Novel autosomal dominant TNNT1 mutation causing nemaline myopathy

Abstract Background Nemaline myopathy (NEM) is one of the three major forms of congenital myopathy and is characterized by diffuse muscle weakness, hypotonia, respiratory insufficiency, and the presence of nemaline rod structures on muscle biopsy. Mutations in troponin T1 (TNNT1) is 1 of 10 genes known to cause NEM. To date, only homozygous nonsense mutations or compound heterozygous truncating or internal deletion mutations in TNNT1 gene have been identified in NEM. This extended family is of historical importance as some members were reported in the 1960s as initial evidence that NEM is a hereditary disorder. Methods Proband and extended family underwent Sanger sequencing for TNNT1. We performed RT‐PCR and immunoblot on muscle to assess TNNT1 RNA expression and protein levels in proband and father. Results We report a novel heterozygous missense mutation of TNNT1 c.311A>T (p.E104V) that segregated in an autosomal dominant fashion in a large family residing in the United States. Extensive sequencing of the other known genes for NEM failed to identify any other mutant alleles. Muscle biopsies revealed a characteristic pattern of nemaline rods and severe myofiber hypotrophy that was almost entirely restricted to the type 1 fiber population. Conclusion This novel mutation alters a residue that is highly conserved among vertebrates. This report highlights not only a family with autosomal dominant inheritance of NEM, but that this novel mutation likely acts via a dominant negative mechanism.

TNNT1-related NEM (NEM5, MIM 605355) is a recessive disorder that, until recently, was known to occur only in the Old Order Amish of Pennsylvania. In this population all patients carry a homozygous nonsense founder mutation, c.538G>T (p.E180X), resulting in a premature stop codon in exon 11 (Johnston et al. 2000). Amish nemaline myopathy presents with tremors of the jaw and lower limbs in the neonatal period that gradually subside with age, development of progressive proximal muscle contractures, diffuse atrophy, delays in gross motor development, pectus carinatum, and respiratory insufficiency by the second year of life (Johnston et al. 2000). The p.E180X mutation results in complete loss of troponin T1 in muscle (Jin et al. 2003). In 2014, van der Pol et al. described the first case of NEM5 known outside of the Old Order Amish in a Dutch family with a compound heterozygous splice site c.309+1G>A mutation and an exon 14 deletion in TNNT1. The c.309+1G>A mutation in splice donor site of exon 8 results in abnormal skipping of this exon and the production of a shortened mRNA transcript (van der Pol et al. 2014). Deletion of exon 8 was shown to alter the function of tropomyosinbinding site 1 and the deletion of exon 14 is predicted to destabilize the TNNT1 protein, thereby reducing binding affinity and incorporation into the thin filament (Amarasinghe et al. 2016). The Dutch clinical phenotype was similar to Amish NEM with hypotonia, delayed gross motor milestones, progressive contractures, diffuse weakness with atrophy, absent reflexes, pectus carinatum, severe kyphosis, spinal rigidity, and absence of tremors, eventually causing respiratory insufficiency requiring home ventilation at age 2 (van der Pol et al. 2014). Marra et al. (2015) reported a second case of NEM5 in a Hispanic boy born from consanguineous parents and carrying a homozygous nonsense c.323C>G (p.S108X) mutation. This variant occurs in exon 9 and results in a truncated troponin T1 protein with 155 missing amino acids at its C-terminus, thereby damaging tropomyosin-binding site 1 (Amarasinghe et al. 2016). The clinical phenotype of this Hispanic male is reminiscent of the Amish NEM with hypotonia, delayed motor development without achieving crawling or independent walking and dysarthria with mild expressive language delay without cognition abnormality by 2.5 years of age (Marra et al. 2015). Other salient features include a high-arched palate, pectus carinatum, thoracic kyphoscoliosis, hip and knee contractures, facial weakness with tented mouth, prominent head lag, prominent proximal muscle weakness, and need for noninvasive ventilation and gastrostomy tube placement by 2.5 years (Marra et al. 2015). Finally, Abdulhaq et al. described nine Palestinian patients from seven unrelated families all carrying a similar homozygous c.574_577 indel TAGTGCTGT (L203*) in TNNT1 leading to a C-terminal truncation of the protein which reduces affinity of troponin T1 protein to tropomyosin (Abdulhaq et al. 2015;Amarasinghe et al. 2016). The Palestinian patients' phenotype also resembled the Amish population with the exception of rigid spine with kyphosis, stiff neck, and a transient tremor (Abdulhaq et al. 2015).
