Genotype-phenotype correlations and characterization of medication use in inherited myotonic disorders

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

By

Alayne Primavera Meyer

Graduate Program in Genetic Counseling

The Ohio State University

2019

Thesis Committee

William Arnold, MD, Advisor

Jennifer Roggenbuck, MS, LGC

Samantha LoRusso, MD

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Copyrighted by

Alayne Primavera Meyer

2019

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Abstract

Objective: The purpose of this study is to characterize the genetic and phenotypic profile of patients with myotonic disorders, compare symptom profiles for significant clinical differences and summarize use of antimyotonia agents.

Background: Myotonic disorders are characterized by hyperexcitability and delayed relaxation of muscle. Variants in CLCN1 and SCN4A cause non-dystrophic myotonia while pathogenic expansions in DMPK and CNBP cause dystrophic myotonia, clinically distinguished by presence of progressive muscle deterioration. Symptoms may include stiffness, weakness, cramping and pain and can be exacerbated by a variety of environmental factors. Combination of clinical examination, laboratory workup, electromyography (EMG) results and genetic testing aid in diagnosis of these patients.

Methods: A total of 142 patients at The Ohio State University were identified to have a myotonia diagnostic code assigned to their medical record and a variant or expansion in

CLCN1, SCN4A, DMPK or CNBP in themselves or a family member. Data collected from the electronic medical record included demographics, symptom history, clinical examination, family history, lab work, EMG results, genetic testing results and medication history. Descriptive statistics and Fisher’s exact tests were utilized in data analysis.

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Results: The final cohort consisted of 27 individuals with CLCN1-related myotonia (23 dominantly inherited, 4 recessively inherited), 15 with SCN4A-related myotonia, 89 with myotonic dystrophy I and 11 with myotonic dystrophy 2. Patient reported weakness was found in the majority of our non-dystrophic (ND) cohort (65.2% CLCN1, 69.2% SCN4A), while clinically identified weakness was only found in 20%. Pain was reported by the majority of our overall cohort (69% CLCN1, 53% SCN4A, 59.5% DMPK, 100% CNBP).

Frequency of patient reported-weakness, clinical myotonia, percussion myotonia and clinical weakness were found in significantly more individuals in our dystrophic (D) cohort compared to our ND cohort while cold exacerbation was found in significantly more individuals in the ND cohort. The only significant difference between our CLCN1 and SCN4A cohorts were the proportion of individuals reporting stiffness (100% CLCN1,

78.6% SCN4A). Only 50% of our overall cohort had trialed an antimyotonia agent and only 28% were currently taking one. Our ND cohort was more likely to have trialed and be currently utilizing an antimyotonia agent than our D cohort (73% ND, 39% D).

Conclusions: The results of this study suggest that weakness may be a more significant symptom in ND myotonias than previously identified and may not be detectable via standard manual muscle testing. Additionally, pain may be a more prevalent symptom in all myotonia disorders than previously identified, particularly in CLCN1. Differentiation between ND patients by phenotype may not be feasible due to significant phenotypic overlap. Most individuals with myotonic disorders are not currently taking an antimyotonia agent despite the presence of symptoms that may be aided by these medications, but ND patients were more likely to do so than D patients.

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Dedication

For my parents who have supported me in all of my endeavors and who raised me to be

the person I am today.

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Acknowledgments

I would like to thank Dr. William Arnold for his amazing support and guidance throughout this process, Jennifer Roggenbuck and Dr. Samantha LoRusso for their expertise and wisdom, Dr. John Kissel for his aid in the study idea formulation, Rachel

Smith for her assistance with statistical analysis, The Ohio State University Center for

Clinical and Translational Science (CCTS) for their financial assistance, The Ohio State

University Neuroscience Research Institute (NRI) for their financial assistance both with this study as well as with travel to the American Academy of Neurology to present the findings of this study, the OSU GCGP faculty who have supported me throughout the past two years and my classmates for their endless support and encouragement.

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Vita

May 2011…………………………………………….Thomas Worthington High School

May 2015………………………………B.S. Biological Engineering, Purdue University

May 2019………………………………M.S. Genetic Counseling, The Ohio State

University, in progress

Fields of Study

Major Field: Genetic Counseling

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Table of Contents

Abstract ...... ii Dedication ...... iv Acknowledgments...... v Vita ...... vi List of Tables ...... viii List of Figures ...... ix Chapter 1. Background and Aims ...... 1 Background ...... 1 Myotonia overview ...... 1 Disease Pathophysiology and Phenotype...... 3 Clinical Diagnostic Testing...... 13 Antimyotonia Agents and Treatment ...... 18 Study Aims...... 21 Chapter 2. Methods and Data Analysis...... 22 Methods...... 22 Data Analysis ...... 29 Chapter 3. Results ...... 31 Aim 1 ...... 33 Aim 2 ...... 41 Aim 3 ...... 47 Chapter 4. Discussion, Implications, Limitations and Future Direction...... 50 Discussion ...... 50 Clinical Implications ...... 56 Limitations ...... 58 Future Direction ...... 59 Bibliography ...... 60 Appendix: Additional Figures and Tables...... 64

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List of Tables

Table 1. Diagnostic Codes Utilized in Feasibility Analysis ...... 23 Table 2: Chart Review Data Collection Summary ...... 28 Table 3. Additional Findings on EMG ...... 38 Table 4. Creatine Kinase Levels ...... 39 Table 5. Participant Demographics ...... 64 Table 6. Patient Reported Symptoms...... 65 Table 7. Physical Examination Results ...... 66 Table 8. Genotype and Myotonia Phenotype for CLCN1 Cohort ...... 67 Table 9. Genotype and Myotonia Phenotype for SCN4A Cohort ...... 69 Table 10. Patient Reported Symptoms in Dystrophic and Non-dystrophic Cohorts ...... 70 Table 11. Physical Examination Results in Dystrophic and Non-dystrophic Cohorts ..... 71 Table 12. Patient Reported Symptoms in CLCN1 and SCN4A Cohorts ...... 72 Table 13. Physical Examination Results in CLCN1 and SCN4A Cohorts ...... 73 Table 14. Summary of Trialed Medications by Genotype ...... 74 Table 15. Duration of Medications Currently Utilized in Months ...... 75 Table 16. Reasons for Medication Discontinuation ...... 76

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List of Figures

Figure 1. Chart Review and Sorting Process ...... 25 Figure 2. Age of Symptom Onset ...... 32 Figure 3. Patient Reported Symptoms ...... 34 Figure 4. Physical Examination Results ...... 36 Figure 5. Proportion of Muscles with Myotonia and Spontaneous Activity on EMG ..... 38 Figure 6. Patient Reported Symptoms in Dystrophic versus Non-dystrophic Patients .... 42 Figure 7. Physical Examination Results in Dystrophic versus Non-dystrophic Patients . 43 Figure 8. Patient Reported Symptoms in CLCN1 versus SCN4A Patients ...... 45 Figure 9. Physical Examination Results in CLCN1 versus SCN4A Patients ...... 46 Figure 10. Medication Trial Status by Genotype ...... 48 Figure 11. Currently Utilized Medication by Genotype ...... 49

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Chapter 1. Background and Aims

Background

Myotonia overview

Myotonia is a phenomenon of skeletal muscle hyperexcitability that impairs muscle relaxation following contraction. Individuals affected by myotonia may have symptoms of muscle stiffness, impaired motor control, weakness or pain. Myotonia is characterized clinically by delayed muscle relaxation following formation of a fist, eye closure, percussion of muscles, or electrically by repetitively firing muscle fiber action potentials of waxing and waning amplitudes and frequencies on electromyography (EMG) studies

(Heatwole & Moxley, 2007; Tang & Chen, 2011; Timothy Miller, 2008; Jeroen Trip et al., 2008). In some forms of myotonia, electrical myotonia may be readily apparent while clinical myotonia is not noted (Timothy Miller, 2008).

There are both acquired and genetic causes of muscle myotonia. Acquired causes include hypothyroidism, inflammatory myopathies, severe denervation and toxic myopathies

(Chad R. Heatwole, Jeffrey M. Statland, & Eric L Logigian, 2013). In general, these acquired forms of myotonia are most often associated with findings of electrical myotonia without overt clinical myotonia. Genetic myotonic disorders are generally divided into two major categories – dystrophic and non-dystrophic – with the major

1 differentiating factor being the presence of progressive muscle degeneration in patients with dystrophic type myotonias (Heatwole & Moxley, 2007; Matthews et al., 2010, p.;

Timothy Miller, 2008).

The dystrophic disorders are further divided into myotonic dystrophy type I (DM1) and myotonic dystrophy type II (DM2, also known as proximal myopathic myotonia or

PROMM) with the main clinical difference being the pattern of weakness seen in the patients. Patients with DM2 generally have more proximal limb weakness while patients with DM1 have more prominent distal limb weakness (Timothy Miller, 2008).

The non-dystrophic disorders are related to mutations of specific skeletal muscle ion channels and are usually grouped or categorized on the basis of the ion channel affected as well as the inheritance pattern and clinical features. The two skeletal muscle ion channels that are associated with non-dystrophic disorders include chloride (CLCN1) and sodium (SCN4A) channels. The non-dystrophic disorders are known to be highly variable in expression, making them a challenge to diagnose clinically (Heatwole & Moxley,

2007; Trivedi, Cannon, & Griggs, 2014). Andersen Tawil Syndrome is a syndromic cause of myotonia caused by potassium ion channel defects (KCNJ2, KCNJ18) as well as periodic paralysis, cardiac dysfunction, and skeletal anomalies (Cannon, 2017; Michael

K. Hehir & Eric L. Logigian, 2013).

Other inherited diseases that can cause electrical myotonia include myotubular myopathy, myofibrillar myopathy, caveolinopathies, Pompe disease, McArdle disease and

Debrancher deficiency (Chad R. Heatwole et al., 2013; Michael K. Hehir & Eric L.

Logigian, 2013)

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Disease Pathophysiology and Phenotype

Non-dystrophic Disorders

Chloride Channelopathies

Myotonia congenita (MC) is the most common inherited muscle channelopathy

(Matthews et al., 2010). It is caused by dominant and recessive pathogenic variants in

CLCN1 which encodes for the chloride channel CLC-1. The conductance through CLC-1 is responsible for 80% of resting membrane conductance in skeletal muscle (Tang &

Chen, 2011). Pathogenic variants in CLCN1 lead to loss of function in the channel that causes reduced chloride conductance and membrane hyper excitability (Tang & Chen,

2011; Timothy Miller, 2008; Jeroen Trip et al., 2008). There are hypotheses as to how the underlying pathophysiology of dominant versus recessive disease differs; however, given that the same mutation can cause dominant disease in one family, while causing recessive disease in another leads these theories to be questioned. One common theory is that recessive disease is caused by mutations that prevent the normal chloride channel complex from forming, leading to a reduced number of normal chloride channels. A single mutation would allow 50% of the normal complexes to form and individuals are free of disease. In dominant disease it is theorized that dominant negative mutations cause the mutant monomer to be incorporated into the chloride channel which affects the normal function of all channels formed (Lehmann-Horn & Rüdel, 1995; Tang & Chen,

2011).

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Dominant CLCN1 mutations cause Thomsen disease which has an incidence of 1 in

25,000 (Heatwole & Moxley, 2007). Classically, this disorder is said to cause transient muscle stiffness that usually begins in early childhood and can be mistaken by parents as weakness or clumsiness (Heatwole & Moxley, 2007; Timothy Miller, 2008). Stiffness is most common in the leg muscles, with facial muscle involvement being rare. This can be alleviated by repetitive muscle activity, a phenomenon called “warm up” (Heatwole &

Moxley, 2007; Matthews et al., 2010; Tang & Chen, 2011; Trivedi et al., 2014). Pain associated with myotonia has been reported, in one study being found in 28% of patients with dominant and recessive MC (Matthews et al., 2010). Other triggers for stiffness include emotional surprise and cold temperatures. In addition, pregnancy can trigger or worsen symptoms. The presence of muscle weakness and systemic symptoms are uncommon (Heatwole & Moxley, 2007).

