Metabolic and Muscle-Derived Serum Biomarkers Define CHCHD10-Linked Late-Onset Spinal Muscular Atrophy

medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Metabolic and muscle-derived serum biomarkers define

CHCHD10-linked late-onset spinal muscular atrophy

Julius Järvilehto, MB1, Sandra Harjuhaahto, MSc1, Edouard Palu, MD2, Mari Auranen, MD, PhD2,

Jouni Kvist, PhD1, Henrik Zetterberg, MD, PhD3,4,5,6, Johanna Koskivuori, MSc7, Marko Lehtonen,

PhD7, Anna Maija Saukkonen, MD8, Manu Jokela, MD, PhD9,10, Emil Ylikallio, MD, PhD1,2*,

Henna Tyynismaa, PhD1,11,12*

1Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland 2Clinical Neurosciences, Neurology, Helsinki University Hospital, Helsinki, Finland 3Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden 4Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden 5Department of Neurodegenerative Disease, UCL Institute of Neurology, London, United Kingdom 6UK Dementia Research Institute at UCL, London, United Kingdom 7School of Pharmacy, University of Eastern Finland, Kuopio, Finland 8Department of Neurology, Central Hospital of Northern Karelia, Joensuu, Finland 9Division of Clinical Neurosciences, Turku University Hospital and University of Turku, Turku, Finland 10Department of Neurology, Neuromuscular Research Center, Tampere University Hospital and Tampere University, Tampere, Finland 11Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland 12Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland

*Corresponding Author: Henna Tyynismaa, Biomedicum Helsinki, Haartmaninkatu 8, 00014 University of Helsinki,

Finland. Email: [email protected]

Word count: 2319

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 2

ABSTRACT

Objective To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular

atrophy Jokela type (SMAJ) for disease monitoring and for understanding of pathogenic

mechanisms.

Methods We collected serum samples from a cohort of 49 SMAJ patients, all carriers of the

heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age- and sex-matched

serum samples obtained from Helsinki Biobank. Neurofilament light (NfL) and glial fibrillary

acidic protein (GFAP) were measured with Single molecule array (Simoa), and fibroblast growth

factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) with enzyme-linked

immunosorbent assay. For nontargeted serum metabolite profiling, samples were analyzed with

liquid chromatography–high resolution mass spectrometry. Disease severity was evaluated

retrospectively by calculating a symptom-based score.

Results Axon degeneration marker NfL was unexpectedly not altered in the serum of SMAJ

patients, whereas astrocytic activation marker GFAP was slightly decreased. Creatine kinase was

elevated in most patients, in particular males. We identified six metabolites that were significantly

altered in SMAJ patients’ serum compared to controls: increased creatine and pyruvate, and

decreased creatinine, taurine, N-acetyl-carnosine and succinate. Creatine correlated with disease

severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial

dysfunction, however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 were not

changed.

Conclusions Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role

for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 3

mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected in the serum

of SMAJ patients.

INTRODUCTION

Spinal muscular atrophy Jokela type (SMAJ, MIM#615048) is a progressive adult-onset lower

motor neuron disease (1). Muscle cramps are a common initial manifestation in SMAJ, typically

appearing after age 30-40 (2). Increasing lower limb predominant muscle weakness, hyporeflexia

and difficulties in walking follow as the disease progresses. Mild distal sensory defects occur in

about half of the patients later in the disease course (2). Life expectancy is within normal range (2).

SMAJ patients have normal nerve conduction velocities but EMG shows widespread neurogenic

alterations (1,3). Muscle biopsies also show characteristic neurogenic findings, including fiber type

grouping and group atrophy, whereas only some patients have mild signs of mitochondrial

myopathy such as a few COX-negative muscle fibers (3). Elevated creatine kinase (CK) has been

reported in SMAJ (1,2). Some patients have received a clinical diagnosis of amyotrophic lateral

sclerosis (ALS) or Charcot-Marie-Tooth (CMT) neuropathy (4,5,6), but a gene test of a single

heterozygous variant, c.197G>T p.G66V, in coiled-coil-helix-coiled-coil-helix domain containing

10 (CHCHD10) sets the diagnosis of SMAJ (2). Other more severe CHCHD10 variants can cause

ALS and frontotemporal dementia (FTD) (5) or mitochondrial myopathy (7).

