The genetic cause for an unusual form of nemaline myopathy solved by RNA sequencing

Nemaline myopathy is a congenital muscle disease that is caused by variants in at least 12 different genes. Many years ago, we received samples from a Finnish patient with an unusual form of nemaline myopathy. The clinical symptoms and histopathology suggested TPM3-caused nemaline myopathy, which is rare.

We embarked on mutation analysis of the known nemaline myopathy-causing genes and found a variant in the first intron of the TPM3 gene. The variant was predicted to alter splicing of the TPM3 mRNA, and hence, be disease causing.

The patient had inherited the variant from his healthy father, meaning that he needed a second disease-causing variant in TPM3 from his healthy mother to be sick. Extensive analysis of the TPM3 gene did not, however, reveal a second clearly disease-causing variant in TPM3. By this time novel sequencing technologies had become available, and we took advantage of those. Whole exome and whole genome sequencing revealed additional interesting variants in other genes, but these were ruled out as disease causing by further analysis. As a last resort we extracted RNA from cultured muscle cells of the patient and sequenced the mRNA pool by massively parallel sequencing. It was immediately evident from the results that intron 1a of TPM3 was aberrantly spliced in all transcripts, also in those expressed from the maternal allele.

It turned out that the patient had inherited a point mutation deep in intron 1a from his mother, in addition to the splice site mutation in the same intron from his father. The maternal variant had been overlooked and predicted to be harmless, but in fact, it activates a cryptic splice site and cause aberrant splicing. Mis-splicing caused by both variants leads to aberrant mRNAs and reduced or no TPM3 protein in the patient’s muscles. We could confirm low TPM3 protein levels by Western blot analysis.

This case illustrates the power of novel sequencing technologies, such as RNA sequencing in disease-gene identification.

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
Pelin K, Sagath L, Lehtonen J, Kiiski K, Tynninen O, Paetau A, Johari M, Savarese M, Wallgren-Pettersson C, Lehtokari VL. Journal of Neuromuscular Diseases.