Congenital muscular dystrophy type 1D (MDC1D) due to a large intragenic insertion/deletion, involving intron 10 of the LARGE gene.

Mutation of the LARGE gene is the rarest of the six known genetic causes of α-dystroglycanopathy. We report further a family with MDC1D due to a complex genomic rearrangement that was not apparent on standard sequencing of LARGE. Two sisters in a consanguineous family had moderate mental retardation and cerebellar malformations, together with dystrophic changes and markedly reduced α-dystroglycan glycosylation staining on muscle biopsy. There was homozygous linkage to the LARGE locus but sequencing of LARGE coding regions was normal. Analysis of LARGE cDNA showed an abnormal sequence inserted between exons 10 and 11, in most of the transcripts, predicted to introduce a premature stop codon. The abnormal sequence mapped to a spliced EST (DA935254) of unknown function, normally located at 100 kb centromeric of LARGE on chromosome 22q12.3. Quantitative PCR analysis of the EST and adjacent regions showed twice the normal copy number in patients' genomic DNA samples, consistent with a large intra-chromosomal duplication inserted into intron 10 of LARGE in a homozygous state. This insertion was associated with deletion of a central region of intron 10, but the exact break points of the deletion/duplication were not found, suggesting that an even more complex rearrangement may have occurred. The exact function of LARGE, a golgi protein, remains uncertain. POMT and POMGnT enzyme activities were normal in patients' lymphoblast cells, suggesting that defects in LARGE do not affect the initiation of O-mannosyl glycans.

In vitro analysis of rod composition and actin dynamics in inherited myopathies.

Rods are the pathological hallmark of nemaline myopathy, but they can also occur as a secondary phenomenon in other disorders, including mitochondrial myopathies such as complex I deficiency. The mechanisms of rod formation are not well understood, particularly when rods occur in diverse disorders with very different structural and metabolic defects. We compared the characteristics of rods associated with abnormalities in structural components of skeletal muscle thin filament (3 mutations in the skeletal actin gene ACTA1) with those of rods induced by the metabolic cell stress of adenosine triphosphate depletion. C2C12 and NIH/3T3 cell culture models and immunocytochemistry were used to study rod composition and conformation. Fluorescent recovery after photobleaching was used to measure actin dynamics inside the rods. We demonstrate that not all rods are the same. Rods formed under different conditions contain a unique fingerprint of actin-binding proteins (cofilin and alpha-actinin) and display differences in actin dynamics that are specific to the mutation, to the cellular location of the rods (intranuclear vs cytoplasmic), and/or to the underlying pathological process (i.e. mutant actin or adenosine triphosphate depletion). Thus, rods likely represent a common morphological end point of a variety of different pathological processes, either structural or metabolic.