Physical examination often reveals muscle hypertrophy of the lower and upper limbs as well as the facial muscles which gives patients an athletic appearance (Heatwole &

Moxley, 2007). Myotonia can be demonstrated through percussion and grip testing

(Heatwole & Moxley, 2007; Trivedi et al., 2014). Myotonia can also be seen in paraspinal and proximal muscles. Reflexes, cerebellar function, sensation and muscle strength are usually normal (Heatwole & Moxley, 2007).

Recessive CLCN1 mutations cause Becker disease which typically has a more severe phenotype than Thomsen disease (Heatwole & Moxley, 2007; Timothy Miller, 2008;

Trivedi et al., 2014). Symptoms typically begin in childhood, but often have later onset than individuals with Thomsen disease (Heatwole & Moxley, 2007). Features

4 differentiating Becker disease include slowly progressive muscle weakness as well as transient weakness of the proximal limbs that can improve with exercise (Heatwole &

Moxley, 2007; Timothy Miller, 2008; Trivedi et al., 2014). Symptom triggers include cold temperatures, prolonged exercise, menses, pregnancy and emotional tension

(Heatwole & Moxley, 2007).

Physical exam reveals myotonia as well as lower limb weakness in more severe cases.

This combination of stiffness and weakness can make initiation of ambulation difficult, but warm up is still experienced so this improves within a few steps. Some individuals experience persistent lower limb weakness, not corrected by warm up. Grip myotonia is more common than tongue or eyelid myotonia. Muscle hypertrophy is most common in the calves as well as lower and proximal muscle groups and is more severe in male patients. Atrophy of distal muscle groups can occur and tendon reflexes may be reduced

(Heatwole & Moxley, 2007).

Sodium Channelopathies

Pathogenic variants in SCN4A cause several different myotonic disorders including paramyotonia congenita (PMC), sodium channel myotonias (SCM, historically referred to as potassium aggravated myotonias) which include myotonia fluctuans, myotonia permanens and acetazolamide-sensitive myotonia and hyperkalemic periodic paralysis with myotonia. These conditions are the result of gain of function in the sodium channel within the muscle which leads to excessive inward sodium ion current through the channel. This can cause hyperexcitability which leads to myotonia or transient loss of

5 excitability which leads to periodic paralysis (Cannon, 2017). All of these conditions are dominantly inherited and affect skeletal muscle excitability (Loussouarn et al., 2016).

PMC is a highly penetrant condition that also shows highly variable expression, even among family members sharing the same pathogenic variant (Heatwole & Moxley, 2007;

Matthews et al., 2010). Age of onset of symptoms is typically before the age of ten

(Heatwole & Moxley, 2007). The main clinical features of PMC include myotonia that is induced by cold or repeated muscle activity and is most prominent in the face, tongue, neck and distal upper extremity muscles (Cannon, 2017; Lehmann-Horn & Rüdel, 1995;

Loussouarn et al., 2016; Matthews et al., 2010). Myotonia is often prolonged, lasting minutes to hours, and painful (Heatwole & Moxley, 2007; Trivedi et al., 2014). The stiffness is not responsive to warm up and instead, worsens with repeated muscle contraction, a phenomenon called paramyotonia (Cannon, 2017; Lehmann-Horn &

Rüdel, 1995; Loussouarn et al., 2016; Timothy Miller, 2008). There is overlap between

PMC and hyperkalemic periodic paralysis in that PMC patients also experience episodes of weakness, lasting hours to days, triggered by exercise (especially in a cold environment), potassium ingestion or fasting (Heatwole & Moxley, 2007; Lehmann-Horn

& Rüdel, 1995; Matthews et al., 2010).

Physical examination of these patients classically will show paramyotonia of the eyelids.

Percussion and handgrip myotonia can also be observed, but are less common (Heatwole

& Moxley, 2007).

SCM is characterized by pure myotonia without attacks of weakness as seen in PMC.

Myotonia is typically aggravated by potassium, but not cold temperatures. The three

6 subtypes are myotonia fluctuans, myotonia permanens and acetazolamide responsive myotonia (Heatwole & Moxley, 2007; Loussouarn et al., 2016; Matthews et al., 2010).

Myotonia fluctuans is defined by five classic features – myotonia that fluctuates in severity, presence of warm up phenomenon, absence of weakness, presence of myotonia after exercise or potassium ingestion and absence of cold induced myotonia. Although potassium ingestion triggers myotonia, it does not cause weakness as in hyperkalemic periodic paralysis. Myotonia following exercise can be delayed, occurring approximately

30 minutes after exercise, and may be severe enough to cause temporary immobilization.

Three mutations in SCN4A are responsible for this phenotype – Val1589Met,

Gly1306Ala and Ser804Phe (Heatwole & Moxley, 2007; Lehmann-Horn & Rüdel, 1995).

Myotonia permanens is a rare and severe form of disease caused by Gly1306Glu in

SCN4A. Patients present with persistent clinical myotonia in the face, arm, leg and respiratory muscles which can cause pulmonary compromise. Muscle hypertrophy is more pronounced in these patients than in other forms of NDMs. Symptoms are present even at rest and can worsen with exercise, potassium ingestion and occasionally with cold temperatures (Heatwole & Moxley, 2007; Lehmann-Horn & Rüdel, 1995; Loussouarn et al., 2016).

Acetazolamide responsive myotonia is named based on symptom improvement seen with acetazolamide treatment. Symptoms are triggered by potassium, cold temperatures or fasting and is often painful in nature. Symptoms progress during childhood, but weakness is not noted in these patients (Heatwole & Moxley, 2007; Loussouarn et al., 2016).

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Periodic paralyses are characterized by recurrent episodes of weakness. Episodes may be triggered by exercise, diet or emotional stress and can last from hours to days before recovery. These patients can develop a slowly progressive and permanent weakness later in life (Cannon, 2017; Lehmann-Horn & Rüdel, 1995). Hyperkalemic periodic paralysis

(hyperPP) is characterized by periodic bouts of weakness triggered by potassium ingestion or elevated serum potassium. These patients usually have myotonia, especially during a period of weakness, but myotonia is usually less obvious clinically and evident only on EMG testing (Cannon, 2017). Hypokalemic periodic paralysis (hypoPP) is another form of periodic paralysis that is associated with mutations in both SCN4A and the CACNA1S, but hypoPP is not associated with clinical or electrical myotonia (Cannon,

2017; Yoshinaga et al., 2015). SCN4A can also rarely cause a form of neuromuscular junction transmission failure as a phenotype of congenital myasthenic syndrome, but this is also not associated with features of muscle hyperexcitability.

Dystrophic Disorders

Myotonic Dystrophy Type I (DM1)

DM1 is the most common adult onset muscular dystrophy, affecting approximately 3-15 in 100,000 individuals (Turner & Hilton-Jones, 2014). It is caused by expansion of unstable trinucleotide (CTG) repeat sequences in DMPK which cause expansions in the product mRNA molecules. These mRNA molecules are thought to inactivate a protein that is necessary for proper splicing of CLCN1 pre-mRNA. Altered splicing is the cause of reduced chloride conductance in the muscle, causing myotonia (Lueck, Mankodi,

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Swanson, Thornton, & Dirksen, 2007; G. Meola, 2000; Thornton, 2014). DM1 is an autosomal dominant disorder that demonstrates intergenerational expansion causing younger generations to have earlier age of symptom onset and more severe phenotype, a phenomenon called genetic anticipation (G. Meola, 2000; Thornton, 2014; Turner &

Hilton-Jones, 2014). Intergeneration expansion is more common when the diseased allele is inherited maternally and on average increases by greater than 200 repeats each generation (Thornton, 2014). Cases of intergenerational contraction have been reported, but are rare, occurring in less than 5% of transmissions (G. Meola, 2000; Thornton,

2014).

A normal CTG repeat length ranges from 5 to 37 repeats. Individuals with 38 to 49 repeats are premutation carriers and are at risk to have children with disease, but will not have disease themselves. Individuals with 50 or greater repeats have fully penetrant disease (“classic” DM1) and individuals with greater than 1000 repeats are at risk to have the congenital form of the disease (Thornton, 2014; Turner & Hilton-Jones, 2014). There is some correlation between repeat size and age of onset of symptoms in disease, but the presence of somatic mosaicism in these patients due to instability in somatic tissues limits the strength of this correlation (G. Meola, 2000; Thornton, 2014; Turner & Hilton-Jones,

2014). Instability varies based on cell type, but has been shown to be more unstable in non-dividing cells such as skeletal muscle, heart and brain (G. Meola, 2000; Thornton,

2014).

Congenital disease accounts for approximately 15% of DM1. It has been seen in individuals with as few as 750 repeats, but more commonly is seen in those with greater

9 than 1000. Hallmark features include neonatal hypotonia, feeding difficulties and respiratory difficulties (G. Meola, 2000; Thornton, 2014). Patients surviving past the neonatal period experience delayed motor milestones, intellectual disability, learning disabilities, a tented appearance to the upper lip and significant dysarthria (G. Meola,

2000; Thornton, 2014). Because of genetic anticipation, over half of infants born with congenital DM1 do not have a mother carrying a diagnosis of DM1, but this is often due to mild or unrecognized symptoms that are present (Thornton, 2014).

Symptoms in classic DM1 typically present between the ages of 20 and 40 (Thornton,

2014). Skeletal muscle symptoms include atrophy, progressive weakness and myotonia, with grip myotonia often manifesting prior to muscle weakness. Myotonia and stiffness more commonly affects the distal arm, hand, tongue and jaw and warm up phenomenon is present. Muscle atrophy and weakness occurs predominantly in the facial, trunk and distal limb muscles but also affect the neck and ocular muscles (G. Meola, 2000;

Thornton, 2014). Muscle atrophy and myotonia in the jaw and tongue can lead to dysarthria and dysphagia. Atrophy of facial muscles leads to a characteristic facial appearance in these patients that includes ptosis, atrophy of the temporal muscles and long facial shape. Also contributing to appearance is the common finding of premature frontal balding (Thornton, 2014; Turner & Hilton-Jones, 2014).

Systemic complications include progressive cardiac conduction defects, respiratory insufficiency, premature cataracts, daytime hypersomnolence, gastrointestinal and endocrine dysfunction, cognitive and behavioral deficits and increased malignancy rates

(G. Meola, 2000; Thornton, 2014; Turner & Hilton-Jones, 2014). Cardiac and respiratory

10 complications account for 70% of premature death in these patients (G. Meola, 2000;

Thornton, 2014).

Myotonic Dystrophy Type II (DM2)

DM2 is estimated to affect less than one-fifth the number of individuals affected by DM1 in the United States (Giovanni Meola & Cardani, 2017; Thornton, 2014). DM2 is caused by a tetranucleotide (CCTG) repeat expansion in CNBP. Similarly to DM1, this causes mRNA splicing abnormalities which lead to the systemic disease. This is inherited in an autosomal dominant manner and does show somatic expansion; however, intergenerational expansion, anticipation and congenital forms of disease have not been described (Lehmann-Horn & Rüdel, 1995; G. Meola, 2000; Giovanni Meola & Cardani,

2017; Thornton, 2014; Turner & Hilton-Jones, 2014). Repeat expansion length usually contracts in younger generations which may explain the lack of congenital-onset disease

(Giovanni Meola & Cardani, 2017). Additionally, repeat size is weakly correlated with disease severity (Thornton, 2014).