CHCHD10 is a mitochondrial of unknown exact function, and although it is located in the

intermembrane space, both its absence and dominant pathogenic mutations affect mitochondrial

respiration (8,9,10,11,12). Recent genetically modified Chchd10 mice have displayed

neuromuscular junction (NMJ) and motor neuron degeneration (9,14). Mice with ALS variant

p.S59L developed mitochondrial dysfunction in muscle before NMJ degeneration (9). Toxic gain-

of-function effect of mutant Chchd10 aggregates were suggested to induce mitochondrial integrated medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 4

stress response (ISR) (8), which was also reported in fibroblasts of ALS patients with the same

mutation (14). Besides fibroblasts, studies using samples from patients with CHCHD10 mutations

have so far been rare.

Currently, no serum-based tests for diagnostic use or for monitoring disease progression are

available for SMAJ. Here we present an investigation of serum biomarkers in SMAJ, an adult-onset

non-5q spinal muscular atrophy of mitochondrial origin. We evaluate neurofilament light (NfL) and

glial fibrillary acidic protein (GFAP) as neuronal injury and astrocytic activation markers, and

fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) as

mitochondrial stress markers, and perform a nontargeted metabolite screen for unbiased biomarker

discovery.

PATIENTS AND METHODS

This study was approved by the medical ethics committee of Helsinki University Hospital (project

code U1024NEURO).

In total 49 individuals who were symptomatic, genetically verified carriers of CHCHD10 p.G66V

variant were available and recruited for the study. We used a questionnaire and review of medical

charts to collect clinical data. Fasting serum samples were collected and stored at -80°C until the

analysis. As controls we used anonymized samples from Helsinki Biobank, which were requested to

match the sex and age distributions of the study population, but with exclusion of neurological

disease diagnosis (ICD-10 code starting with G). The number of samples used in each analyses

varied depending on availability at the time of the measurement (Table 1).

Disease severity was determined by using a symptom-based score, which we modified from the

motor and sensory defect part of a more comprehensive CMT neuropathy score (15) (Table 2).

Sufficient patient records were available for assessing the score for 43 patients. Certain patients’ medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 5

symptoms were not strong enough to merit positive scores on this scale (e.g. only cramps), and thus

received a symptom score of 0. As SMAJ is a progressive disease, the symptom score correlated

with age (r=0.56, p<0.001).

Serum measurements

Serum NfL and GFAP concentrations were measured with Single molecule array (Simoa)

technology on an HD-X Analyzer using commercially available NF-light Advantage and GFAP

Discovery kits (Quanterix, Billerica, MA). Serum FGF-21 and GDF-15 levels were measured with

R&D Systems Quantikine ELISA kits (DF2100 and DGD150, respectively; R&D Systems,

Minneapolis, MN). Creatinine and CK were measured in accredited laboratory HUSLAB using a

photometric enzymatic method.

Nontargeted metabolite profiling

Nontargeted metabolite profiling on serum samples was performed at the LC-MS metabolomics

center (University of Eastern Finland, Kuopio) using ultra-high performance liquid chromatography

coupled to high-resolution mass spectrometry. Before the analysis, samples were randomized and

deproteinized with acetonitrile. A small volume of each sample was pooled for quality control (QC)

sample and were analyzed at the beginning and after every 12th sample of the instrument worklist.

To meet the wide diversity of sample components, all samples were analyzed using two different

chromatographic techniques; i.e., reversed phase (RP) and hydrophilic interaction chromatography

(HILIC). In addition, data were acquired in both mass spectrometric electrospray ionization

polarities; i.e., ESI+ and ESI- (16).

Data processing and statistical analysis medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 6

Statistical analysis was performed using GraphPad Prism 8.42 (GraphPad; La Jolla, CA). Patient

and control serum NfL, GFAP, FGF-21 and GDF-15 concentrations, as well as metabolite

abundances were compared using Mann-Whitney U-test analysis. Correlations were assessed using

Spearman correlation coefficients. P-value < 0.05 was considered significant, except in metabolome

profiling where adjusted p-value < 0.05 was considered significant.

Data availability statement

Anonymized data will be shared by request from any qualified investigator.

