13 research outputs found
A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System
The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans
EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia.
The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease
Exome sequencing and disease-network analysis of a single family implicate a mutation in KIF1A in hereditary spastic paraparesis
Whole exome sequencing has become a pivotal methodology for rapid and cost-effective detection of pathogenic variations in Mendelian disorders. A major challenge of this approach is determining the causative mutation from a substantial number of bystander variations that do not play any role in the disease etiology. Current strategies to analyze variations have mainly relied on genetic and functional arguments such as mode of inheritance, conservation, and loss of function prediction. Here, we demonstrate that disease-network analysis provides an additional layer of information to stratify variations even in the presence of incomplete sequencing coverage, a known limitation of exome sequencing. We studied a case of Hereditary Spastic Paraparesis (HSP) in a single inbred Palestinian family. HSP is a group of neuropathological disorders that are characterized by abnormal gait and spasticity of the lower limbs. Forty-five loci have been associated with HSP and lesions in 20 genes have been documented to induce the disorder. We used whole exome sequencing and homozygosity mapping to create a list of possible candidates. After exhausting the genetic and functional arguments, we stratified the remaining candidates according to their similarity to the previously known disease genes. Our analysis implicated the causative mutation in the motor domain of KIF1A, a gene that has not yet associated with HSP, which functions in anterograde axonal transportation. Our strategy can be useful for a large class of disorders that are characterized by locus heterogeneity, particularly when studying disorders in single families
Variable Myopathic Presentation in a Single Family with Novel Skeletal RYR1 Mutation
We describe an autosomal recessive heterogeneous congenital myopathy in a large consanguineous family. The disease is characterized by variable severity, progressive course in 3 of 4 patients, myopathic face without ophthalmoplegia and proximal muscle weakness. Absence of cores was noted in all patients. Genome wide linkage analysis revealed a single locus on chromosome 19q13 with Zmax = 3.86 at theta = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Direct sequencing of the main candidate gene within the candidate region, RYR1, was performed. A novel homozygous A to G nucleotide substitution (p.Y3016C) within exon 60 of the RYR1 gene was found in patients. ARMS PCR was used to screen for the mutation in all available family members and in an additional 150 healthy individuals. This procedure confirmed sequence analysis and did not reveal the A to G mutation (p.Y3016C) in 300 chromosomes from healthy individuals. Functional analysis on EBV immortalized cell lines showed no effect of the mutation on RyR1 pharmacological activation or the content of intracellular Ca(2+) stores. Western blot analysis demonstrated a significant reduction of the RyR1 protein in the patient's muscle concomitant with a reduction of the DHPRalpha1.1 protein. This novel mutation resulting in RyR1 protein decrease causes heterogeneous clinical presentation, including slow progression course and absence of centrally localized cores on muscle biopsy. We suggest that RYR1 related myopathy should be considered in a wide variety of clinical and pathological presentation in childhood myopathies
A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System
The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans
Effect of the pY3016C mutation on RyR1 protein expression on patient and control muscle biopsies.
<p>The western blot shows a dramatic decrease of the RyR1 protein and of the DHPRalpha 1.1 expression in the patient’s biopsy compared to control’s biopsy (P<0.05). Protein expression levels were quantified by densitometric analysis and normalized to the expression of myosin heavy chain. The bar plot on the right shows the mean % protein content (± SEM; of 3 different western blots) in biopsies from a control and patient Y3016C−/− (P<0.05 by the Student <i>t</i> test).</p
Linkage analysis of the family suffering from autosomal recessive atypical congenital myopathy.
<p>(<b>A</b>) Pedigree of the family. Filled and unfilled symbols represent affected and unaffected individuals, respectively. The arrows denote individuals whose DNA samples were analyzed by SNP250K. (<b>B</b>) Multipoint linkage analysis using SNP data showing LOD score <i>Z<sub>max</sub></i> = 3.86 at θ = 0.0 on chromosome 19q13. X-axis: genetic distance in cM., Y-axis: Lod score.</p
Histological analysis of patients muscle biopsies.
<p>Frozen sections of 3 cases (V28, V31 & V36) that were available for pathological review display non-specific dystrophic-like changes, consistent with muscular dystrophy. H&E stained sections (A–C) show marked variation in myofiber-diameter, in random distribution. The number of internally displaced nuclei (white arrows) is markedly increased. There are no clear-cut signs of necrosis, regeneration or any other specific structural change in the myofibers. There is focal endomysial fibrosis (black arrows). There is no inflammatory infiltrate. The blood vessels are unremarkable. NADH histochemical stain (D–F) is not showing significant changes in the cytoarchitecture, except for occasional moth-eaten-like fibers (red arrows) and overstaining of atrophic fibers (yellow fibers). (Original magnification ×40; Bars = 50 μm).</p
Analysis of <i>RYR1</i> at the DNA level in patients and controls.
<p>(<b>A</b>) Sequence of the <i>RYR1</i> gene revealed a homozygous A to G nucleotide substitution leading to an amino acid change (p.Y3016C) within exon 60 in patient (arrow). (<b>B</b>) Analysis of the mutation in family members and control. Left panel: PCR amplification products of <i>RYR1</i> from exon -60 to intron-60 using primers specific to the mutated allele (MUT Primers, product size = 210 bp). Right panel: PCR amplification products of <i>RYR1</i> from exon-60 to intron – 60 using primers specific to wild type allele (WT Primers, product size = 210 bp). This test confirms the cosegregation of the <i>RYR1</i> mutation with the phenotype and haplotypes in the family. C: control. Affected individuals are underlined.</p
Clinical features and laboratory findings in the patients with myopathy.
<p>F: female; M: male; N: normal; FSV: fiber-size variation; IDN: internally-displaced nuclei; EF: endomysial fibrosis; Y:yes; No: No.</p