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# Modifier genes: Moving from pathogenesis to therapy

Molecular Genetics and Metabolism, September 2017, Volume 122, Issue 1-2, Pages 1-3

### Abstract

This commentary will focus on how we can use our knowledge about the complexity of human disease and its pathogenesis to identify novel approaches to therapy. We know that even for single gene Mendelian disorders, patients with identical mutations often have different presentations and outcomes. This lack of genotype-phenotype correlation led us and others to examine the roles of modifier genes in the context of biological networks. These investigations have utilized vertebrate and invertebrate model organisms. Since one of the goals of research on modifier genes and networks is to identify novel therapeutic targets, the challenges to patient access and compliance because of the high costs of medications for rare genetic diseases must be recognized. A recent article explored protective modifiers, including plastin 3 (PLS3) and coronin 1C (CORO1C), in spinal muscular atrophy (SMA). SMA is an autosomal recessive deficit of survival motor neuron protein (SMN) caused by mutations in SMN1 . However, the severity of SMA is determined primarily by the number of SMN2 copies, and this results in significant phenotypic variability. PLS3 was upregulated in siblings who were asymptomatic compared with those who had SMA2 or SMA3, but identical homozygous SMN1 deletions and equal numbers of SMN2 copies. CORO1C was identified by interrogation of the PLS3 interactome. Overexpression of these proteins rescued endocytosis in SMA models. In addition, antisense RNA for upregulation of SMN2 protein expression is being developed as another way of modifying the SMA phenotype. These investigations suggest the practical application of protective modifiers to rescue SMA phenotypes. Other examples of the potential therapeutic value of novel protective modifiers will be discussed, including in Duchenne muscular dystrophy and glycerol kinase deficiency. This work shows that while we live in an exciting era of genomic sequencing, a functional understanding of biology, the impact of its disruption, and possibilities for its repair have never been more important as we search for new therapies.

### 1 Complexity of genetic disorders: roles of modifiers and networks

During the late 1990s there was a distinction that was being made between single gene disorders and complex traits. Our group and others began to recognize that the phenotypes of patients with “simple,” single gene, Mendelian disorders are complex traits [1] , and we explored the roles of: thresholds, focusing on enzyme activities, but with principles that could be applied to non-enzymatic proteins; modifiers, including genetic and environmental influences; and systems dynamics, focusing on biological networks that provide stability to biological systems, while also revealing their vulnerabilities 1 2 3 .

We originally conceptualized a discrete threshold model, as had others, in which functional protein expression, including enzyme activity, below a certain level or threshold would be associated with disease and above that threshold would be associated with the absence of disease [1] . We recognized that this discrete conceptual model was too simplistic, and the more appropriate model was non-discrete with an indeterminate range between health and disease. Modifiers would have their influences within this indeterminate range.

Work on identifying modifier genes and fundamental mechanisms have utilized vertebrates, primarily mice, and invertebrates, focused on Drosophila and Caenorhabditis elegans , as well as yeast 4 5 . These influences have been recognized as “background” effects in model organisms in which identical mutations can manifest very different phenotypes in different strains of the model organism [4] . The role of polymorphisms in central metabolic genes has been proposed as a mechanism for genetic modifiers [5] , implicating the metabolome in phenotypic modification. Epigenetic effects have been identified 4 6 , and the “moonlighting” functions (in which enzymes exhibit functional activities unrelated to those for which they were originally named) of proteins have been discussed as modifiers 5 7 . Modifiers are targets for therapeutic consideration 4 6 . Since modifiers may have such impressive impacts on Mendelian disorders, the phenotypes of these traditional “single” gene disorders are being reconsidered as polygenic complex traits 1 4 .

Among many examples of modifiers that have been identified primarily using model organisms are the following. The Multiple intestinal neoplasia (Min) mouse has a mutation in the murine Apc gene and consequently develops adenomas in the intestine. Numbers of Modifier of Min ( Mom ) genes have been identified and mapped, and syntenic regions between the murine and human chromosomes and gene interaction networks have been investigated 8 9 10 . Mouse models have been used to identify and to investigate Modifier of von Willebrand factor or Mvwf genes 11 12 13 and human genome-wide association studies have elucidated modifiers influencing levels of von Willebrand factor [14] . Modifiers of hearing loss have been identified in mice and humans 15 16 . The power of invertebrate model organisms for identifying modifier genes has been demonstrated with the motor neuron disease, amyotrophic lateral sclerosis, using Drosophila [17] , and the tauopathies, which include Alzheimer disease, frontotemporal dementia with Parkinsonism, progressive supra nuclear palsy and Pick disease, using Drosophila and C. elegans [18] .