Normal repeat length is typically less than 26 repeats and disease has been documented in an individual with as few as 75 repeats. Individuals with 27 to 74 repeats are considered grey zone carriers, with no associated clinical symptoms reported. DM2 patients have been reported to have anywhere from 75 to 11,000 repeats (Giovanni Meola & Cardani,

2017). Most patients with DM2 (>90%) will have an expansion greater than one thousand repeats (Thornton, 2014).

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Symptoms of DM2 are typically milder than in DM1, with the median age of onset being

48 years. Early symptoms include muscle pain, stiffness and fatigue (Lehmann-Horn &

Rüdel, 1995; G. Meola, 2000; Thornton, 2014; Turner & Hilton-Jones, 2014). Myotonia can be extremely variable in severity and is only present in approximately 50% of patients (Giovanni Meola & Cardani, 2017). Weakness and atrophy is milder than in

DM1 and affects predominantly proximal limb muscles (G. Meola, 2000; Thornton,

2014). Facial and ankle weakness in less common than in DM1 and calf hypertrophy can be observed (Giovanni Meola & Cardani, 2017). Muscle pain is a prominent symptom affecting 50-80% of patients and can be the first clinical sign of disease. Pain is not triggered by exercise and can be episodic which can lead to misdiagnoses of fibromyalgia, chronic fatigue, arthritis or other muscle disease in these patients (Giovanni

Meola & Cardani, 2017).

Percussion myotonia of the forearm is the most sensitive examination for myotonia in

DM2. Grip myotonia can also be present, especially in patients with expansions in CNBP and a heterozygous CLCN1 mutation. In patients with severe or early onset symptoms, suspicion for a mutation in a second myotonia gene should be considered, as this has been observed in patients having both a CNBP expansion and SCN4A mutation (Giovanni

Meola & Cardani, 2017).

Systemic symptoms are also present in patients with DM2, but have not been studied as extensively as in DM1. Cardiac conduction defects, early onset cataracts, endocrine dysfunction, hypersomnolence, gastrointestinal symptoms and increased malignancy rates have all been reported in these patients (G. Meola, 2000; Giovanni Meola &

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Cardani, 2017; Turner & Hilton-Jones, 2014; Win, Perattur, Pulido, Pulido, & Lindor,

2012). Cardiac involvement, cognitive and behavioral symptoms are all less common and milder than in DM1 patients (G. Meola, 2000; Giovanni Meola & Cardani, 2017; Turner

& Hilton-Jones, 2014).

Clinical Diagnostic Testing

Electromyography (EMG) and Nerve Conduction Studies (NCS)

EMG and nerve conduction studies are useful tools for the diagnosis of myotonic disorders. EMG is a sensitive test for myotonic disorders. Electrical myotonia or myotonic potentials are spontaneous trains of muscle fiber action potentials that wax and wane in amplitude and frequency (Heatwole & Moxley, 2007; Michael K. Hehir & Eric

L. Logigian, 2013; Tan et al., 2011). Electrical myotonia is recorded during an EMG examination by inserting a recording electrode into a muscle and, typically, small brisk movements of the needle are made to attempt to trigger a myotonic potential. Electrical myotonia can also be triggered and recorded with EMG following muscle contraction or percussion. (Timothy Miller, 2008).

NCS can also be used to assess muscle excitability. During NCS, compound muscle action potential (CMAP) amplitude changes following exercise or cooling of the muscle and can be used determine altered excitability (Matthews et al., 2010). The patterns of

CMAP amplitude change can be helpful in distinguishing between different myotonic disorders (Heatwole & Moxley, 2007). Three NCS testing protocols have been used for evaluation of patients with myotonia – the short exercise test (SET), long exercise test

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(LET) and provocative cold test (Timothy Miller, 2008). These are generally recorded from a small hand muscle such as the abductor digiti minimi following supramaximal ulnar nerve stimulation. In all three tests a baseline CMAP amplitude is obtained prior to exercise or cold stimuli introduction. In the SET, the patient performs a brief maximal isometric contraction for 10 to 20 seconds, and then CMAP amplitudes are recorded post- exercise at 10 second intervals over a period of a minute or until amplitude stabilizes, depending on the protocol used. In the LET, the patient performs a maximal isometric contraction for 5 minutes and CMAP amplitudes are assessed for up to an hour following exercise. Both SET and LET can be performed at room temperature or post-cooling of the muscle. Provocative cold testing requires the patient place the tested limb in cold water and CMAP amplitudes are taken before and after cooling (Matthews et al., 2010;

Timothy Miller, 2008). Responses to these tests are designated by one of five patterns – named Fournier patterns. In Fournier pattern I there is a gradual decrement of CMAP amplitude following exercise that worsens with repeated exercise and may or may not be increased by cooling of the muscle. In Fournier pattern II there is an immediate decrement of CMAP amplitude following exercise with quick recovery. With repetition of muscle contraction, the decrement is less severe. Fournier pattern III is a normal response without clinically significant decrement post-exercise (Fournier et al., 2004;

Matthews et al., 2010). Fournier pattern IV is characterized by immediate increase in amplitude after exercise followed by delayed decrement. Fournier pattern V shows similar delayed decrement in amplitude as pattern IV, but without an immediate change in amplitude (Fournier et al., 2004).

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Patients with Thomsen disease have diffuse myotonia on EMG with myotonia sometimes lasting longer with cold exposure. Fibrillation potentials and positive waves can also occur (Heatwole & Moxley, 2007). SET is usually normal, showing little to no decrement

(Fournier pattern III). Sometimes abnormalities in SET can be elucidated with cooling, with an early drop in CMAP amplitude with fast recovery and reduction in severity of the decrement upon repeated trials (Fournier pattern II) (Matthews et al., 2010).

Patients with Becker disease have diffuse myotonia on EMG. On exercise testing they will show early decrement in CMAP amplitude with fast recovery and reduction in severity on repeated trial (Fournier pattern II). Cooling typically has little effect on these results (Fournier et al., 2004; Heatwole & Moxley, 2007; Matthews et al., 2010).

In patients with chloride channelopathies, including those with myotonic dystrophy, the

LET is often inconsistent in results or normal (Fournier pattern III) (Timothy Miller,

2008).

Patients with PMC will have diffuse myotonia on EMG, most commonly in distal muscle groups (Heatwole & Moxley, 2007). They also show a gradual and sustained reduction of greater than 20% in CMAP amplitude on SET that will worsen with repeated trials

(Fournier pattern I), with slow recovery. Cooling will cause an even more significant decrement (Fournier et al., 2004; Heatwole & Moxley, 2007; Matthews et al., 2010;

Timothy Miller, 2008). LET yields similar results of decrement followed by slow recovery, often taking approximately an hour (Heatwole & Moxley, 2007; Timothy

Miller, 2008).

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The only abnormality observed in patients with SCM is often EMG myotonia (Matthews et al., 2010; Timothy Miller, 2008). SET reveals no significant decrement in CMAP amplitude with and without cooling (Fournier pattern III) and LET is inconsistent or normal (Fournier et al., 2004; Timothy Miller, 2008).

Patients with hyperPP may have an increase in CMAP amplitude during SET. LET can be crucial for diagnosis as a decrement in CMAP amplitude may not occur for 20 to 40 minutes post-exercise (Fournier et al., 2004; Heatwole & Moxley, 2007; Timothy Miller,

2008).

Patients with hypoPP lack myotonia on EMG. SET is normal (Fournier pattern III) and

LET can yield similar results to those seen in patients with hyperPP, excluding the immediate increase in CMAP amplitude (Fournier et al., 2004; Timothy Miller, 2008).

Phenotypic Algorithms

Several diagnostic algorithms have been created to aid in the differentiation of the myotonic disorders. These were especially useful prior to widespread availability of reliable and affordable genetic testing. These utilize differentiating symptoms and exercise testing patterns to aid in identification of the type of myotonic disorder that may be affecting a patient (Hans G. Kortman, Jan H. Veldink, & Gea Drost, 2012; Tan et al.,

2011; Timothy Miller, 2008).

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Genetic Testing

In recent years, access and affordability to genetic testing panels has changed the approach for diagnosis in patients with myotonic disorders. Both CLCLN1 and SCN4A are tested through sequencing technology which allows for them to be tested simultaneously, often for no additional cost to the patient, while testing for expansions in

DMPK and CNBP are each separate tests due to differences in technology. Detection rate of pathogenic expansions in DMPK and CNBP is nearly 100% in individuals carrying clinical diagnoses of DM1 and DM2 (Michael K. Hehir & Eric L. Logigian, 2013).

Limited research has been performed on the detection rate of variants in CLCN1 and

SCN4A in patients with clinical diagnoses of non-dystrophic myotonic disorders, but the false-negative rate has been reported to be as high as 20% (Michael K. Hehir & Eric L.

Logigian, 2013). Detection of variants in CLCN1 in patients with clinical diagnoses of

MC has been shown to vary between 40% and 75%. Additionally, a study investigating yield of genetic testing in CLCN1 and SCN4A found that 20% of patients in one cohort with suspicion for chloride channelopathy were found to have negative CLCN1 sequencing, but positive SCN4A sequencing. Overall yield of mutation detection in testing both genes was 93% for this cohort (Jeroen Trip et al., 2008).

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Antimyotonia Agents and Treatment

Overview of Treatment

There is a high level of variability in symptoms experienced by patients with myotonic disorders. Symptoms may be mild enough to not require any type of pharmacologic treatment. Additionally, avoidance of triggers such as cold temperatures, strenuous exercise or potassium rich foods may reduce symptoms, making pharmacologic treatment unnecessary (Matthews et al., 2010). For patients who pursue treatment for myotonic symptoms, many find significant benefit with medication and in some cases treatment of the myotonia aids in relief of other symptoms, like pain (Chad R. Heatwole et al., 2013).

There are currently no FDA approved treatments for non-dystrophic myotonia and according to a multinational study only 40% of patients with non-dystrophic myotonia were on a treatment for their myotonia (Trivedi et al., 2014). In 2006, the Cochrane review published a report summarizing the literature available on treatment for myotonia and they concluded that there was a lack of randomized trials to determine the safety and efficacy of treatments currently being utilized for this patient population (Chad R.

Heatwole et al., 2013; J Trip, GG Drost, BGC van Engelen, & CG Faber, 2006).

Treatments utilized for myotonia include anti-arrhythmic, anti-epileptic and anti- depressant medications which have shown some clinical benefit, usually in small case series or single case reports.

18

Mexiletine

Mexiletine is a sodium channel blocker and is often considered the first line of treatment for myotonia. It has been shown to relieve symptoms in patients with myotonia congenita, paramyotonia congenita and the dystrophic myotonias. It has proven to relieve patient reported stiffness as well as quantitatively improve myotonia on clinical exam

(Andersen et al., 2017; Chad R. Heatwole et al., 2013; Heatwole & Moxley, 2007).

Mexiletine is an anti-arrhythmic drug and can cause elongation of QT interval, although reports of this are rare (Chad R. Heatwole et al., 2013; Matthews et al., 2010).

Sodium Channel Blocking Medications

Other sodium channel blocking medications that have been utilized for the treatment of myotonia include Tocainide, Carbamazepine, Lamotrigine, Phenytoin, Quinine and

Procainamide (Andersen et al., 2017; Chad R. Heatwole et al., 2013; Heatwole &

Moxley, 2007; Matthews et al., 2010). Tocainide showed potential antimyotonia effects in both non-dystrophic and dystrophic myotonias, but was removed from the U.S. market due to the risk of potentially fatal side effects (Chad R. Heatwole et al., 2013; Heatwole

& Moxley, 2007; Matthews et al., 2010). In small studies, Carbamazepine has shown benefit in patients with disease due to SCN4A, CLCN1 and DMPK (Chad R. Heatwole et al., 2013). More recently, Lamotrigine has been shown to reduce myotonia in patients with non-dystrophic myotonias (Andersen et al., 2017). Phenytoin has been shown to reduce myotonia in some patients, but carries risk for neurotoxicity (Chad R. Heatwole et al., 2013). Quinine and Procainamide were both drugs trialed in patients with myotonia

19 initially, but both carry risk of serious side effects and are no longer utilized in this patient population (Chad R. Heatwole et al., 2013; Matthews et al., 2010).

