Retrospective Cohort Analysis of Clinical, Molecular, and Histopathologic Characteristics of 275 Patients With Nemaline Myopathy

Abstract

Background and Objectives

Nemaline myopathy (NM) is a congenital myopathy with a wide severity spectrum, from severe, generalized muscle weakness and respiratory failure in the neonatal period to mild, distal weakness in young adulthood. Eleven genes have been definitively established to cause the condition. Although some recurrent variants have been identified, the overall correlation of genotype with clinical severity in NM remains poor. This poses challenges when counseling families about prognosis after a diagnosis of NM is made. Better clinical and molecular predictors of outcome are needed for clinical trial readiness.

Methods

A retrospective cohort analysis of 275 patients with a histopathologic diagnosis of NM and/or pathogenic variants in NM-associated genes was performed to identify relationships between early clinical findings and long-term outcomes, including need for respiratory and feeding support.

Results

Early clinical predictors of long-term outcomes were identified: patients with hypotonia at birth had increased odds of requiring gastrostomy tubes, and patients with respiratory distress at birth had increased odds of requiring both gastrostomy tubes and invasive ventilation. Individuals with ACTA1-NM were more likely to require feeding tubes and invasive ventilation in the first year of life compared with those with NEB-related NM, but the odds of requiring invasive ventilation were similar after the first year of age. Additional clinical information is presented by genotype and severity class.

Discussion

Neonatal findings of individuals with NM are correlated with long-term clinical outcomes, and there are some relationships between genotype and disease severity. Prospective longitudinal studies are needed to confirm these findings and evaluate for additional early clinical predictors of prognosis.

Introduction

Nemaline myopathy (NM) is a nonprogressive or slowly progressive congenital myopathy first defined by the presence of so-called nemaline rods over a half century ago.^1,2 At the severe end of the spectrum, infants can present with fractures and contractures at birth and may never gain respiratory independence or substantial antigravity movements. Individuals with a milder clinical picture experience motor delays but may have normal respiratory function. Bulbar weakness affecting speech and swallowing function is variable. Diagnostic criteria were initially established by a European Neuromuscular Centre workshop in 1998.³ Diagnosis is based on histopathologic findings of nemaline rods on muscle biopsy, clinical features of myopathy, and molecular testing of genes implicated in NM.

Nemaline rods are focal collections of Z-disc–derived proteins noted on light microscopy of Gomori trichrome–stained muscle and are readily apparent on electron microscopy as dark, electron dense collections near Z-discs. Other characteristic histopathologic findings include fiber size variation and type 1 fiber predominance.⁴ According to ClinGen, there is moderate or definitive evidence that pathogenic variants in 11 genes may cause NM: ACTA1, CFL2, KBTBD13, KLHL40, KLHL41, LMOD3, MYPN, NEB, TNNT1, TPM2, TPM3.^5,6 The final common pathogenic pathway is dysregulation of muscle thin filaments and thereby cross-bridge cycling.^7–10 There is also evidence for myopathy with NM features in some patients with pathogenic variants in TNNT3, ADSS1, MYO18B, CAP2, RYR1, and RYR3,^11–16 although none of these has been classified as having moderate or definitive evidence for NM.⁶

Diagnosis can be complicated by differences in histopathologic findings between different muscle samples from the same patient, or changes over time if a repeat biopsy is obtained. Molecular testing to identify the genetic basis is critical because of the extensive genetic heterogeneity as well as clinical and pathologic variability, including the absence of rods in some biopsies and patients. Predicting prognosis in patients with newly diagnosed NM is difficult because few reproducible genotype-phenotype correlations have been established.¹⁷ This is exemplified by the finding of marked variation in severity even within the same family.^18,19 A clinical classification (“2000 NM Classification Scheme”) was initially proposed in 2000 (eTable 1) to categorize the broad spectrum of clinical severity.²⁰ In addition to severe, intermediate, typical, and mild classes categorized by motor symptoms and respiratory impact, 2 additional classifications included a rapidly progressive adult-onset class and an “other” category with uncommon findings (ophthalmoplegia, cardiomyopathy, unusual distribution of weakness, intranuclear rods). Adult-onset NM remains incompletely understood but likely represents a distinct condition with unique pathophysiology that appears to involve immune or inflammatory dysregulation and may not be a monogenic condition.^21,22 A revised classification was proposed in 2019 (“Sewry NM Classification Scheme”) that eliminated the “intermediate” and “other” categories but conflated genotypes with phenotypes into 1 scheme with several gene-specific categories and several clinical ones²³ (eTable 1). After a further revision in 2021,²⁴ a numerical severity scoring system (“Amburgey score”) incorporating ambulation, respiratory support, and feeding support was proposed based on a prospective assessment of 57 individuals with NEB, ACTA1, or TPM2 pathogenic variants or histopathologic diagnosis of NM.²⁵ Nevertheless, characterization of multiple cohorts with each of these classifications or scoring schemes has not yielded reliable predictors of long-term outcomes in NM.

