Gut microbiome and serum metabolome alterations in isolated dystonia

neurological movement disorder characterised involuntary muscle contractions. The relationship between the gut microbiota and isolated dystonia Methods We collected faeces and blood samples to study the microbiome and the serum metabolome from a cohort of 57 drug-naïve isolated dystonia patients and 27 age- and environment-matched healthy individuals. We rst sequenced the V4 regions of the 16S rDNA gene from all faeces samples. Further, we performed metagenomic sequencing of gut microbiome and non-targeted metabolomics proling of serum from dystonia patients with signicant dysbiosis. One that anti-clostridial neurological in one sporadic case of which might provide clues to the mechanisms driving dystonia and potentially other neurological conditions with a gut microbial component. However, the relationship between the gut microbiota and isolated dystonia remains poorly explored. Our results demonstrated the alterations of gut microbiome in isolated dystonia. The most pronounced high-level correlation was a general overrepresentation of Clostridiales and underrepresentation of Bacteroidetes in dystonia compared with Healthy controls. Furthermore, genes related to the citrate cycle, vitamin B6 and glycan metabolism were less abundant in dystonia, while genes linked to purine and tryptophan biosynthesis were more abundant. Altered levels of several serum metabolites were found to be associated with microbial changes, such as D-tyrosine and glutamate, indicating differences in neurotransmitter metabolism. Integrative analysis metabolome suggest that 5-HT system dysfunction may be a possible pathway by which the gut mcirobiome participates in the development of dystonia. The gut microbiome changes provide new insight on suggesting new potential therapeutic directions.


Introduction
Dystonia is a complex, highly variable neurological movement disorder characterised by sustained or intermittent muscle contractions 1 . It can be the manifestation of many neurological disorders, either in isolation (isolated dystonia) or in combination with additional symptoms (combined dystonia) 2 . Isolated dystonia includes syndromes in which dystonia is the sole phenotypic manifestation, with the exception of tremor 1 . Until now, the etiology and pathogenesis of isolated dystonia had not been fully elucidated. Most patients with common adult-onset dystonia tend to arise sporadically with no clear etiology, though several associated genes were identi ed as the cause of childhood-and adolescent-onset dystonia 3 .
The gut microbiome contains a complex community of microbes that live within the gastrointestinal tract. Gut microbes communicate with the central nervous system through nervous-, endocrine-and immune-signalling mechanisms. The brain-gut-microbiome axis allows the brain to control gut function, but also provides an opportunity for the gut microbiome to in uence the brain 4 . Emerging evidence has implicated the gut microbiome as an important player in neurodevelopment, as well as in brain diseases such as PD 5 , Alzheimer's disease (AD) 6 and multiple sclerosis 7 . Interestingly, it was reported that anticlostridial agents were capable of reversing both gastrointestinal and neurological symptoms in one sporadic case of myoclonic-dystonia 8 , which provides clues to the mechanisms driving dystonia and potentially other neurological conditions with a gut microbial component. However, the relationship between the gut microbiota and isolated dystonia remains poorly explored.
In the present study, we rst investigated gut microbial characteristics in drug-naïve patients with isolated dystonia using 16S rDNA amplicon sequencing. We also evaluated whether the parameters correlated with clinically relevant features of dystonia, such as age at disease onset, disease duration, direction of rotation, and dystonia severity. Furthermore, we performed shotgun metagenomic sequencing and nontargeted metabolomics pro ling of serum from patients with signi cant gut dysbiosis in 16S rDNA analysis to investigate the microbial functional alteration and the association of the altered gut microbiome with serum metabolites in the host.