Anti-depressants

Clomipramine, Amitriptyline and Imipramine are all anti-depressants that have been trialed in patients for relief of myotonia with variable success. Small trials of all three medications have shown them to have some effect in patients with myotonic dystrophy type I. Amitriptyline has been trialed in patients with SCN4A-related myotonia and had little to no effect (Chad R. Heatwole et al., 2013)

Other Medications

Acetazolamide is a diuretic that has been shown to reduce myotonia in patients with myotonia congenita, hyperkalemic periodic paralysis, myotonia fluctuans, acetazolamide responsive myotonia and paramyotonia congenita (Heatwole & Moxley, 2007; Matthews et al., 2010). It is also used in non-dystrophic patients to prevent weakness, pain and paralytic attacks. A small study of its use in patients with myotonic dystrophies showed no improvement. Its efficacy in patients with dystrophic myotonia is unclear (Chad R.

Heatwole et al., 2013).

Ranolazine, an anti-anginal medication, has been shown in small trials to improve symptoms in both patients with myotonia congenita and paramyotonia congenita. Both groups of patients had reduction in the duration of myotonia potentials on EMG and in self-reported symptoms of stiffness. In addition, paramyotonia congenita patients

20 reported improvements in symptoms of weakness and pain (Arnold et al., 2017; Lorusso et al., 2019).

Study Aims

Due to the rarity of some of the myotonic disorders, most reviews of genotype-phenotype data to date have been in the format of single case reports or small series of patients.

The purpose of this study was to retrospectively characterize the genotypes and phenotypes of patients diagnosed with clinical or electrical myotonia at The Ohio State

University Wexner Medical center who had received genetic testing as a result of this diagnosis.

There were three primary research objectives of this project:

1. To characterize the genetic and phenotypic profile of patients diagnosed with

myotonia at The Ohio State Wexner Medical Center through review of data in the

electronic medical record.

2. To compare symptom profiles of myotonic disorders to determine significant

clinical differences.

3. To summarize usage of commonly prescribed antimyotonia agents.

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Chapter 2. Methods and Data Analysis

Methods

This retrospective chart review utilized clinical data of patients diagnosed with myotonia at The Ohio State University Wexner Medical Center prior to December 2018. A feasibility analysis was performed to determine the number of patients potentially holding a relevant diagnosis. This was performed utilizing the Ohio State University

Research Data Repository i2b2 system. This system is an IRB-approved database sourced from the Wexner Medical Center’s electronic health records. A list of relevant diagnostic

(ICD-9 and ICD-10) codes was used for this i2b2 search. A complete list of codes included in this search are outlined in Table 1.

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Table 1. Diagnostic Codes Utilized in Feasibility Analysis

ICD-9/ICD-10 Diagnosis 359.39/G71.19 Myotonia fluctuans

359.21/G71.11 Myotonia atrophica

359.23/G71.13 Myotonia chondrodystrophica

359.22/G71.12 Myotonia congenita

728.85, 319, 756.50/M62.89, F79, Myotonia with intellectual disability and Q78.9 skeletal anomaly 359.24, E980.5/G71.14 Myotonia, drug-induced

359.3/G72.3 Periodic myotonia

794.17/R94.131 Myotonic changes present on EMG

V83.89/Z14.8 Carrier of myotonic dystrophy

271/E74.02 Pompe disease

796.4/R89.0 Low acid maltase in muscle determined by biopsy 792.9/R89.0 Low acid maltase levels in fibroblasts

359.0, V84.89/G71.2, Z15.89 Autosomal dominant centronuclear myopathy associated with mutation in DMN2 gene 359.89, 359.0/G72.89, G71.2 Myofibrillar myopathy

23

This search identified that these codes had been assigned 700 times to patients at the

Wexner Medical Center. It was predicted that some patients would carry more than one of these codes and that the total number of patients that would meet all study inclusion criteria would be less than this number. Patient medical record numbers (MRNs) were linked to a coded patient number which was saved on a secure server.

Charts were reviewed for myotonia diagnostic codes to determine eligibility for a complete chart review. Charts with the codes R94.131 and 794.17 were excluded after review of 45 of these charts yielded no patients with myotonia related diagnoses. The remaining unique charts (n=349) were then reviewed based on the following exclusion and inclusion criteria for eligibility:

Inclusion Criteria:

1. Genetic testing revealing a variant in CLCN1, SCN4A, DMPK or CNBP in the

patient or a first degree relative.

Exclusion Criteria:

2. Absence of clinical and electrical myotonia.

3. Finding of a pathogenic or likely pathogenic variant in a second gene related to

neuromuscular disease.

Figure 1 depicts the sorting of these 351 charts based on these criteria.

24

Figure 1. Chart Review and Sorting Process *4 with Pompe disease, 2 with CACNA1S variants, 7 with a variant in a second NM gene in addition to a gene of interest, 2 with variants of interest but no myotonia, 2 with myotonia but (-) genetic testing, 1 with clinical diagnosis of PMC but no genetic testing

Charts for Initial Review (n = 351)

Charts for Final Myotonia + (-) Review: Myotonia Genetic Testing OR No myotonia + Genetic Testing ø testing (n = 117) (n = 160) (n = 74)

Final Data Charts Excluded Analysis Group from Analysis* (n = 142) (n = 18)

CLCN1 SCN4A Variant Variant(s) DMPK Expansion CNBP Expansion (n = 15) (n = 27) (n = 89) (n = 11)

25

Chart review was performed utilizing electronic medical records of patients with a myotonia diagnosis. Patient age, gender and ethnicity were recorded from the

“demographics” tab in the medical record. Myotonia related diagnoses was recorded from the “problem list” tab. Review of neurology chart notes was performed by reviewing the oldest note available and the most recent note available (if there had been multiple appointments). If necessary information was not included in these notes, additional notes were reviewed, until information was obtained or it was determined that the information had not been recorded. Through these note reviews, age of symptom onset, patient reported symptoms (stiffness, weakness, pain, cramping), exacerbating factors and presence of warm up phenomenon were obtained. Family history information was obtained in combination of this note review and the “history” tab of the medical record.

Physical examination data including myotonia (grip, eye closure, percussion and tongue), hypertrophy, muscle weakness and sensory abnormalities, were obtained through review of chart notes. Muscle weakness was ascertained by review of manual muscle testing performed during a clinic appointment. Muscle weakness was considered to be present if the patient scored a “4” or less in any muscle group at any reviewed appointment.

Sensory abnormalities were determined by presence of abnormal response to sensory testing including pinprick, vibration and temperature. Lab values, EMG reports and genetic testing were obtained from the “results review” tab in the medical record. For individuals with more than one CK or TSH result, the highest value was selected.

Reference ranges used to determine number of individuals with abnormal values were the ranges utilized at the Wexner Medical Center: 30-220 U/L for men and 30-184 U/L for

26 women. EMG data was collected by review of all EMG results reports available. Trialed medications and duration of use were determined by review of the “medications” tab in the medical record. Medications that were recorded in the database included:

Acetazolamide, Clomipramine, Diazapam, Dichlorphenamide, Dispyramide, Imipramine,

Lamotrigine, Mexiletine, Nifedipine, Phenytoin, Prednisone, Procainamide, Quinine,

Ranolazine, Taurine, Thiazides and Tocainide. Effectiveness and side effects experienced were determined through chart note review. Summary of chart data collected is provided in Table 2. Review of charts was all performed by one individual to ensure consistency in the method of data collection. All data from the chart review was entered into the

Research Electronic Data Capture (REDCap) database. Patients coded identifiers were utilized in the REDCap system and no personal identifiers were included in the database.

27

Table 2: Chart Review Data Collection Summary

Data Collection Data Collected

Field

Demographics Age (of age of death), gender, ethnicity, myotonia diagnoses, age

of symptom onset

Patient Reported Presence of stiffness, weakness, pain and cramping, muscle groups

Symptoms affected, exacerbating factors, presence or absence of warm up

phenomenon

Family Medical Presence of myotonia in a family member, apparent pattern of

History inheritance, history of other neuromuscular disease in the family

Physical Clinical myotonia, hypertrophy, muscle weakness (including which

Examination muscle groups are affected), sensory abnormalities

Lab Work CK, TSH, acid alpha-glucosidase

EMG Data Number of EMG studies, number of muscles tested, number of

muscles with myotonia and/or spontaneous activity, exercise test

results, presence of other abnormalities on EMG

Genetic Testing Gene with mutation, type of mutation, repeat length (for DMPK),

Results mutation classification, c.DNA, p.DNA

Medication Medications utilized for myotonia, effectiveness, side effects,

History duration of use, current status of use

28

Data Analysis

The Center for Biostatistics and Bioinformatics at The Ohio State University provided statistical support and data analysis for this project. Descriptive statistics were used to create the phenotypic profile for each myotonic disorder. Fisher’s exact tests were performed in determining associations between genotype and phenotypic features.

Aim 1: To characterize the genetic and phenotypic profile of patients diagnosed with myotonia at The Ohio State Wexner Medical Center through review of data in the electronic medical record.

Descriptive statistics were utilized to create the phenotypic profile for each genotype.

Mean values were utilized for participant age and age of symptom onset. For the remaining chart review data, percentages were utilized to depict the incidence of a given phenotypic characteristic by genotype (ex. what percent of participants with a CLCN1 variant reported stiffness?).

Aim 2: To compare symptom profiles of myotonic disorders to determine significant clinical differences.

Comparison of the patients with non-dystrophic phenotypes and patients with dystrophic phenotypes was performed by combining the data from the first aim for patients with variants in CLCN1 and SCN4A to create the “non-dystrophic” group and data from patients with expansions in DMPK and CNBP to create the “dystrophic” group. Statistical comparisons between these two groups were made utilizing a Fisher’s exact test to 29 determine which phenotypic measures were significantly different based on the patient having a dystrophic or non-dystrophic diagnosis.

Comparison of patients with CLCN1 and SCN4A variants was performed utilizing data from the first aim. Statistical comparisons between these two groups were made utilizing

Fisher’s exact test to determine which phenotypic measures were significantly different based on the patient having a CLCN1 variant or a SCN4A variant.

Aim 3: To summarize usage of commonly prescribed antimyotonia agents.

Descriptive statistics were utilized to determine the proportion of each genotype group who had trialed, discontinued and were currently taking an antimyotonia agent. Within these descriptive groups, data was divided by the type of medication. Information on discontinuation was recorded as the number, regardless of genotype, discontinuing a medication for each reason (cost, side effects, lack of effectiveness, etc.). Average duration of each medication was also recorded for medications found in the chart review.

30

Chapter 3. Results

Study Participants

A total of 160 patients were identified through an initial screening as possible candidates for the study. Of the 160 identified, 27 had one or more variants in CLCN1, 15 had a variant in SCN4A, 89 had an expansion in DMPK and 11 had an expansion in CNBP. Of those remaining, 4 had a molecular diagnosis of Pompe disease, 2 had variants in

CACNA1S, 7 had variants in DMPK, CNBP, CLCN1, or SCN4A and a variant in a second neuromuscular gene, 2 had myotonia but negative genetic testing, 2 had variants in a gene of interest, but no myotonia and 1 had a clinical diagnosis of PMC but had not had confirmatory genetic testing. These 18 individuals were excluded from analysis.

