Novel Compound Heterozygous Splice-Site Variants in TPM3 Revealed by RNA Sequencing in a Patient with an Unusual Form of Nemaline Myopathy: A Case Report.

BACKGROUND: Pathogenic variants in the TPM3 gene, encoding slow skeletal muscle α-tropomyosin account for less than 5% of nemaline myopathy cases. Dominantly inherited or de novo missense variants in TPM3 are more common than recessive loss-of-function variants. The recessive variants reported to date seem to affect either the 5' or the 3' end of the skeletal muscle-specific TPM3 transcript. OBJECTIVES: The aim of the study was to identify the disease-causing gene and variants in a Finnish patient with an unusual form of nemaline myopathy. METHODS: The genetic analyses included Sanger sequencing, whole-exome sequencing, targeted array-CGH, and linked-read whole genome sequencing. RNA sequencing was done on total RNA extracted from cultured myoblasts and myotubes of the patient and controls. TPM3 protein expression was assessed by Western blot analysis. The diagnostic muscle biopsy was analyzed by routine histopathological methods. RESULTS: The patient had poor head control and failure to thrive, but no hypomimia, and his upper limbs were clearly weaker than his lower limbs, features which in combination with the histopathology suggested TPM3-caused nemaline myopathy. Muscle histopathology showed increased fiber size variation and numerous nemaline bodies predominantly in small type 1 fibers. The patient was found to be compound heterozygous for two splice-site variants in intron 1a of TPM3: NM_152263.4:c.117+2_5delTAGG, deleting the donor splice site of intron 1a, and NM_152263.4:c.117 + 164 C>T, which activates an acceptor splice site preceding a non-coding exon in intron 1a. RNA sequencing revealed inclusion of intron 1a and the non-coding exon in the transcripts, resulting in early premature stop codons. Western blot using patient myoblasts revealed markedly reduced levels of the TPM3 protein. CONCLUSIONS: Novel biallelic splice-site variants were shown to markedly reduce TPM3 protein expression. The effects of the variants on splicing were readily revealed by RNA sequencing, demonstrating the power of the method.

A Large Deletion Affecting TPM3, Causing Severe Nemaline Myopathy.

BACKGROUND AND OBJECTIVES: Nemaline myopathy may be caused by pathogenic variants in the TPM3 gene and is then called NEM1. All previously identified disease-causing variants are point mutations including missense, nonsense and splice-site variants. The aim of the study was to identify the disease-causing gene in this patient and verify the NM diagnosis. METHODS: Mutation analysis methods include our self-designed nemaline myopathy array, The Nemaline Myopathy Comparative Genomic Hybridisation Array (NM-CGH array), whole-genome array-CGH, dHPLC, Sanger sequencing and whole-exome sequencing. The diagnostic muscle biopsy was investigated further by routine histopathological methods. RESULTS: We present here the first large (17-21 kb) aberration in the α-tropomyosinslow gene (TPM3), identified using the NM-CGH array. This homozygous deletion removes the exons 1a and 2b as well as the promoter of the TPM3 isoform encoding Tpm3.12st. The severe phenotype included paucity of movement, proximal and axial weakness and feeding difficulties requiring nasogastric tube feeding. The infant died at the age of 17.5 months. Muscle biopsy showed variation in fibre size and rods in a population of hypotrophic muscle fibres expressing slow myosin, often with internal nuclei, and abnormal immunolabelling revealing many hybrid fibres. CONCLUSIONS: This is the only copy number variation we have identified in any NM gene other than nebulin (NEB), suggesting that large deletions or duplications in these genes are very rare, yet possible, causes of NM.

A new common mutation in the cardiac beta-myosin heavy chain gene in Finnish patients with hypertrophic cardiomyopathy.