RESULTS

Serum NfL is not elevated in SMAJ

The axonal injury protein NfL has shown to be a potent serum biomarker in a number of

neurological conditions (17), including ALS, CMT and SMA (18,19,20). However, in our

measurements, the level of serum NfL was not significantly different in SMAJ patient samples

(median 14.9 pg/ml) compared to controls (13.6 pg/ml) (Fig 1A). GFAP was also not elevated in

SMAJ, in contrast decreased level was observed in SMAJ patients’ serum (median 118 pg/ml) as

compared to controls (177 pg/ml) (p = 0.006, Fig 1B). GFAP, a marker of astrogliosis (21), is

centrally expressed and an autopsied individual with SMAJ did not have spinal cord abnormalities

(4). The levels of NfL and GFAP increased as a function of age in both patient and control groups,

but neither marker correlated with the symptom score (Table 3).

Serum biomarkers of muscle-origin define SMAJ

Previous studies had indicated that serum CK may be elevated in SMAJ (1,2). We measured CK in

serum samples of patients included in this study, and found that indeed CK was markedly higher in

SMAJ patients as compared to the reference values (Fig 2A). Of the female SMAJ patients, 60% medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 7

had CK values above reference (mean 318 U/l, reference range 35-210 U/l in females over 18),

whereas nearly all (95%) male SMAJ patients had elevated CK values (mean 711 U/l, reference

range 40-280 U/l in males over 50). CK values did not, however, correlate with the symptom score

(r = 0.16, p = 0.35) (Fig 2D).

Next we performed a nontargeted metabolite screen with patient and control serum samples to

identify metabolites that could be used as markers of SMAJ pathogenesis. We focused on six

metabolites that were significantly altered in SMAJ samples in comparison to controls: creatine

(Fold change 2.09, adjusted p-value 6.13E-07), creatinine (FC -1.30, adj.p 1.20E-07), taurine (FC -

1.36, adj.p. 1.08E-06), N-acetyl-L-carnosine (FC -1.77, adj.p 8.20E-05), pyruvate (FC 1.60, adj.p

0.00337), succinate (FC -1.37, adj.p 0.0127). Interestingly, many of the identified metabolites

suggested muscle origin of pathogenesis.

Creatinine, the secreted breakdown product of creatine, has been found to correlate with disease

severity in SMA and spinal and bulbar muscular atrophy (SBMA) (22,23). Our metabolite profiling

indicated that creatinine was decreased also in SMAJ (Fig 2B), whereas creatine was increased (Fig

2C). We validated the metabolite profiling result by measuring creatinine levels in an accredited

laboratory and found that the two measurements correlated well (r = 0.74, p < 0.001). Similarly to

CK, creatinine levels did not correlate with the symptom score (r = -0.11, p = 0.52) (Fig 2E).

However, creatine levels correlated with the symptom score (r = 0.43, p = 0.009) (Fig 2F), and also

with CK (r = 0.34, p = 0.03) (Fig 2G). We also detected declined levels of N-acetyl-L-carnosine

(Fig 2H) and taurine in SMAJ patients’ serum (Fig 2I). These changes are likely to reflect

alterations of muscle origin in SMAJ, and indicate muscle damage as well as decreased muscle

mass.

Indications of altered mitochondrial metabolism in SMAJ

Metabolite profiling showed elevated levels of pyruvate (Fig 3A), whereas succinate was decreased

in SMAJ in comparison to controls (Fig 3B), indicating alterations in mitochondrial metabolism.

Interestingly, however, lactate was not changed between the two groups (p = 0.34) (Fig 3C). These

findings suggested that the mitochondrial origin of SMAJ contributed to the serum metabolite

profile in patients. Thus, we tested if the previously characterized serum biomarkers of

mitochondrial myopathy, FGF-21 or GDF-15 (24,25), were increased in SMAJ. However, we did

not find the level of either marker to differ between patient and control groups (FGF-21 SMAJ

median 162 pg/ml, controls 90 pg/m, [p = 0.15]; GDF-15 SMAJ median 653 pg/ml, controls 574

pg/ml, [p = 0.29]) (Fig 3D, E). None of the mitochondrial markers correlated with symptom score.

DISCUSSION

Serum biomarkers are required as measures of treatment benefit in clinical trials of rare

neurological diseases, which are challenged by small numbers of patients, and genetic and

phenotypic heterogeneity. In addition, serum markers reveal features of pathogenesis, tissue

involvement, and progression. Here we aimed at serum biomarker characterization in a unique

homogeneous cohort of SMAJ patients with the identical pathogenic CHCHD10 variant G66V, a

founder mutation in Finland (26), surpassing ALS in prevalence in certain regions of the country

(27). Our study resulted in the following main findings: 1) Unlike in several other motor neuron

diseases, the neurodegeneration serum biomarkers NfL and GFAP are not elevated in SMAJ

patients. 2) Serum metabolites of muscle-origin suggest that skeletal muscle pathogenesis is a major

contributor to SMAJ. Of these, serum creatine correlated with symptom severity in SMAJ. 3)

Abnormal CHCHD10 alters mitochondrial metabolite levels in serum (Fig 4).