Participant Characteristics

The average age of symptom onset for individuals with CLCN1, SCN4A, DMPK and

CNBP variants was 16.5 years (SD 12.1), 23.6 years (SD 21.0), 28.0 years (SD 15.9) and

40.7 (SD 12.0), respectively. Participants were further classified by age category

(neonatal, childhood, adulthood) and this data is summarized in Figure 2.

31

Figure 2. Age of Symptom Onset

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Neonatal Childhood Adulthood

CLCN1 SCN4A DMPK CNBP

The majority of participants were Caucasian and female, with 83.8% and 64.8% of all participants being identified as such in the medical record, respectively.

A total of 13 individuals included in the study were deceased at the time of chart review.

Eleven of these individuals were patients with DMPK expansions. Average age of death among this cohort was 54.3 years (SD 10.4).

Refer to Table 5 in the appendix for full demographic information on the study participants.

32

Aim 1

Patient Reported Symptoms

Patient reported history of stiffness, weakness, pain, cramping and cold as an exacerbating factor for symptoms were recorded as either being present or being absent.

Stiffness was most commonly reported in individuals with variants in CLCN1 with 100% of our cohort reporting this symptom (n = 24). Reported stiffness in the other three groups were similar with 78.6% (n = 11), 78.5% (n = 62) and 80% (n = 8) of individuals with SCN4A, DMPK and CNBP variants reporting this, respectively. Weakness was reported in a similar proportion of individuals with non-dystrophic myotonias with

65.2%, (n= 15) and 69.2% (n = 9) of individuals with CLCN1 variants and SCN4A variants reporting this. Weakness was also reported in a similar proportion of individuals with dystrophic myotonia with 90.8% (n = 79) and 90% (n = 9) of individuals with

DMPK and CNBP expansions reporting this, respectively. History of pain was reported in

100% (n = 10) of individuals with CNBP expansions. In the other three groups, pain was reported in a similar proportion of individuals, affecting 69.2% (n = 18) of individuals with CLCN1 variants, 53.3% of individuals with SCN4A variants (n = 8) and 59.5% (n =

50) of individuals with DMPK expansions. Presence of muscle cramping was most common in individuals with SCN4A variants with 50% (n = 6) reporting this symptom.

Individuals in the other three groups reported this symptom at a similar proportion:

34.8% (n = 8) with CLCN1 variants, 37.5% (n = 30) with DMPK expansions and 30% (n

= 3) with CNBP expansions. Cold as an exacerbating factor for symptoms was most commonly reported in individuals with non-dystrophic myotonias, with 40.7% (n = 11) 33 of individuals with CLCN1 variants and 46.7% (n = 7) of individuals with SCN4A variants reporting this. This was reported in 36.4% (n = 4) of individuals with CNBP expansions and in 7.9% (n = 7) of individuals with DMPK expansions. This data is summarized in Figure 3 and in the appendix in Table 6.

Figure 3. Patient Reported Symptoms

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Stiffness Weakness Pain Cramping Cold Exacerbation

CLCN1 SCN4A DMPK CNBP

34

Physical Examination

Presence of clinical myotonia, hand grip myotonia, percussion myotonia, muscle weakness and sensory abnormalities on physical exam were recorded as either present or absent in our cohort. For individuals who were not tested for one of these areas, they were not included in the statistical analysis for that variable. Most individuals in the overall cohort had at least one form of clinical myotonia with 73.9% (n = 17) of individuals with CLCN1 variants, 69.2% (n = 9) of individuals with SCN4A variants,

93.8% (n = 75) of individuals with DMPK expansions and 77.8% (n = 7) of individuals with CNBP expansions having this present on physical examination. Hand grip myotonia and percussion myotonia were reported in similar proportions across the entire cohort, being reported in 77.7% (n = 87) and 85.8% (n = 91), respectively. Hand grip myotonia was least common in individuals with expansions in CNBP with only 16.7% (n = 1) having this noted in the medical record. The other three groups had this finding in similar proportions with 70% (n = 14) of individuals with CLCN1, 75% (n = 9) of individuals with SCN4A variants and 85.1% (n = 63) individuals with DMPK expansions having this found on exam. Percussion myotonia was most commonly identified in individuals with

DMPK expansions with this being reported in 94.6% (n = 70) of these individuals. The remaining three groups had this finding reported in similar proportions with 70.6% (n =

12) of individuals with CLCN1 variants, 57.1% (n = 4) of individuals with SCN4A variants and 62.5% (n = 5) of individuals with CNBP expansions being positive for this finding. Muscle weakness was not surprisingly more common in the individuals with dystrophic myotonic disorders, with 91.8% (n = 78) of individuals with DMPK

35 expansions and 54.5% (n = 6) of individuals with CNBP expansions having weakness on exam. However, 16% (n = 4) of individuals with CLCN1 variants and 26.7% (n = 4) of individuals with SCN4A variants were also found to have weakness in at least one muscle group. Sensory abnormalities were present in at least one individual with each gene variant, with 29.2% (n = 7) of individuals with CLCN1 variants, 7.7% (n = 1) of individuals with SCN4A variants, 30.4% (n = 24) of individuals with DMPK expansions and 36.4% (n = 4) of individuals with CNBP expansions having at least one abnormal sensory evaluation. This data is summarized in Figure 4 and in the appendix in Table 7.

Figure 4. Physical Examination Results

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Clinical Myotonia Hand Grip Myotonia Percussion Weakness Sensory Myotonia abnormalities

CLCN1 SCN4A DMPK CNBP

36

EMG Data

EMG data recorded for each patient included the number of EMGs performed, the number of muscles tested, the number of muscles with myotonic potentials, the number of muscles with spontaneous activity (including myotonia) and whether there were other findings on EMG in addition to myotonia. In this cohort, 70.4% (n = 19) of individuals with CLCN1 variants, 73.3% (n = 11) of individuals with SCN4A variants, 43.8% (n =

39) of individuals with DMPK expansions and 100% (n = 11) of individuals with CNBP expansions had at least one EMG performed. The proportion of muscles with myotonia and spontaneous activity present by genotype is summarized in Figure 5. Among the subset of the cohort that had an EMG, additional EMG findings were identified in 11.1%

(n = 2, both myopathy) of individuals with CLCN1 variants, 15.4% (n = 2, 1 myopathy, 1 other) of individuals with SCN4A variants, 73% (n =27, 22 myopathy, 5 neuropathy, 3 other) of individuals with DMPK expansions and 100% (n = 11, 7 myopathy, 4 neuropathy) of individuals with CNBP expansions. Refer to Table 3 for a full summary of the additional EMG findings.

37

Figure 5. Proportion of Muscles with Myotonia and Spontaneous Activity on EMG

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% CLCN1 SCN4A DMPK CNBP

Myotonia Spontaneous

Table 3. Additional Findings on EMG

EMG Findings CLCN1 SCN4A DMPK CNBP Total

Other findings on Yes 2 (11.1%) 2 (15.4%) 27 (73.0%) 11 (100.0%) 42 (53.2%) EMG? No 9 (50.0%) 7 (53.8%) 8 (21.6%) - 24 (30.4%)

Unknown 7 (38.9%) 4 (30.8%) 2 (5.4%) - 13 (16.5%)

Neuropathy Yes 0 0 7 4 9

Myopathy Yes 2 1 22 7 32

Radiculopathy Yes 1 1 2 0 5

38

Creatine Kinase (CK) Level

CK levels were available for 81 participants. Values were similar for the non-dystrophic groups, with the average being 157.8 U/L (SD 153.6) for individuals with CLCN1 variants and 168.6 U/L (SD 137.8) for individuals with SCN4A variants. Values were higher for the individuals with DMPK and CNBP expansions with the averages being

243.4 U/L and 345.6 U/L, respectively. The proportion of individuals with an abnormal

CK level was 23.1% (n = 3) of individuals with CLCN1 variants, 37.5% (n = 3) of individuals with SCN4A variants, 54% (n = 27) of individuals with DMPK expansions and 40% (n = 4) of individuals with CNBP expansions. This data is summarized in Table

4.

Table 4. Creatine Kinase Levels

Level CLCN1 SCN4A DMPK CNBP Total CK Level Mean (SD) 157.8 (153.6) 168.6 (137.8) 243.4 (203.6) 349.6 (343.4) 235.4 (216.7) [N] {range} {64.0, 580.0} {47.0, 455.0} {13.0, 1155.0} {41.0, 963.0} [n=81] Normal vs. Abnormal 3 (23.1%) 3 (37.5%) 27 (54.0%) 4 (40.0%) 37 (45.7%) Abnormal Normal 10 (76.9%) 5 (62.5%) 23 (46.0%) 6 (60.0%) 44 (54.3%)

39

Genetic Testing

There were a total of 27 individuals with at least one variant in CLCN1. Of these, 23 individuals had one variant with apparent autosomal dominant inheritance and 4 had two variants with apparent autosomal recessive inheritance of disease. A total of 13 unique variants were identified in these individuals with 8 being classified as pathogenic, 2 being classified as likely pathogenic and 3 being classified as variants of uncertain significance

(VUS). Table 8 provides a complete list of variants identified in this cohort.

There were a total of 15 individuals with one variant in SCN4A. No individuals were identified to have more than one variant in this gene. Of these individuals, 9 unique variants were identified with 8 being classified as pathogenic and one being classified as a VUS. Table 9 provides a complete list of variants identified in this cohort.

There were 89 individuals with a pathogenic expansion identified in DMPK. Expansion length ranged from 74 repeats to 2450 repeats and 50.6% (n = 45) of reports noted mosaicism in the sample. Among individuals in this cohort, 5.6% (n = 5) were identified as having less than 100 repeats, 67.4% (n = 60) were identified as having between 100 and 1000 repeats and 27% (n = 24) were identified as having greater than 1000 repeats in at least part of the sample (in the case of mosaicism). There were 11 individuals identified with pathogenic expansions in CNBP.

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Aim 2

Non-dystrophic versus Dystrophic Myotonia

The average age of onset of symptoms was significantly younger in the non-dystrophic cohort compared to the dystrophic cohort. Individuals in the non-dystrophic cohort had an average age of onset of 18.6 years (SD 15.1) compared to 29.9 years (SD 16.0) for the dystrophic cohort (p = 0.0037).

Proportion of participants reporting stiffness, pain and cramping between these two cohorts was not significantly different. Stiffness was reported in 92.1% (n = 35) and

78.7% (n = 70) in the non-dystrophic group and dystrophic group, respectively (p =

0.0770). Pain was reported in 63.4% (n = 26) of individuals in the non-dystrophic group and 63.8% (n== 60) of individuals in the dystrophic group (p = 1.000). Muscle cramps were reported in 40% (n = 14) of individuals in the non-dystrophic group and 36.7% (n =

33) of individuals in the dystrophic group (p = 0.8374). Exacerbation of symptoms with cold temperatures and presence of muscle weakness were both significantly different among these patients. Cold was an exacerbating factor in 42.9% (n = 18) of individuals with non-dystrophic myotonia and in 11% (n = 11) of individuals with dystrophic myotonia (p < 0.0001). Muscle weakness was more commonly reported in individuals with dystrophic myotonia with 90.7% (n = 88) reporting this compared to 66.7% (n = 24) in the non-dystrophic group (p = 0.0022). Summary of patient reported symptom data between these two cohorts is included in Figure 6 and in the appendix in Table 10.