All five TNNT1 mutations described so far have involved the functionally important and evolutionarily highly conserved C-terminal region of the troponin T1 protein (Jin et al. 1998;Johnston et al. 2000;van der Pol et al. 2014;Marra et al. 2015;Mondal and Jin 2016).
We report a novel autosomal dominantly inherited missense mutation in TNNT1 gene (c.311A>T, p.E104V) with considerable intrafamilial clinical heterogeneity in a large family with Ashkenazi Jewish ancestry residing in the United States. This family is of historical importance since various members of the family were clinically and pathologically described by Spiro and Kennedy (1965) and Gonatas et al. (1966) as the first cases of hereditary nemaline myopathy.

Patient cohort
Members of an extended family with Ashkenazi Jewish ancestry, living in distant parts of the United States, had their blood samples drawn after obtaining informed consent. Information regarding the history, clinical manifestations and rate of progression, age of onset, and genetic relationships were collected. Some of the participants were evaluated personally by one of the authors (CGK, indicated by asterisk on pedigree, Fig. 1 . LMOD3 was not evaluated as it was identified as a cause of NEM after this genetic analysis had been completed (Yuen et al. 2014). The proband also underwent analysis of NEB exon 55 deletion using appropriate primers that flank or lie within the deleted exon (Anderson et al. 2004). To determine which variants found in the proband segregated with disease, the remaining 14 subjects were subsequently analyzed by PCR amplification and sequencing for TNNT1 exon 9 and NEB exon 62. Primer sequences used are available on request.

Muscle pathology
Vastus lateralis open muscle biopsies of three affected family members ( Fig. 1; subjects IV.16 [proband], IV.15, III.9) were analyzed at time of presentation or if genetic analysis indicated presence of the suspected segregating mutation. One specimen was frozen in isopentane precooled by liquid nitrogen and a small piece of muscle was fixed in 4% glutaraldehyde. Standard techniques were applied for hematoxylin and eosin (H&E) and enzyme histochemical staining. Biopsies from subjects III.9 and IV.15 underwent further structural analysis via electron microscopy using standard techniques at the Medical College of Wisconsin (MCW) Electron Microscopy Core Facility.

RNA isolation and expression analysis via reverse transcription and PCR
Total RNA was isolated from human muscle tissue using Trizol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). Genomic DNA was removed from RNA by incubation with TURBO DNA-free kit (Thermo Fischer Scientific, Vilnius, Lithuania) at 37°C for 30 min. Using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, CA, USA), 400 ng of total RNA was reverse transcribed to generate cDNA. PCR was performed with Taq polymerase, one denaturation step at 98°C for 5 min, 28 cycles of amplification at 98°C for 15 sec, 67°C for 30 sec, 72°C for 1 min, and a final step at 72°C for 5 min using the specific human primers for TNNT1 exon 5 (forward: GGCTCAGCCTCAAGATTCAC, reverse: TCCAGCAGGTCTTTCTCCAT), for TNNT1 exon 9 (forward: TGGAGCTGCAGACACTCATC; reverse: TTACCACGCTTCTGTTCTGC), and for TNNT1 exon 12  (forward: GCAGAACAGAAGCGTGGTAA, reverse: GCC ATCAGGTCGAACTTCTC). The PCR products were analyzed by electrophoresis with 10% polyacrylamide native gel, stained with SYBR gold nucleic acid gel stain (Thermo Fisher Scientific, Eugene, OR, USA, 1:10,000) and visualized with gel imaging system (Bio-Rad, Hercules, CA, USA). The imaging quantification of each PCR product was performed using ImageJ quantification. The muscle protein extracts were resolved on 14% Laemmli gel with an acrylamide to bis-acrylamide ratio 180:1 and transferred onto PVDF membrane. After blocking with 5% nonfat dry milk (NFM) in Tris-buffered saline (TBS, 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5), the membrane was either incubated with mouse monoclonal antibody anti-slow troponin T (clone CT3, Santa Cruz, sc-20025, 1:500) or with rabbit monoclonal anti-GAPDH (clone 14C10, Cell Signaling, 2118, 1:2000). Then, the membrane was washed three times and incubated with anti-mouse or anti-rabbit peroxidase antibody (GE Healthcare Life Sciences, 1:10,000) for 1 h in TBS-5% NFM, followed by revelation using ECL chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA).

