Introduction

Myofibrillar myopathy (MFM) is a clinically and genetically heterogeneous group of hereditary muscle diseases characterized by ectopic protein aggregates and a distinct pattern of myofibrillar disorganization. The disintegration of the myofibrils commences in the immediate proximity of the Z-disc and results in Z-disc streaming. This is followed by abnormal accumulation of degraded filamentous material in various patterns in the myofibril-free fiber regions around nuclei and under the sarcolemma. The abnormally accumulated proteins include desmin, myotilin, α-B-crystallin, filamin C, Bag3, actin, plectin, dystrophin, sarcoglycans, neural cell adhesion molecule (NCAM), gelsolin, ubiquitin, syncoilin, synemin, ** muscles, and proper muscle and heart performance. Development 142:994–1005. https://doi.org/10.1242/dev.115352 " href="/article/10.1007/s13353-018-0463-4#ref-CR67" id="ref-link-section-d34606543e1347">2015).

In addition, proteins encoded by BAG3, DNAJB6, and also HSPB8 are involved in chaperone-assisted selective autophagy (CASA). The CASA complex facilitates degradation of damaged Z-disc and is required to prevent protein aggregation (Arimura et al. 2011; Ulbricht et al. 2015). Also, sequestosome 1, itself not a chaperone, plays a role in the autophagy of ubiquitinated inclusions and thus prevents accumulation of abnormally folded proteins.

A recently described PYROXD1 myopathy (O’Grady et al. 2016), despite being initially associated with the Z-disc in co-localization studies, demonstrates that altered cellular redox regulation could be another pathomechanism in myopathies.

Overall, in the majority of MFM patients, the molecular dysfunction is associated with proteins involved in forming the structure and/or providing homeostasis of the Z-disc in the muscle fiber. It is anticipated that also potential novel genes involved in MFM yet to be identified might encode structural or chaperone proteins. Particular attention should be given to genes encoding proteins known to interact with the known MFM-associated proteins.

Potential non-monogenic inheritance

In about 50% of the MFM patients, the causative gene defect remains unknown. This might be partially due to the monogenic-centered concept of inherited diseases relying on Sanger sequencing of one or a few candidate genes at a time. However, di- and oligogenic heterozygosity has already been proposed as a potential disease mechanism for some metabolic and neurological diseases (Vockley et al. 2000; Schaaf et al. 2011). In some patients co-morbid, incomplete defects in multiple proteins of the same or of several independent pathways may lead to a disease, even though no causal mutation has been identified so far (Fig. 1). Such cases could be explained by the “epistatic effect” when one genetic variant is modulated by another one giving either a synergistic or an antagonistic effect on the phenotype (Phillips 2008). Another explanation is “synergistic heterozygosity,” when variants in two or more genes do not have a modulatory effect on each other but can cause partial defects in the same or intersecting pathways. In myopathies, several benign variants may accumulate in genes encoding proteins involved in a specific pathway (i.e., protein folding quality control) or in a single subcellular structure (such as the Z-disc or extrasarcomeric cytoskeleton). The cumulative effect of these variants, if exceeding a certain threshold, could affect muscle functioning and result in pathology such as MFM. We believe that such a cumulative effect of seemingly benign variants (with small effect when present in isolation) could explain the genetic background of a phenomenon observed in some families with children displaying a more severe disease phenotype than the one of their parents (Li et al. 2017). In addition, common polymorphisms might be disease course-modifying factors, such as LTPB4 variant in dystrophinopathy (Flanigan et al. 2013). We have observed a similar case in one MFM family, in which initial Sanger sequencing revealed a causative Q348P mutation in desmin co-segregating with the disease phenotype (Fichna et al. 2014). Subsequent whole exome sequencing revealed an additional S206L DNAJB6 mutation only in one patient with disease onset two decades earlier than of his mother’s, who also presented with a milder course of the disease (Fichna et al., manuscript in preparation/data not shown).

Fig. 1
figure 1

Pictorial comparison and difference between possible inheritance modes. a Typical monogenic inheritance with a pathogenic causal mutation in one gene. b Digenic inheritance, where mutations affecting the function of two genes are needed to produce the phenotype. c Oligogenic inheritance, where a number of mutations cause minor defects that together result in pathology. d Rare variant load, where accumulation of seemingly benign variants, including both mutations and polymorphisms, can lead to disease. In c and d, variants cause partial defects either in one pathway or in intersecting pathways, which together produce a cumulative effect leading to the disease. Circle symbolizes a gene; Asterisk stands for mutation/genetic variant

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On the other hand, typical digenic inheritance has been demonstrated in facioscapulohumeral muscular dystrophy (Lemmers et al. 2012), in congenital myasthenic syndrome (Lam et al. 2017), and in calpainopathy (Sáenz and López de Munain 2017). Digenic inheritance has also been described in MFM-like cases caused by mutations in SQSTM1 and TIA1 (Niu et al. 2018).