Clear evidence of neurogenic muscle injury in SMAJ has been shown in EMG measurements as

well as in muscle biopsies (1). Moreover, fasciculations indicate a neurogenic defect. Nevertheless, medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 9

unlike in many neurodegenerative diseases, symptomatic SMAJ patients did not have an elevation

of serum NfL despite of having a chronic injury process of spinal motor neurons (1). This contrasts

with the high levels of neurofilaments, both NfL and pNFH (phosphorylated neurofilament heavy),

in serum and CSF of patients with ALS (28,29), and in infants and young children with recently

diagnosed 5q SMA, being also responsive to Nusinersen treatment (30,31). On the other hand,

adults and adolescents with SMA disease did not have consistently increased NfL in CSF

(32,33,34). As such, active ongoing motor neuron damage and broad anatomical involvement may

be needed to produce a significant leakage of neurofilaments in a peripheral nervous system

disease. The fact that SMAJ is slowly progressive and largely restricted to lower limbs may thus

mean that the number of actively degenerating spinal motor neurons is not sufficient to elevate

serum NfL. However, this hypothesis is in contrast with the elevation of serum NfL in patients with

inherited peripheral neuropathies, including CMT1, CMT2 and hereditary sensory autonomic

neuropathy (35).

An alternative explanation for the lack of NfL elevation among SMAJ patients is that the

pathophysiology of SMAJ differs from neuropathies. An interesting parallel is provided by SBMA,

in which the clinical picture includes slowly progressive weakness and wasting of bulbar and limb

muscles (36). Similarly to SMAJ, in SBMA serum NfL was normal, but CK and creatine were

increased whereas creatinine was decreased (22,37). These findings suggested that

neurodegeneration in SBMA is a muscle-driven process (38). Thus it is possible that the primary

cause of neuronal injury in SMAJ is not in the spinal motor neuron itself but driven non-cell

autonomously by abnormalities in neuromuscular junction or muscle. Indeed, myopathic changes

on muscle biopsy are more prominent in SMAJ and SBMA than in ALS (3). In addition to

creatinine, decreased levels of taurine and N-acetyl-L-carnosine may also be indicators of decreased

muscle mass in SMAJ patients. CK did not correlate to the clinical severity in SBMA or ALS (22),

but creatinine predicted the severity in both SBMA and SMA, particularly the types 1 and 2 of the medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 10

latter (22,23). Creatinine also correlated to therapeutic outcome of Nusinersen in SMA (39).

However, only creatine correlated with the disease severity in SMAJ.

The absence of GFAP increase in SMAJ serum suggests that astrocytic injury or activation is not an

important part of the disease process; the significantly lower levels of GFAP in SMAJ patients

compared with controls may suggest impaired astrocytic activation but more studies are needed

before conclusions regarding this can be drawn.

Altered pyruvate has been previously linked to primary mitochondrial myopathies (40,41).

Increased pyruvate together with decreased succinate suggest a block in the TCA cycle, and thus

glycolysis may be upregulated. The altered metabolites may thus be directly involved in

compensatory pathways for balancing an energy deficiency in SMAJ. Interestingly, a recent study

of ALS-linked CHCHD10 variant reported similar findings for pyruvate and succinate in patient

fibroblasts (14). The same study, as well as Chchd10 mouse models have indicated upregulation of

ISR as part of pathogenesis, which leads to increased expression of muscle-secreted FGF-21 and

GDF-15 (42). However, here we have shown that in the serum of patients with CHCHD10-linked

disease, these growth factors are not increased. It is, however, possible that since p.G66V produces

a milder phenotype than the other CHCHD10 mutations, it does not initiate the ISR.

As a limitation of this study, although a large proportion of all SMAJ patients were included in the

study, number of analyzed samples was still relatively small for a biomarker study. In addition, the

symptom scoring system that we used was quite coarse, consisting of only three measurable

sections of retrospectively collected data.

