DOI: 10.5061/dryad.z08kprrb9
title: A cross-sectional study of nemaline myopathy supplementary figures

description: Objective:  Nemaline myopathy (NM) is
 a rare neuromuscular condition with clinical and genetic
 heterogeneity. To establish disease natural history,
 we performed a cross-sectional study of NM, complemented by longitudinal assessment and exploration of pilot outcome measures.   Methods: Fifty-seven individuals with NM were recruited at two family workshops, including 16 examined at both time points. Subjects were evaluated by clinical history and physical examination. Functional outcome measures included the Motor Function Measure (MFM), pulmonary function tests (PFTs), myometry, goniometry, and bulbar assessments.  Results: The most common clinical classification was “typical congenital” (54%), whereas 42% had more severe presentations. 58% of individuals needed mechanical support, with 26% requiring wheelchair, tracheostomy, and feeding tube. The MFM scale was performed in 44/57 participants and showed reduced scores in most with little floor/ceiling effect. Of the 27 individuals completing PFTs, abnormal values were observed in 65%. Lastly, bulbar function was abnormal in all patients examined, as determined using a novel outcome measure. Genotypes included mutations in ACTA1 (18), NEB (20), and TPM2 (2). Seventeen individuals were genetically unresolved. Patients with pathogenic ACTA1 and NEB variants were largely similar in clinical phenotype. Patients without genetic resolution had more severe disease.  Conclusion: In all, we present a comprehensive cross-sectional study of NM. Our data identify significant disabilities and support a relatively stable disease course. We identify a need for further diagnostic investigation for the genetically unresolved group. Lastly, MFM, pulmonary function tests, and the slurp test were identified as promising outcome measures for future clinical trials. 
description: Our cohort included male (n=27) and female (n=30)
 participants ages 1 to 57 years old (the age at study entry was unknown
 for one participant). The diagnosis of NM was based on genetic
 confirmation of ACTA1, NEB or TPM2 pathogenic variants (n=40) or a
 clinical diagnosis of nemaline myopathy based on a muscle biopsy
 consistent with the presence of nemaline rods (n=16) or clinical features
 of nemaline myopathy with a family history of nemaline rods on muscle
 biopsy (n=1). Of note, 33/40 of the genetically established cases also had
 muscle biopsy, and all biopsies contained nemaline rods.  
 Participants were recruited at nemaline myopathy family
 conferences hosted by A Foundation Building Strength in 2009 and
 2016 (White Plains, New York). A subset of participants attended both
 conferences during which longitudinal data was collected (n=16). The
 remainder of subjects participated at one time point exclusively, either
 2009 (n=16) or 2016 (n=25). Subjects participating exclusively in 2009
 were not re-contacted, and therefore the study team could not verify
 current living status.  At study entry, one deceased individual was
 enrolled. Ethnicity was captured for 41 participants (32 White/Caucasian,
 3 Native American, 1 Middle Eastern, 4 Hispanic, 1 Asian, 1 Black/African
 American, and 8 Ashkenazi Jewish.  9/41 reported multiple
 ethnicities.   Standard Protocol Approvals,
 Registrations, and Patient Consents 
 Written informed consent was obtained according to
 the Declaration of Helsinki from all participants or a parent/legal
 guardian. The cross-sectional study was approved by the Institutional
 Review Board of Boston Children’s Hospital (IRB# M09-08-0403) (2009) and
 the Research Ethics Board at the Hospital for Sick Children (REB#
 1000053925) (2016). All participants were de-identified and provided with
 unique study identifiers.     Study Measures 
 Cross-sectional data were collected on written source
 documentation (2009) or uploaded directly into two electronic
 systems, REDCap and PhenoTips (2016). At the family conferences, the
 following measures were assessed: medical questionnaire (including
 pedigree, pregnancy, birth, developmental and medical histories), physical
 examination, motor assessments (Gowers’ maneuver, 10 meter run test, stair
 climb test, Motor Function Measure 20 or 32 (MFM20,
 MFM32)23, and myometry), goniometry, and pulmonary function
 testing.   Timed Gowers’ measured the time taken to
 rise from lying on the floor to standing. Reference values for the timed
 Gowers’ were for individuals rising from seated on the floor to standing
 and were calculated from an average of the mean values from ages 2-12
 years old 24. Reference values for the 10 meter run test for healthy
 children 2 – 12 years old (range: 4.63 – 7.95 seconds) were
 included 24. The MFM20 was used in 2009 and the MFM32 was used in 2016,
 with MFM20 values extracted from the larger MFM32 dataset to facilitate
 comparison. The MFM reflects testing in three domains, standing and
 transfers (domain one), axial and proximal motor function (domain two),
 and distal motor function (domain three). Direct muscle strength was
 measured using Medical Research Council (MRC) grading. Measure of facial
 and trunk muscle strength is not performed with MRC grading. Myometry was
 preformed to quantify muscle strength at the elbow, knee and hip joints.
 Normalized myometry values were defined by the study physiotherapist
 (calculated using weight, sex, age, and handedness). Goniometry scores
 were measured for the elbows, knees, and ankles. Categories were defined
 by the study physiotherapist (Grade 0: no
 contracture/hyperflexibility to Grade 4: most severe
 contracture). Reference values for pulmonary function testing were
 included
 for forced vital capacity (FVC) 25, maximal inspiratory pressure (MIP) 26,
 and peak cough flow (PCF) 27. Participants were excluded from FVC%
 Predicted calculation if height and weight were not captured, or if they
 were unable to perform the assessment.   Bulbar
 function was assessed via two scales: the drooling rating scale28 and the
 drooling impact scale29 and one quantitative test. For the drooling rating
 scale, drooling severity and frequency are ranked on a scale
 of 1 (severity: dry, frequency: never) to 5 (severity: profuse, frequency:
 constant). The drooling impact scale rates the extent of drooling over the
 past week using 10 questions on a scale of one (not at all) to ten (a
 lot). The slurp test was also administered which involves measuring the
 time to drink 4 ounces of water through a straw30.   A
 severity scoring system was developed by the study team to
 incorporate ambulatory, respiratory, and feeding status. Scores
 were calculated for each participant using the following formula
 (6*Ambulatory (No = 1, Yes =0)) +(2*Respiratory Support (Invasive = 3,
 Non-Invasive day and night = 2, Non-Invasive night only = 1, No support =
 0)) + (Feeding Tube (Yes = 1, No = 0)) = Severity Score.  Scores
 were categorized as mild (0-2) or not mild (3-13). Ambulatory status was
 only assessed in participants &gt;2 years of age. Clinical
 classifications were based on the criteria from Ryan et al, 2001. Two
 participants from the 2016 cohort were excluded from clinical
 classification due to incomplete data.   Data
 Analysis  Missing data was excluded from
 analyses. Analysis mainly consisted of descriptive statistics while a
 subset of data was analyzed using T- tests to determine statistical
 significance based on two-sided p-values being &lt;0.05. Data analysis
 was completed at the Hospital for Sick Children.
description: See Methods