BACKGROUND: In the nationwide FinHCM Study including 306 Finnish patients with hypertrophic cardiomyopathy (HCM), we have previously identified two founder mutations in the alpha-tropomyosin (TPM1-D175N) and myosin-binding protein C (MYBPC3-Q1061X) genes, accounting for 18% of all cases. Objective. To screen additional mutations, previously identified in eastern Finnish cohorts with HCM, in the FinHCM Study population. PATIENTS AND METHODS: Ten mutations in the beta-myosin heavy chain gene (MYH7), TPM1, and MYBPC3 were screened. RESULTS: MYH7-R1053Q was found in 17 of 306 patients (5.6%). No carriers of MYH7-R719W or N696S were found. A novel TPM1-D175G mutation was found in a single patient. MYBPC3 mutations were found in 14 patients: IVS5-2A-C in two, IVS14-13G-A in two, K811del in six, and A851insT in four patients. Altogether, a HCM-causing mutation was identified in 32 patients, accounting for 10.5% of all cases. In addition, two MYBPC3 variants R326Q and V896M with uncertain pathogenicity were found in eight and in 10 patients, respectively. CONCLUSION: Combining the present findings with our previous results, a causative mutation was identified in 28% of the FinHCM cohort. MYH7-R1053Q was the third most common mutation, and should be screened in all new cases of HCM in Finland.

Novel TPM3 mutation in a family with cap myopathy and review of the literature.

Cap myopathy is a rare congenital myopathy characterized by the presence of caps within muscle fibres and caused by mutations in ACTA1, TPM2 or TPM3. Thus far, only three cases with TPM3-related cap myopathy have been described. Here, we report on the first autosomal dominant family with cap myopathy in three-generations, caused by a novel heterozygous mutation in the alpha-tropomyosin-slow-encoding gene (TPM3; exon 4; c.445C>A; p.Leu149Ile). The three patients experienced first symptoms of muscle weakness in childhood and followed a slowly progressive course. They presented generalized hypotrophy and mild muscle weakness, elongated face, high arched palate, micrognathia, scoliosis and respiratory involvement. Intrafamilial variability of skeletal deformities, respiratory involvement and mild cardiac abnormalities was noted. Muscle MRI revealed a recognizable pattern of fatty muscle infiltration and masseter muscle hypertrophy. Subsarcolemmal caps were present in 6-10% of the fibres and immunoreactive with anti-tropomyosin antibodies. We conclude that the MRI-pattern of muscle involvement and the presence of masseter muscle hypertrophy in cap myopathy may guide molecular genetic diagnosis towards a mutation in TPM3. Regular respiratory examinations are important, even if patients have no anamnestic clues. We compare our findings to all cases of cap myopathy with identified mutations (n=11), thus far reported in the literature.

Genetic variations of α-cardiac actin and cardiac muscle LIM protein in hypertrophic cardiomyopathy in South India.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a disease of the heart muscle, with an autosomal dominant mode of inheritance. It is also known as the 'disease of the sarcomere', and is a major cause of morbidity and mortality worldwide. Mutations in the sarcomeric genes have been largely implicated in the manifestation of HCM. Modifier genes and environmental factors, along with causative mutation, add to the cumulative effect of the disease. METHODS: In the present study, the role of the cardiac actin gene and the cardiac muscle LIM protein as contributors to HCM - through genetic variation - has been elucidated by screening the entire coding region in 100 control and 100 HCM subjects through polymerase chain reaction-based single-strand conformation polymorphism analysis and direct sequencing. RESULTS: The authors could not find any novel or reported exonic variations in any of the genes in the studied population; however, intronic variations were revealed in the cardiac actin gene through direct sequencing. A case of compound heterozygosity was observed in a patient with a variation in intron 1, along with a novel heterozygous mutation in exon 7 (S215L) of α-tropomyosin. CONCLUSIONS: The particular genes are highly conserved, and account for only 1.5% of HCM cases. They do not seem to play a major role in the genesis of HCM in the present population, thus confirming earlier reports of conserved sequences and ethnicity.