs. We first performed an Alcian blue staining of zebrafish larvae at 5 days post fertilization (dpf) to determine whether they bore craniofacial abnormalities upon expression of the human wt or mutated RNA. We observed a significantly increased distance between the ceratohyal and Meckel's cartilages (Fig. 6a) upon overexpression of the wt mRNA, compared with age-matched controls (Fig. 6b). Strikingly, we observed an exacerbated effect on the cartilage distance upon overexpression of the vimentin p.(Leu387Pro) mRNA compared with the wt mRNA (Fig. 6b).
We next performed a whole-mount immunostaining with an anti-acetylated tubulin antibody on zebrafish larvae at 3 dpf to visualize the well-organized architecture of peripheral axons that extend from the notochord and innervate the myotomes via a fork-like termination (Fig. 6c). We scored the number of peripheral axons with sprouting and/or pathfinding defects on eight fixed consecutive metameres. Although overexpression of both the wt and the mutant vimentin p.(Leu387Pro) RNAs led to a significant number of axonal defects compared with controls, the phenotypic severity is exacerbated in the mutant condition (i.e., no larva injected with the wt mRNA has been scored in the categories "with defects in 5, 6, or 8 axons") (Fig. 6d).
Finally, to measure body fat distribution as a readout for lipodystrophy in zebrafish larvae, we stained lipids of mRNA-injected larvae at 5 dpf with Oil Red O as described [15]. Overexpression of human wt or vimentin p.(Leu387Pro) RNA showed a significant lipid accumulation in the gut when compared with controls (Fig. 6e, f). However, no significant difference was observed between wt and mutant at this developmental stage. Taken together, these data suggested that overexpression of wt vimentin or vimentin p.(Leu387Pro) RNAs disturbed the development of the face, the sprouting, and pathfinding of peripheral axons, and affected fat distribution, with vimentin p.(Leu387Pro) displaying exacerbated phenotypes.

3T3-L1/Vim p.(Leu387Pro) -HA adipocytes accumulate less lipid droplets and perilipin than wt vimentinexpressing cells
To gain insight into mechanisms underlying the lipodystrophy, we investigated the impact of vimentin p.(Leu387Pro) during adipogenesis in vitro using 3T3-L1 preadipocytes engineered to express vimentin p.(Leu387Pro) -HA and wt vimentin-HA (3T3-L1/Vim-HA cells and 3T3-L1/ Vim p.(Leu387Pro) -HA cells), in addition to non-transduced controls. Preadipocytes were subjected to an adipogenic differentiation protocol followed by expression analysis of major differentiation genes by reverse-transcriptase quantitative PCR (C/EBPα, β, δ, PPARγ transcription factors, the insulin-regulated glucose transporter GLUT4 and perilipin) and immunoblotting (for C/EBPα, β, and PPARγ). [16] Expression levels of these genes were comparable in all cell lines analyzed ( Supplementary  Figs. S4A and S6B), indicating that vimentin p.(Leu387Pro) did not affect adipogenic differentiation at the transcriptional level [17]. However, the analysis of lipid droplets revealed a significantly reduced lipid quantity in mutant compared with wt vimentin-expressing and control cells (Fig. 7a, b). Thus, decreased lipid accumulation in cellulo is reminiscent of the patient's lipodystrophy.

Discussion
We report here a novel missense variant in VIM responsible for a syndrome associating developmental and aging-related features. The vimentin p.(Leu387Pro) mutant impaired craniofacial development, peripheral axon branching, and fat distribution in zebrafish. We also demonstrate that the vimentin p.(Leu387Pro) mutant reduced lipid droplet size and numbers, and negatively regulated the abundance of perilipin during in vitro adipogenesis. Finally, we present evidence for enhanced proteolytic processing and turnover of vimentin p.(Leu387Pro) in patient-derived iPS and in two types of transfected cells.
Unlike most other disease-associated variants in cytoplasmic IF genes reported so far [2,21], vimentin p.(Leu387Pro) likely affects development. In fact, variants found in KRT8, KRT18, or KRT19, which are expressed before vimentin [22,23], only predispose to a mild phenotype with adult onset [2,21]. Moreover, variants in epidermal keratins and desmin, which are responsible for the integrity of strainexposed cells and tissues [24,25], manifest perinatally or later, upon the onset of mechanical load.
The Leucine residue at position 387 in vimentin is located in an "e" position along the α-helical wheel of the coil2 subdomain, which is important for early steps of vimentin oligomerization [3]. This amino acid is highly conserved among IF proteins and its substitution in KRT17 and desmin causes steatocystoma multiplex with oligodontia (MIM: 184500) and myofibrillar myopathy (MIM: 601419), respectively [12,26] (Fig. 2a). Although consequences for protein stability have not been reported for corresponding KRT17 and desmin variants, we found a lower abundance of the vimentin p.(Leu387Pro) variant likely due to augmented proteolytic processing and proteasomal turnover (Figs. 4 and 5, and Supplementary Figs. S1, S2). The latter could be caused by an improper folding of vimentin p.(Leu387Pro) protein, as it formed aggregate-like structures in IF-free transfected cells (Figs. 4a, c and Supplementary Fig. S2A).