Genotype-phenotype correlation is important to provide families with an estimate of prognosis in the newborn period when critical decisions about level of respiratory support are made. When genotype is an insufficient predictor of long-term outcome, other information available in the first few months of life should be assessed for predictive power. A comprehensive understanding of the natural history of NM and a reliable way to predict disease severity are crucial in designing clinical trials for this condition. To assess the relative values of clinicopathologic and genetic diagnoses, this study reviews a large cohort of patients with clinicopathologic diagnoses of NM and/or pathogenic variants in NM-related genes. We describe and quantify common clinical characteristics and complications of individuals with NM by genotype and severity. Analysis of this cohort reveals that certain genotypes, as well as early clinical findings, are associated with increased medical interventions, including need for respiratory and feeding support.

Methods

Standard Protocol Approvals, Registrations, and Patient Consents

This study was approved by the Institutional Review Board of Boston Children's Hospital (IRB protocol 03-08-128R). Inclusion criteria required either a clinicopathologic or molecular diagnosis of NM with pathogenic variants in one of the 11 genes with moderate or definitive evidence for NM disease association as determined by the ClinGen Congenital Myopathies Gene Curation Expert Panel.⁶ A positive clinicopathologic diagnosis is defined as a clinical history/examination consistent with congenital myopathy and muscle biopsy diagnostic for NM in that individual or a similarly affected relative. Thirty-two individuals had pathologic diagnosis other than NM and were included on the basis of having mutations in one of the recognized NM genes (eTables 2–4). Patients were ascertained by physician or self-referral from multiple centers and not independently examined by study clinicians. Ascertainment biases are discussed in limitations. Patients were consented and enrolled from 1990 to 2019, and clinical data were collected from referring physicians, by medical record release procedures, and/or from the patients/families themselves. Paper charts and electronic research database records of eligible patients were reviewed. Where necessary and possible, patients were recontacted to provide missing information and clinical data were updated over time when available. Data across 5 major categories were collected: demographic information, medical history, neurologic assessments, histopathology, and molecular testing. Three hundred forty-seven charts were reviewed, and 275 contained sufficient information for analysis and were thus included in the study. Individually identifiable information on 97 patients was previously included in other published studies (eTables 2–4).^{1,4,7,8,17,19,21,25–55}

Statistical Analysis

Statistical analyses, software packages, methods, and models are summarized in eTable 5. For each analysis, all cases with available information were included (no exclusion of outliers).

Qualitative data included medical history, developmental history, examination findings, and histology findings, and the data dictionary used to encode these for statistical analysis is provided as an eAppendix. Motor development was defined as delayed if independent ambulation was achieved after 14 months of age. Patients who died before 1.5 years of age were excluded from the analysis of motor and ambulation status. Patients were classified using the 2000 NM Classification Scheme,²⁰ as well as the updated Sewry NM Classification Scheme.²³ The mean age at last clinical evaluation was 14.6 years (range birth to 76 years). Six patients were excluded from certain outcome analyses because either the pregnancy was terminated or the child was stillborn or died within hours of birth. These cases were included in comparisons of genotype with clinical severity because all 6 had severe features identified on prenatal imaging or birth examination.

Molecular Testing

Two hundred thirty-nine patients had clinical or research-based genetic testing while 36 had no testing reported. Variants underwent variant interpretation according to the American College of Medical Genetics 2015 guidelines,⁵⁶ supplemented by reports in ClinVar,⁵⁷ where available, and unpublished functional and other data are provided in eTables 2–4. Given the difficulty in identifying NEB variants because of complexity and size of the gene, patients with clinicopathologic diagnoses of NM and only 1 identifiable pathogenic or likely pathogenic variant in the NEB gene, with no evidence of disease-causing variants in other known NM genes, were classified as cases of NEB-NM for purposes of subsequent analysis.

Data Availability

Deidentified data not published within this article will be made available on request from any qualified investigator.

