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ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1480

New Biomarkers forNeuromuscular Function andMyasthenia Gravis

CARL JOHAN MOLIN

ISSN 1651-6206ISBN 978-91-513-0390-1urn:nbn:se:uu:diva-356116

Dissertation presented at Uppsala University to be publicly examined in Auditorium Minus,Gustavianum, Akademigatan 3, Uppsala, Friday, 21 September 2018 at 13:00 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin English. Faculty examiner: Professor Jan Verschuuren (Department of Neurology, LeidenUniversity Medical Center).

AbstractMolin, C. J. 2018. New Biomarkers for Neuromuscular Function and Myasthenia Gravis.Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine1480. 84 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0390-1.

Myasthenia gravis (MG) is an autoimmune disorder, which is caused by autoantibodies againstthe acetylcholine receptor (AChR). The cardinal symptom is muscle fatigue, which can rangefrom slight weakness of the extraocular muscles (causing droopy eyelids or double vision),to paralysis of the respiratory muscles. Antibodies towards other muscle proteins have beendiscovered, and MG is now considered a very heterogeneous disease with several subgroups.The severity of symptoms in MG patients is often fluctuating, and the antibody titers do notcorrelate with disease severity or treatment response. Therefore, there is a great need for reliablebiomarkers in MG, both for assessing neuromuscular function, but also for clinical aspects suchas disease progression and subgrouping.

In Study I, the use of compound motor action potential (CMAP) as a biomarker for musclestatus was examined in trained and untrained individuals. We found that trained individualshave a higher CMAP in proximal muscles, and the CMAP value in the biceps correlate withmuscle strength in these individuals, indicating that CMAP can be used as a biomarker formuscle function. In Study II, subjects from study I were examined with ultrasound to assess theeffect of high-resistance strength training (HRST) on peripheral nerves, and to compare musclethickness. We did not find a difference in nerve cross-sectional area between the two groups.Trained individuals had thicker biceps muscles. The results from study I and II has led to CMAPand ultrasound being used to evaluate the result of physical exercise as an intervention in MGpatients.

In Study III, the expression of inflammatory proteins in the sera of MG patients wascompared to healthy controls, in search for possible biomarkers. We found eleven proteins tobe elevated, which provide new insight to the inflammatory response in MG and have possiblefunctions as new biomarkers of inflammatory activity.

In Study IV, the effect of thymectomy on the potential microRNA MG biomarkersmiR-150-5p and miR-21-5p was examined. A decrease in miR-150-5p was seen 24 months afterthymectomy, which further validate the use of miR-150-5p as a disease-specific biomarker forclinical outcome in AChR positive MG patients.

Keywords: Biomarkers, myasthenia gravis, neuromuscular function, neurophysiology

Carl Johan Molin, Department of Neuroscience, Clinical Neurophysiology, Akademiskasjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.

Carl Johan Molin 2018

ISSN 1651-6206ISBN 978-91-513-0390-1urn:nbn:se:uu:diva-356116 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-356116)

To my family

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Molin, C.J. & Punga, A.R. (2016) Compound Motor Action Poten-tial: Electrophysiological Marker for Muscle Training. J Clin Neu-rophysiol, 33(4), 340-345.

II Molin, C.J., Widenfalk, J. & Punga, A.R. (2017) High-resistance strength training does not affect nerve cross sectional area - An ul-trasound study. Clinical Neurophysiology Practice 2, 163-169.

III Molin, C.J.*, Westerberg, E.* & Punga, A.R. (2017) Profile of up-regulated inflammatory proteins in sera of Myasthenia Gravis pa-tients. Sci Rep, 7, 39716.

IV Molin, C.J., Sabre, L., Weis, C.-A., Punga, T. & Punga, A. R. (2018) Thymectomy lowers the myasthenia gravis biomarker miR-150-5p. Neurology - Neuroimmunology Neuroinflammation 5.

* Equal first authors.

Reprints were made with permission from the respective publishers.

Related papers published during the PhD period

1. Westerberg, E., Molin C.J., Lindblad. I., Emtner, M. & Punga, A.R. (2017) Physical exercise in myasthenia gravis is safe and improves neuromuscular parameters and physical performance-based measures: A pilot study. Muscle Nerve 56, 207-214.

2. Westerberg, E., Molin, C.J., Sprndly Nees, S., Widenfalk, J. & Punga, A.R. (2018) The impact of physical exercise on functional muscle measures in myasthenia gravis patients - a single subject de-sign study. In press, Medicine.

Contents

Introduction ................................................................................................... 11Myasthenia gravis ..................................................................................... 11

Historical background .......................................................................... 11Pathophysiology ................................................................................... 12The thymus gland, autoimmunity, and inflammation .......................... 16Clinical signs and symptoms of MG .................................................... 18Diagnosis .............................................................................................. 19Treatment ............................................................................................. 21Epidemiology and genetics .................................................................. 23

Physical activity ........................................................................................ 24General recommendations and health benefits .................................... 24Muscle physiology of high-resistance strength training ...................... 25

Biomarkers ................................................................................................ 25Biomarkers for muscle training and neuromuscular function .............. 26Biomarkers for clinical use in MG ....................................................... 28

Aims .............................................................................................................. 31Specific aims ............................................................................................. 31

Methods ......................................................................................................... 32Patients and subjects ................................................................................. 32Ethics ........................................................................................................ 33Methods .................................................................................................... 34

Electrophysiological and muscle strength measurements .................... 34Ultrasound examinations ..................................................................... 35Inflammatory protein analysis ............................................................. 37miRNA analysis ................................................................................... 37Thymic lymphofollicular hyperplasia grading ..................................... 38

Statistics .................................................................................................... 38

Results ........................................................................................................... 41Study I CMAP as an electrophysiological marker for muscle training . 41

Subjects and training habits ................................................................. 41CMAP and muscle strength recordings ............................................... 42

Study II Nerve and muscle ultrasound in trained and untrained individuals ................................................................................................ 43

Subjects ................................................................................................ 43

Ultrasound of nerves and muscles ....................................................... 44Study III Profile of upregulated inflammatory proteins in sera of MG patients ...................................................................................................... 46

Patients and subjects ............................................................................ 46Inflammatory protein expression ......................................................... 46

Study IV miRNA in sera of MG patients in the MGTX study .............. 48Patients and clinical data ...................................................................... 48miRNA analysis ................................................................................... 49

Discussion ..................................................................................................... 53Neurophysiological biomarkers ................................................................ 53

CMAP as a biomarker for muscle training .......................................... 53Effect of HRST on peripheral nerves ................................................... 54Role of CMAP and ultrasound in exercise studies .............................. 55Drawbacks ...........................................................

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