The general goals of identifying modifier genes are to elucidate more completely the networks involved in the pathogenesis of disorders and to identify novel therapeutic targets among these networks. However, there are concerns that these therapies may be expensive and that this cost may limit access to and compliance with treatment. For example, among the reasons physicians provided for why patients with urea cycle disorders received less than the ideal dose of the small molecule, sodium phenylbutyrate, included “high cost.” [19] Enzyme replacement therapies (ERTs) for lysosomal storage disorders are often cited as examples of expensive therapeutics that limit access with the average annual cost per patient for ERT of $90,000–$720,000 20 21 22 23 .

Modifier genes and their roles in biological networks are increasingly recognized as critical in the pathogenesis of disease, and protective modifiers reduce pathogenicity of mutations considered primary to the disease [24] . Could specific modifiers be translated into interventions?

### 2 Protective Modifiers in Spinal Muscular Atrophy (SMA)

Hosseinibarkooie and colleagues recently described how protective modifiers might improve endocytosis and rescue the SMA phenotype [25] . They summarized the pathogenesis of SMA, which is due to the autosomal recessive deficit of the “survival muscular neuron 1” (SMN1) protein. SMN1 is a ubiquitous housekeeping protein involved in small nuclear ribonucleoprotein (snRNP) complex assembly. Humans have two SMN genes, SMN1 and SMN2 , which are nearly identical. Due to mis-splicing of SMN2 , only approximately 10% of SMN2 protein is expressed.

Most individuals with SMA have mutations of SMN1 and the severity of an individual’s SMA is determined primarily by the number of SMN2 copies [25] . This results in significant phenotypic variability:

• SMA1 (MIM 253300) is the severe form with onset before 6 months of age, and these patients are unable to sit or to walk and live less than 2 years;

• SMA2 (MIM 253550) is the intermediate form with onset before 6 months of age, and they are able to sit, but not to walk;

• SMA3 (MIM 253400) is the mild form with onset after 18 months of age, and these patients are able to sit and to walk; and

• SMA4 (MIM 271150) is the adult form with onset after 20 years.

In general, individuals with: SMA1 have two copies of SMN2 , SMA2 have three copies of SMN2 , SMA3 have four copies of SMN2 , and SMA4 have four to six copies of SMN2 .

These investigators used this knowledge, as well as additional research, to consider the practical application of protective modifiers to rescue SMA phenotypes [25] . They had previously shown that the plastin 3 ( PLS3 ) gene was a protective modifier by examining its expression in families discordant for SMA phenotype [26] . PLS3 was differentially expressed in siblings who were asymptomatic and those with SMA2 or SMA3, but the siblings had identical homozygous SMN1 deletions and equal numbers of SMN2 copies. PLS3 was upregulated in lymphoblastoid cell lines from the individuals who were asymptomatic. Overexpression of PLS3 improved neuromuscular junction (NMJ) endocytosis in SMA mice [25] .

This group next interrogated the PLS3 interactome [25] . They found that coronin 1C (CORO1C) interacted directly with PLS3 and overexpression of CORO1C rescued endocytosis in cells deficient for SMN. When they investigated Smn morphant zebrafish, the morphants were rescued by PLS3 and CORO1C mRNAs. F-actin is critical for cellular integrity and processes including endocytosis, is reduced in cells with low SMN, and binds PLS3 and CORO1C. In animal models of SMA, F-actin-dependent processes are disrupted, including axonal growth and connectivity at NMJs, neurotransmission, and synaptic vesicle recycling. These processes improved with PLS3 overexpression.

Humans with PLS3 mutations have osteoporosis and associated fractures [27] . This suggests a role of PLS3 in bone development and remodeling. Although the cellular mechanism leading to osteoporosis is unknown, fibroblasts from a male hemizygous for an Xq23 PLS3 mutation showed abnormal endocytosis [25] .

A C. elegans screen of an SMA model identified additional modifiers that had roles in endocytosis [28] . The investigators concluded, therefore, that the aberrant endocytosis might not be solely due to decreased presynaptic F-actin and additional research would be required [25] .

Hosseinibarkooie and colleagues stated that their work showed “the power of genetic modifiers and their ability to unravel key cellular mechanisms and protein networks that counteract disease-causing processes,” and, “Most importantly, this knowledge might open new therapeutic avenues in the treatment of individuals with SMA, by allowing the use of genetic modifiers involved in endocytosis.” [25]

Next generation sequencing is identifying known pathogenic mutations in individuals who do not show signs or symptoms of the disorder [24] , the so-called “healthy knockouts.” [29] The work on genetic modifiers in SMA suggests that this approach should be expanded to identify disease-related networks and new therapeutic opportunities.