Results

Cohort Characteristics

Two hundred seventy-five patients, representing 233 unrelated kindreds, met inclusion criteria for this study (Table 1). Of these, 70 had a clinicopathologic diagnosis of NM without molecular confirmation of a genetic cause, because either they did not undergo genetic testing or genetic testing was nondiagnostic, 171 had a clinicopathologic and molecular diagnosis, and 7 had a molecular diagnosis without having undergone a muscle biopsy themselves or in any affected family member. Thirty-two individuals had likely pathogenic or pathogenic variants in an NM gene, but other pathologic diagnoses, including congenital fiber-type disproportion (20), cardiomyopathy (3), multiminicore disease (2), cap myopathy (3), and congenital myopathy with nonspecific histologic findings (4). Ninety-five individuals had NEB-NM, 79 had ACTA1-NM, 18 had TPM3-NM, 9 had TPM2-NM, 5 had LMOD3-NM, 7 had TNNT1-NM, 3 had KLHL40-NM, 2 had KLHL41-NM, and 2 had CFL2-NM (eTables 2–4). Sixty-three were classified as severe congenital, 59 as intermediate, 117 as typical congenital, 24 as mild/juvenile onset, and 12 as adult onset (eTable 6). Two patients with milder symptoms who fit best in the typical/congenital category did have contractures at birth (pseudocamptodactyly, talipes equinovarus, and finger contractures), and 1 had a fracture at birth (clavicle).^51,58 Scoliosis was seen in each severity class except for the adult-onset group. Fifty-six had atypical features; 6 of 236 patients with relevant information had ophthalmoplegia reported in the medical record, 36 of 219 had unusual distribution of weakness, 12 of 226 had cardiomyopathy, and 7 patients had intranuclear rods. Atypical genetic findings included 2 patients with autosomal recessive ACTA1 loss-of-function variants, 5 with autosomal recessive TPM3-NM, and 1 with de novo dominant NEB mutation presenting as congenital fiber type disproportion (Pt. 1304-1, eTables 3 and 7). Recessive cases of ACTA1-NM were invariably severe. In addition, parental mosaicism was questioned in 2 families with ACTA1-NM, but mosaicism studies were not available on these parents. One hundred fourteen patients (42% of the total cohort) had a family member with NM. There were 19 individuals born from consanguineous unions (Table 1). eTable 8 provides the total number of patients with variants in each gene and selected clinical characteristics.

Table 1.

Overall Cohort Characteristics

Total patients included	275
Ancestry, n (%)a
African American	15 (6)
Ashkenazi Jewish	24 (8)
Asian	16 (6)
European	193 (70)
Hispanic	39 (14)
Middle Eastern	13 (5)
Native American	13 (5)
Other	6 (2)
Unknown	23 (8)
More than 1 ethnicity	64 (24)
Consanguinity, n (%)b
Yes	19 (7)
Sex, n (%)
M	131 (48)
F	144 (52)
Family history, n (%)c
No	143 (52)
Yes	114 (42)
Unknown	18 (6)
Status, n (%)
Living	209 (75)
Deceased	61 (22)
Unknown/NA	5 (2)
Birth, y (mean, median, range)d	269 (1999, 2005, 1939–2017)
Age in 2021 for all living patients, n (mean, median)	205 (37.3, 32)
Age at last evaluation,e n (mean, median)	267 (14.7, 8)
Clinicopathologic diagnosis, n (%)
Nemaline myopathy	244 (89)
Cap myopathy	2 (1)
Cardiomyopathy	3 (1)
Congenital myopathy NOS	4 (1)
Congenital fiber-type disproportion	20 (7)
Multiminicore disease	2 (1)
Method of ascertainment, n (%)
Neurology clinic/consult	130 (47)
NICU	75 (27)
Affected family member	31 (11)
Other	10 (4)
Unknown	29 (11)

Abbreviation: NA = not applicable; NICU = neonatal intensive care unit; NM = nemaline myopathy; NOS = not otherwise specified.

^aSelf-reported identities. Several individuals had multiple ancestries; therefore, individual percentages do not add to 100.

^bInformation on consanguinity was not available for 141 cases.

^cFamily history of diagnosed NM or family member with consistent symptoms.

^dIncludes 3 terminations.

^eEither clinical examination or study questionnaire.

Age at Onset and Age at Diagnosis

The age at symptom onset and age at diagnosis were ascertained by genotype and severity class for patients with available information (Figure 1). The median age at onset was similar for patients with the most common molecular causes of NM: NEB, ACTA1, TPM2, and TPM3. The median at diagnosis was lowest for ACTA1, KLHL40, and LMOD3 groups. The median age at symptom onset was similar for patients with severe, intermediate, and typical/congenital NM, but the severity of these symptoms varied. Except for 1 patient with adult-onset TNNT1-NM with a dominant pathogenic variant, all patients with variants in TNNT1, KLHL40, KLHL41, LMOD3, and CFL2 had congenital onset. Patients with KLHL40-NM and LMOD3-NM were generally diagnosed shortly after birth. Cases of TNNT1-NM, KLHL41-NM, and CFL2-NM often had delayed diagnosis despite onset in the neonatal period.

Figure 1. Ages at Onset and Diagnosis by Genotype and Clinical Severity Scores.

