GFAP mutations, age at onset, and clinical subtypes in Alexander disease.

OBJECTIVE: To characterize Alexander disease (AxD) phenotypes and determine correlations with age at onset (AAO) and genetic mutation. AxD is an astrogliopathy usually characterized on MRI by leukodystrophy and caused by glial fibrillary acidic protein (GFAP) mutations. METHODS: We present 30 new cases of AxD and reviewed 185 previously reported cases. We conducted Wilcoxon rank sum tests to identify variables scaling with AAO, survival analysis to identify predictors of mortality, and χ(2) tests to assess the effects of common GFAP mutations. Finally, we performed latent class analysis (LCA) to statistically define AxD subtypes. RESULTS: LCA identified 2 classes of AxD. Type I is characterized by early onset, seizures, macrocephaly, motor delay, encephalopathy, failure to thrive, paroxysmal deterioration, and typical MRI features. Type II is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, bulbar symptoms, and atypical MRI features. Survival analysis predicted a nearly 2-fold increase in mortality among patients with type I AxD relative to those with type II. R79 and R239 GFAP mutations were most common (16.6% and 20.3% of all cases, respectively). These common mutations predicted distinct clinical outcomes, with R239 predicting the most aggressive course. CONCLUSIONS: AAO and the GFAP mutation site are important clinical predictors in AxD, with clear correlations to defined patterns of phenotypic expression. We propose revised AxD subtypes, type I and type II, based on analysis of statistically defined patient groups.

Decreased platelet expression of myosin regulatory light chain polypeptide (MYL9) and other genes with platelet dysfunction and CBFA2/RUNX1 mutation: insights from platelet expression profiling.

We have reported on a patient with thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, associated with a heterozygous mutation in transcription factor CBFA2 (core binding factor A2, RUNX1 or AML1). To obtain insights into the abnormal platelet mechanisms and CBFA2-regulated genes, we performed platelet expression profiling in four control subjects and the patient using the Affymetrix U133 GeneChips. In the patient, 298 probe sets were significantly downregulated at least 2-fold. MLC regulatory polypeptide (MYL9 gene) was decreased approximately 77-fold; this is an important finding because agonist-stimulated MLC phosphorylation is decreased in patient platelets. Genes downregulated > or = 5-fold include those involving calcium binding proteins (CABP5), ion transport (sodium/potassium/Ca exchanger, SLC24A3), cytoskeletal/microtubule proteins (erythrocyte membrane protein band 4.1-like 3, EPB41L3; tropomyosin 1, TPM1; tubulin, alpha 1, TUBA1), signaling proteins (RAB GTPase activating protein 1-like, RABGAP1L; beta3-endonexin, ITGB3 BP) and chemokines (platelet factor 4 variant 1, PF4V1; chemokine CXCL5, CXCL5). These and other downregulated genes are relevant to the patient's platelet defects in function and production. These studies provide the first proof of concept that platelet expression profiling can be applied to obtain insights into the molecular basis of inherited platelet defects.

Discrepancy between neuroimaging findings and clinical phenotype in Alexander disease.

We present a case of infantile-onset Alexander disease (AD) with a novel glial fibrillary acidic protein mutation but without clinical evidence of neurologic deterioration. Brain MRI studies showed typical AD findings and increasing size of frontal cavitations. Serial proton MR spectroscopy demonstrated high levels of myo-inositol and lactic acid and decreasing levels of N-acetylaspartate. The degree of demyelination and the timing of the axonal degeneration may determine phenotypic severity of the disease. Conventional neuroimaging techniques cannot always predict the outcome.

Indian Agarwal megalencephalic leukodystrophy with cysts is caused by a common MLC1 mutation.

