Nemaline Myopathy group

Nebulin expressionNebulin super-repeat panelMurine modelYBX3
NMD-CGH-arrayLong-read sequencingSegmental duplication ddPCR

Functional studies

Nebulin expression

Nebulin (600-900 kDa) is one of the largest proteins known. One nebulin molecule spans nearly the entire length of the thin filament in the skeletal muscle sarcomere. The nebulin gene (NEB) has 183 exons, giving rise to a theoretical full-length mRNA of 26 kb, but due to extensive alternative splicing, a great number of isoforms exist. We have studied the expression of the nebulin RNA extensively in different leg muscles and brain (Laitila et al. 2012). However, no muscle specific isoforms were identified on the RNA level. We are currently studying the expression of nebulin isoforms at the RNA and protein level in different myofibres of the same muscle. There seems to be some variation in the expression of the alternative isoforms studied, but the reason for this variation remains to be elucidated (Lam et al., submitted).

Nebulin super-repeat panel

Nebulin protein structure is highly modular, consisting of over 200 simple repeats, each binding to actin. Most of the simple repeats are further organized into seven simple-repeat containing super repeats, also harbouring a tropomyosin binding site. Studying the interactions between nebulin and its binding partners is hindered by the enormous size of the protein. To overcome this problem, we have constructed a complete panel of nebulin super repeats, to allow studying the interactions using shorter protein fragments. We have studied the nebulin-actin interaction, revealing a pattern of strong and weak binding along the length of nebulin (Laitila et al., manuscript submitted). The super-repeat panel is going to be a powerful tool in elucidating nebulin function in health and disease. We are currently also investigating the nebulin interaction with tropomyosin along the super-repeat panel.

Compound heterozygous disease model

Although many roles for nebulin have been suggested, much about its function is still unknown. Due to its enormous size, studying the effects of mutations on disease pathogenesis is difficult.

The murine models published so far have each provided new pieces of knowledge about nebulin function and the potential pathogenesis of nemaline myopathy (NM). However, none of them had a compound heterozygous genotype (two different mutations in nebulin), typical for nemaline myopathy with nebulin mutations (NEB-NM). Furthermore, the need remains for a model recapitulating the typical form of NM. Therefore, in collaboration with Dr. Kristen Nowak (Harry Perkins Institute of Medical Research, Western Australia), we have characterised a mouse strain with compound heterozygous Neb variants, one a missense within a conserved actin-binding site and the other a nonsense variant, matching the genetics of most patients with typical NEB-NM.

This new model, along with the corresponding parental lines with only the missense or the nonsense mutation, will be useful in deciphering the pathogenetic mechanisms of NEB-NM. Moreover, this mouse model will be suitable for evaluating therapeutic approaches, including gene-based therapies for the disorders caused by mutations in NEB. (Laitila et al., manuscript in preparation).

The candidate gene YBX3

Variants in the Y-box binding protein 3 (YBX3) gene have been connected to Nemaline myopathy. To better understand the impact of these variants, we are working on localization and interaction studies of the wild-type protein and the variants. Part of the work is being done in collaboration with Stephan Lange's group in California.

Molecular genetics studies

Custom CGH-arrays

We have published two custom Comparative Genomic Hybridisation array designs - one including the known Nemaline Myopathy genes ("the NM-CGH-array", Kiiski et al. 2013) and an extended version with 176 additional genes related to other neuromuscular disorders ("the NMD-CGH-array", Sagath et al. 2018). The NM-CGH-array is built as a 8×60k design, and the NMD-CGH-array as a 4×180k design. The arrays constitute a robust method for copy number variant analysis for diagnostics of neuromuscular disorder patients, and also covers the segmental duplication regions of both nebulin and titin. These are regions that most commercial arrays do not include and are challenging to analyse by next generation sequencing based methods. We have run over 400 samples from approximately 300 families, and have found pathogenic variants in 12% of these.

Long-read sequencing

Nanopore is a sequencing method for long reads. We were hoping it would solve our problems of sequencing nebulin, but turns out things aren't always as simple as you'd hoped.

Droplet Digital PCR for Segmental Duplications

Titin and nebulin, two gigantic sarcomeric proteins, both contain segmental duplication regions in their sequences, and harbor both normal and pathogenic copy number variation. These blocks are challenging to analyze by both Comparative Genomic Hybridization array methods - therefore we are developing ddPCR based assays for the analysis of these regions. The method is under validation, and will be subject for a manuscript in the near future.


Folkhälsan Research Center
Department of Medical Genetics
Biomedicum Helsinki
Haartmaninkatu 8, PB63
FI-00014 University of Helsinki

© Nemaline Myopathy Group 2018