New techniques and novel challenges

The strictly Mendelian concept of inheritance, with one mutation causing one clinical phenotype, is an oversimplification, as it was already elegantly presented on the example of phenylketonuria, the classical recessive disease (Scriver and Waters 1999). It was then proposed that modeling the interaction of a limited number of genes and understanding the molecular consequences of such interactions are a prerequisite for understanding genomic basis of phenotypic complexity of Mendelian disorders (Badano and Katsanis 2002). Nowadays, with the development and widespread application of various next-generation sequencing methods, it has become clear that not only a single gene can be associated with a specific disease (locus heterogeneity), but different variants in one gene can cause different phenotypes (allelic heterogeneity), and some variants can even be associated with multiple clinical phenotypes (Keith et al. 2014). The availability of exome and whole genome data for various conditions has challenged the classical definition of genetic causality and the concept of strictly monogenic disorders, providing evidence for the roots of heterogeneity and complexity of the human genome (Katsanis 2016). Many diseases with overlap** phenotypes have multiple genetic associations. Interestingly, to date, there have been over 400 genes associated with various neuromuscular disorders, with 407 associated only with “myopathy” in the OMIM database (omim.org).

Myopathies and muscular dystrophies seem to form a disease continuum; thus, the causative gene identification in one disease impacts the genetic etiology of the others. It could be hypothesized that complex patterns of inheritance, including oligogenic inheritance, account for a sizable fraction of myopathy cases, particularly the atypical ones, overlap** with other muscular disease forms. Specific genetic background, in particular comprising the genes for elements of pathways or structures involved in the pathogenesis of MFM, although difficult to pinpoint, may likely influence the phenotype. Modifying variants and even co-causal mutations may also explain the observed inter- and intrafamilial variability between patients harboring the same main causal mutation (Fichna et al. 2014). In MFM patients with an unknown genetic defect, the pathogenic and co-causative mutations (including copy number variants), as well as the modifying variants, could be located in non-coding, e.g., regulatory or deep intronic regions, not resolved by the whole exome sequencing approach, which highlights the superiority of the whole genome sequencing approach (Itan and Casanova 2015).

The concept of oligogenic inheritance should influence not only the approach to gene identification, but also genetic testing and counseling. NGS results are too complex to be easily interpreted by the health care professionals, who also struggle to pass comprehensive information on to the patients (Niemiec et al. 2018). However, one of the biggest challenges remains the discrimination between possible co-causative or modifying variants and the thousands of insignificant variants present in any genome. Advanced bioinformatic assessment of identified variants and comparison of worldwide NGS results offer some solution of this conundrum (Chakravorty and Hegde 2018).

A recent comprehensive genetic analysis using whole exome sequencing in a group of patients with various muscle disorders has shown that each patient bears over 30 rare (minor allele frequency, MAF < 1%) genetic variants potentially influencing the structure of relevant proteins and putatively related to the myopathic phenotype (Fichna et al. 2018). Interestingly, more of those rare variants were shared between patients with MFM and limb-girdle muscular dystrophy (LGMD) than between LGMD or MFM and a control group (Fichna et al., unpublished results). The hypothesis-driven approach used in the above study could have resulted in missing variants in genes not yet related to any muscle phenotype. Therefore, it was augmented by applying an additional filtering pipeline relating to muscle physiology or structure. It was aimed at the identification of very-rare variants (MAF < 0.1%) in genes expressed in the muscle and in genes coding for proteins from a broad interactome of muscle disease-related proteins. Using this combined approach, we were able to detect numerous variants often in genes coding for proteins involved in sarcomere structure and assembly, signal transduction, protein’s glycosylation, and folding or/and previously implicated in diverse muscle disorder (Fichna et al. 2018). Among these variants, potentially compromising protein structure and/or function could be novel pathogenic or phenotype-modifying ones. Therefore, we proposed that those variants could be part of a “variant burden,” contributing to deterioration of muscle cell molecular homeostasis and facilitating or modifying effects of the well-established causative MFM mutations. Moreover, mutations in genes encoding chaperones (CRYAB, BAG3, HSPB8, and DNAJB6) may likely unmask the burden of variants with minor effect. Chaperones rescue proteins misfolded by environmental stresses, but could also stabilize mutated proteins (Tomala and Korona 2008). Even if many MFM cases can be easily attributed clinically to mutations in a single gene, the high number of variants associated with myopathy and sometimes with specific phenotype features suggests that the variant load may be important even in patients with a well-defined primary pathogenic cause (Fichna et al. 2018). It is likely that in MFM-like cases with mutations in genes typically associated with other disorders (like ACTA1, HSPB8, LMNA, or SQSTM1), it is the variant load that ultimately determines the features of myofibrillar pathology.

The superiority of high-scale bioinformatic analysis over focused genetic studies lies in the possibility of repeating the analysis and applying novel knowledge, updated databases, better algorithms, and prediction tools. Therefore, genetic testing of MFM should be combined not only with bioinformatic analyses and deep phenoty**, but also with comprehensive analyses of transcripts and protein isoforms to pinpoint novel causal, co-causal, and modifying variants (Hennekam and Biesecker 2012). Identification of the modifying variants requires high-throughput analyses of combined genomic and clinical data on large groups of ethnically diverse patients with various muscle diseases and should be followed by functional studies. Such an approach will enable examination of groups of genes encoding entire pathways and cellular modules (McCarthy and MacArthur 2017). Better understanding of the importance of the modifying variants will inevitably transform the traditional descriptive classification of muscle diseases into a systemic and pathway-based view of clinical phenotypes (Thompson and Straub 2016).

Conclusions

There is mounting evidence that MFM and other myopathies should be viewed as oligogenic disorders in which variable clinical presentation can result from the combined effect of mutations in many genes. In this view, the inter- and intrafamilial variability could reflect a specific genetic background and the presence of sets of phenotype-modifying, co-causal mutations (variant burden). The concept of non-monogenic inheritance should influence not only the current approach to gene identification, but also genetic testing and counseling.