BACKGROUND: A distinct clinical syndrome characterized by megalencephaly, mild to moderate cognitive decline, slowly progressive spasticity, ataxia, occasional seizures, and extensive white matter changes with temporal cysts by imaging studies has been described in a particular ethnic group (Agarwals) in India. This disorder is very similar to megalencephalic leukoencephalopathy with subcortical cysts (MLC), a newly characterized leukodystrophy whose molecular basis was recently shown to be mutations in a gene (KIAA0027) that has been renamed MLC1. OBJECTIVE: To determine if this disorder among the Agarwals is due to mutations in MLC1 by a mutation screening study conducted on affected Agarwal patients. METHODS: Genomic DNA from these Indian leukodystrophy patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the MLC1 gene. RESULTS: Thirty-three affected individuals whose clinical and imaging presentations were consistent with MLC were screened. All were from northern India and included 31 known Agarwals, 1 non-Agarwal, and 1 adopted patient whose ethnicity is unknown. All 31 Agarwal patients tested positive for a homozygous insertion of a cytosine in exon 2. The adopted patient was homozygous for A157E. No mutation in the coding region was found in the non-Agarwal patient. CONCLUSIONS: Indian patients with megalencephaly and MRI changes that show extensive white matter changes with temporal cysts should raise suspicion for MLC. Members of the Agarwal ethnic group affected with the disorder present with a mildly progressive course and show a common mutation (320insC) in the MLC1 gene, suggesting a founder effect.

Molecular findings in symptomatic and pre-symptomatic Alexander disease patients.

BACKGROUND AND OBJECTIVE: Alexander disease is a slowly progressive CNS disorder that most commonly occurs in children. Until recently, the diagnosis could only be established by the histologic finding of Rosenthal fibers in brain specimens. Mutations in the glial fibrillary acidic protein (GFAP) gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease. A prospective study on patients suspected to have Alexander disease was conducted to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. METHODS: Patients who showed MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening were included in this study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the GFAP gene. RESULTS: Twelve of 13 patients (approximately 90%) were found to have mutations in GFAP. Seven of those 12 patients presented in infancy with seizures and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that changed the charge of the mutant protein. Four of the nine changes were novel mutations. CONCLUSIONS: In symptomatic and asymptomatic patients with a predominantly frontal leukoencephalopathy by MRI, GFAP gene mutation analysis should be included in the initial diagnostic evaluation process for Alexander disease.

Mutations in the sarcoglycan genes in patients with myopathy.

BACKGROUND: Some patients with autosomal recessive limb-girdle muscular dystrophy have mutations in the genes coding for the sarcoglycan proteins (alpha-, beta-, gamma-, and delta-sarcoglycan). To determine the frequency of sarcoglycan-gene mutations and the relation between the clinical features and genotype, we studied several hundred patients with myopathy. METHODS: Antibody against alpha-sarcoglycan was used to stain muscle-biopsy specimens from 556 patients with myopathy and normal dystrophin genes (the gene frequently deleted in X-linked muscular dystrophy). Patients whose biopsy specimens showed a deficiency of alpha-sarcoglycan on immunostaining were studied for mutations of the alpha-, beta-, and gamma-sarcoglycan genes with reverse transcription of muscle RNA, analysis involving single-strand conformation polymorphisms, and sequencing. RESULTS: Levels of alpha-sarcoglycan were found to be decreased on immunostaining of muscle-biopsy specimens from 54 of the 556 patients (10 percent); in 25 of these patients no alpha-sarcoglycan was detected. Screening for sarcoglycan-gene mutations in 50 of the 54 patients revealed mutations in 29 patients (58 percent): 17 (34 percent) had mutations in the alpha-sarcoglycan gene, 8 (16 percent) in the beta-sarcoglycan gene, and 4 (8 percent) in the gamma-sarcoglycan gene. No mutations were found in 21 patients (42 percent). The prevalence of sarcoglycan-gene mutations was highest among patients with severe (Duchenne-like) muscular dystrophy that began in childhood (18 of 83 patients, or 22 percent); the prevalence among patients with proximal (limb-girdle) muscular dystrophy with a later onset was 6 percent (11 of 180 patients). CONCLUSIONS: Defects in the genes coding for the sarcoglycan proteins are limited to patients with Duchenne-like and limb-girdle muscular dystrophy with normal dystrophin and occur in 11 percent of such patients.

Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy.

Recently, mutations in the genes encoding several of the dystrophin-associated proteins have been identified that produce phenotypes ranging from severe Duchenne-like autosomal recessive muscular dystrophy to the milder limb-girdle muscular dystrophies (LGMDs). LGMD type 2C is generally associated with a more severe clinical course and is prevalent in northern Africa. A previous study identified a single base pair deletion in the gene encoding the dystrophin-associated protein gamma-sarcoglycan in a number of Tunisian muscular dystrophy patients. To investigate whether gamma-sarcoglycan gene mutations cause autosomal recessive muscular dystrophy in other populations, we studied 50 muscular dystrophy patients from the United States and Italy. The muscle biopsies from these 50 patients showed no abnormality of dystrophin but did show diminished immunostaining for the dystrophin-associated protein alpha-sarcoglycan. Four patients with a severe muscular dystrophy phenotype were identified with homozygous, frameshifting mutations in gamma-sarcoglycan. Two of the four have microdeletions that disrupt the distal carboxyl-terminus of gamma-sarcoglycan yet result in a complete absence of gamma-and beta-sarcoglycan suggesting the importance of this region for stability of the sarcoglycan complex. This region of gamma-sarcoglycan, like beta-sarcoglycan, has a number of cysteine residues similar to those in epidermal growth factor cysteine-rich regions.

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Recruitment of mast cells to muscle after mild damage.

We followed the response of muscle following mild intentional injury to determine a temporal sequence of cellular events involved in muscle repair. We found that intramuscular saline injection induced mild damage to muscle which resulted in the gradual recruitment of mast cells. Around the needle track, mast cells appear around 8 h post-injection. Mast cell accumulation were most dramatic immediately neighboring the posterior tibial vessels supplying the injured muscle. Dystrophin-deficient mdx muscle showed mast cell accumulations 3-fold higher than normal muscle, and this number did not change after saline injection. Additionally, we show that stem cell factor (SCF), a known mast cell chemoattractant, is expressed in both normal and mdx muscle at high levels. This steady-state level did not appear to be influenced by injury or dystrophin status. The implications of these findings are discussed as they relate to the repair of injured muscle and to their possible significance in the pathophysiology of Duchenne muscular dystrophy.

Phospholipase A2 activity in dystrophinopathies.

Phospholipase A2 activity in human muscle with or without dystrophin abnormality was studied. The results showed an increased phospholipase A2 activity in Duchenne muscular dystrophy (DMD) patients (1160 +/- 160, P < 0.01) compared to controls (< 200 U mg-1). DMD fetal muscle showed normal levels, but levels then increased dramatically postnatally. Highest levels were found at 5 yr of age (10 times normal) and then declined to 1.5-2 times normal by age 10. Steroid treatment did not change the phospholipase A2 levels significantly. In patients with abnormal dystrophin, i.e. Becker muscular dystrophy, phospholipase A2 activity was increased in the age group 3-15 (920 +/- 230 U mg-1, P < 0.01), while older patients (17-49) showed a non-significant (220 +/- 60 U mg-1) increase. The lack of phospholipase A2 activation in fetuses with DMD, indicates that activation is not a direct consequence of dystrophin deficiency. Phospholipase A2 activity has been shown to be connected to the formation of several inflammatory mediators such as prostaglandins, leukotriens, platelet activating factor and lysophospholipids. Phospholipase A2 activation may therefore play an important role in the development of inflammation and necrosis, with subsequent fibrosis and massive loss of muscle function, which develops in Duchenne and Becker muscular dystrophy.

Dystrophin-deficient myofibers are vulnerable to mast cell granule-induced necrosis.

