Myoferlin regulation by NFAT in muscle injury, regeneration and repair.

Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.

Myoferlin is required for insulin-like growth factor response and muscle growth.

Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.-Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth.

The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion.

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.

Detection and calibration of microdeletions and microduplications by array-based comparative genomic hybridization and its applicability to clinical genetic testing.

PURPOSE: Genome-wide telomere screening by fluorescence in situ hybridization (FISH) has revealed that approximately 6% of unexplained mental retardation is due to submicroscopic telomere imbalances. However, the use of FISH for telomere screening is labor intensive and time consuming, given that 41 telomeres are interrogated. We have evaluated the use of array-based Comparative Genomic Hybridization (aCGH) as a more efficient tool for identifying telomere rearrangements. METHODS: In this study, 102 individuals with unexplained mental retardation, with either normal or abnormal FISH results, were selected for a blinded retrospective study using aCGH. Results between the two methodologies were compared to ascertain the ability of aCGH to be used in a clinical diagnostics setting. RESULTS: We detected 100% of all imbalances previously identified by FISH (n = 17) and identified two additional abnormalities, a 10q telomere duplication and an interstitial duplication of 22q11. Interphase FISH analysis verified all abnormal array results. We also demonstrated that aCGH can accurately calibrate the size of telomere imbalances by using an array with "molecular rulers" for the telomeric regions of 1p, 16p, 17p, and 22q. CONCLUSION: This study demonstrates that aCGH is an equivalent methodology to telomere FISH for detecting submicroscopic deletions. In addition, small duplications that are not easily visible by FISH can be accurately detected using aCGH. Because aCGH allows simultaneous interrogation of hundreds to thousands of DNA probes and is more amenable to automation, it offers an efficient and high-throughput alternative for detecting and calibrating unbalanced rearrangements, both of the telomere region, as well as other genomic locations.

Diverse fates of paralogs following segmental duplication of telomeric genes.

The telomeric region of chromosome 9p is paralogous to the pericentromeric regions of chromosome 9 as well as to 2q13, the site of an ancestral telomere-telomere fusion. These paralogous regions span approximately 200 kb and contain seven transcriptional units, including the previously identified CBWD, FOXD4, PGM5, F379, CXYorf1, and two human Unigene clusters, Hs.115173 and Hs.189160. Within these gene duplicates, the number of expressed paralogous loci varies, from one in PGM5 to all three in CBWD and Hs.115173. FOXD4 shows the most dramatic changes among its paralogs. Two independent insertion/deletion changes created four different carboxy ends of these intronless genes, two of which are within the 2q13 locus. A comparison of KA/KS values among functional paralogs shows these genes evolved rapidly in primates. This study shows the importance of paralogous regions in the generation of transcriptional diversity and highlights the significance that large-scale telomeric duplication may play in this process.

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages.

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.