Age at onset and age at diagnosis by genotype (A) and 2000 NM Classification Scheme (B). The vertical lines represent the interquartile range, and the horizontal line represents the median. (A) The median age at onset was 0.01 years for patients with NEB-NM (n = 95, IQR 0.01–0.5), ACTA1-NM (n = 77, IQR 0.01–0.07), TPM2-NM (n = 9, IQR 0.01–2.7), and TPM3-NM (n = 17, IQR 0.01–0.28). The median age at diagnosis for patients with NEB-NM was 2.0 years (n = 92, IQR 0.42–5.0), ACTA1-NM was 0.35 years (n = 74, IQR 0.1–3.5), TPM2-NM was 13.0 years (n = 7, IQR 2.5–50.0), and TPM3-NM was 1.3 years (n = 17, IQR 0.75–5.7). (B) The median age at onset was 0.01 years for patients with severe NM (n = 60, IQR 0.01–0.01), intermediate NM (n = 58, IQR 0.01–0.01), and typical/congenital NM (n = 112, IQR 0.01–0.25); 3.0 years for those with mild/juvenile NM (n = 22, IQR 2.0–4.25); and 46.0 years for those with adult-onset NM (n = 12, IQR 42–58). The median age at diagnosis for patients with severe NM was 0.08 years (n = 59, IQR 0.04–0.2), intermediate NM was 0.6 years (n = 57, IQR 0.76–8.75), typical/congenital NM was 2.6 years (n = 104, IQR 0.76–8.75), mild/juvenile NM was 11.0 years (n = 23, IQR 4.0–39.0), and adult-onset NM was 56.0 years (n = 12, IQR 45–62.8). IQR = interquartile range.

Survival and Loss of Ambulation

Time to death and time to loss of ambulation were evaluated using Spearman correlation. Two hundred sixty-three patients were live-born and had sufficient information available for inclusion in survival analysis. Among 61 patients who died, the mean age at death was 6.1 years and the median age at death was 0.2 years. Of 8 individuals who had information available past 65 years, 2 individuals died at age 66 and 6 lived past age 66 (Figure 2A). Survival between genotype groups with enough informative cases (NEB-NM, ACTA1-NM, and TPM3-NM groups and those without a molecular diagnosis) was compared (Figure 2B). Survival durations differed between the 4 groups of patients (p = 0.02). Although sample sizes were too small for meaningful comparisons, no patients with TPM2-NM died (age at last evaluation 7–58 years), 1 patient with TNNT1-NM died at 66 years, 2 patients with KLHL40-NM died in infancy, neither of the patients with KLHL41-NM died (follow-up ages 16 and 17 years), 3 patients with LMOD3-NM died in infancy, and neither of the patients with CFL2-NM died (follow-up ages 2.25 and 9 years).

Figure 2. Survival and Time-to-Event Analyses of Censored Data by Diagnosis and Clinical Findings.

(A) Survival of the overall cohort of patients with available information (dark line, n = 263), compared with survival of the general US population (gray line). Dashed lines represent 96% CI. Of survivors past the age of 60, 1 died of complications related to thyroid cancer and another passed in their sleep because of unknown causes. (B) Survival by genotype: no molecular cause identified (solid dark line, n = 50), NEB (dashed dark line n = 95), ACTA1 (solid gray line, n = 76), and TPM3 (dashed gray line, n = 18), among patients with available information. Of 15 patients with TPM3-NM still alive at last known follow-up, age at last follow-up ranged from 1 year to 36 years with an average of 15.6 years. Pairwise comparisons (using Benjamini-Hochberg correction to adjust for multiple testing) showed that the group without molecular diagnosis (18 of total 50 patients died) and those with NEB-NM (13 of 95 patients died) showed a difference in survival, with the NEB-NM group having higher rates of survival (p = 0.0076). (C) Survival by degree of respiratory assistance before 1 year of age: no respiratory assistance (solid dark line, n = 134), invasive respiratory assistance (dashed line, n = 51), and continuous positive airway pressure or bilevel positive airway pressure (dashed/dotted line, n = 11), among patients with available information. This analysis was based on 196 patients (5 excluded because of missing last follow-up/death time). (D) Survival by finding of respiratory failure at birth: no respiratory failure (dark line, n = 209) and presence of respiratory failure (gray line, n = 54), among patients with available information. Two hundred sixty-three patients were included in analyses (5 with missing information and 4 terminated or stillborn patients were excluded). (E) Time to complete or partial loss of ambulation of the cohort, in patients with available information (n = 212). Additional 53 patients had missing age at loss of ambulation or last follow-up and were excluded from analyses. A total of 153 patients were included in the analyses. Dashed lines represent 96% CI. These analyses omit 3 cases of TNNT1-NM.

Comparison of survival between individuals requiring invasive ventilation in the first year of life and those with positive pressure support (noninvasive ventilation), and those without respiratory support, showed different survival durations between the 3 groups of patients (p < 0.001), with patients requiring no respiratory assistance surviving longer. The median survival time in the group with invasive ventilation was 0.75 years, with 31 of 51 patients dying. Median survival time in patients with no respiratory assistance up to 1 year of age and those with positive pressure ventilation cannot be assessed because only 9 of 134 patients died in the no-support group and 2 of 11 died in the positive pressure group (Figure 2C). Survival durations were compared between patients with respiratory failure and those without this finding, showing a difference between the 2 groups of patients (p < 0.001), with patients with no respiratory failure surviving longer. The median survival time in patients with respiratory failure was 0.68 years; for patients without respiratory failure, it could not be assessed because only 26 of 209 patients died (Figure 2D).