Clinical features
The proband, an 18-year-old Ashkenazi Jewish male (Fig. 1, Table 1, IV.16) born to nonconsanguineous parents, presented at 8 years of age with mild facial and palatal weakness and minor fatigue of his lower extremity muscles when swimming. He exhibited very mild progression of proximal weakness with age, manifesting as fatigue in the legs during prolonged exercise. Examination showed a slender build, high-arched palate, elongated facies, pectus carinatum, mild scoliosis, mild bilateral isolated Achilles contractures, myopathic facies, mild quadriceps and ankle dorsiflexion weakness, poor jump, and reduced reflexes only in the upper extremities. Of note, his intellect, extraocular muscle function, cardiac function, and breathing and swallowing functions were normal. EMG performed at age 11 years demonstrated normal nerve conductions with evidence of a nonirritable myopathy in proximal and distal lower extremity muscles (data not shown). Creatine kinase (CK) was mildly elevated at 249 (reference 30-150 IU/L). The family history of the proband revealed multiple cases of myopathy in three different generations and a history of consanguinity with individual I.1 being the half-uncle of I.2 and individuals III.8 and III.9 being third cousins (Fig. 1). Table 1 summarizes the age of onset, presenting symptoms, associated clinical features, and relevant studies of studied members of this large family.
Symptoms in the proband's brother (IV.15) and father (III.9) started at 15 and 20 years of age, respectively, both of whom represented milder phenotypes than the proband with self-reported complaints of poorer endurance in the legs during weight training compared to their peers ( Fig. 1 and Table 1). Both exhibited no focal weakness and were fairly athletic, although mild myopathic facies with high-arched palates were noted. The proband's brother could not heel walk and had reduced reflexes in the upper extremities. The proband's father could heel walk and had intact reflexes, but developed intermittent mild dysphagia at 65 years of age with normal levels of creatine kinase (CK) at 176 IU/L (reference 30-220 IU/L).
A broad spectrum of clinical heterogeneity was observed in this family. The earliest onset of weakness was in childhood ranging from ages 5 to 10 years for subjects IV.16 (proband; onset at age 8 years), II.1 (onset at age 8 years), III.2 (onset at age 10 years), III.4 (onset at age 5 years) ranging from a Gower's maneuver for subject III.4 to mild waddling gait and inability to walk on heels for subject III.2 (Table 1). The oldest affected member of the family, subject II.2 died at age 80 years from presumably unrelated stroke and complications of heart failure, despite having onset of muscle weakness at 8 years of age (Table 1). The overall pattern of weakness was proximal, manifesting as a Trendelenburg gait, Gower's maneuver, or difficulty in performing repeated exercises that require proximal muscle strength. However, isolated ankle dorsiflexion weakness was a common early distal manifestation. Several subjects exhibited pectus carinatum (III.5, III.6, IV.16; Table 1). Mild scoliosis or kyphosis was seen in IV.16 and III.6 with onset in the teens (Table 1). High-arched palate and/or myopathic elongated facies was seen in subjects IV.16, III.4, III.6, III.9, IV.13, and IV.15 (Table 1). The weakness progressed slowly over time and the most severely affected members were still independently ambulatory in their 50-60s (III.2, II.4, III.5) and subject II.1 was ambulatory over short distances with a walker until her time of death at age 80 years (Table 1). Subjects IV.16 and III.9 reported subjective symptomatic improvement in exercise endurance after taking L-tyrosine 2 g/day for at least 1 year (Table 1).
