<p>(A–F) Immunohistochemical analysis of quadriceps muscles from 9–10 month old wild-type (... more <p>(A–F) Immunohistochemical analysis of quadriceps muscles from 9–10 month old wild-type (A–C) and VCP<sup>R155H/+</sup> knock-in mice (D–F) were stained with a ubiquitin-specific FK1 antibody (A,D) and a TDP-43-specific antibody (B,E). (C) shows the overlay of (A) and (B), and (F) is the overlay of (D) and (E). Ubiquitin- and TDP-43-positive, cytoplasmic inclusion body is shown by an arrow in (D–F). Nuclei were stained with DAPI. Magnification: 630×.). (G) Expression of TDP-43 and ubiquitinated proteins. Proteins were harvested from the quadriceps muscle of 2 littermates of wild-type and knock-in mice and analyzed by Western blotting using TDP-43 (upper panel) and ubiquitin/FK1 (lower panel) antibodies. Each membrane was re-probed with actin to confirm equal protein loading in each lane. Protein bands are indicated on the left and molecular weights of marker bands for the ubiquitin blot on the right. Genotypes are shown above. Wild-type and knock-in samples are from two litters (indicated above the figure) (N = 4 WT and 4 R155H animals).</p
<p>(A–C) Quadricep muscles from 9–10 month-old wild-type and VCP<sup>R155H/+</sup&... more <p>(A–C) Quadricep muscles from 9–10 month-old wild-type and VCP<sup>R155H/+</sup> knock-in mice were analyzed by H&E staining. (B) An enlarged vacuole in the mutant tissue is shown by white arrows and (C) centrally located nuclei and rimmed vacuoles are revealed in the mutant mice shown by white arrows. (D) Quadriceps muscles from 15-month old VCP<sup>R155H/+</sup> knock-in mice, centrally located nuclei shown by white arrows. (E–F) Modified Gomori Trichrome staining of muscle fibers from wild type and VCP<sup>R155H/+</sup> knock-in mice. Magnification: 630×. (G–L) Electron microscopy analyses of the mouse quadricep muscles. Vacuolization and loss of myofilament organization are observed in quadriceps muscle from 10-month-old VCP<sup>R155H/+</sup> knock-in mice (G–I), but not in wild-type mice (J–L). Sarcomeric direction is indicated by white double ended arrow (H,K). Swollen mitochondria are also observed in the mutant tissue (L). Black arrows in (K) and (L) indicate accumulation of vacuoles. Size bars are shown in the lower left corner of each image. Mt = mitochondria. Magnifications: E+H = 900×, F+I = 2,950×, G+J = 11,500×. (N = 3 WT and 3 R155H animals).</p
<p>(A–B) micro CT images showing sclerotic lesions at anterior tibia and posterior femur sh... more <p>(A–B) micro CT images showing sclerotic lesions at anterior tibia and posterior femur shown by white arrows in 15-month old knock-in mice. (C–D) Transverse sections of decalcified 6<sup>th</sup> lumbar vertebra, white arrowheads indicate red colored TRAP-positive osteoclasts (Magnification 10×). (E–G) OCLs formed from non-adherent marrow cell cultures for WT or VCP<sup>R155H/+</sup> cultured for 9 days in the presence of 10 ng/ml M-CSF and varying concentrations of RANKL. The cells were then fixed and stained for TRAP activity. Results represent TRAP positive cells containing ≥ three nuclei and are expressed as the mean ± SEM for duplicate cultures.*P<0.05 compared with results from wt cell cultures. MNC = multi-nuclear cell. (G) Large TRAP positive multinuclear OC-like cell from knock-in bone marrow derived macrophages (BMDM). Differentiated in 100 ng/ml RANKL and 50 ng/ml M-CSF then fixed and TRAP stained on day 9 of differentiation. (H) Osteoclastogenesis cytokine sensitivity was determined with increasing concentrations of RANKL. TRAP-expressing multi-nucleated OCs was scored (N = 4 WT and 4 R155H animals).</p
<p>Significant differences were identified in the trabecular pattern factor and cortical wa... more <p>Significant differences were identified in the trabecular pattern factor and cortical wall thickness.</p
<p>(A–B) H&E staining of 15 month old wild type and knock-in mouse of hippocampus and n... more <p>(A–B) H&E staining of 15 month old wild type and knock-in mouse of hippocampus and neuronal injury shown by white arrows (Magnification 400×). (C–D) IBA1 staining of frontal cortex from wild-type and R155H knock-in mice shown by white arrows Magnification: 630× (N = 3 WT and 3 R155H animals).</p
Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible... more Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible evidence. Here, we present three DNA methylation-based age estimators (epigenetic clocks) for verifying age claims of centenarians. The three centenarian clocks were developed based on n = 7039 blood and saliva samples from individuals older than 40, including n = 184 samples from centenarians, 122 samples from semi-supercentenarians (aged 105 +), and 25 samples from supercentenarians (aged 110 +). The oldest individual was 115 years old. Our most accurate centenarian clock resulted from applying a neural network model to a training set composed of individuals older than 40. An epigenome-wide association study of age in different age groups revealed that age effects in young individuals (age < 40) are correlated (r = 0.55) with age effects in old individuals (age > 90). We present a chromatin state analysis of age effects in centenarians. The centenarian clocks are expected to be ...