Analysis of loss of ambulation (either complete or partial) was based on 212 individuals who survived or were followed up after age 1.5 years with known ambulation status. Sixty of them were nonambulant (average age at last evaluation 23 years), 17 required occasional use of a wheelchair or other mobility assistance, and 129 were ambulatory. Median time to complete or partial loss of ambulation in patients able to walk cannot be assessed because only 25 of 153 patients who were previously ambulatory lost ambulation (Figure 2E).

Odds Ratios

Odds ratios were used to investigate relationships between clinical findings (hypotonia at birth, respiratory distress at birth) and long-term outcomes (Figure 3A), as well as genotype and clinical findings (Figure 3B) and birth examination findings and genotype (Figure 3C).

Figure 3. Odds Ratio Analyses.

Odds ratios, modeled using the Fisher exact test, for relationships between clinical findings and long-term outcomes (A), genotype and clinical findings (B), and birth findings and genotype (C). Outcomes for each reported association are indicated on Y axes. Dots represent odds ratio, and lines represent 95% CI. Because multiple comparisons were conducted, the expected number of significant results by chance was compared with the number observed. With 18 comparisons and alpha set at 0.05, 0.9 comparisons would be expected to reject the null hypothesis (no difference in odds) by chance. The analysis found 7 comparisons whose 95% CIs exceeded the odds ratio of one, rejecting the null hypothesis. GT = gastrostomy tube.

Patients whose medical records indicated hypotonia at birth were more likely to require a gastrostomy tube (GT) (OR = 2.2, 95% CI 1.3–3.7) than patients without hypotonia at birth but were not more likely to require invasive ventilation in the first year of life or later in life. Patients whose medical records indicated respiratory distress at birth compared with those without this finding were more likely to require a GT (OR = 4.8, 95% CI 2.6–8.6), invasive ventilation in the first year of life (OR = 2.8, 95% CI 1.5–5.5), and invasive ventilation after the first year (OR = 3.3, 95% CI 1.8–6.1). Of 105 patients who required a GT, 21 eventually had the GT removed. Of the patients who required GT placement, there was no difference in odds of later removal between those with hypotonia and those with no hypotonia at birth, or between patients with respiratory distress and those with no respiratory distress at birth (Figure 3A).

Genotypic analysis reveals that severe congenital NM was more likely in patients with ACTA1-NM compared with patients with NEB-NM (OR = 5.4, 95% CI 2.31–12.11). Patients with ACTA1-NM were more likely to require invasive ventilation in the first year of life compared with patients with NEB-NM (OR = 4.7, 95% CI 1.9–11.0), but not later in life, and were more likely to require a GT (OR = 2.2, 95% CI 1.2–3.9). Patients with NEB-NM vs ACTA1-NM did not appear to have different odds of presenting with NM symptoms at birth, but patients with NEB-NM and the common exon 55 deletion were more likely to be symptomatic at birth than patients with other variants in NEB (OR = 7.15, 95% CI 1.2–78.9). In patients with a GT, there was no difference in odds of GT removal between patients with NEB-NM and those with ACTA1-NM (Figure 3B). Patients presenting with hypotonia at birth did not have higher odds of having pathogenic NEB vs ACTA1 variants identified. Similarly, patients with respiratory distress at birth did not have higher odds of having pathogenic NEB vs ACTA1 variants identified, and there was also no difference in odds of identifying pathogenic TPM2 vs TPM3 variants (Figure 3C).

Clinical Severity Scores

Amburgey et al.²⁵ calculated severity scores according to the following formula: (6 × ambulatory [no = 1, yes = 0]) + (2 × respiratory support [invasive = 3, noninvasive day and night = 2, noninvasive night only = 1, no support = 0]) + (feeding tube [yes = 1, no = 0]) = severity score. To assess utility, Amburgey scores in our cohort were compared within the following analysis groups: diagnosis category (clinical/histologic, clinical/histologic and molecular, molecular only), genotype, and 2000 NM Classification category for individuals with enough available information to calculate a score. Because data distributions were non-Gaussian, Kruskal-Wallis analysis with Dunn correction for multiple comparisons was used to compare medians. The median score of severe NM (n = 13, median = 13) was not statistically different from that of intermediate NM (n = 41, median = 9), but there were differences in median scores between severe and typical (n = 104, median = 0), severe and mild/juvenile (n = 24, median = 0), and severe and adult onset (n = 11, median = 0), as well as intermediate and typical/congenital (Figure 4A). There was no difference in median scores of typical and mild/juvenile NM. There were no differences in median scores when comparing Amburgey scores by genotype (Figure 4B).

Figure 4. Clinical Severity Scores Broken Down by Clinical Category and Genotype.

