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Changes in cytoplasmic Ca(2+) concentration, resulting from activation of intracellular Ca(2+) channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca(2+) channels, inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca(2+) channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP(3)Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest.

The dystroglycanopathies are a group of inherited muscular dystrophies that have a common underlying mechanism, hypoglycosylation of the extracellular receptor alpha-dystroglycan. Many of these disorders are also associated with defects in the central nervous system and the eye. Defects in alpha-dystroglycan may also play a role in cancer progression. This review discusses the six dystroglycanopathy genes identified so far, their known or proposed roles in dystroglycan glycosylation and their relevance to human disease, and some of animal models now available for the study of the dystroglycanopathies.

Mutations in human genes encoding proteins involved in α-dystroglycan glycosylation result in dystroglycanopathies: severe congenital muscular dystrophy phenotypes often accompanied by CNS abnormalities and ocular defects. We have identified the zebrafish orthologues of the seven known genes in this pathway and examined their expression during embryonic development. Zebrafish Large, POMT1, POMT2, POMGnT1, Fukutin, and FKRP show in situ hybridization patterns similar to those of dystroglycan, with broad expression throughout early development. By 30 h postfertilization (hpf), transcripts of all these genes are most prominent in the CNS, eye, and muscle, tissues that are predominantly affected in the dystroglycanopathies. In contrast, Large2 expression is more restricted and by 30 hpf is confined to the lens, cerebellum, and pronephric duct. We show that the monoclonal antibody IIH6, which recognizes a glycoform of dystroglycan, also detects the zebrafish protein. Injection of morpholino oligonucleotides against zebrafish Large2 resulted in loss of IIH6 immunostaining. These data indicate that the dystroglycan glycosylation pathway is conserved in zebrafish and suggest this organism is likely to be a useful model system for functional studies.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed “DUX4,” containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism.

The splicing of nascent mRNA precursors is an essential step for the expression of all intron-containing eukaryotic genes. Removal of intron sequences from nascent transcripts is mediated by the spliceosome, a large multicomponent complex. We describe here the identification of two genes encoding related, putative splicing factors on human chromosome 19p13.11, SF4 (splicing factor 4) and SFRS14 (splicing factor arginine/serine-rich 14). Both genes encode proteins containing a SURP motif; this domain is found in several splicing proteins including Drosophila alternative splicing regulator, suppressor-of-white-apricot (SWAP) and the yeast splicing factor, prp21p. In addition, SF4 and SFRS14 contain a G-patch domain at their C-termini, a motif present in a large number of eukaryotic RNA-binding proteins. SFRS14 also contains an N-terminal region that is rich in arginine/serine residues, suggesting SFRS14 is a novel member of the SR-related family of pre-mRNA processing factors. We have also identified the mouse orthologues of SF4 and SFRS14 , based on conserved domain organization and high sequence similarity. Interestingly, SFRS14 undergoes alternative 3′-end processing events that are conserved between human and mouse, suggesting a functional significance.

The myodystrophy (myd) mutation arose spontaneously and has an autosomal recessive mode of inheritance. Homozygous mutant mice display a severe, progressive muscular dystrophy. Using a positional cloning approach, we identified the causative mutation in myd as a deletion within the Large gene, which encodes a putative glycosyltransferase with two predicted catalytic domains. By immunoblotting, the alpha-subunit of dystroglycan, a key muscle membrane protein, is abnormal in myd mice. This aberrant protein might represent altered glycosylation of the protein and contribute to the muscular dystrophy phenotype. Our results are discussed in the light of recent reports describing mutations in other glycosyltransferase genes in several forms of human muscular dystrophy.

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b , the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087 bp encoding a protein of 1961 amino acids was isolated. By RT–PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3′ UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33 bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.

The distal end of chromosome 4q contains the locus involved in facioscapulohumeral muscular dystrophy (FSHD1). Specific genomic deletions within a tandem DNA repeat (D4Z4) are associated with the disease status, but no causal genes have yet been discovered. In a systematic search for genes, a 161-kb stretch of genomic DNA proximal to D4Z4 was sequenced, analyzed for homologies, and subjected to gene prediction programs. A major fraction (45%) of the subtelomeric region is composed of repeat sequences attributable mainly to LINE-1 elements. Apart from the previously identified FRG1 and TUB4q sequences, several additional potential coding regions were identified by analyzing the sequence with exon prediction programs. So far, we have been unable to demonstrate transcripts by RT-PCR or cDNA library hybridization. However, several retrotransposed pseudogenes were identified. The high density of pseudogenes and repeat elements is consistent with the subtelomeric location of this region and explains why previous transcript identification studies have been problematic.