One of the key clinical features of the patient carrying the vimentin p.(Leu387Pro) variant is a progressive peripheral neuropathy, in line with its early and widespread expression in development [27]. We hypothesize that the early developmental expression of vimentin p.(Leu387Pro) in nearly all neuronal precursors in vivo, before its replacement by neurofilaments shortly after the immature neurons become Fig. 6 Overexpression of p.(Leu387Pro) vimentin RNA in zebrafish larvae leads to craniofacial and axonal guidance defects, and disturbs their lipid storage. a Representative ventral images of zebrafish larvae injected with sham (control), wt, or vimentin p.(Leu387Pro) RNAs at 5 days post fertilization, stained with Alcian blue to visualize craniofacial cartilages. b Boxplots of the measured distance between the ceratohyal (CH) and Meckel's (MK) cartilages for each condition tested. Note the increased distance of wt compared with controls (pvalue = 0.0009174) and of vimentin p.(Leu387Pro) compared with controls (p-value = 6.56.10 −12 ) or wt (p-value = 0.0003561). T-test was conducted between pairs of conditions. c Representative lateral images of zebrafish larvae injected with sham (control), wt, or vimentin p. post-mitotic, sets the trigger for multiple pathological alterations. Vimentin is also known to support astrocyte migration and the initiation of neurite outgrowth [28,29]. In our study, we indeed showed that overexpression of human vimentin p.(Leu387Pro) mRNA in zebrafish larvae leads to an altered sprouting and pathfinding of the peripheral axons (Fig. 6c, d). This is in line with recent studies of vimentin KO mice showing a cerebellar defect, impaired motor coordination, hypermyelination, and impaired microglia [27,[30][31][32]. Moreover, deletion of vimentin in glial fibrillary acidic protein KO mice aggravated a range of glial and neuronal phenotypes [2]. The clinical presentation of the patient also suggested a role of human vimentin during adipogenesis. In line with this, we found that vimentin p.(Leu387Pro) reduced the abundance of perilipin and of large lipid droplets in the wellestablished 3T3-L1 model ( Fig. 7 and Supplementary Figs.  S4, S5), without affecting the transcription of key genes governing adipogenesis. It suggests that the decrease in fulllength vimentin, which possibly interacts with perilipin through Arg residues located in the N-terminal vimentin head domain [18], destabilizes perilipin. In support, expression of a dominant-negative acting vimentin mutant in 3T3-L1 preadipocytes caused disruption of lipid droplets and increased turnover of triglycerides [33], consistent with the importance of the cage-like vimentin scaffolds engulfing lipid droplets during adipogenesis [18,19]. Our in vitro data were further confirmed by direct observation of lipid distribution defects in zebrafish (Fig. 6e, f), which is reminiscent of the fat accumulation defects observed in vimentin KO mice [8]. The increase in lipodystrophy upon injection of wt vimentin mRNA is compatible with the notion that transgenic overexpression of wt vimentin caused pathological anomalies in mice [34].
Premature aging remains incompletely understood at the molecular level. A subset of LMNA variants cause Hutchison-Gilford Progeria Syndrome (HGPS). The patient described here shares several features with HGPS, such as alopecia, oligodontia, and lipodystrophy, albeit in a milder form. In addition, early onset of neuropathy, deafness, stroke, and glaucoma also clinically evoked a progeroid syndrome.
Remarkably, vimentin knockin mice in which all Ser residues phosphorylated during mitosis were mutated to Ala (vimentin SA ) also developed a phenotype compatible with an aging phenotype partially overlapping with our patient's symptoms [9][10][11]. The vimentin SA mice showed increased neural progenitor differentiation, subcutaneous fat loss, delayed skin wound-healing, elevated expression of senescence markers, aneuploidy, and chromosomal instability. Ultimately, defects in lens epithelial cells resulted in a cataract, reminiscent of the one described earlier in mice transgenic for vimentin p.(Arg113Cys) [7]. Of note, vimentin SA  Collectively, the data strongly support the view that the complex pathology that results from expression of vimentin p.(Leu387Pro) is much more severe than that of vimentin loss-of-function phenotypes, even if they occur together with another type III IF protein. Apart from a disturbed lipid metabolism, vimentin KO mice and cells do not share obvious phenotypes with any of the reported vimentin mutant mouse models, human diseases, or the vimentin p.(Leu387Pro) variant. We therefore favor the hypothesis that the p.(Leu387Pro) variant causes primarily dominant effects. Therefore, our data suggest that VIM p.(Leu387Pro) probably acts as a dominant-negative or gain-of-function variant that affects the development and/or homeostasis of a wide range of tissues and organs. We showed that this variant enhances vimentin turnover, which possibly triggers deterioration of multiple organs. The complex pathology resulting from a single vimentin variant likely results from its widespread expression and its heteromeric association with type III and type IV IF proteins, features that set it apart from tissuerestricted IF proteins [2]. Moreover, we presently cannot exclude that some of the other rare variants detected in the patient could contribute to the vimentin p.(Leu387Pro) syndrome described here. However, the occurrence of multisystem disorders following single gene variants is not unique but a hallmark of laminopathies and additional disorders, such as RASopathies [35,36]. Given the widespread expression of vimentin, we hypothesize that other vimentin variants may trigger similar and additional syndromes, affecting particularly mesoderm-derived tissues. We anticipate that the identification of other vimentin variants by exome or genome analyses will extend the phenotypic spectrum associated with vimentin defects.
Author contributions BC, J-EB, CG, TMM, and BI co-designed the study and wrote the manuscript. BC designed and performed exome analysis. J-EB designed and performed the experiments involving mammalian cells and sequence alignments. CG and GH designed and performed the zebrafish experiments. SP, DK, XL, TB, and SK performed molecular biology experiments. PB and BI performed clinical evaluation of the patient. LD, AC, and KS performed patient's cells reprogramming.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.