<p>(A) Decline of physical performance by Rotarod analysis in knock-in mice. (B) Progressiv... more <p>(A) Decline of physical performance by Rotarod analysis in knock-in mice. (B) Progressive impairment of muscle strength measured by grip strength meter in knock-in mice. Ages of mice are shown in the X-axis and results are indicated in the Y-axis as relative values when compared to the wild-type mouse values. The following numbers of mice have been used to analyze physical performance test at different time points: 3 months (25 wild-type, 9 knock-in), 6 months (51 wild-type, 20 knock-in), 9 months (48 wild-type, 18 knock-in), 12 months (24 wild-type, 9 knock-in), and 15 months (20 wild-type, 8 knock-in). Note * * in the figure represents p value <0.05.</p
<p>(A–B) Quadricep muscles from 9–10 month old wild type and VCP<sup>R155H/+</sup&... more <p>(A–B) Quadricep muscles from 9–10 month old wild type and VCP<sup>R155H/+</sup> knock-in mice were stained with an LC3-II-specific antibody. Nuclei were stained with DAPI (Magnification: 630×). (C) Protein expression of LC3-II is increased in knock in mice as compared with wild type litter mates. (D–E) DAPI and TUNEL staining of the quadriceps section from a wild-type and VCP<sup>R155H/+</sup> knock-in mouse models (N = 3 WT and 3 R155H animals). Apoptotic nuclei of the mutant tissue are shown by white arrows. Magnification: 400×. (F) Caspase-3 activity was measured from the quadriceps muscle lysates of wild-type and VCP<sup>R155H/+</sup> knock-in mice. Specific activities are shown in the Y-axis and genotypes in the X-axis (N = 4 WT and 4 R155H animals).</p
Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of... more Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso-1-cytochrome c. Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine. Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy. Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the rn-value, the rate of change of free energy with respect to denaturant concentration, to different degrees. Changes in rn-values are indicative of changes in the equilibrium folding mechanism of a protein. Decreases in rn-values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state. When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the rn-value for a given variant appear to depend upon the substructure in which the mutation is made. These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein.
Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy ass... more Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. Xray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP R155H/+ knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMP... more Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a progressive, fatal genetic disorder with variable penetrance, predominantly affecting three main tissue types: muscle (IBM), bone (PDB), and brain (FTD). IBMPFD is caused by mutations in the ubiquitously expressed valosin-containing protein (VCP) gene, a member of the AAA-ATPase superfamily. The majority of individuals who develop IBM have progressive proximal muscle weakness. Muscle biopsies reveal rimmed vacuoles and inclusions that are ubiquitinand TAR DNA binding protein-43 (TDP-43)-positive using immunohistochemistry. PDB, seen in half the individuals, is caused by overactive osteoclasts and is associated clinically with pain, elevated serum alkaline phosphatase, and X-ray findings of coarse trabeculation and sclerotic lesions. FTD diagnosed at a mean age of 55 years in a third of individuals is characterized clinically by comprehension deficits, dysnomia, dyscalculia, and social unawareness. Ubiquitin-and TDP-43positive neuronal inclusions are also found in the brain. Genotype-phenotype correlations are difficult with marked intra-familial and inter-familial variations being seen. Varied phenotypes within families include frontotemporal dementia, amyotrophic lateral sclerosis, Parkinsonism, myotonia, cataracts, and anal incompetence, among others. Cellular and animal models indicate pathogenetic disturbances in IBMPFD tissues including altered protein degradation, autophagy pathway alterations, apoptosis, and mitochondrial dysfunction. Currently, mouse and drosophila models carrying VCP mutations provide insights into the human IBMPFD pathology and are useful as tools for preclinical studies and testing of therapeutic strategies. In this review, we will explore the pathogenesis and clinical phenotype of IBMPFD caused by VCP mutations.
VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal... more VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype-phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring ten missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and non-carriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter-familial variation; and significant genotype-phenotype correlations were difficult because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with earlier onset of myopathy and Paget (p=0.03). Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p=0.03). We identified amyotrophic lateral sclerosis (ALS) in thirteen individuals (8.9%) and Parkinson's disease in five individuals (3%); however
Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD... more Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder caused by mutations in the Valosin Containing Protein (VCP) gene on chromosome 9p12-13. Patients demonstrate limb girdle muscle weakness, which eventually progresses to involve respiratory muscles, and death from respiratory and cardiac failure. This is the first investigation to analyze key molecular mediators and signaling cascades in skeletal muscle causing myopathy by global gene microarray in hopes of understanding the dysregulated genes and molecular mechanisms underlying IBMPFD and the hope of finding novel therapeutic targets. We determined expression profiles using Human Genome Array microarray technology in Vastus lateralis muscles from patients and their first degree relatives. We analyzed gene annotations by DAVID and identified differentially dysregulated genes with roles in several novel biological pathways, including regulation of actin cytoskeleton, ErbB signaling, cancer, in addition to regulation of autophagy, and lysosomal signaling, known disrupted pathways in VCP disease. In this report, we present data from the first global microarray analyzing IBMPFD patient muscles and elucidating dysregulated pathways to further understand the pathogenesis of the disease and discover potential therapeutics.
Background— The intermediate filament protein desmin is encoded by the gene DES and contributes t... more Background— The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. Methods and Results— We identified the 2 novel variants DES -p.A120D (c.359C>A) and DES -p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in ...
Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medi... more Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.
Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of... more Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia [IBMPFD] syndrome) is caused by mutations in the gene encoding VCP classically affecting the muscle, bone and brain. Although the genetic cause has been identified, details regarding the pathogenesis of IBMPFD have not been fully determined. Muscle wasting observed in VCP disease is suggestive of cytokine imbalance. We hypothesized that dysfunctional protein homeostasis caused by VCP mutations leads to cytokine imbalances thereby contributing to the muscle wasting phenotype. Circulating levels of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF a) and epidermal growth factor (EGF) were measured in plasma of patients with VCP disease or controls. TNF a and EGF were significantly altered in VCP disease as compared to control. TNF a was up-regulated, consistent with a cachexia phenotype and EGF levels were increased. No significant differences were observed in IL-4 and IL-6. Cytokine imbalances may be associated with VCP disease and may play a contributory role in VCP myopathy. Further understanding of how VCP dysfunction leads to aberrant protein homeostasis and subsequent cytokine imbalances may also aid in the understanding of other proteinopathies and in the development of novel treatments.
BACKGROUND: Progress in science and technology have created the capabilities and alternatives to ... more BACKGROUND: Progress in science and technology have created the capabilities and alternatives to symptom-driven medical care. Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority we address using advanced screening detection. METHODS: We enrolled active adults for early detection of risk for age-related chronic disease associated with premature mortality. Whole genome sequencing together with: global metabolomics, 3D/4D imaging using non-contrast whole body magnetic resonance imaging and echocardiography, and 2-week cardiac monitoring were employed to detect age-related chronic diseases and risk for diseases. RESULTS: We detected previously unrecognized age-related chronic diseases requiring prompt (<30 days) medical attention in 17 (8%, 1:12) of 209 study participants, including 4 participants with early stage neoplasms (2%, 1:50). Likely mechanistic genomic findings correlating with clinical ...
ABSTRACTThere is a significant interest in the standardized classification of human genetic varia... more ABSTRACTThere is a significant interest in the standardized classification of human genetic variants. The availability of new large datasets generated through genome sequencing initiatives provides a ground for the computational evaluation of the supporting evidence. We used whole genome sequence data from 8,102 unrelated individuals to analyze the adequacy of estimated rates of disease on the basis of genetic risk and the expected population prevalence of the disease. Analyses included the ACMG recommended 56 gene-condition sets for incidental findings and 631 genes associated with 348 OrphaNet conditions. A total of 21,004 variants were used to identify patterns of inflation (i.e. excess genetic risk). Inflation, i.e., misclassification, increases as the level of evidence in ClinVar supporting the pathogenic nature of the variant decreases. The burden of rare variants was a main contributing factor of the observed inflation indicating misclassified benign private mutations. We als...