There is evidence of multiple copies of the FSHD Region Candidate Gene 1 (FRG1) in humans. Analysis of human FRG1 ESTs showed many of them to be non-processed pseudogenes dispersed throughout the genome. To determine when the amplification of FRG1 occurred, we used a PCR-based approach to identify FRG1 sequences from great apes, chimpanzee, gorilla and orang-utan, and an Old World monkey, Macaca mulatta. In common with humans, multiple copies of FRG1 were detected in the great apes. However, in Macaca mulatta, only two FRG1 loci were identified, one presumed to be the homologue of the human chromosome 4q gene. This is strikingly similar to the distribution of a dispersed 3.3-kb repeat family in primates. A member of this family, D4Z4, maps to the subtelomeric region of 4q, in close proximity to FRG1. We propose that an ancestral duplication of distal 4q included FRG1. This duplication is present in Macaca mulatta whose divergence from hominoids is thought to have occurred at least 33 million years ago. We propose that this telomeric region then underwent further amplification and dispersion events in the great ape lineage, with copies of FRG1 and the 3.3-kb repeats being localized in heterochromatic regions.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35. FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4. D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs. In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease. However, no expressed sequences from D4Z4 have been identified. We have previously shown that there are other loci similar to D4Z4 within the genome. In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat. Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes. D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies. The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed. We propose the name 3.3-kb repeat for this family of repetitive sequence elements.

Recently, probe p13E-11 (D4F104S1) was shown to identify de novo DNA rearrangements, which are associated with the development of facioscapulohumeral muscular dystrophy (FSHD). These rearrangements are likely to become instrumental in cloning the FSHD gene itself. Analysis by pulsed-field gel electrophoresis demonstrates that p13E-11 recognizes two highly polymorphic loci, with HindIII restriction fragments ranging in size from about 30 to 320 kb. Haplotype analysis unambiguously assigned one of the two loci to chromosome 4q35. The detection of identical NotI or Nru I fragments with both CEB8 (D4F35S1) and p13E-11 demonstrated that the DNA rearrangements are deletions that are restricted to the HindIII fragments detectable by p13E-11. In two cases, the sizes of the deletion could be established and were found to be 25 and 85 kb in length, respectively. So far, we have been able to define the parental origin of the mutation in seven different patients and have found that in five cases the maternal allele was involved.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder. The disease affects specific muscles of the face, shoulder-girdle and upper arm. The biochemical defect underlying FSHD is unknown and there are no specific tests that are diagnostic of FSHD. Genetic linkage studies have mapped the FSHD gene to chromosome 4q35. A DNA marker (p13E-11; D4F104S1, formerly D4S810) has been isolated which recognizes two highly polymorphic loci detectable by EcoRI or HindIII; one locus maps to chromosome 4q35 and shows fragments between about 50 and 320 kb. In FSHD patients deletions occur within this EcoRI/HindIII fragment, yielding fragments that are usually smaller than 28 kb. Characterization of the polymorphic fragments demonstrates that they consist of a 3.2 kb tandem repeat; their number can range between approximately 12 and 96 within the 4q35-specific fragments. In FSHD patients, an integral number of these tandem repeats are deleted, leaving at maximum eight copies.

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5′ untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3–10 encode the remainder of the coding and 3′ noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human × mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.

Alpha-dystroglycan ( α DG) is an extracellular, glycosylated protein attached to the outer plasma membrane by its interaction with the transmembrane protein, beta-dystroglycan. Genetic mutations that alter the glycosylation state of α DG cause a number of inherited disorders, including Fukuyama MD and certain congenital and limb-girdle MDs. Many of the pathogenic mutations are in proteins that carry out glycosylation of α DG and not in the DG gene itself. These enzymes are usually located in the Golgi where proteins are modified before export to the extracellular space. Amino-acid mutations in α DG itself can also cause congenital MD when sugar attachment to the α DG backbone is affected. Antibodies against dystroglycans immunostain the sarcolemma in a similar manner to dystrophin antibodies. It would be useful to the muscle pathologist to have monoclonal antibodies (mAbs) that recognize specific glycosylated forms of α DG associated with different mutations or disease states, since such mAbs would be useful for diagnosis using muscle biopsy sections. There are problems of reproducibility with existing mAbs against α DG, but it is still unclear whether those problems are attributable to the antibodies themselves or to the nature of the glycosylated epitopes on α DG. To determine whether it is possible to obtain improved mAbs for diagnostic use, we have begun a collaborative programme, supported by LGMD2I and CureCMD, to produce new mAbs against glycosylated and non-glycosylated forms of α DG. As a first step, we now describe a new mAb raised against bacterial recombinant α DG core sequence, which gives good staining of the sarcolemma in human muscle biopsy sections. We have mapped the epitope recognized by this mAb to short sequence that appears to be accessible to antibody in BOTH glycosylated and non-glycosylated forms of α DG.

We have generated novel monoclonal antibodies (mAbs) against alpha-dystroglycan to immunolabel the sarcolemma in human muscle biopsies. For one of these, DAG-6F4, a seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with a disruption in the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was generally less reduced than staining by IIH6 (an antibody commonly used to identify glycosylated alpha-dystroglycan), though the extent of the differences between the two antibodies varied between different patients, some biopsies showing reductions in core protein as well as its glycosylation. A second mAb, DAG-3H9, gave similar results, but recognised a different epitope, GDRAP, closer to the C-terminus. There are currently few antibodies available against core alpha-dystroglycan, so DAG-6F4 represents a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.