Molecular genetics studies

Long-read sequencing

Oxford Nanopore MinION is a modern sequencing device capable of analyzing extremely long sections of the genome at once, facilitating the sequencing of previously inaccessible regions - such as the triplicate reigion of the nebulin gene.

Our work with the MinION device began even before its official release, as participants in the MinION Early Access Programme. Since then we have established the basic operations of the Nanopore sequencing protocols for general DNA and tissue samples. We are also building our own analysis pipeline concurrently with the production of the sequencing data.

We are also examining other possibilities of the MinION sequencer, such as using RNA and cDNA as materials. This could help us understand the intricacies of the nebulin gene expression better. The testing on these methods is still in the early phases, but may be incorporated as a part of our complete MinION sequencing pipeline in the future.

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 causative variants in 12% of these.

Since January 2022, the NMD-CGH-array is available as a diagnostic service at the Diagnostic Laboratory of the Helsinki University Hospital.

Droplet Digital PCR for Segmental Duplications

Titin and nebulin, two gigantic sarcomeric proteins, both contain segmental duplication regions in their sequences, and harbour both normal and pathogenic copy number variation. These blocks are challenging to analyze by both Comparative Genomic Hybridization array methods. We have therefore developed custom Droplet Digital PCR assays for the copy number variant screening in both NEB (Sagath et al. 2022a) and TTN (Sagath et al. 2022b).

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. 2018).

Nebulin super-repeat panel

Nebulin's 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., 2019). 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.

Functional studies of novel CASQ1 variants

Variants in calsequestrin-1 (CASQ1) have been associated with a late-onset, slowly progressive myopathy. We are studying novel CASQ1 variants to better understand how they affect the calcium-binding ability and stability of the protein.

Elucidating myosin dysfunction and altered energy metabolism in NM

One of the pathogenetic mechanisms commonly occurring in NM muscle, regardless of the causative gene, is defective myosin function (Ochala et al. 2011). In the muscle tissue of NM patients, myosin binding to actin filaments is often disrupted, limiting the intrinsic force-generating capacity. It is hypothesised that a shift in the equilibrium between the different states of the myosin heads occurs in the NM muscle, leading to increased energy consumption.

Dr. Jenni Laitila is visiting Prof. Julien Ochala's lab (University of Copenhagen, Denmark) to study this hypothesis. The methodology includes a combination of structural and functional experiments on single muscle fibres from NM patient and healthy control samples.

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., 2020).

Nutritional status and functioning of patients

We have embarked on a novel line of research to unravel the effects of nutrition on the wellbeing and lived experience of functioning of persons with nemaline myopathy (NM) and related disorders (NMR). For healthy persons, much knowledge exits about the effects of nutrition, but in disabled people, medical research has focused mainly on studying the disease itself in terms of cause, mechanisms, and treatment. Until very recently, little, or no scientific attention has been paid explicitly to the lived experience of functioning of disabled people. The same is true for the impact of eating and nutrition on the daily life of patients. Persons with NM and related disorders often have weakness of the facial and bulbar muscles and secondary structural abnormalities of the oral region, complicating eating and swallowing, and likely affecting their nutritional state. Many patients use tube-feeding. In addition, many patients need respiratory support, which might affect eating. We conducted a pilot study in patients with NM or NMR in Finland to start building knowledge on nutrition, food intake and eating, and whether they affect the physical, psychological and social functioning, and wellbeing of the persons. This multi-professional study included specialists in adult and pediatric neurology, a dietitian, a nutritionist, and an expert on research into function.

Physical, psychological, and social functioning of the patients with NM and NMR in Finland

To elucidate the self-reported physical, psychological, and social functioning as well as eating and dining related functioning in the daily lives of adult persons with NM or NMR, we designed a survey using questions (items) primarily from the Patient Reported Outcomes Measurement Information System, PROMIS®. In addition, we used items from e.g. FinSote Survey and designed some of the questions in our research group together with five Finnish NM patients. In total, 20 (62.5%) out of 32 invited persons resident in Finland participated in the study; 12 had NM and 8 NMR, 15 were women and 5 men aged 19-75 years. Sixteen (80%) were ambulatory and 4 (20%) NM patients used wheelchairs. The results indicated that non-ambulatory patients of this study faced more challenges in all areas of functioning than ambulatory ones, but the differences were smaller in the domains measuring psychological and social functioning than in physical functioning. In addition, the COVID-19 pandemic adversely affected the functioning of non-ambulatory patients more than that of ambulatory patients. The results indicate vulnerability of non-ambulatory patients being at higher risk to a decrease in general functioning during global or national exceptional periods. The interindividual differences were, however, noticeable.

Nutritional status of patients with nemaline myopathy and related congenital myopathies in Finland: a pilot study

Comprehensive research on the possible difficulties in eating or the nutritional status of persons with nemaline myopathy (NM) and related disorders (NMR) has not been done despite the fact that the muscle weakness in these disorders often affects the muscles used for eating and dining. We conducted a study among adult Finnish NM or NMR patients to investigate their food consumption, nutrient intakes, selected nutrient-related laboratory parameters in blood, and self-assessed functioning at dining and eating and of the gastrointestinal tract. The methods included a survey including eating and dining related questions, a food frequency questionnaire (FFQ), food diaries, and laboratory analyses from blood samples. The same 20 persons, who participated and returned the survey mentioned above, returned the FFQ, as well. Food diaries were returned by 17, and blood samples were obtained from 16. The inter-individual differences were large in food consumption as well as in nutrient and energy intake. Energy intakes, and intakes of vitamin D, calcium, dietary fiber, vitamin C, folate, and iron as well as consumption of healthy foods (such as fruits, vegetables, and whole grains) were low, especially in non-ambulatory participants, who also reported challenges in eating and dining-related functioning. The severity of the muscle weakness, however, determine the quality of diet, and by selecting healthy foods easy to eat (e.g. porridges and smoothies), a good diet was achievable. The laboratory parameters did not indicate severe undernourishment in any of the participants. Thus, evaluation of food consumption and nutrient intakes were needed to find patients at risk of undernourishment. The results underline the importance of monitoring adequate intake of calcium and vitamin D in this group of patients, especially considering the immobility-induced risk of osteoporosis.