Participants
Fifty-seven patients with isolated dystonia (15 male, 42 female) and 27 age-and sex-matched healthy participants (nine male, 18 female) were enrolled. Among the patients, 44 had focal dystonia, 11 had segmental dystonia, and two had general dystonia. All patients, from 12 provinces across the country, admitted to the Movement Disorders and Botulinum Toxin-A Treatment center underwent a standardised neurological examination by a specialist in movement disorders. All dystonia patients were newly diagnosed and did not receive oral medicine or botulinum toxin injection. None of the participants had a family history of dystonia. All patients were negative for the genetic testing of DYT1 (TOR1A), DYT4 TUBB4 ,DYT6 (THAP1) ,DYT23 (CIZ1),DYT24 ANO3 ,DYT25 GNAL and COL6A3. Subjects who have no neurological or psychiatric disease were enrolled as Healthy controls (HCs). Subjects were excluded if they had gastrointestinal diseases, malignant tumours, autoimmune disorders, infectious diseases, a history of gastrointestinal surgery, or were administered antibiotics or laxatives or immunosuppressive agents in the past three months. No subjects were or are on any antianxiety or antidepressant medication. No relevant constipation was present in all subjects; dietary and smoking habits did not differ between groups. No detailed dietary plan was requested prior to the feces collection and samples were collected as rst bowel movement of the day. Demographic information (gender, age, age of disease onset, disease duration, height and weight,) and severity evaluation scores, including Global Dystonia Rating Scale (GDS) and Uni ed Dystonia Rating Scale (UDRS) were collected from all patients. Evaluation for anxiety and depression including Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD), were assessed for all subjects. Written informed consent was obtained from all individuals. The study was approved by the ethics committees of Beijing Tiantan Hospital.
Analysis of Fecal Microbiota by 16s rDNA Ampli con Sequencing Stool samples freshly collected from each participant were immediately transported to the laboratory and frozen at -80°C. Bacterial DNA was extracted and the V4 region of the 16S rRNA gene was ampli ed by PCR using barcoded universal fusion primers 515 and 806. PCR products were sequenced and for taxonomic assignment. The number of reads in each sample was standardised to 37,666 for further analysis. Dirichlet multinomial mixtures (DMM) modeling examines taxa frequencies and determines how many microbial community states (MCS) exist within a dataset using the DirichletMultinomial 9 .
Please refer to the Supplemental Methods section for further details on the laboratory protocols.

Functional Analysis of Microbiome by Shotgun Metagenomic Sequencing
Total DNA from the 26 fecal samples (13 cases and 13 controls) were subject to shotgun metagenomic sequencing. Sheared DNA (350 bp) was used for Illumina library construction and index codes were added to attribute sequences to each sample. Functional annotation was carried out by BLASTP searches against the Kyoto Encyclopedia of Genes and Genomes (KEGG database (e-value ≤1e-5 and high-scoring segment pair scoring >60). For each functional feature (KO in the KEGG database), we estimated its abundance by accumulating the relative abundance of all genes belonging to that feature.
Please refer to the Supplemental Methods for further details.
Untargeted metabolomics analysis of serum samples All serum samples were thawed on ice and a quality control (QC) sample, made by blending equal volumes (20 µl) of each serum sample, was used to estimate a mean pro le representing all the analytes encountered during analysis. Metabolite extraction was performed by mixing 100 µl thawed serum and 400 µl ice-cold 80% methanol and 0·1% formic acid, vortexing for 30 s, and keeping at −20 °C for one hour. After centrifuged at 14,000 g and 4 °C for 20 min some of supernatant was diluted to nal concentration containing 53% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 14,000 g, 4°C for 20 min. Finally, the supernatants were subjected to metabolomics pro ling by high performance liquid chromatograph (HPLC)-MS in both positive and negative ionization modes. The raw data les were processed using the Compound Discoverer 3·1 (CD3·1, ThermoFisher). Peak intensities were normalized to the total spectral intensity. All data were mean-centred and unit variance (UV)-scaled before multivariate statisti cal analysis, and partial least squares discriminant analysis (PLS-DA) was performed. Signi cantly different metabolites were determined by a combination of VIP value >1 from the PLS-DA model and p-values <0·05 from two-tailed Student's t tests. Please refer to the Supplemental Methods for further details.

Spearman's multi-omics correlation analysis
Spearman's correlation coe cients were computed for relationships between the relative abundance of identi ed dystonia-associated species and normalised individual metabolomic features. A scaled heatmap was constructed for the correlation matrix, including cladogram classi cation of variables, using the default clustering method. Visual presentation of multiple omics correlations was performed using the Corrplot package in R (v3·5 ·3). Signi cant microbiota-metabolite correlations were determined based on an r value <-0·3 or >0·3 and a false discovery rate (FDR)-adjusted p-value <0·05.

Demographics and clinical data
Fifty-seven isolated dystonia patients and 27 HCs were recruited and an integrative analysis of the gut microbiome and serum metabolome was performed. Clinical details of the study cohort are shown in Table 1.