In conclusion, our study has established new insights into the pathophysiology of SMAJ in human

patients and identified disease-associated metabolites, which may be beneficial in monitoring of

treatment trials. Normal serum NfL may be clinically useful to differentiate SMAJ from early-stage medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 11

ALS. Our findings on skeletal muscle may be relevant in the future studies of SMAJ and suggest

that therapies designed for primary mitochondrial diseases may be worth testing also in SMAJ.

TABLES Table 1. Demographics of the study groups in different analyses.

Analysis Patients Controls n (M/F) Age median n (M/F) Age median (range) (range)

S-NFL 49 (20/29) 65 (39-86) 45 (17/28) 64 (40-86) S-GFAP

S-FGF-21 49 (20/29) 65 (39-86) 24 (10/14) 63 (47-85) S-GDF-15

Metabolite profiling 43 (19/24) 65 (39-86) 48 (18/30) 65 (34-86)

S-CK 49 (20/29) 65 (39-86) - - S-Creatinine (reference (reference values) values)

Symptom score 43 (16/27) 65 (39-85) - -

Table 2. Symptom score for SMAJ patients, modified from the motor and sensory defect part of a

more comprehensive CMT-score (10).

Parameter 0 1 2 3 4

Sensory None Symptoms below Symptoms up to Symptoms up to Symptoms above the symptoms or at ankle bones the distal half of the proximal half knee (above the top the calf of the calf, of the patella) including knee

Motor None Trips, catches Ankle support or Walking aids Wheelchair symptoms toes, slaps foot, stabilization (cane, walker) (legs) shoe inserts (AFOs), foot surgery

Motor None Mild difficulty Severe difficulty Unable to cut Proximal weakness, symptoms with buttons or unable to do most foods (affect movements (arms) buttons involving the elbow and above)

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity.

Table 3. Correlations between different markers doi: https://doi.org/10.1101/2021.04.07.21254960

Symptom N-acetylL- Age Sex Creatine Creatinine CK Taurine Pyruvate Succinate FGF-21 GDF-15 GFAP NfL score carnosine r = 0.40 r = 0.43 r = -0.16 r = 0.34 r = 0.27 r = - 0.25 r = 0.038 r = 0.17 r= 0.32 r = 0.10 r = 0.41 r = 0.27 Creatine p = 0.056 p = 0.009 p = 0.009 p = 0.31 p = 0.03 p = 0.08 p = 0.11 p = 0.81 p = 0.27 p = 0.04 p = 0.52 p = 0.008 p = 0.09 r = 0.22 r = - 0.11 r = -0.16 r = 0.15 r = 0.015 r = 0.60 r = 0.18 r = 0.0078 r = 0.0088 r = 0.014 r = 0.047 r = 0.10 Creatinine p = 0.12 All rightsreserved.Noreuseallowedwithoutpermission. p = 0.16 p = 0.52 p = 0.31 p = 0.36 p = 0.92 p < 0.001 p = 0.24 p = 0.96 p = 0.96 p = 0.93 p = 0.77 p = 0.53

r = 0.079 r = 0.16 r = 0.34 r = 0.15 r = -0-0014 r = 0.022 r = - 0.10 r = 0.28 r = -0.14 r = - 0.10 r = - 0.18 r = - 0.12 CK p < 0.0001 p = 0.62 p = 0.35 p = 0.03 p = 0.36 p > 0.99 p = 0.89 p = 0.52 p = 0.07 p = 0.34 p = 0.48 p = 0.21 p = 0.43

r = 0.15 r = -0.33 r = 0.27 r = 0.015 r = -0-0014 r = -0.019 r = -0.19 r = 0.37 r = 0.27 r = -0.0094 r = 0.22 r = 0.30 Taurine p = 0.17 p = 0.32 p = 0.05 p = 0.08 p = 0.92 p > 0.99 p = 0.90 p = 0.23 p = 0.01 p = 0.09 p = 0.95 p = 0.17 p = 0.06

r = - 0.032 r = - 0.10 r = - 0.25 r = 0.60 r = 0.022 r = -0.019 r = 0.084 r = 0.066 r = - 0.083 r = 0.21 r = - 0.050 r = -0.12 ;