The Amburgey scoring system related to the 2000 NM Classification Scheme (A) and genotype (B) (NEB-NM n = 80, median = 1.5, mean = 4.65; ACTA1-NM n = 53, median = 0, mean = 2.76; TPM2-NM n = 9, median = 0, mean = 3; TPM3-NM n = 14, median = 1, mean = 3.79; TNNT1-NM n = 3, median = 0, mean = 6.33; KLHL40-NM n = 0; KLHL41-NM n = 2, median = 6.5, mean = 3.5; LMOD3-NM n = 2, median = 3, mean = 3; CFL2-NM n = 2, median = 0, mean = 0). *p = 0.048, ****p < 0.0001, ns = not significant. The blue vertical lines represent the interquartile range, and the horizontal line represents the median. NM = nemaline myopathy.

The 2000 NM Classification Scheme³ was compared with the updated Sewry NM Classification Scheme²³ using Spearman correlation (p < 0.0001) (Figure 5). Using the Fisher exact test, hypotonia at birth and the original severity classes were found not to be independent (p < 0.001, OR 0.34, 95% CI 0.29–0.68) and respiratory distress at birth was also not independent of the severity class (p < 0.001, OR 0.29, 95% CI 0.17–0.48). Clinical birth findings appeared to correlate with the 2000 NM Classification Scheme as expected (Figure 6).

Figure 5. Concordance Analysis of Alternate NM Classification Schemes.

Comparison of the 2000 NM Classification Scheme²⁰ and the updated Sewry NM Classification Scheme proposed in 2019.²³ The 2 schemes are significantly correlated (p 0.0001). Color legend and numbers in each box indicate the number of overlapping cases for each severity class. NM = nemaline myopathy.

Figure 6. Clinical Presentations at Birth Among Infants Eventually Categorized According to the 2000 NM Classification Scheme.

The relationship between birth respiratory distress (A) and birth hypotonia (B) with the 2000 NM Classification Scheme. NM = nemaline myopathy.

Descriptive Results

Detailed clinical and neurologic findings, histology, and information on adult-onset cases can be found in eSupplement 1 and eTables 6–11, respectively.

Discussion

Clinical severity classes are helpful to describe the degree of system involvement in individuals with NM but often have to be applied retroactively once certain milestones are achieved or missed. An ideal severity score would be predictive, but currently, there is limited information on early predictors of long-term outcome in NM. As rapid genetic diagnosis is becoming increasingly common in the prenatal and neonatal period, providing accurate prognostic information for families is important. Swift molecular diagnosis of critically ill neonates demonstrates consistent high yield,^59,60 and parents especially value the prognostic information that early and rapid molecular diagnosis can provide.^61,62 In this study, we detail clinical findings and outcomes in a large cohort of patients with NM to identify the range of clinical outcomes associated with specific genotypes and early clinical presentations.

The most common presenting symptom in this cohort of 275 patients was hypotonia (n = 87, 32%). Frequent complications included need for respiratory support and feeding support. Seventy-three patients (27%) required invasive ventilation, and an additional 40 (15%) required some degree of positive pressure ventilation while 105 patients (38%) required a GT at some point in their lives. Sixty individuals (22%) were not ambulatory at the time of data collection (at an age where ambulation would be expected), and 17 (6%) required some assistance with ambulation. Of 131 ambulatory patients, 13 required invasive ventilation and 17 required positive pressure ventilation at some time during recorded follow-up. Of the 13 patients requiring invasive ventilation, 3 were able to discontinue invasive ventilation in childhood (eSupplement 1).

The Amburgey composite scoring system is useful in prospectively following patients to determine severity over time. However, while the scoring system was able to differentiate severe from typical, mild, or adult-onset disease and intermediate from typical disease according to the 2000 NM Classification Scheme, it was not able to clearly differentiate between severe and intermediate NM or typical and mild NM in this cohort. It is important to have early clinical predictors of long-term outcomes, but to be scored, a child must be of walking age, so predicting long-term outcomes is not possible during the first years of life. Our analysis identified several genetic and early clinical risk factors that increase the odds of eventually requiring feeding or respiratory support. Patients with ACTA1-NM were more likely to require invasive ventilation in the first year of life than patients with NEB-NM. Early clinical predictors of need for invasive ventilation included respiratory distress at birth, and early clinical predictors of requiring a GT included respiratory distress and hypotonia at birth. However, once GT was placed, neither early clinical signs nor NEB vs ACTA1 variant status predicted whether GT could later be removed, perhaps in part because of variable clinical practice as some clinicians and families may opt to leave a GT in place for use in times of illness. A previous study showed equal rates of respiratory and feeding support among 8 individuals with NEB-NM and 17 with ACTA1-NM,⁶³ but another study reported 67% feeding tube use among 18 patients with ACTA1-NM and 32% among 19 with NEB-NM, and need for invasive ventilation in 39% and 25%, respectively, of these patients with ACTA1-NM and NEB-NM.²⁵ Similar to this latter study, we show that rates for invasive ventilation (any time in life) were 34% in ACTA1-NM and 23% in NEB-NM and patients with ACTA1-NM were more likely to require invasive ventilation in the first year of life compared with patients with NEB-NM. The rate of feeding tube use was 57% in ACTA1-NM and 37% in NEB-NM. Our study also confirms that individuals with ACTA1-NM are more likely to have severe disease than individuals with NEB-NM (OR 5.4). The impact of the Ashkenazi founder NEB exonic deletion (ΔExon55) was evaluated and revealed that patients with NEB-NM who were symptomatic at birth were more likely to have the ΔExon55 than other variants in NEB, as suggested previously.^27,46 Death was reported more frequently in children with NEB-NM because of deletion of exon 55 (3/13 or 23%) than in those with NEB-NM because of other pathogenic variants (10/83 or 12%), but this did not meet statistical significance (OR 2.2, 95% CI 0.56–8.99). Prospective studies analyzing both genotype and early clinical predictors will need to be conducted to determine the power of these findings.

