Hereditary Inclusion Body Myopathy Patients Research Articles

P4.47 Hereditary inclusion body myopathy therapy: Assessment of an adeno associated virus AAV2/8 based GNE gene delivery system

Hereditary inclusion body myopathy (HIBM) is a unique neuromuscular disorder characterized by adult onset and slowly progressive distal and proximal muscle and a typical histology including rimmed vacuoles and filamentous inclusions. The disease is caused by recessive mutations in the UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase gene (GNE), encoding the key enzyme in the biosynthetic pathway of sialic acid. In an attempt to develop a possible treatment for HIBM patients, we have establish a platform for gene therapy trials based on adeno associated virus (AAV 8) vectors harboring the wild type GNE gene.

P2.01 Phenotypic variability of knock in mice carrying the M712T mutation in GNE

Mutations in GNE cause hereditary inclusion body myopathy (HIBM), an adult onset, slowly progressive distal and proximal primary myopathy. GNE is well known as the key enzyme for the biosynthesis of sialic acid, but the exact mechanism of the disease is still an enigma. GNE knock out results in lethality of mice embryos at day E8.5. We have generated a knock in mouse by introducing the founder mutation in Middle Eastern HIBM patients, M712T, by homologous recombination.

Novel GNE Mutations in Hereditary Inclusion Body Myopathy Patients of Non-Middle Eastern Descent

Autosomal recessive hereditary inclusion body myopathy (HIBM or IBM2) is a progressive adult onset muscle wasting disorder characterized by sparing of the quadriceps. IBM2 is also known as distal myopathy with rimmed vacuoles or nonaka myopathy. IBM2 is associated with mutations in the UDP-GlcNAc 2-Epimerase/ManNAc Kinase gene (GNE). GNE is the rate-limiting enzyme of N-Acetylneuraminate (Neu5Ac, Sialic acid) biosynthesis. The GNE coding region of 64 symptomatic patients were sequenced. Twenty-eight patients were found to bear GNE mutations. Ten novel mutations were identified among nine patients, including four nonsense (p.R8X, p.W204X, p.Q436X, and p.S615X) and five missense (p.R71W, p.I142T, p.I298T, p.L556S, and p.E2G) variations spanning both the epimerase and kinase domains of GNE. Additionally, a synonymous variation (p.Y591Y, codon tac > tat) was seen in a patient bearing compound heterozygous nonsynonymous mutations (p.S615X and p.Y675H). Six of the nine are Caucasian, one patient is Taiwanese, one patient is Asian Indian, and one patient is of European descent. These findings further expand the clinical and genetic spectrum of IBM2.

Hereditary inclusion body myopathy: single patient response to GNE gene Lipoplex therapy

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult onset myopathy. It is characterized by mutations of the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Afflicted patients have no therapeutic options. In preclinical testing, we have previously demonstrated the ability to correct GNE gene function and the safety of delivery of wild type GNE gene using a liposomal delivery vehicle. A single patient (subject #001) with severe HIBM treated by compassionate investigational new drug received four doses of GNE gene Lipoplex via intramuscular injection. GNE transgene expression, downstream induction of sialic acid, safety and muscle function were evaluated. Significant durable improvement in locoregional skeletal muscle function was observed in the injected left extensor carpi radialis longus of #001 in correlation with GNE transgene upregulation and local induction of sialic acid. Other than transient low grade fever and pain at the injection site, no significant toxicity was observed. Proof of principle for manufacturing of 'clinical grade' GNE gene Lipoplex, clinical safety and activity are demonstrated with GNE gene Lipoplex. Further assessment will involve intravenous administration and subsequent phase I trial involving additional but less severely afflicted HIBM patients.

