ABSTRACT
Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain disease. A common feature of these disorders is the variable reduction in the glycosylation of skeletal muscle alpha-dystroglycan. In order to gain insight into the pathogenesis and clinical variability, we have generated two lines of mice, the first containing a missense mutation and a neomycin cassette, FKRP-Neo(Tyr307Asn) and the second containing the FKRP(Tyr307Asn) mutation alone. We have previously associated this missense mutation with a severe muscle-eye-brain phenotype in several families. Homozygote Fkrp-Neo(Tyr307Asn) mice die soon after birth and show a reduction in the laminin-binding epitope of alpha-dystroglycan in muscle, eye and brain, and have reduced levels of FKRP transcript. Homozygous Fkrp(Tyr307Asn) mice showed no discernible phenotype up to 6 months of age, contrary to the severe clinical course observed in patients with the same mutation. These results suggest the generation of a mouse model for FKRP related muscular dystrophy requires a knock-down rather than a knock-in strategy in order to give rise to a disease phenotype.
Subject(s)
Muscular Dystrophies/genetics , Mutation, Missense , Proteins/genetics , Animals , Blotting, Southern , Cell Movement , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Chimera , Dystroglycans/metabolism , Female , Gene Targeting , Genotype , Male , Mice , Mice, Knockout , Mice, Mutant Strains , Models, Animal , Muscular Dystrophies/metabolism , Muscular Dystrophies/physiopathology , Pentosyltransferases , Phenotype , Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , TransferasesABSTRACT
The mechanism of disease in forms of congenital and limb girdle muscular dystrophy linked to mutations in the gene encoding for Fukutin-related protein (FKRP) has previously been associated with the mis-localisation of FKRP from the Golgi apparatus. In the present report, we have transfected V5-tagged Fukutin-related protein expression constructs into differentiated C2C12 myotubes and the tibialis anterior of normal mice. The transfection of either wild type (WT) or several mutant constructs (P448L, C318Y, L276I) into myotubes consistently showed clear co-localisation with GM130, a Golgi marker. In contrast, whilst WT and the L276I localised to the Golgi of Cos-7 cells, the P448L and C318Y was mis-localised in the majority of these undifferentiated cells. The injection of the same constructs into the tibialis anterior of mice resulted in similar localisation of both the WT and all the mutants. Immunolabelling of FKRP in the muscle of MDC1C and LGMD2I patients was found to be indistinguishable from normal controls. Overall, these data suggest that retention in the endoplasmic reticulum of FKRP is not the main mechanism of disease but that this may instead relate to a disruption of the functional activity of this putative enzyme with its substrate(s) in the Golgi.
Subject(s)
Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Proteins/genetics , Animals , COS Cells , Chlorocebus aethiops , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Muscle, Skeletal/physiology , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology , Mutation , Myoblasts/physiology , Pentosyltransferases , Proteins/metabolism , Transfection , TransferasesABSTRACT
We report two novel mutations in three cases of spinal muscular atrophy (SMA), including two distant cousins who followed an unexpectedly severe course. Diagnosis was confirmed by reduced SMN protein and full-length SMN mRNA levels. Sequencing of the non-deleted SMN1 gene revealed a single G insertion at the end of exon 1 in the two cousins and a novel G275S exon 6 missense mutation in the milder case.