Most patients in this cohort were symptomatic in childhood (median 0.13 years, mean 2.8 years), with diagnosis often occurring several years later (median 5.2, mean 8.2 years). Possible contributors to the overall delay in diagnosis may include limitations in genetic testing for patients born in the 20th century and early 2000s and delay in obtaining muscle biopsies, requiring sedation, in less severely affected children. Now that definitive molecular diagnosis is possible earlier, muscle biopsies are often deferred. In this cohort, most patients in each severity class had rods on muscle biopsy and it did not appear that there were major differences in histology among the severity classes (eTable 10).

The median survival of this cohort was 66 years. Survival appeared bimodal with noticeable dropoffs in the first year of life, as well as after age 60 years. Although survival for NEB-NM and ACTA1-NM groups was not statistically different, it does appear that death occurs earlier in the ACTA1-NM group. Decreased survival in patients without a molecular diagnosis compared with patients with NEB-NM may reflect that the former group was part of an older cohort when molecular testing was less available and life-prolonging medical interventions were less successful. Predictably, survival in patients with respiratory distress at birth or need for invasive ventilation in the first year of life was lower than in patients without these findings.

A previous study including 91 patients overlapping with the present cohort, but published in 2001 when molecular testing was largely limited to some ACTA1-NM and TPM3-NM cases, revealed that cardiac disease outside of neonatal critical illness is rare.⁵⁴ In our cohort, cardiomyopathy was reported in 12 of 226 informative cases (2 with NEB-NM, 5 with ACTA1-NM, 1 with TPM2-NM, 4 without molecular diagnosis). That same 2001 study⁵⁴ indicated that while respiratory failure in the newborn period was predictive of early mortality, hypotonia at birth was not. This is supported by findings in our extended cohort that neonatal respiratory distress increases odds of need for invasive ventilation and results in concomitant increase in mortality, but neonatal hypotonia did not. In the 2001 cohort, creatine kinase (CK) was infrequently elevated and EMG showed abnormalities in approximately two-thirds of tested patients. Similarly, our study showed elevated CK in only 16 of 116 informative cases and abnormal EMG in 78 of 132 (approximately two-thirds) of informative cases.

The analysis of this cohort faced several limitations because of the retrospective nature of the study. Patient data were collected over a long period and from many different institutions leading to a diversity in treatment approaches, variable recording of clinical assessments (interexaminer variability), challenges with data coding, and missing data. Clinical outcomes were grouped into broad categories to address the variability of clinical terminology in medical records from different institutions. The standard workup, including availability of molecular genetic testing, and treatment evolved over the period of this study and may have affected the characterization of patients and skewed certain outcome measurements. Survival analyses were also likely affected by the long enrollment period as the degree and success of medical management have increased. Not all patients were followed into adulthood, affecting ascertainment of long-term outcomes. Patients entered the study via physician or self-referral, which may have led to a selection bias for more severe cases. Clinical data were collected using a standardized document completed by a physician or family member, which may have introduced recall bias and missing data given the length between events and the time of reporting. Another limitation is the differing diagnostic basis for inclusion because some patients were molecularly defined while others were included on the basis of clinicopathologic data alone. While this may make it difficult to compare groups for certain analyses, it does reflect the real diagnostic process of NM, which should be considered when evaluating potential long-term predictors of clinical outcomes.

Despite the relatively large size of this NM cohort the significant genotypic variability makes it difficult to ascertain genotype-phenotype relationships because of the many smaller and underpowered subgroup comparisons that would be required. A larger international registry with prospective longitudinal data collection will be necessary to further define genotype-phenotype relationships and could aid in clinical trial recruitment. This study did not assess perceptions of carrying a diagnosis of NM or having a child with NM. It will be important to investigate perceived quality of life among the different severity classes and to involve families with NM in determining meaningful clinical outcomes for potential therapies. As therapies for NM become available, accurate early assessment of severity and prognosis will be crucial to determine eligibility. It will be important to ensure equitable access to care for these therapies. Determination of barriers to access of care and new therapies among individuals with NM should be investigated as new therapies are developed.

