Genotype‐phenotype study in patients with valosin‐containing protein mutations associated with multisystem proteinopathy

Mutations in valosin‐containing protein (VCP), an ATPase involved in protein degradation and autophagy, cause VCP disease, a progressive autosomal dominant adult onset multisystem proteinopathy. The goal of this study is to examine if phenotypic differences in this disorder could be explained by the specific gene mutations. We therefore studied 231 individuals (118 males and 113 females) from 36 families carrying 15 different VCP mutations. We analyzed the correlation between the different mutations and prevalence, age of onset and severity of myopathy, Paget's disease of bone (PDB), and frontotemporal dementia (FTD), and other comorbidities. Myopathy, PDB and FTD was present in 90%, 42% and 30% of the patients, respectively, beginning at an average age of 43, 41, and 56 years, respectively. Approximately 9% of patients with VCP mutations had an amyotrophic lateral sclerosis (ALS) phenotype, 4% had been diagnosed with Parkinson's disease (PD), and 2% had been diagnosed with Alzheimer's disease (AD). Large interfamilial and intrafamilial variation made establishing correlations difficult. We did not find a correlation between the mutation type and the incidence of any of the clinical features associated with VCP disease, except for the absence of PDB with the R159C mutation in our cohort and R159C having a later age of onset of myopathy compared with other molecular subtypes.


| INTRODUCTION
Inclusion body myopathy (IBM) associated with Paget's disease of bone (PDB) and frontotemporal dementia (FTD) (IBMPFD) or multisystem proteinopathy is an adult-onset progressive, autosomal dominant ultimately lethal disease that involves degeneration of 3 main organ systems: muscle, bone, and brain. Most cases are caused by heterozygous missense mutations in valosin-containing protein (VCP). 1,2 As awareness increases we are realizing that VCP disease is not as rare as previously considered as it is often misdiagnosed as related disorders.

| Pathology of IBMPFD
IBM is characterized by progressive weakness and atrophy of skeletal muscles of pelvic and shoulder girdle muscles. Ultimately, patients die from respiratory failure, cardiomyopathy and cardiac failure. 3 Histologically, IBM consists of cytoplasmic rimmed vacuoles containing the same proteins that aggregate in the brains of patients with neurodegenerative diseases: tau, amyloid, and TAR DNA-binding protein-43 (TDP-43). 4 PDB is a unique skeletal disease caused by overactive osteoclasts and an imbalance between osteoclast and osteoblast function. The result is a gain in bone mass, but the new bone is disorganized, weak, and prone to fractures. Typical radiological findings of PDB include coarse trabeculation, cortical thickening and spotty sclerosis. Clinical features include bone pain, bone enlargement, bowing of long bones, large head, fractures, hearing loss due to auditory foramen narrowing, and arthritis. Rare complications include kidney stones, osteosarcoma, and high-output heart failure due to the formation of arteriovenous shunts in bone. 5 FTD is an early-onset type of dementia that is typically diagnosed in younger patients roughly 60% occurring in people 45 to Ebaa Al-Obeidi and Sejad Al-Tahan contributed equally to the manuscript. 64 years old, 6 however, in VCP disease is associated with an earlier age of onset. Degeneration and atrophy of the frontal and temporal lobes of the brain results in changes in personality and progressive loss of language. Brain histology in patients with IBMPFD affected by FTD is characterized by gliosis, spongiosis, and neuronal intranuclear inclusions. 7 TDP-43 aggregates are commonly associated with VCPassociated FTD as well as in amyotrophic lateral sclerosis (ALS). 4,8,9 TDP-43 is a DNA/RNA-binding protein involved in various cellular processes including RNA transcription and splicing. [10][11][12] We have previously shown that the presence of 1 or 2 APOE4 alleles is associated with an increased risk of developing FTD in patients with VCP disease. 13 Mutations in VCP have also been associated with a spectrum of other diseases including ALS, 14 hereditary spastic paraplegia, 15 Charcot-Marie-Tooth type 2 (CMT2) disease. 16 Other common disorders that have an overlap with VCP disease include facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy (LGMD), scapuloperoneal muscular dystrophy (SPMD), inclusion body myositis, and distal myopathy/oculopharyngeal muscular dystrophy. 17 1.2 | Structure and function of VCP VCP has 4 domains: an N-terminal ubiquitin-binding domain, 2 ATPase domains (D1 and D2), and a C-terminal region. 18 Valosin is a 25 amino acid peptide named after its N-terminal valine and C-terminal tyrosine, and was originally isolated from the porcine gut. 19 That peptide sequence is present in VCP, which is a highly abundant ATPase found in all cells where it interacts with various adaptor proteins to carry out many essential cellular processes. Among them are endoplasmic reticulum-associated degradation, 20 transcription factor processing, 21 nuclear envelope reconstruction, 22 membrane fusion, 23 post-mitotic golgi reassembly, 24 spindle disassembly, 25 and cell cycle control. 26 Several of these activities are associated with the ubiquitin-proteasome system in which VCP helps deliver ubiquitylated substrates to the 26S proteasome for degradation. 27 VCP's roles in protein degradation and autophagy is implicated in the pathogenesis of IBMPFD, and may account for the cytoplasmic inclusions observed in muscle, bone, and neuronal tissue. 1,27 The hypothesis is that the location of the mutation may influence certain roles of VCP and thus lead to variation in the phenotype seen in VCP.

| Statistical analysis
One-way analysis of variance (ANOVA) and Bonferroni post-hoc analysis were performed using SPSS statistical package (v. 21). Only mutation groups with more than 5 patients were included in the 1way ANOVA. Population standard deviations are reported as this is a rare disease and this study represents the largest assembly of patients published to date.   Although VCP multisystem disorder is associated with a triad of symptoms, only 10% of the patients in our study presented with all three main features of the disorder (Figure 1). Myopathy was the most common symptom, presenting in 89% of patients and as an isolated feature in 36%. PDB was diagnosed in 43% of patients, and in 5% it was the sole clinical feature. A total of 29% patients had FTD, and in 3% this was the sole feature.

| Genotype-phenotype studies
To elucidate the effect of different mutations on phenotypic variations, the families were divided into 15 groups according to their  No statistically significant differences were identified for PDB or FTD. We also did not see a higher incidence of ALS or PD with any specific mutation types. The G97E mutation was reported to be associated with CMT2 35 ; however, we did not find any case of CMT in our cohort of patients.

| DISCUSSION
VCP disease is an autosomal dominant syndrome associated with progressive inclusion body limb-girdle type myopathy, PDB, FTD (IBMPFD), and ALS. We report genotype-phenotype studies in patients bearing 1 of the 15 mutations in this report to determine whether correlations exist between a patient's mutation and the age of onset of their symptoms and associated manifestations. The primary findings are the general lack of genotype-phenotype correlations because of the enormous phenotypic heterogeneity within and between families. We found an incidence of 90% for the myopathy, 42% for PBD, 30% for FTD, 9% for classic ALS, and 4% for PD (  Among families of European ancestry, patients of German, 17,43 Italian, 41,44,45 Spanish, 46 Austrian, 47 Belgian, 48 French, 49 Irish, 50 and British 51-53 backgrounds have been identified. In Asia, Korean, 54 Japanese, 55 and Chinese, 56