Subject IV.7 was unavailable for clinical or molecular testing, however, his mother reported weakness (Table 1). Subject II.5 died at the age of 55 years of encephalitis and II.6 died at the age of 40 years from glomerulonephritis prior to developing any significant symptoms (Table 1).

Muscle pathology
Skeletal muscle biopsies of the vastus lateralis in subjects III.9, IV.15, and IV.16 identified severe type 1 fiber hypotrophy (diameter 10-70 microns), with numerous red/purple-staining rod-like structures on Gomori trichrome exclusively in type 1 fibers ( Fig. 2A-C, muscle histopathology). These inclusions were distributed unevenly throughout the sarcoplasm. Type 2 fibers in the specimens were hypertrophic (diameter 90-130 microns), and there was type 1 fibers predominance (ranging from 60% to 90% type 1 fibers) across all three biopsies. Mild increased internal nucleation was present. There were no cores, inflammation, or increased endomysial connective tissue. Electron microscopy for III.9 and IV.15 confirmed the presence of electron dense bodies consistent with nemaline rods in the former, however, not in the latter (likely due to sampling; Fig. 2D). Unlike the patients studied here (III.9, IV.15, and IV.16), histopathology of subjects II.1 and III.2 published in 1965 demonstrated presence of nemaline rods in both type 1 and 2 fibers (Fig. 1, Table 1) (Spiro and Kennedy 1965). In contrast, muscle pathology for subjects III.5 and III.6 revealed nemaline rods mostly in type 1 and rarely in type 2 fibers with type 2 fiber hypertrophy, recapitulating our findings (Fig. 1, Table 1) (Spiro and Kennedy 1965;Gonatas et al. 1966).

Mutation detection
Genetic testing in the proband demonstrated the presence of a novel heterozygous missense variants of unknown clinical significance in exon 9 of the gene TNNT1 c.311A>T (p.E104V) (NM_003283.4) ( Fig. 3A and B, electropherogram). The TNNT1 c.311A>T change is located at the 3 0 splicing site of the evolutionarily conserved exon 9 among vertebrates (Jin et al. 1998). This variant was absent from the NHLBI EVS and the 1000 Genomes database (Genomes Project C, Abecasis, et al., 2012) and the Exome Aggregation Consortium (ExAC, Figure 2. Pathological analysis of quadriceps muscle (A-D) reveals considerable fiber size disproportion and presence of nemaline rods. H&E (A) demonstrates clusters of atrophic type 1 fibers and hypertrophic type 2 fibers with nemaline rods as dark red inclusions (arrowheads). These same inclusions are better visualized on Gomori trichrome (B) as purple/blue inclusions (arrowhead) almost exclusively localized to the atrophic type 1 fibers. The ATPase stain (C) at pH 4.3 demonstrates absence of staining in the location of rods (yellow circles) in the hypotrophic type 1 fibers (dark staining). Electron microscopy (D) demonstrates the electron dense nemaline rods (arrowhead). Scale bar = 50 lm for panels A-C and 500 nm for panel D. Cambridge, MA; URL: http://exac.broadinstitute.org). Furthermore, molecular analysis in the proband revealed no significant sequence variants or unknown variants in ACTA1, NEB, CFL2, TPM2, TPM3, KBTD13, KLHL40, and KLHL41 genes. PCR detection using primers that flank the documented deletion of NEB exon 55, an Ashkenazi Jewish founder mutation, was also negative (Anderson et al. 2004). Subjects III.2, III.4, III.5, III.6, and III.9, all of whom have known nemaline rods on pathology, also have the TNNT1 c.311A>T (p.E104V) change (Table 1). Subject II.2 was deceased at the time of this study, thus mutation analysis was not possible. None of the six unaffected family members tested had the TNNT1 c.311A>T mutation. Two variants already reported in individuals not affected with congenital myopathy, NEB c.8734 T>C (p.S2912P) and c.8801G>A (p.R2934H), were found in several individuals of the family but did not segregated with disease. Therefore, only the TNNT1 mutation segregated with clinical and pathological phenotype.