Duchenne muscular dystrophy is the most common inherited lethal X-linked disorder of mankind and is caused by dystrophin deficiency. The steps involved in the dystrophin-deficiency-induced cascade which lead to myofiber necrosis, progressive muscle wasting in humans and dogs and prominent muscle hypertrophy in mice and cats are obscure. Dystrophin is an intracellular component of the membrane cytoskeleton and its absence would be expected to cause necrosis of isolated myofibers (cell autonomous defect). However, all dystrophin-deficient muscles characteristically show simultaneous degeneration of large groups of muscle fibers (grouped necrosis). This implies that cell death may be mediated by extracellular, non-cell autonomous factors which occur as a secondary consequence of dystrophin deficiency. We have proposed a model where tissue pathology may be mediated by infiltrating mast cells (Gorospe et al., J Neurol Sci 1994). Here we show that intramuscular injections of purified mast cell granules induce widespread myofiber necrosis in dystrophin-deficient mdx mice, but not in normal mice. These data support the hypothesis that dystrophin acts as a plasma membrane stabilizer and that its deficiency renders myofibers more susceptible to damage from mast cell proteases. Moreover, our results support the hypothesis that mast cell degranulation may be a trigger for myofiber death in dystrophin-deficient muscle.

A role for mast cells in the progression of Duchenne muscular dystrophy? Correlations in dystrophin-deficient humans, dogs, and mice.

Dystrophin deficiency has been shown to be the underlying cause of Duchenne muscular dystrophy. Although dystrophin-deficient homologous animal models have been identified (dog, mouse, and cat), the clinical expression of the biochemical defect is species-specific. Thus, while the genetics and biochemistry of Duchenne dystrophy is understood, the pathophysiological cascade leading to muscle weakness in only humans and dogs remains obscure. To begin to dissect the pathophysiology at the histological level, we undertook a systematic study of mast cells in normal and dystrophin-deficient muscle. Mast cells have been implicated in the development of fibrosis in other disorders, and progressive fibrosis has been hypothesized to mediate the failure of muscle regeneration in human and dog dystrophin deficiency. Our results show a strong correlation between mast cell content and localization, and the clinico-histopathological progression in humans, dogs and mice. The mast cell increases were disease specific: other dystrophic myopathies with normal dystrophin generally did not show substantial increases in mast cell content or degranulation. Our data suggest that mast cell accumulation and degranulation may cause the grouped necrosis characteristic of dystrophin deficiency in all species.

Duchenne muscular dystrophy.

Advances in the understanding of the genetic basis for Duchenne muscular dystrophy over the past 4 years has led to the quick application of molecular diagnostics. More recently, attention has turned towards acquiring a better understanding of dystrophin biochemistry and the pathophysiologic consequences of dystrophin deficiency.

Dystrophin deficiency causes lethal muscle hypertrophy in cats.

Two 5-month-old male Domestic Shorthair littermates showed general skeletal muscle hypertrophy, multifocal submucosal lingual calcification with lingual enlargement, and excessive salivation. Both cats had a reduced level of activity, walked with a stiff gait, and tended to "bunny hop" when they ran. These clinical features were similar to those of previously reported dystrophin-deficient cats. Using multiple dystrophin antibodies, we found that the cats described in this report also showed marked dystrophin deficiency. The histopathology was remarkable for hypertrophy and splitting of fibers, and progressive accumulation of calcium deposits within the muscle. There was little or no endomysial fibrosis at 2 years of age. The natural history of dystrophin-deficiency in cats has not been described: both previous cats had been euthanized at 2 years of age prior to experiencing any life-threatening problems. At 6 months of age, one of the new cats developed megaesophagus because of severe progressive hypertrophy of the diaphragmatic muscles. The diaphragm completely occluded the esophagus, and the cat was euthanized for humane reasons. The second cat remained in good condition until age 18 months when it developed acute renal failure attributed to severe prolonged dehydration and hyperosmolality. The cat recovered after receiving supportive treatment but was unable to maintain fluid homeostasis. The insufficient water intake was attributed to glossal hypertrophy and dysfunction. At age 2 years, the cat received regular subcutaneous injections of low-sodium fluids to maintain proper hydration. The clinical consequence of dystrophin deficiency in cats is lethal muscle hypertrophy. We have called the feline disease "hypertrophic feline muscular dystrophy" (HFMD).