Gut microbiota revealed by 16s rDNA amplicon sequencing
Although gut microbial α-diversity was not signi cantly different as calculated by Shannon and Simpson indices ( Figure 1A), NMDS analysis demonstrated a signi cant difference between isolated dystonia and HCs ( Figure 1B, PERMANOVA, R 2 = 0·157, p = 0·002). We also observed higher β-diversity in the microbiota of dystonia patients based on ANOSIM analysis, indicating a more heterogeneous community structure among dystonia patients compared with HCs ( Figure 1C). LEfSe analysis showed that Clostridiales and Bacteroidetes were the key taxa distinguishing isolated dystonia from HCs. Dystonia patients were signi cantly enriched with Blautia, Bi dobacterium and unidenti ed Lachnospiraceae, while depleted with Bacteroidetes and unidenti ed Clostridiales as compared with HCs ( Figure 1D). The relative abundance of the most abundant taxa (≥1%) at different taxonomic levels was further compared (Supplementary Table S2 and Figure S2). At the species level, Eubacterium hallii, Blautia obeum and Dorea longicatena were enriched in dystonia subjects, while Bacteroides vulgatus, Bacteroides_plebeius and Fusobacterium varium were enriched in HCs ( Figure 1E).

Strati cation of bacterial community in dystonia patients
Given more heterogeneity, we applied DMM to our cohort to investigate possible gut microbial subgroups in dystonia, and identi ed two signi cantly distinct MCSs (n = 8 and n = 49; R 2 = 0·138; p <0·001) using a Laplace approximation (Figure 2A). Speci c co-occurring bacterial families were characteristically enriched in these groups; the MCS1 (n = 8) microbiota was characteristically enriched by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae. The second larger group, which we designated MCS2A (n = 20) and MCS2B (n = 29), respectively exhibited a sharp decreased abundance of Bacteroidaceae, especially in MCS2B ( Figure 2B). NMDS analysis con rmed a strong and signi cant relationship between MCS class and bacterial β-diversity (PERMANOVA, R 2 = 0·29, p <0·001), corroborating the existence of compositionally distinct microbial states ( Figure 2C). These distinct microbial states exhibited signi cant differences in diversity (Shannon, Kruskal-Wallis tests, p = 0·002; Figure 2D), with MCS2B exhibiting the lowest index. We also investigated whether speci c factors (illness duration, age at onset, BMI and severity evaluation scores) that was differentially associated with microbial community states. No signi cant correlation was detected.
Functional analysis of the isolated dystonia by metagenomic sequencing As shown above, 29 of 57 (51%) the isolated dystonia underwent a gut microbial dysbiosis. To further explore microbial functional feature of dystonia, we performed metagenomic sequencing of a subgroup consisting of 13 patients from MCS2B and 13 gender-and age-matched HCs. An average of 10·7 ± 2·1 Gb clean reads were generated per sample except for one HC sample removed due to low depth of sequencing (Supplementary Table S3).
Consistent with the 16S rDNA analysis, B. obeum, D. longicatena and E. hallii were signi cantly increased in dystonia, while B. vulgatus and B. plebeius were decreased ( Figure 1F and Supplementary Figure S3). We estimated the abundance of metabolic pathways using metagenomic reads mapped to functional orthologs from the KEGG databases to explore differences in the metabolic potential of gut microbiomes between dystonia and HCs. Dystonia communities were functionally different from healthy communities, and less closely clustered together among individuals, suggesting that inter-individual functional variation was higher in dystonia than in HCs ( Figure 3A).
Furthermore, we traced the contributing genes and determined their likely taxonomic origin to determine which bacteria are involved in these pathways ( Figure 3B, C).

Associations between gut microbial species and serum metabolites
To investigate the extent to which the altered microbiome in the dystonia patients was associated with serum metabolites in the host, we performed non-targeted metabolomics pro ling of serum from the 13 dystonia patients and the 12 HCs in metagenomic analysis, and yielded 1543 features (Supplementary   Table S5). A supervised PLS-DA using two components (R 2 X = 0.525, R 2 Ycum = 0·99, Qcum 2 = 0·975, p <0·001) was performed, resulting in some separation tendencies between dystonia cases and HCs ( Figure 4A). Based on the PLS-DA models of metabolite pro ling data, 242 metabolites were found to be signi cantly different in abundance between dystonia cases and HCs, with 87 metabolites having a higher concentration and 169 metabolites having a lower concentration in dystonia compared with HCs (p <0·05, VIP >1 and FC >2 or <0·5, Figure 4B, Supplementary Table S5).
Subsequently, spearman's correlation coe cients were computed for relationships between the relative abundance of the identi ed dystonia-associated species and the different 242 normalised individual metabolomic features. Metabolites were grouped into two clusters depending on the correlations, and correlation coe cients with signi cant p-values (<0·01) are shown in Figure 4C. Clostridiales species enriched in dystonia patients correlated positively with the rst metabolite cluster, including L-glutamic acid, phenylalanylphenylalanine and taurine. While Bacteroides species with decreased abundance in dystonia patients correlated positively with the second cluster, including D-tyrosine, D-(-)-aspartic acid and N, N-dimethylacetamide (DMAc).