As development of therapies for NM progresses, it is important to understand the clinical history of the disease and determine findings detectable early in life that can predict prognosis. This retrospective cohort analysis revealed several features present on examination at birth, such as respiratory distress and hypotonia, which are related to long-term clinical outcomes. Data suggest that patients with ACTA1-NM are more likely to have severe congenital disease than patients with NEB-NM. This may prioritize therapy development for this group, as these individuals would be most likely to derive measurable benefit from novel therapies. We also show that the Amburgey scoring system can differentiate between clinically distinct severity classes. Because this score cannot be applied until after expected ambulation, early clinical predictors such as those identified in this cohort may help guide selection of candidates for new therapies. The creation of an NM registry would facilitate recruitment for a large prospective longitudinal natural history study, an important next step to inform the design of upcoming clinical trials.

Glossary

CK

creatine kinase

GT

gastrostomy tube

NM

nemaline myopathy

ΔExon55

exonic deletion

Author Contributions

C. Hildebrandt: drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; analysis or interpretation of data. C.A. Genetti: major role in the acquisition of data. T. Logvinenko: analysis or interpretation of data. W. Win: major role in the acquisition of data. P. Barraza-Flores: major role in the acquisition of data. L.H. Hayes: drafting/revision of the manuscript for content, including medical writing for content. S. Rockowitz: major role in the acquisition of data. V.-L. Lehtokari: major role in the acquisition of data. S.T. Iannaccone: major role in the acquisition of data. B.T. Darras: major role in the acquisition of data. H. Topaloglu: major role in the acquisition of data. C. Wallgren-Pettersson: major role in the acquisition of data. A.H. Beggs: drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data.

Study Funding

Subject ascertainment, enrollment, and data collection were supported by MDA602235 from the Muscular Dystrophy Association USA, R01HD075802 from the National Institute of Child Health and Human Development of NIH, A Foundation Building Strength for NM, and generous support from the Lee and Penny Anderson Family Foundation. DNA sequencing and variant analysis for some participants utilized the resources of the Boston Children's Hospital Intellectual Developmental Disabilities Research Center Molecular Genetics Core Laboratory funded by P50HD105351 from the National Institutes of Child Health and Human Development of the NIH, UM1HG008900 and U01HG011755 from the National Human Genome Research Institute of the NIH, and from Boston Children's Hospital institutional support for the Children's Rare Disease Cohorts initiative. Support for nebulin gene analysis of some participants was derived from research grants from the Sigrid Jusélius Foundation, the Academy of Finland, the Association Francaise contre les Myopathies, Muscular Dystrophy UK, the Finska Läkaresällskapet, the Medicinska understödsföreningen Liv och Hälsa and the Jane and Aatos Erkko Foundation. All funders had no role in the design and conduct of the study.

Disclosure

B.T. Darras has served as an ad hoc scientific advisory board member for Audentes Therapeutics, AveXis/Novartis Gene Therapies, Biogen, Pfizer, Sarepta, Vertex and Roche/Genentech; was a Steering Committee Chair for Roche FIREFISH and MANATEE studies and data safety monitoring board member for Amicus Inc. and Lexeo Therapeutics; he has no financial interests in these companies. He has received research support from the NIH/NINDS, the Slaney Family Fund for SMA, the Spinal Muscular Atrophy Foundation, CureSMA, and Working on Walking Fund and has received grants from Ionis Pharmaceuticals, Inc., for the ENDEAR, CHERISH, CS2/CS12 studies; from Biogen for CS11; and from AveXis, Sarepta Pharmaceuticals, Novartis (AveXis), PTC Therapeutics, Roche, Scholar Rock, and Fibrogen and has also received royalties for books and online publications from Elsevier and UpToDate, Inc. S. Iannaccone recieves research funding from NIH and DoD W81X WH 2010293, Cure SMA and industry (Astellas, RegenxBio, Capricor, Novartis, Biogen, Sarepta, PTC Therapeutics and Scholar Rock). She has served on medical advisory boards for Novartics, Biogen and Sarepta and as consultant for Genetech. L.H. Hayes reports research funding from Vertex Pharmaceuticals, Astellas Pharma, Novartis, and Biohaven Pharmaceuticals, and recieves consulting fees from Biohaven Pharmaceuticals. A.H. Beggs reports grants or contracts for studies of skeletal muscle and diagnosis and treatment of congenital myopathies from NIH, MDA (USA), A Foundation Building Strength, and the Chan Zuckerberg Initiative, and from AFM Telethon, Alexion Pharmaceuticals Inc., Avidity, Dynacure SAS, Kate Therapeutics, and Pfizer Inc. He has received consulting fees from Astellas Pharma, F. Hoffmann-La Roche, GLG, Guidepoint Global, and Kate Therapeutics; has received support for attending meetings from Kate Therapeutics and Muscular Dystrophy Association; holds equity in Kate Therapeutics and Kinea Bio; and is an inventor on a US patent describing a method for gene therapy of X-linked myotubular myopathy. The remaining authors report no disclosures relevant to the manuscript.Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.