41

Figure 6. Patient Reported Symptoms in Dystrophic versus Non-dystrophic Patients *Differences reaching statistical significance (p<0.05)

100.0% * 90.0%

80.0%

70.0%

60.0%

50.0% * 40.0%

30.0%

20.0%

10.0%

0.0% Stiffness Weakness Pain Cramping Cold Exacerbation

Non-dystrophic Dystrophic

On clinical examination, presence of hand grip myotonia and sensory abnormalities were not significantly different between the two cohorts. Hand grip myotonia was found in

71.9% (n = 23) of individuals in the non-dystrophic group compared to 80% (n = 64) of individuals in the dystrophic group (p = 0.4514). Sensory abnormalities were identified in

21.6% (n = 8) of individuals in the non-dystrophic group compared to 31.1% (n = 28) of individuals in the dystrophic group (p = 0.3864). Clinical myotonia, percussion myotonia and muscle weakness were all significantly different between these groups. Clinical myotonia and hand grip myotonia were more prevalent in the dystrophic group. Clinical myotonia was seen in 92.1% (n = 82) of individuals with dystrophic myotonia compared to 72.7% (n = 26) of individuals with non-dystrophic myotonia (p = 0.0073) and 42 percussion myotonia was seen in 91.5% (n = 75) versus 66.7% (n = 16) of individuals with non-dystrophic versus dystrophic myotonia respectively (p = 0.0051). Muscle weakness identified by MMT was found in more individuals with dystrophic myotonia compared to non-dystrophic myotonia with 87.5% (n = 84) compared to 20% (n = 8) being identified respectively (p < 0.0001). Summary of clinical exam data between these two cohorts is included in Figure 7 and in the appendix in Table 11.

Figure 7. Physical Examination Results in Dystrophic versus Non-dystrophic Patients *Differences reaching statistical significance (p<0.05)

100.0%

90.0% * * *

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Clinical Myotonia Hand Grip Myotonia Percussion Weakness Sensory Myotonia abnormalities

Non-dystrophic Dystrophic

43

CK levels were not significantly different when comparing average CK level and when comparing number of individuals with abnormal results. Average CK level was 161.9

U/L (SD 144.3) in individuals with non-dystrophic myotonia versus 261.1 U/L (SD

232.4) in individual with dystrophic myotonia (p = 0.0708). Abnormal CK levels were found in 28.6% (n = 6) individuals with non-dystrophic myotonia compared to 51.7% (n

= 31) individuals with dystrophic myotonia (p = 0.0797).

CLCN1 versus SCN4A Variants

Age of symptom onset was not significantly different among individual with CLCN1 versus SCN4A variants with the average ages being 16.5 years (SD 12.1, n = 17) and 23.6 years (SD 21.0, n = 7), respectively (p = 0.3068).

No significant differences in patient reported symptoms of weakness, pain or muscle cramping or in exacerbation with cold were identified. Symptoms exacerbation with cold was reported in 40.7% (n = 11) of individual with CLCN1 variants compared to 46.7% (n

= 7) of individuals with SCN4A variants (p = 0.7540). Weakness was reported in 65.2%

(n = 15) and 69.2% (n = 9) of individuals with CLCN1 and SCN4A variants, respectively

(p = 1.0000). Pain was reported in 69.2% (n = 18) of individual with CLCN1 variants versus 53.3% (n = 8) of individuals with SCN4A variants (p = 0.3357). Cramping was reported in 34.8% (n = 8) compared to 50% (n = 6) individuals with CLCN1 variants compared to SCN4A variants, respectively (p = 0.4769). A significant difference was identified in the number of patients reporting stiffness, with individuals with CLCN1 variants more likely to experience stiffness. In our cohort, 100% (n = 24) of individuals

44 with CLCN1 variants reported stiffness compared to 78.6% (n = 11) of individuals with

SCN4A variants (p = 0.0431). Summary of patient reported symptom data between these two cohorts is included in Figure 8 and in the appendix in Table 12.

Figure 8. Patient Reported Symptoms in CLCN1 versus SCN4A Patients *Differences reaching statistical significance (p<0.05)

100.0% *

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Stiffness Weakness Pain Cramping Cold Exacerbation

CLCN1 SCN4A

No significant differences were identified between these two cohorts on physical examination. Clinical myotonia was found in 73.9% (n = 17) individuals with CLCN1 variants compared to 69.2% (n = 9) of individuals with SCN4A variants (p = 1.0000).

Hand grip myotonia and percussion myotonia were seen in 70% (n = 14) versus 75% (n =

9) and in 70.6% (n = 12) versus 57.1% (n = 4) individuals with CLCN1 variants versus

SCN4A variants, respectively (p = 1.0000, p = 0.6466). Eyelid myotonia and tongue 45 myotonia were not separated from the clinical myotonia group due to low patient numbers. Muscle weakness was exhibited in 16% (n = 4) of individuals with CLCN1 variants compared to 26.7% (n = 4) of individuals with SCN4A variants (p = 0.4439).

Sensory abnormalities were found in 29.2% (n = 7) of individuals with CLCN1 variants versus 7.7% (n = 1) of individuals with SCN4A variants (p = 0.2164). Summary of clinical exam data between these two cohorts is included in Figure 9 and in the appendix in Table 13.

Figure 9. Physical Examination Results in CLCN1 versus SCN4A Patients

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Clinical Myotonia Hand Grip Myotonia Percussion Weakness Sensory Myotonia abnormalities

CLCN1 SCN4A

Number of individuals with non-myotonia EMG findings were not significantly different.

In this cohort, 18.2% (n = 2) of individuals with CLCN1 variants and 22.2% (n = 2) of individuals with SCN4A variants were found to have at least one non-myotonia finding on EMG (p = 1.0000). 46

Neither CK levels nor number of individuals with abnormal levels were found to be significantly different between these two groups. Average CK levels were 157.8 U/L (SD

153.6) compared to 168.6 U/L (SD 137.8) in individuals with CLCN1 variants compared to SCN4A variants (p = 0.872). Comparing number of individuals with abnormal CK levels, 23.1% (n = 3) of individuals with CLCN1 variants compared to 37.5% (n = 3) of individuals with SCN4A variants had abnormal results (p = 0.6311).

Aim 3

The use of medications for myotonia was least common in individuals with DMPK expansions with 27% (n = 24) of these individuals having ever trialed an antimyotonia agent. Medications had been trialed in 85.2% (n = 23), 93.3% (n = 14) and 63.6% (n = 7) of individuals with CLCN1, SCN4A and CNBP variants or expansions, respectively. The most commonly trialed medication across all four groups was Mexiletine at 40.4% (n =

42) of individuals trialing any medication. A summary of medication use status by genotype can be found in Figure 10 and a summary of trialed medications can be found in the appendix in Table 14. Average duration of medication use for a single medication is summarized in the appendix in Table 15.

47

Figure 10. Medication Trial Status by Genotype

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% CLCN1 SCN4A DMPK CNBP

Trialed Currently Taking

More individuals with non-dystrophic myotonias were currently taking at least one medication for myotonia with 51.9% (n=14) of individuals with CLCN1 variants and

80.0% (n=12) of individuals with SCN4A variants compared to 13.5% (n=12) of individuals with DMPK expansions and 18.2% (n=2) of individuals with CNBP expansions currently doing so. See Figure 11 for summary of currently utilized medication data by genotype.

48

Figure 11. Currently Utilized Medication by Genotype

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0% Mexiletine Ranolazine Acetazolamide Lamogotrine Phenytoin

CLCN1 SCN4A DMPK CNBP

Out of the patients trialing at least one medication, none with SCN4A variants had discontinued all medication use for myotonia. Patients with dystrophic myotonias more commonly had discontinued all antimyotonia agents with 50.0% (n=12) patients with

DMPK expansions and 71.4% (n=5) patients with CNBP expansions having done so compared to 17.4% (n=4) of individuals with CLCN1 variants. Summary of reasons for discontinuation can be found in Table 16 in the appendix.

49

Chapter 4. Discussion, Implications, Limitations and Future Direction

Discussion

In this retrospective investigation, we were able to perform a comprehensive review of the medical record data from a large group with genetically confirmed dystrophic and non-dystrophic myotonic syndromes. Utilizing this data we were able to characterize phenotype profiles as well as compare the phenotypic features of these diseases.

Additionally, we reviewed antimyotonia treatment in our cohort, an aspect that has been minimally studied for this group of disorders.

Interestingly, we found that a majority of patients with non-dystrophic myotonic disorders complained of weakness and that there was no significant difference in the occurrence of these symptoms with respect to genotype (65.2% CLCN1 and 69.2%

SCN4A, p = 1.0000). This was especially surprising given that the majority of our

CLCN1 cohort had dominant mutations, typically not associated with weakness. Patient- reported weakness in non-dystrophic myotonia has been identified previously in two prospective studies; however, the proportion of individuals manifesting weakness differed by genotype. Trip et al. found that patient-reported weakness was almost twice as common in individuals with CLCN1 variants (75%) compared to SCN4A variants

(36.7%) while Trivedi et al. found episodic weakness to be approximately twice as

50 common in individuals with SCN4A variants (76.5%) compared to individuals with

CLCN1 variants (37.5%) (J. Trip et al., 2009; Trivedi et al., 2013). These disparate findings may be due to the different proportion of individuals with dominant versus recessive inheritance in the CLCN1 cohorts. The majority of the Trip et al. cohort had recessive CLCN1 mutations (previously reported associated with a higher incidence of muscle weakness), while the cohort in the Trivedi et al. study had approximately equivalent proportions of dominant and recessive mutations (Fialho et al., 2007; J. Trip et al., 2009; Trivedi et al., 2013). However, in our cohort, most patients (85%) of patients with CLCN1-related disease had dominant mutations, with the majority reporting weakness. Alternatively, some of these differences could be due to differences in symptom ascertainment. Trip et al. queried about the presence of muscle weakness, while

Trivedi et al. queried about “episodic” weakness. In future studies, it may be helpful to ascertain patient-reported weakness in several different ways to better characterize the weakness each cohort is experiencing. Regardless of these differences, our study identified that weakness is an under-recognized phenotypic feature for individuals with dominant CLCN1 mutations.

Evaluation of weakness through MMT revealed that 20% of our non-dystrophic cohort had clinically identifiable weakness. Clinical weakness has previously been described for a cohort with SCN4A variants by Matthews et al. which revealed that four out of seventeen (23.5%) had weakness, none of whom had strength of less than 4/5 (Matthews et al., 2008). This is similar to our finding for SCN4A of 26.7%. Similar studies correlating weakness identified on MMT with CLCN1 variants has not been published.

51

The discrepancy in reported and clinically detectable weakness is present in reference literature as well as in the current study. It is possible that individuals experiencing episodic weakness or weakness with a specific trigger could be missed during standard strength testing which could account for a portion of this discrepancy.

Pain was reported in a high proportion across our entire cohort, affecting 63.7%, including both the non-dystrophic and dystrophic phenotypes. Results of our study found that a similar proportion of patients with CLCN1 variants and SCN4A variants experience pain (69% vs 53%, p = 0.3357). Although painful myotonia has long been an accepted clinical feature of SCN4A-related myotonia, CLCN1 related myotonia was originally described as usually being painless (Heatwole & Moxley, 2007). Previous reports of the incidence of pain in non-dystrophic patients have been varied, but have shown higher levels of pain in individuals with SCN4A variants. In two studies, proportions ranged from 28 to 53% of individuals with CLCN1 variants and 57 to 82% of individuals with

SCN4A variants (J. Trip et al., 2009; Trivedi et al., 2013). In patients with dystrophic myotonia, we found pain in 100% of DM2 patients and 59.5% of patients with DM1.

Presence of pain is often considered more common in individuals with DM2, which our data supports. However, this differs from several published studies. In two studies, pain was present in 57% to 88% of patients with DM1 and 55% to 86% of patients with DM2, suggesting similar prevalence of pain in these groups (Parmova, 2014; Peric et al., 2015).