The glutamic acid residue at position 104 is highly conserved in human slow skeletal muscle troponin T1, cardiac troponin T2, and fast skeletal muscle troponin T3 (Jin and Chong 2010;Wei and Jin 2016). The E104 is located in the highly conserved tropomyosin-binding site 1 region among vertebrates of the troponin T1 protein, suggesting its importance in the structure and/or the function of troponin T1 (Amarasinghe et al. 2016;Mondal and Jin 2016;Wei and Jin 2016).

Splicing and protein level analysis
TNNT1 consists of 14 exons and encodes for a 30-to 35-kDa protein with variable N-terminal regions and conserved middle and C-terminal regions (Jin et al. 2003). The mutation c.311A>T is located in exon 9, two nucleotides from the intron 8-exon 9 junction at the splice acceptor site (Figs. 3B, 4A). We first determined whether the mutation was associated with abnormal splicing of exon 9 in RNA extracted from vastus lateralis muscle biopsies of the proband (IV.16), affected father (III.9), and two age-matched controls (Fig. 4B). By reverse transcriptase PCR using primers in exons 8 and 11, we found that TNNT1 exon 9 was normally included in TNNT1 transcripts from both patients ( Fig. 4B; upper panel). We then examined the inclusion of exon 5 and exon 12 0 (a longer version of exon 12) which were reported to be alternatively spliced exons (Gahlmann et al. 1987;Samson et al. 1994;Jin et al. 1998;Zhang et al. 2014). Indeed, previous work reported that resistance training increased expression of TNNT1 mRNA isoforms without exons 5 and 12 0 in vastus lateralis, whereas a sedentary lifestyle increased the inclusion of these exons (Zhang et al. 2014). In addition, Larsson et al. determined that expression of the TNNT1 isoform missing exon 5 was increased in the demyelinating forms of Charcot-Marie-Tooth (CMT type 1) but not in axonal form of the disease (CMT type 2) (Larsson et al. 2008). We did not observe any inclusion of exon 12 0 in vastus lateralis muscle biopsies of the proband (IV.16) and his affected father III.9 ( Fig. 4B; lower panel). In contrast, transcripts without exon 5 were increased in both patients compared to agematched controls (Fig. 4B, middle panel and Fig. 4C). Consistent with this result, immunoblot of proteins extracted from vastus lateralis muscle biopsies identified increased levels of a low-molecular-weight band in both patients (Fig. 4D). This short isoform of troponin T1 protein was previously shown to result from skipping of exon 5 (Larsson et al. 2008). Notably, the c.311A>T (p.E104V) was not associated with reduced level of the protein in muscles from patients compared to controls (Fig. 4D).

Discussion
This study describes the first TNNT1 mutation that transmits in an autosomal dominant fashion to cause nemaline myopathy. TNNT1 encodes troponin T type 1, which is exclusively found in slow skeletal muscle (type 1 fibers) and serves to anchor the troponin complex (along with troponin C and I) onto the tropomyosin-actin thin filaments (Nadal-Ginard and Mahdavi 1989;Jin et al. 2003;Wei et al. 2014). Three homologous genes encode different isoforms of troponin T in specific tissues: slow skeletal muscle troponin T (TNNT1), fast skeletal muscle troponin T (TNNT3), and cardiac troponin T (TNNT2) with alternative splicing of each gene conferring protein variants with slightly different functional abilities (Samson et al. 1994;Jin et al. 2003;Wei et al. 2014). Troponin T1 regulates the conformational changes in the thin filaments during excitation-contraction-coupling of slow skeletal muscle (Wei et al. 2014;Wei and Jin 2016).