Discussion
An integrative analysis of the gut microbiome and serum metabolome was employed to explore the relationship between the gut microbiome and isolated dystonia. We found that more than half of the isolated patients (29/57, 51%) got a gut microbial dysbiosis in 16S rRNA analysis, showing a general overrepresentation of Clostridiales and underrepresentation of Bacteroidetes in dystonia compared with HCs. Further analysis thus for this subgroup using shotgun metagenomic sequencing and non-targeted metabolomics pro ling of serum was performed to gain an insight into the links between gut dysbiosis and dystonia, and indicated alterations in several genes and serum metabolites. Genes related to the citrate cycle, vitamin B6 and glycan metabolism were less abundant in dystonia, while genes linked to purine and tryptophan biosynthesis were more abundant. Altered levels of several serum metabolites associated with microbial changes, such as D-tyrosine and glutamate, were found, indicating differences in neurotransmitter metabolism.
Evidence is accumulating for a potential role of the microbiota in the pathogenesis of neurological disorders [5][6][7][8] . One myoclonic dystonia case was reported to present a 90% improvement in tremor and abnormal posture after taking anti-clostridial antibiotics, indicating a possible correlation between the pathogenesis of dystonia and the gut microbiome, underlying the potential role of Clostridial species 8 . In this study, intestinal dysbiosis was characterised by signi cantly increased Clostridiales and decreased Bacteroidetes in dystonia patients, which might explain the neurological improvement in the abovementioned case after taking anti-clostridial agents, suggesting that Clostridiales may have a potential pathogenic role in the pathogenesis of dystonia.
Blautia and unidenti ed Lachnospiraceae, belonging to the Lachnospiraceae family, contributed principally to the increase in Clostridiales in dystonia patients. The Lachnospiraceae family is known to participate in the breakdown of carbohydrates into short-chain fatty acids (SCFAs) 10 , which are believed to play a key role in microbiota-gut-brain crosstalk 11,12 . SCFAs, with acetate, propionate and butyrate being the most abundant components, may in uence the brain by crossing the blood-brain barrier (BBB) 13 , modulating neurotransmission, in uencing levels of neurotrophic factors, and promoting serotonin (5hydroxytryptamine, 5 HT) biosynthesis 12 . In a cell model, acetate and butyrate promoted serotonin biosynthesis by inducing the expression of tryptophan 5-hydroxylase 1 (the rate-limiting enzyme for 5 HT synthesis) transcription 14 . Another study found that butyrate and propionate play a critical role in promoting host 5 HT biosynthesis, and regulating levels of 5 HT in both colon and serum 15 . Furthermore, they also induce tyrosine hydroxylase gene transcription 16 , which catalyses the rate limiting step in the biosynthesis of dopamine, noradrenaline and adrenaline 17 . A decrease in SCFA production can cause intestinal barrier dysfunction, but whether an increased dose of SCFAs could directly lead to adverse effects on locomotor behaviour has not been elucidated and requires animal studies in the future. In our current study, the increased abundance of Clostridiales may have led to elevated SCFA levels, and a consequent increase in 5 HT.
The abundance of Bacteroides, Parabacteroides and Alistipes was signi cantly decreased in isolated dystonia compared with HCs. A decrease or low level of Bacteroides is believed to be associated with metabolic diseases such as obesity 18 and diabetes 19 , and psychiatric disorders such as depression 20 . Given that no subjects were associated with obesity or diabetes, and there were no differences in bodymass index (BMI) between the two groups, an association between Bacteroides and metabolic factors can be ruled out in our study. Growing evidence suggests that non-motor symptoms are essential components of dystonia, with psychiatric co-morbidity being most prevalent 21 , and a link between dystonia and depression has been con rmed 22 . In our cohort, patients were newly diagnosed and there was no statistical difference of HAMD between the two groups, it is unlikely that depression was a confounding factor in our study. Alistipes species are indole-positive and may thus in uence tryptophan availability 23 . As tryptophan is also the precursor of serotonin, decreased abundance of Alistipes might therefore disrupt the balance of the intestinal serotonergic system. Until now, the pathophysiological basis of dystonia remains poorly understood. Evidence indicates that the pathophysiological basis of dystonia involves dysfunction of the basal ganglia and cortico-striatalthalamo-cortical circuits 24 , hence