N-acetyl L- this versionpostedApril9,2021. carnosine p = 0.35 p = 0.77 p = 0.54 p = 0.11 p < 0.001 p = 0.89 p = 0.90 p = 0.59 p = 0.67 p = 0.61 p = 0.18 p = 0.75 p = 0.44 r = 0.24 r = 0.28 r = 0.038 r = 0.18 r = - 0.10 r = -0.19 r = 0.084 r = 0.082 r = 0.054 r = 0.072 r = 0.14 r = 0.018 Pyruvate p = 0.07 p = 0.13 p = 0.10 p = 0.81 p = 0.24 p = 0.52 p = 0.23 p = 0.59 p = 0.60 p = 0.74 p = 0.65 p = 0.37 p = 0.91 r = 0.22 r = - 0.044 r = 0.17 r = 0.0078 r = 0.28 r = 0.37 r = 0.066 r = 0.082 r = 0.11 r = 0.17 r = 0.046 r = 0.25 Succinate p = 0.86 p = 0.16 p = 0.80 p = 0.27 p = 0.96 p = 0.07 p = 0.01 p = 0.67 p = 0.60 p = 0.50 p = 0.28 p = 0.77 p = 0.11 r = 0.022 r = 0.043 r= 0.32 r = 0.0088 r = -0.14 r = 0.27 r = - 0.083 r = 0.054 r = 0.11 r = 0.35 r = 0.32 r = 0.085 FGF-21 p = 0.96 p = 0.88 p = 0.79 p = 0.04 p = 0.96 p = 0.34 p = 0.09 p = 0.61 p = 0.74 p = 0.50 p = 0.02 p = 0.03 p = 0.57 r = 0.18 r = 0.18 r = 0.10 r = 0.014 r = - 0.10 r = -0.0094 r = 0.21 r = 0.072 r = 0.17 r = 0.35 r = 0.25 r = 0.084 GDF-15 p = 0.22 The copyrightholderforthispreprint p = 0.21 p = 0.26 p = 0.52 p = 0.93 p = 0.48 p = 0.95 p = 0.18 p = 0.65 p = 0.28 p = 0.02 p = 0.08 p = 0.56

r = 0.57 r = 0.25 r = 0.41 r = 0.047 r = - 0.18 r = 0.22 r = - 0.050 r = 0.14 r = 0.046 r = 0.32 r = 0.25 r = 0.59 GFAP p = 0.88 p < 0.001 p = 0.11 p = 0.008 p = 0.77 p = 0.21 p = 0.17 p = 0.75 p = 0.37 p = 0.77 p = 0.03 p = 0.08 p < 0.001

r = 0.55 r = 0.066 r = 0.27 r = 0.10 r = - 0.12 r = 0.30 r = -0.12 r = 0.018 r = 0.25 r = 0.085 r = 0.084 r = 0.59 NfL p = 0.37 p < 0.001 p = 0.68 p = 0.09 p = 0.53 p = 0.43 p = 0.06 p = 0.44 p = 0.91 p = 0.11 p = 0.57 p = 0.56 p < 0.001 medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Acknowledgments

The authors thank the patients involved in the study and their families for participation and support.

Riitta Lehtinen is acknowledged for assistance in ELISA measurements. We appreciate Biocentre

Finland and Biocentre Kuopio for supporting LC-MS laboratory facility.

Contributors

Study concept: JJ, SH, EP, MA, MJ, EY, HT. Data collection: JJ. Analysis and interpretation of

data: JJ, SH, EY, HT. Statistical analysis: JK. NfL and GFAP measurements: HZ. Metabolite

profiling: JK, ML. Patient data collection: AMS, MJ. Drafting of manuscript: JJ, SH, EY, HT.

Critical revision of the manuscript: all authors.

Funding

This study was supported by Academy of Finland, University of Helsinki, Emil Aaltonen

Foundation, Sigrid Juselius Foundation, Neurocenter Finland and HUS Helsinki University

Hospital VTR funds. HZ is a Wallenberg Scholar supported by grants from the Swedish Research

Council (#2018-02532), the European Research Council (#681712), Swedish State Support for

Clinical Research (#ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA

(#201809-2016862), the AD Strategic Fund and the Alzheimer's Association (#ADSF-21-831376-

C, #ADSF-21-831381-C and #ADSF-21-831377-C), the Olav Thon Foundation, the Erling-Persson

Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2019-0228), the

European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-

Curie grant agreement No 860197 (MIRIADE), and the UK Dementia Research Institute at UCL.