Mitochondrial processes are impaired in hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. To elucidate the pathological mechanisms leading from the mutated GNE to the HIBM phenotype, we attempted to identify and characterize early occurring downstream events by analyzing the genomic expression patterns of muscle specimens from 10 HIBM patients carrying the M712T Persian Jewish founder mutation and presenting mild histological changes, compared with 10 healthy matched control individuals, using GeneChip expression microarrays. When analyzing the expression profile data sets by the intersection of three statistic methods (Student's t-test, TNoM and Info score), we found that the HIBM-specific transcriptome consists of 374 differentially expressed genes. The specificity of the HIBM transcriptome was assessed by the minimal transcript overlap found between HIBM and the transcriptome of nine additional muscle disorders including adult onset limb girdle myopathies, inflammatory myopathies and early onset conditions. A strikingly high proportion (18.6%) of the overall differentially expressed mRNAs of known function were found to encode for proteins implicated in various mitochondrial processes, revealing mitochondria pathways dysregulation. Mitochondrial morphological analysis by video-rate confocal microscopy showed a high degree of mitochondrial branching in cells of HIBM patients. The subtle involvement of mitochondrial processes identified in HIBM reveals an unexpected facet of HIBM pathophysiology which could at least partially explain the slow evolution of this disorder and give new insights in the disease mechanism.

G.P.17.05 Predictive role of NCAM in the identification of patients with HIBM due to GNE mutations with atypical clinical phenotype

Hereditary inclusion-body myopathy (HIBM) is an autosomal recessive disorder characterized by onset in the second–third decade of life, associated with mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene that codes for a bifunctional enzyme with a critical role in sialic acid biosynthesis. We now describe four HIBM patients with atypical and severe phenotype, characterized by an early onset in the first decade of life and a rather aggressive progression of muscle weakness.

GNE protein expression and subcellular distribution are unaltered in HIBM

Mutations in GNE encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) cause hereditary inclusion body myopathy (HIBM). To define the role of GNE mutations in HIBM pathogenesis, GNE protein expression was analyzed. GNE protein is expressed at equal levels in HIBM patients and normal control subjects. Immunofluorescence detection of GNE did not reveal any mislocalization of GNE in skeletal muscle. We conclude that impaired GNE function, not lack of expression, may be the key pathogenic factor in HIBM. For diagnostic purposes, direct genetic analysis of the GNE gene in patients with IBM will remain the mainstay and is not aided by immunohistochemistry or immunoblotting using antibodies against the GNE protein.

G.O.5 Impairment of signal transduction pathways in hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is a unique worldwide muscular disorder caused by mutations in the GNE gene. The gene product of GNE is a bifunctional enzyme which is the limiting factor in the biosynthetic pathway of sialic acid. However, there is no evidence of sialylation impairment in most HIBM patients, and the underlying myopathological mechanism leading to the disease phenotype is poorly understood. For further insights in this pathophysiology, we have established a cellular model of human myoblasts, derived from HIBM patients satellite cells, and identified cellular and molecular characteristics of these cells.

564. Correction of Hyposialylation in Hereditary Inclusion Body Myopathy, by Ex-Vivo Therapy

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive disorder that is characterized by a late onset of muscle weakness in upper and lower limbs with sparing of quadriceps and accumulation of vacuoles and amyloid deposits in muscle tissues. Recently, HIBM was associated with mutations of the UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE) gene, located on chromosome 9. This bi-functional enzyme is responsible of the synthesis and regulation of sialic acid, the most common monosaccharide in glycoconjugated molecules of eukaryotes. It is known that sialic acids are important in a lot of cellular functions. Mutations have been identified in both domains of GNE, and in compound heterozygous form. One of the most prevalent mutations in HIBM (M712T) is located in the kinase domain of GNE. The presence of this mutation reduce the GNE enzymatic activity and thus the sialylation in muscles. Although reduced activity is found among mutated GNE gene, the process that causes muscular pathology is not yet understood. There is probability inadequate sialylation or hyposialylation of important functional proteins in HIBM muscles that causes the pathology.To investigate possible therapies of HIBM a knock-in mouse model has been produced by Dr. D. Darvish (Encino, CA). The model contain a GNE gene with the M712T mutation frequently found in HIBM patients. A possible therapy of HIBM is to restore in muscle fibers a normal GNE activity by gene therapy. This could be done by genetically modifying the patient own myoblasts before an autologous transplantation.Myoblasts were grown from normal mouse muscle biopsies and from homozygous knock-in M172T GNE mice. The level of sialylation of these myoblasts was determined by flowcytometry following labelling of the cells with FITC coupled lectins (Limax flavus lectin LFA or MAA). The level of sialylation was found to be inferior on the homozygous M712T mouse myoblasts. We then produced SIN lentiviral vector containing either the normal GNE gene or the mutated M712T GNE gene. The homozygous M712T mouse myoblasts were infected or not with a lentivirus containing the normal GNE gene or the M712T mutated gene. The cells were then selected and proliferated. The level of sialylation was increased following the viral infection with both viral vectors. The level of sialylation was increased up to 2 folds by the lentivirus containing the normal GNEgene.A 1.3 to 1.5 increased level of sialylation was also observed following lentiviral infection of myoblasts grown from a muscle biopsy of an HIBM patient also affected by the M712T mutation.In the future, intramuscular transplantation of the genetically modified myoblasts will be made in the muscles of homozygous M712T mice.Major stock holder in CellGene Inc.