neurotransmitter systems are likely involved. In addition to dopamine, a signi cant neurotransmitter in motor control, serotonin and serotonin-dopamine interactions have received attention 25 . Disturbance of the serotonergic system is known to be involved in extrapyramidal movement and psychiatric disorders. A [ 11 c]DASB positron emission tomography study of cervical dystonia patients revealed a signi cant correlation between serotonin transporter (SERT) binding in the dorsal raphe nucleus and motor and non-motor symptoms 26 . Higher non-displaceable binding potential (BPND) was statistically correlated with motor severity, pain and sleep disturbance, with motor symptom severity being the most important predictor of SERT binding 26 . Since Lachnospiraceae and Alistipes are involved in the production and regulation of 5-HT biosynthesis, the increase in the Lachnospiraceae family and the decrease in Alistipes species observed in our study may lead to imbalance of peripheral levels of 5-HT, which might, in turn, modulate brain function by affecting the immune system 27 or signalling to the brain via 5 HT receptors on vagal afferent bres 28 .
Functional analyses revealed that phenylalanine, tyrosine and tryptophan biosynthesis tend to be more active in dystonia, as reported previously for PD 29 . These aromatic amino acids are important as precursors of 5-HT and catecholamines. Correspondingly, metabolome analysis found that the serum concentration of D-tyrosine, the precursor for serotonin, was signi cantly elevated in dystonia cases. Composition analysis, functional analysis of the gut microbiome, serum metabolome analysis, and imaging all point to a potential role of serotonergic system dysfunction in the pathogenesis of isolated dystonia.
Additionally, the sulphur metabolism pathway appeared to be more active in the microbiome of dystonia patients. In the brain, sulphur metabolism is intimately involved in metabolic cooperation, and provides cells with four major reagents critical for methylation reactions (S-adenosylmethionine), antioxidant capacity (glutathione), signalling (H2S), and cell volume regulation (taurine). An important intermediate in sulphur metabolism is homocysteine (HCY), a neuronal excitotoxic amino acid, and hyperhomocysteinaemia is considered an independent vascular risk factor. Increased HCY has also been observed in movement disorders such as PD 30 , Huntington's disease (HD) 31 and dystonia 32,33 . In patients with isolated dystonia, hyperhomocysteinaemia was observed independently from immune activation 33 . Our study indicates that hyperhomocysteinaemia might, to some extent, be related to an increase in sulphur metabolism caused by disturbance of the gut microbiome.
It should be noted that the diversity of the gut microbiome is in uenced by several factors, including health status, age, diet, and antibiotics 34 . One highlight of our study is the enrollment of drug-naïve patients. Evidence is accumulating on effects that drugs can have on the composition and function of the gut microbiome 35 . A case-control study on cause-effect relationships could be performed in the future by analysing drug-naïve dystonia patients.
Despite being, to our knowledge, the rst study to explore the alterations of gut microbiome in dystonia, the present study has some limitations that should be acknowledged. First, this study was performed at one single centre (Tiantan Hospital). Selection bias and unknown confounding factors cannot be excluded. However, our hospital has a big center for neurological diseases and appeals to the patients across the country. Patients in our corhort came from 12 provinces and are to some extent, representative. Second, our study is an exploratory study and the number of our patients is relatively small. More studies including larger cohort from different regions and ethics will be needed to con rm the role of gut microbiome in isolated dystonia.

Conclusions
This is the rst study to reveal a signi cant gut microbial dysbiosis in isolated dystonia patients, which may be associated with altered serum metabolites. Integrative analysis of microbiome and metabolome suggest that 5-HT system dysfunction may be a possible pathway by which the gut mcirobiome participates in the development of dystonia. The ndings provide new insight on the pathogenesis of dystonia, indicating the potential involvement of the enteric neurotransmitter system in dystonic patients.

Declarations
All participants provided informed consent and the study was approved by the ethics board of Beijing Tiantan Hospital.

Consent for publication
The paper has not been published previously and is not under consideration for publication elsewhere. All co-authors have read and approved the submission.   shown; *p <0·05; **p <0·01; ***p <0·001).