Trivedi et al. reported painful myotonia in 100% of DM2 patients studied, but did not have a DM1 population to compare this finding to (Trivedi et al., 2013).

52

Our study further strengthened the conclusion that individuals with CLCN1 variants are hard to phenotypically distinguish from individuals with SCN4A variants. The only aspect of symptom and clinical exam profile that differed significantly between these two cohorts was the presence of stiffness, with more individuals with CLCN1 variants reporting this symptom than individuals with SCN4A variants (100%, n=24 versus

78.6%, n=11). This differs from Trivedi et al., who found 100% of both CLCN1 and

SCN4A patients reported stiffness (Trivedi et al., 2013). Overall, this supports the findings by Trip et al. that symptom profile may not be a reliable way to diagnose individuals with non-dystrophic myotonias, suggesting that genetic testing should always include both SCN4A and CLCN1 when this phenotype is suspected (J. Trip et al., 2009)

Pharmacologic treatment history has not been extensively studied in patient with myotonia. Overall, 47.9% of our cohort had trialed at least one medication for myotonia and 28.2 % of our cohort was taking an antimyotonia medication at the time of chart review. This is similar to a study by Trivedi et al. reported that 60.6% of their cohort

(CLCN1, SCN4A, CNBP) was currently taking an antimyotonia agent, but did not specify if this value varied by genotype. Although a larger proportion of that cohort reported medications use, DM1 patients, who were least likely to take medication in our study, were not included. This lack of medication utilization cannot be explained by patient- reported symptoms alone, with 83% of our overall cohort reporting stiffness and 64% reporting pain. This gap could be due to lack of patient request for medication to treat

53 these symptoms, due to lack of FDA approved medications or due to lack of charting on medications trialed at other medical centers, but regardless this suggests that there may be a treatment gap for patients who may benefit from utilization of antimyotonia agents.

We found that individuals with non-dystrophic myotonia were significantly more likely to have trialed (88.1% non-dystrophic versus 31% dystrophic) and to be currently taking an antimyotonia agent than were the individuals with dystrophic myotonia (61.9% non- dystrophic versus 14% dystrophic) (p<0.0001). In our study, stiffness was reported in more individuals with non-dystrophic myotonia, but this difference was not statistically different and clinical elucidated myotonia (all forms) were more common in patients with dystrophic myotonias suggesting that the presence of the symptom being treated was not the driving reason for this difference. This difference could be due to providers being more likely to recommend an antimyotonia agent to patients with non-dystrophic phenotypes or that the patients are more likely to request a medication to treat their myotonia. Alternatively, it is possible that the lack of systemic complications found in patients with non-dystrophic disorders could lend toward a focus on the symptoms of myotonia in this cohort.

The percent of individuals trialing medications who remained on a medication has implications for efficacy and compliance if all other possible reasons for discontinuation

(cost, side effects, etc.) are assumed to be unaffected by genotype. A smaller proportion of individuals with dystrophic myotonias remained on a medication as compared to the proportion with non-dystrophic myotonias. In our cohort, 70.3% of individuals with non-

54 dystrophic myotonias who trialed medications were currently taking a medication as compared to 45.2% in the dystrophic myotonia group (p<0.0001). This could be due to several factors including that the efficacy of antimyotonia agents in patients with non- dystrophic myotonias may be greater than that in patients with dystrophic myotonias, that the medications used in higher proportion in the individuals with non-dystrophic myotonia are more effective than the medications used in higher proportion in the individuals with dystrophic myotonia or that compliance in individuals with non- dystrophic myotonias is better than that of individuals with dystrophic myotonias.

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Clinical Implications

This study provided data that confirmed previously recognized symptoms of myotonic disorders as well as expanded the phenotypic profiles for these disorders. Weakness and pain were described differently in our cohort than in previous studies. Patient reported weakness was reported at a similar prevalence in individuals with CLCN1 variants as

SCN4A variants. This finding contradicted prior studies, but additionally, identified weakness as a minimally described symptom of dominant CLCN1 mutations.

Additionally, pain was found in a significant proportion of individuals with all four diseases studied, with more of individuals with CLCN1 variants having pain than those with SCN4A variants, which directly contradicts many studies finding that SCN4A has a higher incidence of pain. This expansion of the phenotypic profile for these diseases can aid health professionals in identification of patients with myotonic disorders, allow better understanding of previously under described or undescribed features of disease and allow for treatment of symptoms that may have not been thought to be significant in this patient population.

The second implication of this study was in strengthening the data suggesting the high level of phenotypic overlap between CLCN1- and SCN4A-related myotonia. This study identified only one statistically significant symptom difference among the two cohorts, with stiffness being more prevalent in CLCN1. This suggests that when ordering genetic testing for individuals with suspected non-dystrophic myotonia, that panel testing

(including both genes) is the best option as the diseases cannot be reliably distinguished phenotypically. 56

Finally, this study has implications for understanding of the use of anti-myotonia agents in all four diseases. This study identified that approximately 50% of this cohort had never trialed an antimyotonia agent, despite the fact that 83% reported stiffness, suggesting that this may be an under treated symptom. Additionally, it was identified that more individuals with non-dystrophic myotonias trialed and maintained use of these medications than those with dystrophic myotonias. Differences in stiffness among these individuals was not significantly different, suggesting that the difference in medication use was not driven by symptoms prevalence. Whether the severity of stiffness or presence of other symptoms could be driving factors in this difference is unknown. However, this study suggests that there could be a gap between patients who may benefit from use of antimyotonia agents and those being prescribed.

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Limitations

Limitations of this study are similar to those of other retrospective chart reviews. These include a lack of consistency in the type and depth of information recorded in the medical record, possibility for data present in chart notes to be missed by the reviewer and therefore not included in the dataset and possibility for the patient to not be truthfully reporting symptoms or treatment response to the physician and therefore introducing false information into the data set. In addition, this study was limited by patient numbers due to the rarity of these diseases. Efforts were made to make data review and entry as consistent as possible. All patients were seen and evaluated at The Ohio State Wexner

Medical Center which limited the total number of physicians recording chart data or running and reporting test results. All data was reviewed and entered by a single individual which ensured the maximum amount of consistency in the method of chart review as well as interpretation of notes and results. Data was entered into a pre-set data entry form which also aided in the consistency of data collection.

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Future Direction

Further work could be performed with the data set collected during this study. Data from the non-dystrophic cohort could be analyzed further to search for correlations between specific genetic variants and phenotype as well as functional domain or exon and phenotype. Data from the entire cohort in this study could be utilized to create a clinical algorithm for genetic testing choice in a patients with myotonia based on their reported symptoms and clinical examination findings. Additionally, a comprehensive phenotype review of myotonic dystrophy type 1 could be performed and incidence of symptoms could be compared to DMPK repeat length which was recorded during this data collection, but not utilized for the purposes of this study. Phenotypic comparison could be attempted for the dystrophic myotonias to search for statistically significant differences among these two sub-groups of patients. Finally, patients presenting with variants in multiple neuromuscular genes could be described in case reports to better understand the implications and effect on myotonic phenotype.

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Appendix: Additional Figures and Tables.

Table 5. Participant Demographics

Level CLCN1 SCN4A DMPK CNBP Total Current Age Mean (SD) 41.5 (13.3) 52.3 (14.6) 45.9 (13.2) 56.9 (14.2) 46.7 (13.9) [N] {21.0, 69.0} {22.0, 73.0} {19.0, 72.0} {36.0, 82.0} [n=120] {range} [n=20] [n=12] [n=78] [n=10] Symptom Adulthood 8 (38.1%) 4 (36.4%) 47 (61.8%) 9 (90.0%) 68 (57.6%) Onset Childhood 11 (52.4%) 7 (63.6%) 26 (34.2%) 1 (10.0%) 45 (38.1%)

Neonatal 2 (9.5%) - 3 (3.9%) - 5 (4.2%)

Missing 6 4 13 1 24

Age at Mean (SD) 16.5 (12.1) 23.6 (21.0) 28.0 (15.9) 40.7 (12.0) 26.7 (16.5) Symptom [N] {0.5, 38.0} {4.0, 61.0} {2.0, 72.0} {20.0, 55.0} [n=85] Onset {range} [n=17] [n=7] [n=52] [n=9] Gender Female 22 (81.5%) 11 (73.3%) 54 (60.7%) 5 (45.5%) 92 (64.8%)

Male 5 (18.5%) 4 (26.7%) 35 (39.3%) 6 (54.5%) 50 (35.2%)

Race & White 17 (63.0%) 12 (80.0%) 79 (88.8%) 11 (100.0%) 119 ethnicity (83.8%) Other 2 (7.4%) - 7 (7.9%) - 9 (6.3%)

Unknown 8 (29.6%) 3 (20.0%) 3 (3.4%) - 14 (9.9%)

Age at death Mean (SD) 68.0 (.) {68.0, 54.3 (10.4) 68.0 (.) {68.0, 56.4 (10.8) [N] 68.0} [n=1] {39.0, 75.0} 68.0} [n=13] {range} [n=11] [n=1]

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Table 6. Patient Reported Symptoms

Label Level CLCN1 SCN4A DMPK CNBP Total History of Yes 24 (100.0%) 11 (78.6%) 62 (78.5%) 8 (80.0%) 105 (82.7%) Stiffness No - 3 (21.4%) 17 (21.5%) 2 (20.0%) 22 (17.3%) Unknown 3 1 10 1 15 History of Yes 15 (65.2%) 9 (69.2%) 79 (90.8%) 9 (90.0%) 112 (84.2%) Weakness No 8 (34.8%) 4 (30.8%) 8 (9.2%) 1 (10.0%) 21 (15.8%) Unknown 4 2 2 1 9 History of Pain Yes 18 (69.2%) 8 (53.3%) 50 (59.5%) 10 86 (63.7%) (100.0%) No 8 (30.8%) 7 (46.7%) 34 (40.5%) - 49 (36.3%) Unknown 1 0 5 1 7 History of Yes 8 (34.8%) 6 (50.0%) 30 (37.5%) 3 (30.0%) 47 (37.6%) Cramping No 15 (65.2%) 6 (50.0%) 50 (62.5%) 7 (70.0%) 78 (62.4%)

Unknown 4 3 9 1 17

Cold Yes 11 (40.7%) 7 (46.7%) 7 (7.9%) 4 (36.4%) 29 (20.4%) Exacerbation? No 16 (59.3%) 8 (53.3%) 82 (92.1%) 7 (63.6%) 113 (79.6%)

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Table 7. Physical Examination Results

Level CLCN1 SCN4A DMPK CNBP Total Clinical Yes 17 (73.9%) 9 (69.2%) 75 (93.8%) 7 (77.8%) 108 (86.4%) myotonia? No 6 (26.1%) 4 (30.8%) 5 (6.3%) 2 (22.2%) 17 (13.6%) Unknown 4 2 9 2 17 Hand grip Yes 14 (70.0%) 9 (75.0%) 63 (85.1%) 1 (16.7%) 87 (77.7%) myotonia? No 6 (30.0%) 3 (25.0%) 11 (14.9%) 5 (83.3%) 25 (22.3%) Unknown 7 3 15 5 30 Percussion Yes 12 (70.6%) 4 (57.1%) 70 (94.6%) 5 (62.5%) 91 (85.8%) myotonia? No 5 (29.4%) 3 (42.9%) 4 (5.4%) 3 (37.5%) 15 (14.2%) Unknown 10 8 15 3 36 Muscle Yes 4 (16.0%) 4 (26.7%) 78 (91.8%) 6 (54.5%) 92 (67.6%) weakness? No 21 (84.0%) 11 (73.3%) 7 (8.2%) 5 (45.5%) 44 (32.4%) Unknown 2 0 4 0 6 Sensory Yes 7 (29.2%) 1 (7.7%) 24 (30.4%) 4 (36.4%) 36 (28.3%) abnormalities? No 17 (70.8%) 12 (92.3%) 55 (69.6%) 7 (63.6%) 91 (71.7%)