It should be noted that as this manuscript was being prepared a drug that increases expression of SMN2 was approved by the U.S. Food and Drug Administration to treat SMA [30] . The drug, Spinraza® (nusinersen) is an antisense RNA-like molecule that reduces mis-splicing from the SMN2 gene, thereby improving protein expression from this gene. Since it has been known that the number of copies of the SMN2 genes and therefore the amount of SMN2 protein modified the phenotype, this drug may be considered a form of modifier therapy.

### 3 Additional examples with therapeutic insights

#### 3.1 Duchenne muscular dystrophy (DMD)

Bello et al. reported a modifier gene that reduced the age at loss of independent ambulation (LoA) among individuals with DMD and suggested a novel therapeutic approach to this disorder [31] . The minor T allele (C > T) at rs1883832 in the 5′-untranslated region of the CD40 locus was associated with earlier LoA in independent international cohorts. Given the role of CD40 in immune function, this study suggested a role for cell-mediated immunity in the pathogenesis of DMD.

Corticosteroids are currently considered “standard of care” for individuals with DMD and are thought to reduce symptoms by reducing inflammation [32] . These steroids have broad effects to inhibit the immune response. Focusing therapy for DMD on cell-mediated immunity would provide a novel therapeutic target [31] and might reduce the side effects associated with corticosteroids.

#### 3.2 Isolated glycerol kinase deficiency (iGKD)

iGKD is caused by mutations within the X-linked GK gene and has two different clinical presentations: the “symptomatic form” associated with episodic acute metabolic and central nervous system (CNS) decompensations in children; and the “asymptomatic form” associated with glyceroluria and hyperglycerolemia, but without symptomatic decompensations [33] . Interestingly, with longer follow-up men with “asymptomatic” iGKD were found to have obesity and reduced insulin tolerance and/or type 2 diabetes mellitus (T2DM) [34] .

Our group showed that the residual GK activity was very similar in cells from those with the symptomatic and asymptomatic forms of iGKD [33] . We concluded that genotype did not predict phenotype for males with iGKD and suggested that the phenotype for this “single” gene disorder was a complex trait influenced by independently inherited genes.

One might argue that there could be a role for environmental modifiers for iGKD. Episodic symptomatic decompensation is precipitated by inter-current viral illnesses. Therefore, if a boy with the potential for the symptomatic form did not have the environmental exposure to an inter-current illness of sufficient severity to cause the decompensation, then he would be considered on clinical grounds to have the asymptomatic form.

The association of T2DM with iGKD and the role of GK in T2DM therapy suggest a potential novel therapeutic approach to the management of episodic metabolic decompensation with iGKD. The thiazolidinediones, drugs that are used to treat individuals with T2DM by improving insulin sensitivity, and increasing GK expression in fat cells with an associated futile cycle involving GK and the reduction of fatty acids 35 36 . It remains to be determined whether thiazolidinediones would increase fat cell GK expression in patients with iGKD, and if they did then whether the increased expression of GK in fat cells of boys with symptomatic iGKD would be sufficient to prevent metabolic and CNS decompositions.

### 4 Summary

The phenotypes of patients with simple, single gene Mendelian disorders are in fact complex traits, and modifiers, both genetic and environmental, play significant roles in this complexity. Identifying protective modifiers can provide novel therapeutic opportunities, and examples, including SMA, DMD and iGKD, have been provided.

We live in an exciting era when genomic sequencing is an important component in our diagnostic tool kit. However, a functional understanding of biology, the impact of its disruption, and the possibilities for its repair have never been more important as we search for new therapeutic strategies

### Acknowledgement

This work was supported in part by salary for ERB McCabe from the March of Dimes .

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Comment by Ellen Sidransky
One of the major challenges in the study of Mendelian disorders is the vast clinical heterogeneity encountered even among individuals with the same DNA mutations. This led to the concept that Mendelian disorders are actually complex traits with one primary gene, but other genes or factors that act as modifiers.
In his commentary piece, Dr. McCabe, the editor of MGM expands on this topic, providing examples where modifier genes have been identified, often from the use of model organisms. He chose Spinal Muscular Atrophy as a case where there are protective modifiers, identified using genomics, C. elegans and interactomes. In addition, he provides illustrative examples where the identification of modifiers has led to new therapeutic strategies.
This is an important area which is likely to become increasingly crucial as genomic sequencing and pathway analyses lead to a better understanding of disease pathogenesis. Stay tuned!