Competing interests

HZ has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed,

Siemens Healthineers, Pinteon Therapeutics, Nervgen and CogRx, has given lectures in symposia medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Järvilehto 15

sponsored by Fujirebio, Alzecure and Biogen, and is a co-founder of Brain Biomarker Solutions in

Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. Other authors report

no disclosures.

Figure legends

Figure 1. Serum neurofilament light is not elevated in SMAJ

Comparison of serum concentrations of NfL and GFAP between SMAJ patients (n=49) and controls

(n=45). Median per group is indicated by bar. ** = p<0.01, Mann-Whitney U-test.

Figure 2. Biomarkers of muscle damage and mass in SMAJ serum

A) Creatine kinase (S-CK) units of enzyme activity per liter of serum in female (n=29) and male

(n=20) SMAJ patients. Blue lines indicate the upper and lower age- and sex-matched laboratory

reference values.

Comparison of serum B) creatinine and C) creatine values between SMAJ patients (n=43) and

controls (n=48) measured with mass spectrometer.

Correlation of C) S-CK, D) creatinine and E) creatine to symptom score by Spearman correlation

test. For all, n = 36.

F) Correlation of creatine to CK Spearman correlation test n = 43. Comparison of serum

G) N-acetyl-L-carnosine and H) taurine values between SMAJ patients (n=43) and controls (n=48)

measured with mass spectrometer.

For comparisons median per group is indicated by bar. *** = p< 0.0001, Mann-Whitney U-test.

Figure 3. Mitochondrial disease biomarkers in SMAJ

Comparison of serum A) pyruvate, B) succinate, and C) lactate values between SMAJ patients

(n=43) and controls (n=48) measured with mass spectrometer.

Comparison of serum D) FGF-21 and E) GDF-15 levels between SMAJ patients (n=49) and

controls (n=24) measured by ELISA assays.

Median per group is indicated by bar. *** = p< 0.0001, Mann-Whitney U-test.

Figure 4. Summary of the SMAJ serum analyses of this study.

SMAJ is caused by a dominant mutation in a gene, which codes for CHCHD10 protein located in

mitochondrial intermembrane space. CHCHD10 defects impair mitochondrial respiration by an

unknown mechanism. Serum analyses of biomarkers and metabolites suggest that SMAJ

pathogenesis originates from skeletal muscle, showing altered muscle damage and mass markers,

and signs of a metabolic shift in response to mitochondrial dysfunction.

References

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medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. NfL All rights reserved. No reuse allowed without permission. GFAP ns ✱✱ 300 1000 200 900 600 60

400 40

S-NfL (pg/ml) S-NfL 20 200 S-GFAP (pg/ml) S-GFAP

0 0 controls SMAJ controls SMAJ A Creatine kinase B Creatinine C Creatine ✱✱✱ ✱✱✱ 2000 2.0 4

medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint 1500(which was not certified by peer review) is the1.5 author/funder, who has granted medRxiv a license3 to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

1000 1.0 2 S-CK (U/l) 500 0.5 1 Relative abundance Relative abundance

0 0.0 0 female SMAJ male SMAJ controls SMAJ controls SMAJ D E F 2500 r = 0.16 1.5 r = -0.11 4 r = 0.43 p = 0.35 p = 0.52 p = 0.009 2000 3 1.0 1500 2 1000 Creatine S-CK (U/l) Creatinine 0.5 1 500

0 0.0 0 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 Symptom score Symptom score Symptom score G H I N-Acetyl-L-Carnosine Taurine ✱✱✱ 4 r = 0.33 ✱✱✱ p = 0.03 6 2.0 5 3 1.5

2 2 1.0 Creatine 1 1 0.5 Relative abundance Relative abundance

0 0 0.0 0 500 1000 1500 2000 2500 controls SMAJ controls SMAJ Creatine kinase A Pyruvate B Succinate C Lactate ✱✱✱ ✱✱✱ 4 medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.212549603 ; this version posted April 9, 2021. The5 copyright holder forns this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 4 3 2 3 2 2 1 1 1 Relative abundance Relative abundance Relative abundance

0 0 0 controls SMAJ controls SMAJ controls SMAJ

C FGF-21 D GDF-15 ns ns 2000 15000 1000 14000 600

500 4000 400 300 2000 200 S-FGF-21 (pg/ml) 100 S-GDF-15 (pg/ml) 0 0 controls SMAJ controls SMAJ medRxiv preprint doi: https://doi.org/10.1101/2021.04.07.21254960; this version posted April 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.