All of the previously described mutations were autosomal recessive cases of TNNT1-related NEM (NEM5) (Johnston et al. 2000;van der Pol et al. 2014;Abdulhaq et al. 2015;Marra et al. 2015). The TNNT1 c.311A>T nucleotide substitution predicts a p.E104V missense mutation which alters the nature of the amino acid from a polar to a nonpolar residue and is predicted to be pathogenic by polyphen (Adzhubei et al. 2010). The amino terminal segment undergoes alternative splicing, with studies indicating that although this region does not bind any other thin filament protein, it plays a regulatory role in the conformational changes in troponin T1, thus modulating muscle contraction and interactions with the myofilament (Jin et al. 1998;Amarasinghe et al. 2016). In contrast to the previously described recessive TNNT1 mutations resulting in truncation or reduction of troponin T1 protein levels, we describe the first heterozygous missense mutation with evidence of intact (albeit apparently functionally altered) TNNT1 protein. Levels of troponin T1 were not affected by the mutation, and the TNNT1 c.311A>T mutation located at the second nucleotide position of exon 9 did not alter the constitutive splicing of TNNT1 exon 9 containing the mutation. Troponin T1 binds tropomyosin to link the troponin complex to actin via two tropomyosin binding sites present in the highly conserved regions corresponding to residues 64 to 108 and residues 180 to 204, respectively (Jin and Chong 2010;Amarasinghe et al. 2016;Mondal and Jin 2016). The p.E104 residue is located within tropomyosin-binding site 1 (Jin and Chong 2010;Mondal and Jin 2016). We hypothesize that the mutation of a glutamic acid residue into valine at this particular position can affect the affinity of troponin T1 for tropomyosin.
A low-molecular-weight (LMW) troponin T1 protein isoform that results from alternative splicing of exon 5 of TNNT1 (Jin et al. 1998;Zhang et al. 2014) was observed in both the affected proband and his father. The shorter troponin T1 is developmentally regulated: troponin T1 LMW is absent in the fetus, appears in term the newborn, and decreases with age in human quadriceps muscle (Jin et al. 1998;Zhang et al. 2014). Larsson et al. (2008) determined that expression of the LMW troponin T1 was increased in the demyelinating forms of Charcot-Marie-Tooth (CMT) type 1, presumably due to compensatory overuse of the muscle, in the setting of a reinnervating and intact axon, in comparison to CMT type 2, which has axonal destruction. We hypothesize that the shorter band identified on the immunoblot from the affected individuals could represent an upregulation of LMW TNNT1 as a physiological adaptation in patients carrying the TNNT1 c.311A>T mutation. If this alternative splicing is a physiological response to nemaline myopathy in patients with the c.311A>T mutation, further investigation is necessary to determine whether this mechanism extends to other TNNT1 mutations or other NEM-related gene mutations.
Clinically, the TNNT1 c.311A>T heterozygous missense autosomal dominant mutation results in a mild phenotype with considerable clinical heterogeneity, whereas the recessive truncating and internal deletion mutations with loss of function are associated with severe, early-onset phenotypes (Mondal and Jin 2016). Variable expression is a common feature of autosomal dominant diseases (Strachan and Read 2011). In our family, severity ranged from mild difficulty with weight-training exercises to a Gower's maneuver in early childhood (age 8). While the severe recessive mutations resulted in respiratory insufficiency and ventilatory assistance by 2 years of age, none of the members of this family have required ventilatory assistance even into their late 60s. Pectus carinatum, a common finding in individuals with the autosomal recessive form of the disease, is found in several members of this family and seem to correlate with increased severity. Although nonspecific, blunted upper extremity reflexes, high-arched palate, ankle dorsiflexion weakness resulting in poor heel walk, and mild myopathic facies were the Schematic representation of the TNNT1 pre-mRNA. The 311A>T mutation is located in the 3 0 splicing site of the constitutive exon 9 of TNNT1 gene, with the boxes representing the exons and the black lines representing the introns. The alternative exon 5, the constitutive exon 9, and the alternative exon 12 0 (partial retention of intron 11) are shown in blue, orange, and green boxes, respectively. Forward (F1, F2, F3) and reverse primers (R1, R2, R3) used for the