<p>(A–F) Immunohistochemical analysis of quadriceps muscles from 9–10 month old wild-type (... more <p>(A–F) Immunohistochemical analysis of quadriceps muscles from 9–10 month old wild-type (A–C) and VCP<sup>R155H/+</sup> knock-in mice (D–F) were stained with a ubiquitin-specific FK1 antibody (A,D) and a TDP-43-specific antibody (B,E). (C) shows the overlay of (A) and (B), and (F) is the overlay of (D) and (E). Ubiquitin- and TDP-43-positive, cytoplasmic inclusion body is shown by an arrow in (D–F). Nuclei were stained with DAPI. Magnification: 630×.). (G) Expression of TDP-43 and ubiquitinated proteins. Proteins were harvested from the quadriceps muscle of 2 littermates of wild-type and knock-in mice and analyzed by Western blotting using TDP-43 (upper panel) and ubiquitin/FK1 (lower panel) antibodies. Each membrane was re-probed with actin to confirm equal protein loading in each lane. Protein bands are indicated on the left and molecular weights of marker bands for the ubiquitin blot on the right. Genotypes are shown above. Wild-type and knock-in samples are from two litters (indicated above the figure) (N = 4 WT and 4 R155H animals).</p
<p>(A–C) Quadricep muscles from 9–10 month-old wild-type and VCP<sup>R155H/+</sup&... more <p>(A–C) Quadricep muscles from 9–10 month-old wild-type and VCP<sup>R155H/+</sup> knock-in mice were analyzed by H&E staining. (B) An enlarged vacuole in the mutant tissue is shown by white arrows and (C) centrally located nuclei and rimmed vacuoles are revealed in the mutant mice shown by white arrows. (D) Quadriceps muscles from 15-month old VCP<sup>R155H/+</sup> knock-in mice, centrally located nuclei shown by white arrows. (E–F) Modified Gomori Trichrome staining of muscle fibers from wild type and VCP<sup>R155H/+</sup> knock-in mice. Magnification: 630×. (G–L) Electron microscopy analyses of the mouse quadricep muscles. Vacuolization and loss of myofilament organization are observed in quadriceps muscle from 10-month-old VCP<sup>R155H/+</sup> knock-in mice (G–I), but not in wild-type mice (J–L). Sarcomeric direction is indicated by white double ended arrow (H,K). Swollen mitochondria are also observed in the mutant tissue (L). Black arrows in (K) and (L) indicate accumulation of vacuoles. Size bars are shown in the lower left corner of each image. Mt = mitochondria. Magnifications: E+H = 900×, F+I = 2,950×, G+J = 11,500×. (N = 3 WT and 3 R155H animals).</p
<p>(A–B) micro CT images showing sclerotic lesions at anterior tibia and posterior femur sh... more <p>(A–B) micro CT images showing sclerotic lesions at anterior tibia and posterior femur shown by white arrows in 15-month old knock-in mice. (C–D) Transverse sections of decalcified 6<sup>th</sup> lumbar vertebra, white arrowheads indicate red colored TRAP-positive osteoclasts (Magnification 10×). (E–G) OCLs formed from non-adherent marrow cell cultures for WT or VCP<sup>R155H/+</sup> cultured for 9 days in the presence of 10 ng/ml M-CSF and varying concentrations of RANKL. The cells were then fixed and stained for TRAP activity. Results represent TRAP positive cells containing ≥ three nuclei and are expressed as the mean ± SEM for duplicate cultures.*P<0.05 compared with results from wt cell cultures. MNC = multi-nuclear cell. (G) Large TRAP positive multinuclear OC-like cell from knock-in bone marrow derived macrophages (BMDM). Differentiated in 100 ng/ml RANKL and 50 ng/ml M-CSF then fixed and TRAP stained on day 9 of differentiation. (H) Osteoclastogenesis cytokine sensitivity was determined with increasing concentrations of RANKL. TRAP-expressing multi-nucleated OCs was scored (N = 4 WT and 4 R155H animals).</p
<p>Significant differences were identified in the trabecular pattern factor and cortical wa... more <p>Significant differences were identified in the trabecular pattern factor and cortical wall thickness.</p
<p>(A–B) H&E staining of 15 month old wild type and knock-in mouse of hippocampus and n... more <p>(A–B) H&E staining of 15 month old wild type and knock-in mouse of hippocampus and neuronal injury shown by white arrows (Magnification 400×). (C–D) IBA1 staining of frontal cortex from wild-type and R155H knock-in mice shown by white arrows Magnification: 630× (N = 3 WT and 3 R155H animals).</p
Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible... more Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible evidence. Here, we present three DNA methylation-based age estimators (epigenetic clocks) for verifying age claims of centenarians. The three centenarian clocks were developed based on n = 7039 blood and saliva samples from individuals older than 40, including n = 184 samples from centenarians, 122 samples from semi-supercentenarians (aged 105 +), and 25 samples from supercentenarians (aged 110 +). The oldest individual was 115 years old. Our most accurate centenarian clock resulted from applying a neural network model to a training set composed of individuals older than 40. An epigenome-wide association study of age in different age groups revealed that age effects in young individuals (age < 40) are correlated (r = 0.55) with age effects in old individuals (age > 90). We present a chromatin state analysis of age effects in centenarians. The centenarian clocks are expected to be ...