Unknown 3 2 10 0 15

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Table 8. Genotype and Myotonia Phenotype for CLCN1 Cohort

ID c. p. Lab reported ClinVar Clinical Electrical classification Classification Myotonia Myotonia

p.Leu157Phefs* 55 c.469delC Pathogenic Pathogenic No Yes (7/11) 13

54 c.501C>G p.Phe167Leu VUS Conflicting Yes Yes (4/10)

9 c.592C>G p.Leu198Val VUS Conflicting Yes Yes (2/5)

3 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Yes (3/3)

33 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Unknown

44 c.689G>A p.Gly230Glu Pathogenic Pathogenic Unknown Yes (3/9)

49 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Unknown

59 c.689G>A p.Gly230Glu Pathogenic Pathogenic No Unknown

68 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Yes (3/3)

71 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Unknown

74 c.689G>A p.Gly230Glu Pathogenic Pathogenic Unknown Unknown

160 c.689G>A p.Gly230Glu Pathogenic Pathogenic Yes Unknown

73 c.929C>T p.Thr310Met Pathogenic Pathogenic No Unknown

Unknown 75 c.929C>T p.Thr310Met Pathogenic Pathogenic Unknown (+ exercise test)

2 c.937G>A p.Ala313Thr Pathogenic Pathogenic Unknown No (0/17)

52 c.937G>A p.Ala313Thr Pathogenic Pathogenic Yes Unknown

c.1167-10 Likely Likely 11 T>C Intronic Yes Yes (8/8) pathogenic pathogenic

Likely Likely 60 c.1444G>C p.Gly482Arg No Yes (10/10) pathogenic pathogenic

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Conflicting 5 c.1655A>G p.Gln552Arg Pathogenic Yes Yes (LP/P)

70 c.2680C>T p.Arg894Ter Pathogenic Conflicting No Unknown

72 c.2680C>T p.Arg894Ter Pathogenic Conflicting Yes Unknown

66 c.2848G>A p.Glu950Lys VUS N/a Yes Yes (1/5)

Pathogenic; Pathogenic; c.689G>A p.Gly482Arg 10 Likely Likely Yes Unknown c.1444G>C p.Gly230Glu Pathogenic Pathogenic c.568G>A p.Gly190Arg VUS Conflicting 8 Unknown Yes c.1238T>G p.Phe413Cys Pathogenic Pathogenic Pathogenic c.979G>A p.Val327Ile Pathogenic 43 Likely Yes Unknown c.1262G>T p.Arg421Leu VUS Pathogenic c.1238T>G p.Phe413Cys Pathogenic Pathogenic 7 Yes Yes (3/5) c.2680C>T p.Arg894Ter Pathogenic Conflicting Likely Likely c.409T>G p.Tyr137Asp 62 Pathogenic; Pathogenic; Yes Yes (18/18) c.1238T>G p.Phe413Cys Pathogenic Pathogenic

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Table 9. Genotype and Myotonia Phenotype for SCN4A Cohort

Lab reported ClinVar Clinical Electrical ID c. p. classification Classification Myotonia Myotonia

45 c.1333G>A p.Val445Met Pathogenic Pathogenic Yes Yes (6/6)

46 c.1333G>A p.Val445Met Pathogenic Pathogenic Yes Yes (2/2)

50 c.1333G>A p.Val445Met Pathogenic Pathogenic No Yes (3/3)

30 c.2078T>C p.Ile693Thr Pathogenic Pathogenic Yes Unknown

53 c.3917G>C p.Gly1306Ala Pathogenic Pathogenic No Unknown

76 c.3917G>C p.Gly1306Ala Pathogenic Pathogenic Yes Unknown

Unknown (+ 78 c.3917G>C p.Gly1306Ala Pathogenic Pathogenic Yes exercise test)

37 c.3938C>T p.Thr1313Met Pathogenic Pathogenic Yes Yes (7/7)

35 c.4343G>A p.Arg1448His Pathogenic Pathogenic Yes Yes (5/5)

Unknown (+ 51 c.4343G>A p.Arg1448His Pathogenic Pathogenic No exercise test)

36 c.4343G>A p.Arg1448His Pathogenic Pathogenic Yes Yes (2/2)

64 c.4372G>T p.Val1458Phe Pathogenic VUS Unknown Yes (1/22)

77 c.4386C>G p.Ile1462Met Pathogenic VUS Unknown Unknown

34 c.4765G>A p.Val1589Met Pathogenic Pathogenic Yes Yes (2/2)

61 c.5126A>G p.Asn1709Ser VUS VUS Unknown Yes (2/4)

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Table 10. Patient Reported Symptoms in Dystrophic and Non-dystrophic Cohorts *Results with statistically significant differences among the two cohorts

Non- P- Label Level Dystrophic Dystrophic Total value Symptoms of No 3 (7.9%) 19 (21.3%) 22 (17.3%) 0.0770 Stiffness Yes 35 (92.1%) 70 (78.7%) 105 (82.7%)

Exacerbation No 24 (57.1%) 89 (89.0%) 113 (79.6%) <.0001 with Cold* Yes 18 (42.9%) 11 (11.0%) 29 (20.4%) History of No 12 (33.3%) 9 (9.3%) 21 (15.8%) 0.0022 Weakness* Yes 24 (66.7%) 88 (90.7%) 112 (84.2%) History of Pain No 15 (36.6%) 34 (36.2%) 49 (36.3%) 1.0000 Yes 26 (63.4%) 60 (63.8%) 86 (63.7%) History of No 21 (60.0%) 57 (63.3%) 78 (62.4%) 0.8374 Cramping Yes 14 (40.0%) 33 (36.7%) 47 (37.6%)

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Table 11. Physical Examination Results in Dystrophic and Non-dystrophic Cohorts *Results with statistically significant differences among the two cohorts

Label Level Non-Dystrophic Dystrophic Total P-value Clinical No 10 (27.8%) 7 (7.9%) 17 (13.6%) 0.0073 myotonia* Yes 26 (72.2%) 82 (92.1%) 108 (86.4%) Hand grip No 9 (28.1%) 16 (20.0%) 25 (22.3%) 0.4514 myotonia Yes 23 (71.9%) 64 (80.0%) 87 (77.7%)

Percussion Yes 16 (66.7%) 75 (91.5%) 91 (85.8%) 0.0051 myotonia* No 8 (33.3%) 7 (8.5%) 15 (14.2%) Muscle Yes 8 (20.0%) 84 (87.5%) 92 (67.6%) <.0001 weakness* No 32 (80.0%) 12 (12.5%) 44 (32.4%) Sensory No 29 (78.4%) 62 (68.9%) 91 (71.7%) 0.3864 abnormalities Yes 8 (21.6%) 28 (31.1%) 36 (28.3%)

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Table 12. Patient Reported Symptoms in CLCN1 and SCN4A Cohorts *Results with statistically significant differences among the two cohorts

Label Level CLCN1 SCN4A Total P-value Symptoms of No - 3 (21.4%) 3 (7.9%) 0.0431 Stiffness* Yes 24 (100.0%) 11 (78.6%) 35 (92.1%) Exacerbation No 16 (59.3%) 8 (53.3%) 24 (57.1%) 0.7540 with Cold Yes 11 (40.7%) 7 (46.7%) 18 (42.9%) History of No 8 (34.8%) 4 (30.8%) 12 (33.3%) 1.0000 Weakness Yes 15 (65.2%) 9 (69.2%) 24 (66.7%) History of Pain No 8 (30.8%) 7 (46.7%) 15 (36.6%) 0.3357 Yes 18 (69.2%) 8 (53.3%) 26 (63.4%) History of No 15 (65.2%) 6 (50.0%) 21 (60.0%) 0.4769 Cramping Yes 8 (34.8%) 6 (50.0%) 14 (40.0%)

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Table 13. Physical Examination Results in CLCN1 and SCN4A Cohorts *Results with statistically significant differences among the two cohorts

Label Level CLCN1 SCN4A Total P-value Clinical Myotonia No 6 (26.1%) 4 (30.8%) 10 (27.8%) 1.0000 Yes 17 (73.9%) 9 (69.2%) 26 (72.2%) Hand Grip No 6 (30.0%) 3 (25.0%) 9 (28.1%) 1.0000 Myotonia Yes 14 (70.0%) 9 (75.0%) 23 (71.9%) Percussion Yes 12 (70.6%) 4 (57.1%) 16 (66.7%) 0.6466 Myotonia No 5 (29.4%) 3 (42.9%) 8 (33.3%) Muscle Weakness Yes 4 (16.0%) 4 (26.7%) 8 (20.0%) 0.4439 No 21 (84.0%) 11 (73.3%) 32 (80.0%) Sensory No 17 (70.8%) 12 (92.3%) 29 (78.4%) 0.2164 Abnormalities Yes 7 (29.2%) 1 (7.7%) 8 (21.6%)

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Table 14. Summary of Trialed Medications by Genotype

Level CLCN1 SCN4A DMPK CNBP Total Trialed any Yes 23 (85.2%) 14 (93.3%) 24 (27.0%) 7 (63.6%) 68 (47.9%) medication? No 4 (14.8%) 1 (6.7%) 65 (73.0%) 4 (36.4%) 74 (52.1%)

# Trialed Mean (SD) [N] 2.0 (1.1) 2.0 (0.9) 1.1 (0.3) 1.0 (0.0) 1.6 (0.9) [n=68] (if yes) {range} Medication Acetazolamide 6 (14.0%) 2 (7.1%) - - 8 (7.7%)

Lamotrigine 2 (4.7%) 3 (10.7%) 3 (11.5%) - 8 (7.7%)

Mexiletine 13 (30.2%) 9 (32.1%) 16 (61.5%) 4 (57.1%) 42 (40.4%)

Nifedipine - - - 1 (14.3%) 1 (1.0%)

Phenytoin 3 (7.0%) 1 (3.6%) 3 (11.5%) 1 (14.3%) 8 (7.7%)

Procainamide 1 (2.3%) - - - 1 (1.0%)

Quinine 2 (4.7%) 1 (3.6%) 1 (3.8%) - 4 (3.8%)

Ranolazine 16 (37.2%) 12 (42.9%) 3 (11.5%) - 31 (29.8%)

Taurine - - - 1 (14.3%) 1 (1.0%)

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Table 15. Duration of Medications Currently Utilized in Months

Medication Mean (SD) {range} [total n] Phenytoin 60.0 (.) {60.0, 60.0} [n=1]

Mexiletine 31.1 (31.5) {1.0, 108.0}

Nifedipine 28.0 (.) {28.0, 28.0} [n=1]

Acetazolamide 49.3 (56.4) {10.0, 114.0}

Ranolazine 24.7 (19.3) {2.0, 63.0}

Lamotrigine 21.4 (23.0) {3.0, 48.0}

Total 30.4 (29.5) [n=37]

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Table 16. Reasons for Medication Discontinuation *No patients discontinued medication due to allergic reaction **Not all fields will add to 100% as not all individuals discontinuing a medication had a reason noted in their char Reason Procainamide Phenytoin Quinine Mexiletine Acetazolamide Ranolazine Lamotrigine Total Cost - - 1 (33.3%) - - 2 (16.7%) - 3 (6.0%) Drug - - - - - 1 (8.3%) - 1 (2.0%) Interactions Efficacy 1 (100.0%) 3 (42.9%) - 4 (21.1%) 4 (80.0%) 4 (33.4%) - 16 (32.0%) Side Effects - 1 (14.3%) - 5 (26.3%) 1 (20.0%) 2 (16.7%) 1 (50.0%) 10 (20.0%)

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