Differential inclusion of NEB exons 143 and 144 provides insight into NEB-related myopathy variant interpretation and disease manifestation.

Biallelic pathogenic variants in the gene encoding nebulin (NEB) are a known cause of congenital myopathy. We present two individuals with congenital myopathy and compound heterozygous variants (NM_001271208.2: c.2079C>A; p.(Cys693Ter) and c.21522+3A>G ) in NEB. Transcriptomic sequencing on patient muscle revealed that the extended splice variant c.21522+3A>G causes exon 144 skipping. Nebulin isoforms containing exon 144 are known to be mutually exclusive with isoforms containing exon 143, and these isoforms are differentially expressed during development and in adult skeletal muscles. Patients MRIs were compared to the known pattern of relative abundance of these two isoforms in muscle. We propose that the pattern of muscle involvement in these patients better fits the distribution of exon 144-containing isoforms in muscle than with previously published MRI findings in NEB-related disease due to other variants. To our knowledge this is the first report hypothesizing disease pathogenesis through the alteration of isoform distributions in muscle.

Recurrent de novo SPTLC2 variant causes childhood-onset amyotrophic lateral sclerosis (ALS) by excess sphingolipid synthesis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the upper and lower motor neurons with varying ages of onset, progression and pathomechanisms. Monogenic childhood-onset ALS, although rare, forms an important subgroup of ALS. We recently reported specific SPTLC1 variants resulting in sphingolipid overproduction as a cause for juvenile ALS. Here, we report six patients from six independent families with a recurrent, de novo, heterozygous variant in SPTLC2 c.778G>A [p.Glu260Lys] manifesting with juvenile ALS. METHODS: Clinical examination of the patients along with ancillary and genetic testing, followed by biochemical investigation of patients' blood and fibroblasts, was performed. RESULTS: All patients presented with early-childhood-onset progressive weakness, with signs and symptoms of upper and lower motor neuron degeneration in multiple myotomes, without sensory neuropathy. These findings were supported on ancillary testing including nerve conduction studies and electromyography, muscle biopsies and muscle ultrasound studies. Biochemical investigations in plasma and fibroblasts showed elevated levels of ceramides and unrestrained de novo sphingolipid synthesis. Our studies indicate that SPTLC2 variant [c.778G>A, p.Glu260Lys] acts distinctly from hereditary sensory and autonomic neuropathy (HSAN)-causing SPTLC2 variants by causing excess canonical sphingolipid biosynthesis, similar to the recently reported SPTLC1 ALS associated pathogenic variants. Our studies also indicate that serine supplementation, which is a therapeutic in SPTLC1 and SPTCL2-associated HSAN, is expected to exacerbate the excess sphingolipid synthesis in serine palmitoyltransferase (SPT)-associated ALS. CONCLUSIONS: SPTLC2 is the second SPT-associated gene that underlies monogenic, juvenile ALS and further establishes alterations of sphingolipid metabolism in motor neuron disease pathogenesis. Our findings also have important therapeutic implications: serine supplementation must be avoided in SPT-associated ALS, as it is expected to drive pathogenesis further.

A comprehensive study of skeletal muscle imaging in FHL1-related reducing body myopathy.

OBJECTIVE: FHL1-related reducing body myopathy is an ultra-rare, X-linked dominant myopathy. In this cross-sectional study, we characterize skeletal muscle ultrasound, muscle MRI, and cardiac MRI findings in FHL1-related reducing body myopathy patients. METHODS: Seventeen patients (11 male, mean age 35.4, range 12-76 years) from nine independent families with FHL1-related reducing body myopathy underwent clinical evaluation, muscle ultrasound (n = 11/17), and lower extremity muscle MRI (n = 14/17), including Dixon MRI (n = 6/17). Muscle ultrasound echogenicity was graded using a modified Heckmatt scale. T1 and STIR axial images of the lower extremity muscles were evaluated for pattern and distribution of abnormalities. Quantitative analysis of intramuscular fat fraction was performed using the Dixon MRI images. Cardiac studies included electrocardiogram (n = 15/17), echocardiogram (n = 17/17), and cardiac MRI (n = 6/17). Cardiac muscle function, T1 maps, T2-weighted black blood images, and late gadolinium enhancement patterns were analyzed. RESULTS: Muscle ultrasound showed a distinct pattern of increased echointensity in skeletal muscles with a nonuniform, multifocal, and "geographical" distribution, selectively involving the deeper fascicles of muscles such as biceps and tibialis anterior. Lower extremity muscle MRI showed relative sparing of gluteus maximus, rectus femoris, gracilis, and lateral gastrocnemius muscles and an asymmetric and multifocal, "geographical" pattern of T1 hyperintensity within affected muscles. Cardiac studies revealed mild and nonspecific abnormalities on electrocardiogram and echocardiogram with unremarkable cardiac MRI studies. INTERPRETATION: Skeletal muscle ultrasound and muscle MRI reflect the multifocal aggregate formation in muscle in FHL1-related reducing body myopathy and are practical and informative tools that can aid in diagnosis and monitoring of disease progression.

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Recognizable Pattern of Arthrogryposis and Congenital Myopathy Caused by the Recurrent TTN Metatranscript-only c.39974-11T > G Splice Variant.

INTRODUCTION: Arthrogryposis is characterized by the presence of multiple contractures at birth and can be caused by pathogenic variants in TTN (Titin). Exons and variants that are not expressed in one of the three major isoforms of titin are referred to as "metatranscript-only" and have been considered to be only expressed during fetal development. Recently, the metatranscript-only variant (c.39974-11T > G) in TTN with a second truncating TTN variant has been linked to arthrogryposis multiplex congenita and myopathy. METHODS: Via exome sequencing we identified the TTN c.39974-11T > G splice variant in trans with one of three truncating variants (p.Arg8922*, p.Lys32998Asnfs*63, p.Tyr10345*) in five individuals from three families. Clinical presentation and muscle ultrasound as well as MRI images were analyzed. RESULTS: All five patients presented with generalized muscular hypotonia, reduced muscle bulk, and congenital contractures most prominently affecting the upper limbs and distal joints. Muscular hypotonia persisted and contractures improved over time. One individual, the recipient twin in the setting of twin-to-twin transfusion syndrome, died from severe cardiac hypertrophy 1 day after birth. Ultrasound and MRI imaging studies revealed a recognizable pattern of muscle involvement with striking fibrofatty involvement of the hamstrings and calves, and relative sparing of the femoral adductors and anterior segment of the thighs. CONCLUSION: The recurrent TTN c.39974-11T > G variant consistently causes congenital arthrogryposis and persisting myopathy providing evidence that the metatranscript-only 213 to 217 exons impact muscle elasticity during early development and beyond. There is a recognizable pattern of muscle involvement, which is distinct from other myopathies and provides valuable clues for diagnostic work-up.

POLRMT mutations impair mitochondrial transcription causing neurological disease.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.