detection of the TNNT1 splicing isoforms by reverse transcriptase RT-PCR are represented with arrows. Primers F1 and R1, F2 and R2, F3 and R3 were used to analyze the splicing of the exons 5, 9, and 12 0 , respectively. (B) RT-PCR analysis of the splicing of TNNT1 exon 9 (upper panel), exon 5 (middle panel), and exon 12 0 (lower panel) in vastus lateralis muscle from control (black) and affected subjects IV.16 and III.9 (red) carrying the 311A>T mutation. Exons 5, 9, and 12 0 inclusions (+5, +9, +12 0 ), exclusions (À5, À9, À12 0 ), and expected sizes of the PCR products are indicated on the right of each panel. (C) Quantification of TNNT1 transcripts with exclusion of exon 5 in vastus lateralis muscle from control and affected subjects IV.16 and III.9 carrying the 311A>T mutation. The percentage of the exon exclusion was calculated by the ratio of the intensity of the upper PCR product relative to the sum of two products on acrylamide gel. (D) Total protein extracted from vastus lateralis muscle biopsies was analyzed by immunoblotting for troponin T1 protein with the monoclonal CT3 antibody. Fulllength protein is observed at the expected molecular weight. A low-molecular-weight troponin T1 isoform reported to result from mRNA with exon 5 skipping (Larsson et al. 2008) is detected (asterisk). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control. most common clinical hallmarks of our affected subjects. The impact on life span appears fairly negligible in this family, since multiple affected family members are independently ambulatory into in their 60s and subject II.1 survived to 80 years of age despite having mild symptoms since childhood. Although contractures were mild and limited to the Achilles tendon in this cohort, they were progressive and present proximally in the recessive forms of the disease. Tremors were characteristically absent in our cohort.
The fiber type disproportion found on most of the muscle biopsies suggests that the abnormality more profoundly impacts type 1 fibers. This is not surprising, as troponin T1 localizes to the type 1 muscle fibers and Wei et al. (2014) demonstrated that TNNT1 deficiency results in approximately 40% reduction in cross-sectional area of type 1 fibers. While the previous reports in the Amish and Dutch NEM5 families recapitulate the fiber type disproportion with type 1 hypotrophy, it is unclear whether the nemaline rods are primarily restricted to type 1 fibers in all individuals with TNNT1 mutations (Johnston et al. 2000;van der Pol et al. 2014). Marra et al. described the presence of nemaline rods in roughly 80% of type 1 and 2 fibers in their case study (Marra et al. 2015). The same pathologic finding of fiber type disproportion with atrophic type 1 fibers has been described in patients with mutations in TPM3, another protein known to cause congenital myopathy and primarily expressed in slow skeletal muscle (Laing et al. 1995;Lawlor et al. 2010;Ottenheijm et al. 2011;Marttila et al. 2014). This report illustrates that the newly identified TNNT1 c.311A>T mutation can produce NEM with hypotrophy and nemaline rods restricted to the type 1 fiber population, and such cases should prompt genetic screening for both the TNNT1 and TPM3 genes in patients. The type 2 fibers in the three cases reported here display marked hypertrophy, which we presume is a compensatory adaptation to hypotrophic type 1 fibers. This compensatory hypertrophy may mitigate symptomatic weakness and suggest a role for exercise or hypertrophic agents in the management of these patients.
Three members of the family (III.9, IV.15, IV.16) reported subjective improvement in endurance after taking L-tyrosine 2-3 g/day for several months. Ryan et al. reported improved sialorrhea and muscle strength in infants with ACTA1 nemaline myopathy, presumably due to increased peripheral catecholamine synthesis improving sympathetically mediated salivary gland function (Ryan et al. 2008). Of note, other sympathetic systemic features, such as tachycardia and hypertension, did not occur in the Australian cohort (Ryan et al. 2008) or in our patients.
TNNT1 appears to be emerging as a causative gene for both recessive and dominant nemaline myopathy outside the Old Order Amish population. A pattern of myofiber hypotrophy and nemaline rods restricted to type 1 fibers was specifically seen in muscle biopsies of these TNNT1 missense mutant patients. In addition to classic infantile NEM, we propose that TNNT1 should be considered in mild cases with nemaline rods and an autosomal dominant pattern of inheritance.