News Release

Title
Ratio of urinary N-terminal titin fragment to urinary creatinine is a novel biomarker for amyotrophic lateralsclerosis
Key Points• Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease showing a rapidprogression.
• Biomarkers for monitoring disease progression of ALS have not been established.
• Our findings indicate that urinary N-terminal titin fragment is a non-invasive measure of muscle damage
in ALS, which could be applied in disease monitoring and prediction of disease progression.
• Urinary N-terminal titin fragment can be used in clinical practice and clinical trials.
Summary
A group of researchers, headed by Prof. Masahisa Katsuno, Department of Neurology, Nagoya University
Graduate School of Medicine have revealed that urinary levels of titin N-terminal fragment normalised withurinary Cr which can be repeatedly measured reflect motor function and could be a marker for survival ofpatients with ALS. This work was published online in Journal of Neurology, Neurosurgery, and Psychiatry
on March 19, 2021.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by rapid progression of
upper and lower motor neuron loss, which causes paralysis and death within an average of 3-4 years from
disease onset. After trials and testing spanning over half a century on over 150 different therapeutic agents orstrategies, only riluzole and edaravone, which have beneficial effects in a limited population of patients withALS, have been approved. This could be attributed to the lack of sensitive, non-invasive biomarkers thatreflect human pathophysiology.
In this study, compared with healthy controls (HC), patients with ALS had significantly increased urinarylevels of titin N-terminal fragment normalised with Cr (titin/Cr), which were correlated with the scores of theRevised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). A Cox proportional hazards
model demonstrated that the high urinary level of titin/Cr was a survival predictor in patients with ALS.
Multivariate analysis of prognostic factors showed that the urinary titin/Cr and serum NfL were independent
factors for poor prognosis.
Our findings indicate that urinary N-terminal titin fragment is a non-invasive measure of muscle damage
in ALS, which could be applied in disease monitoring and prediction of disease progression.
Research BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by rapid progression of
upper and lower motor neuron loss, which causes paralysis and death within an average of 3-4 years fromdisease onset. Most ALS cases are sporadic with unknown aetiology.
Well-designed studies have reported negative results of several candidate drugs for ALS therapy, which
could be attributed to several reasons. Disease progression before motor dysfunction onset attenuates theeffects of disease-modifying therapies that are beneficial in preclinical studies. Great variations in the clinicalcourses of ALS could impede the assessment of the drug effects on disease progression. Moreover, there is alack of reliable, less invasive outcome measures for monitoring of ALS progression, evaluating treatment
efficacy, and exploring potential molecular targets of therapy development.
Therefore, biomarker which predict severity, disease course, and prognosis of ALS are warranted todevelop biomarkers based on the results of clinical studies in patients with sporadic ALS.
Research Results
Skeletal muscle titin, which is also known as connectin, is the largest human protein that works in aspring-like mechanical fashion to connect the Z-disc to the M-line in the sarcomere. Mutations in TTN havebeen reported in various myopathies. Moreover, titin degradation is reported in muscle biopsies obtained
from patients with ALS.
We found that urinary levels of titin N-terminal fragment normalised with urinary Cr levels (titin/ Cr) weresignificantly increased in patients with ALS compared with HC (figure 1A). To examine the utility of urinary
titin N-terminal fragment as a biomarker that reflects ALS severity, we investigated the association of urinarylevels of titin/Cr with the total score of the ALSFRS-R in the patients with ALS. Urinary levels of titin/Cr
were strongly correlated with the total score of ALSFRS-R (Figure 1B).

Subsequently, we examined whether urinary N-terminal titin fragment could predict prognosis in patientswith ALS. Compared with the high titin/Cr group, we found that the low titin/Cr group had longer mediansurvival (Figure 2). The Cox regression analysis also identified urinary titin/Cr that were associated withsurvival period from baseline. These findings indicate that the baseline level of urinary titin/Cr is prognostic
markers for ALS.

Next, we also assessed urinary p75ECD and serum NfL, which are recently developed ALS biomarkers.
There was a correlation of motor function measures with the urinary titin N-fragment and p75ECD levels, but
not with serum NfL. These findings suggest that urinary titin, as well as urinary p75ECD, reflects ALS severity.
ROC curves indicated that the urinary N-terminal titin fragment and serum NfL could discriminate patientswith ALS from healthy controls with a high probability (Figure 3).

Research Summary and Future Perspective
Our findings indicate the potential of urinary titin N-terminal fragment normalised with urinary Cr as a
non-invasive biomarker for evaluation of disease severity and prognosis in ALS and other motor neuron
diseases. Future prospective, multicentre, longitudinal studies are warranted to confirm our findings and toexamine whether urinary titin N-terminal fragment can predict the biological effect of the drug therapeutics

for ALS.

Publication
Shinichiro Yamada, Atsushi Hashizume, Yasuhiro Hijikata, Daisuke Ito, Yoshiyuki Kishimoto, Madoka Iida,
Haruki Koike, Akihiro Hirakawa and Masahisa Katsuno“Ratio of urinary N-terminal titin fragment to urinary creatinine is a novel biomarker for amyotrophic
lateral sclerosis”
Journal of Neurology, Neurosurgery, and Psychiatry, March 19, 2021.
DOI：http://dx.doi.org/10.1136/jnnp-2020-324615
Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Jou_Neuro_Psy_210319.pdf