Single nucleotide polymorphisms in the dystroglycan gene do not correlate with disease severity in hereditary inclusion body myopathy

Aberrant glycosylation of dystroglycan occurs in certain muscular dystrophies, including hereditary inclusion body myopathy (HIBM). HIBM harbors a widely varying clinical severity and age of onset, which raised the suspicion of the presence of disease modifier genes. We considered the highly polymorphic dystroglycan gene ( DAG1) as a feasible candidate modifier gene. DAG1 genomic DNA was sequenced for 32 HIBM patients, mainly of Persian-Jewish descent. Five novel DAG1 single nucleotide polymorphisms (SNPs) were identified, bringing the total number of SNPs to 19. However, no direct correlation between DAG1 SNPs and clinical severity of HIBM could be detected. Several identified SNPs substitute an amino acid and might modulate dystroglycan function or glycosylation status, and deserve further research. These data are valuable for future studies on the role of DAG1 in HIBM and other muscular dystrophies, especially those dystrophies that involve abnormal glycosylation of dystroglycan.

α-Dystroglycan does not play a major pathogenic role in autosomal recessive hereditary inclusion-body myopathy

Mutations of the GNE gene are responsible for autosomal recessive hereditary inclusion-body myopathy (HIBM). In this study we searched for the presence of any significant abnormality of α-dystroglycan (α-DG), a highly glycosylated component of the dystrophin-glycoprotein complex, in 5 HIBM patients which were previously clinically and genetically characterized. Immunocytochemical and immunoblot analysis showed that α-DG extracted from muscle biopsies was normally expressed and displayed its typical molecular mass. Immunoblot analysis on the wheat germ lectin-enriched glycoprotein fraction of muscles and primary myotubes showed a reduced amount of α-DG in 4 out of 5 HIBM patients, compared to normal and other diseased muscles. However, such altered lectin-binding behaviour, possibly reflecting a partial hyposialylation of α-DG, did not affect the laminin binding properties of α-DG. Therefore, the subtle changes within the α-DG glycosylation pattern, detected in HIBM muscles, likely do not play a key pathogenic role in this disorder.

Hypoglycosylation of α-dystroglycan in patients with hereditary IBM due to GNE mutations

Hereditary inclusion body myopathy (HIBM) is an adult onset neuromuscular disorder associated with mutations in the gene UDP- N-acetylglucosamine-2-epimerase/ N-acetylmannosamine kinase ( GNE), whose product is the rate limiting bi-functional enzyme catalyzing the first two steps of sialic acid biosynthesis. Loss of GNE activity in HIBM is thought to impair sialic acid production and interfere with proper sialylation of glycoconjugates, but it remains unclear how such a defect would lead to muscle destruction and muscle weakness. Hypoglycosylation of α-dystroglycan, a central protein of the skeletal muscle dystrophin–glycoprotein complex, results in disturbed interactions with extracellular matrix proteins. This has recently been identified as the pathomechanism involved in several congenital muscular dystrophies. We examined the glycosylation status of α-dystroglycan in muscle biopsies of four HIBM patients of non-Iranian Jewish origin (one American, two Indians, and one Greek). Two of these patients carry novel compound heterozygous GNE mutations on exon 2 and exon 9. All four muscle biopsies showed absent or markedly reduced immunolabeling with two different antibodies (VIA4 and IIH6) to glycosylated epitopes of α-dystroglycan. Normal labeling was found using antibodies to the core α-dystroglycan protein, β-dystroglycan, and laminin α-2. These findings resemble those found for other congenital muscular dystrophies, suggesting that HIBM may be a “dystroglycanopathy,” and providing an explanation for the muscle weakness of patients with GNE mutations.