<p>(A) Decline of physical performance by Rotarod analysis in knock-in mice. (B) Progressiv... more <p>(A) Decline of physical performance by Rotarod analysis in knock-in mice. (B) Progressive impairment of muscle strength measured by grip strength meter in knock-in mice. Ages of mice are shown in the X-axis and results are indicated in the Y-axis as relative values when compared to the wild-type mouse values. The following numbers of mice have been used to analyze physical performance test at different time points: 3 months (25 wild-type, 9 knock-in), 6 months (51 wild-type, 20 knock-in), 9 months (48 wild-type, 18 knock-in), 12 months (24 wild-type, 9 knock-in), and 15 months (20 wild-type, 8 knock-in). Note * * in the figure represents p value <0.05.</p
<p>(A–B) Quadricep muscles from 9–10 month old wild type and VCP<sup>R155H/+</sup&... more <p>(A–B) Quadricep muscles from 9–10 month old wild type and VCP<sup>R155H/+</sup> knock-in mice were stained with an LC3-II-specific antibody. Nuclei were stained with DAPI (Magnification: 630×). (C) Protein expression of LC3-II is increased in knock in mice as compared with wild type litter mates. (D–E) DAPI and TUNEL staining of the quadriceps section from a wild-type and VCP<sup>R155H/+</sup> knock-in mouse models (N = 3 WT and 3 R155H animals). Apoptotic nuclei of the mutant tissue are shown by white arrows. Magnification: 400×. (F) Caspase-3 activity was measured from the quadriceps muscle lysates of wild-type and VCP<sup>R155H/+</sup> knock-in mice. Specific activities are shown in the Y-axis and genotypes in the X-axis (N = 4 WT and 4 R155H animals).</p
Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of... more Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso-1-cytochrome c. Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine. Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy. Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the rn-value, the rate of change of free energy with respect to denaturant concentration, to different degrees. Changes in rn-values are indicative of changes in the equilibrium folding mechanism of a protein. Decreases in rn-values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state. When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the rn-value for a given variant appear to depend upon the substructure in which the mutation is made. These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein.
Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy ass... more Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. Xray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP R155H/+ knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMP... more Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a progressive, fatal genetic disorder with variable penetrance, predominantly affecting three main tissue types: muscle (IBM), bone (PDB), and brain (FTD). IBMPFD is caused by mutations in the ubiquitously expressed valosin-containing protein (VCP) gene, a member of the AAA-ATPase superfamily. The majority of individuals who develop IBM have progressive proximal muscle weakness. Muscle biopsies reveal rimmed vacuoles and inclusions that are ubiquitinand TAR DNA binding protein-43 (TDP-43)-positive using immunohistochemistry. PDB, seen in half the individuals, is caused by overactive osteoclasts and is associated clinically with pain, elevated serum alkaline phosphatase, and X-ray findings of coarse trabeculation and sclerotic lesions. FTD diagnosed at a mean age of 55 years in a third of individuals is characterized clinically by comprehension deficits, dysnomia, dyscalculia, and social unawareness. Ubiquitin-and TDP-43positive neuronal inclusions are also found in the brain. Genotype-phenotype correlations are difficult with marked intra-familial and inter-familial variations being seen. Varied phenotypes within families include frontotemporal dementia, amyotrophic lateral sclerosis, Parkinsonism, myotonia, cataracts, and anal incompetence, among others. Cellular and animal models indicate pathogenetic disturbances in IBMPFD tissues including altered protein degradation, autophagy pathway alterations, apoptosis, and mitochondrial dysfunction. Currently, mouse and drosophila models carrying VCP mutations provide insights into the human IBMPFD pathology and are useful as tools for preclinical studies and testing of therapeutic strategies. In this review, we will explore the pathogenesis and clinical phenotype of IBMPFD caused by VCP mutations.
VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal... more VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype-phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring ten missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and non-carriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter-familial variation; and significant genotype-phenotype correlations were difficult because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with earlier onset of myopathy and Paget (p=0.03). Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p=0.03). We identified amyotrophic lateral sclerosis (ALS) in thirteen individuals (8.9%) and Parkinson's disease in five individuals (3%); however
Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD... more Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder caused by mutations in the Valosin Containing Protein (VCP) gene on chromosome 9p12-13. Patients demonstrate limb girdle muscle weakness, which eventually progresses to involve respiratory muscles, and death from respiratory and cardiac failure. This is the first investigation to analyze key molecular mediators and signaling cascades in skeletal muscle causing myopathy by global gene microarray in hopes of understanding the dysregulated genes and molecular mechanisms underlying IBMPFD and the hope of finding novel therapeutic targets. We determined expression profiles using Human Genome Array microarray technology in Vastus lateralis muscles from patients and their first degree relatives. We analyzed gene annotations by DAVID and identified differentially dysregulated genes with roles in several novel biological pathways, including regulation of actin cytoskeleton, ErbB signaling, cancer, in addition to regulation of autophagy, and lysosomal signaling, known disrupted pathways in VCP disease. In this report, we present data from the first global microarray analyzing IBMPFD patient muscles and elucidating dysregulated pathways to further understand the pathogenesis of the disease and discover potential therapeutics.
Background— The intermediate filament protein desmin is encoded by the gene DES and contributes t... more Background— The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. Methods and Results— We identified the 2 novel variants DES -p.A120D (c.359C>A) and DES -p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in ...
Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medi... more Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.
Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of... more Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia [IBMPFD] syndrome) is caused by mutations in the gene encoding VCP classically affecting the muscle, bone and brain. Although the genetic cause has been identified, details regarding the pathogenesis of IBMPFD have not been fully determined. Muscle wasting observed in VCP disease is suggestive of cytokine imbalance. We hypothesized that dysfunctional protein homeostasis caused by VCP mutations leads to cytokine imbalances thereby contributing to the muscle wasting phenotype. Circulating levels of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF a) and epidermal growth factor (EGF) were measured in plasma of patients with VCP disease or controls. TNF a and EGF were significantly altered in VCP disease as compared to control. TNF a was up-regulated, consistent with a cachexia phenotype and EGF levels were increased. No significant differences were observed in IL-4 and IL-6. Cytokine imbalances may be associated with VCP disease and may play a contributory role in VCP myopathy. Further understanding of how VCP dysfunction leads to aberrant protein homeostasis and subsequent cytokine imbalances may also aid in the understanding of other proteinopathies and in the development of novel treatments.
BACKGROUND: Progress in science and technology have created the capabilities and alternatives to ... more BACKGROUND: Progress in science and technology have created the capabilities and alternatives to symptom-driven medical care. Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority we address using advanced screening detection. METHODS: We enrolled active adults for early detection of risk for age-related chronic disease associated with premature mortality. Whole genome sequencing together with: global metabolomics, 3D/4D imaging using non-contrast whole body magnetic resonance imaging and echocardiography, and 2-week cardiac monitoring were employed to detect age-related chronic diseases and risk for diseases. RESULTS: We detected previously unrecognized age-related chronic diseases requiring prompt (<30 days) medical attention in 17 (8%, 1:12) of 209 study participants, including 4 participants with early stage neoplasms (2%, 1:50). Likely mechanistic genomic findings correlating with clinical ...
ABSTRACTThere is a significant interest in the standardized classification of human genetic varia... more ABSTRACTThere is a significant interest in the standardized classification of human genetic variants. The availability of new large datasets generated through genome sequencing initiatives provides a ground for the computational evaluation of the supporting evidence. We used whole genome sequence data from 8,102 unrelated individuals to analyze the adequacy of estimated rates of disease on the basis of genetic risk and the expected population prevalence of the disease. Analyses included the ACMG recommended 56 gene-condition sets for incidental findings and 631 genes associated with 348 OrphaNet conditions. A total of 21,004 variants were used to identify patterns of inflation (i.e. excess genetic risk). Inflation, i.e., misclassification, increases as the level of evidence in ClinVar supporting the pathogenic nature of the variant decreases. The burden of rare variants was a main contributing factor of the observed inflation indicating misclassified benign private mutations. We als...
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