Myotonic dystrophy type 1 (DM1) clinical subtypes and CTCF site methylation status flanking the CTG expansion are mutant allele length-dependent.

Myotonic dystrophy type 1 (DM1) is a complex disease with a wide spectrum of symptoms. The exact relationship between mutant CTG repeat expansion size and clinical outcome remains unclear. DM1 congenital patients (CDM) inherit the largest expanded alleles, which are associated with abnormal and increased DNA methylation flanking the CTG repeat. However, DNA methylation at the DMPK locus remains understudied. Its relationship to DM1 clinical subtypes, expansion size and age-at-onset is not yet completely understood. Using pyrosequencing-based methylation analysis on 225 blood DNA samples from Costa Rican DM1 patients, we determined that the size of the estimated progenitor allele length (ePAL) is not only a good discriminator between CDM and non-CDM cases (with an estimated threshold at 653 CTG repeats), but also for all DM1 clinical subtypes. Secondly, increased methylation at both CTCF sites upstream and downstream of the expansion was almost exclusively present in CDM cases. Thirdly, levels of abnormal methylation were associated with clinical subtype, age and ePAL, with strong correlations between these variables. Fourthly, both ePAL and the intergenerational expansion size were significantly associated with methylation status. Finally, methylation status was associated with ePAL and maternal inheritance, with almost exclusively maternal transmission of CDM. In conclusion, increased DNA methylation at the CTCF sites flanking the DM1 expansion could be linked to ePAL, and both increased methylation and the ePAL could be considered biomarkers for the CDM phenotype.

Longitudinal increases in somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 are associated with variation in age-at-onset.

In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-dependent, tissue-specific and expansion-biased. These features contribute toward variation in disease severity and confound genotype-to-phenotype analyses. To investigate how the (CTG)n repeat expansion changes over time, we collected three longitudinal blood DNA samples separated by 8-15 years and used small pool and single-molecule PCR in 43 DM1 patients. We used the lower boundary of the allele length distribution as the best estimate for the inherited progenitor allele length (ePAL), which is itself the best predictor of disease severity. Although in most patients the lower boundary of the allele length distribution was conserved over time, in many this estimate also increased with age, suggesting samples for research studies and clinical trials should be obtained as early as possible. As expected, the modal allele length increased over time, driven primarily by ePAL, age-at-sampling and the time interval. As expected, small expansions <100 repeats did not expand as rapidly as larger alleles. However, the rate of expansion of very large alleles was not obviously proportionally higher. This may, at least in part, be a result of the allele length-dependent increase in large contractions that we also observed. We also determined that individual-specific variation in the increase of modal allele length over time not accounted for by ePAL, age-at-sampling and time was inversely associated with individual-specific variation in age-at-onset not accounted for by ePAL, further highlighting somatic expansion as a therapeutic target in DM1.

Abnormal splicing of NEDD4 in myotonic dystrophy type 2: possible link to statin adverse reactions.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2.

A Z-DNA sequence reduces slipped-strand structure formation in the myotonic dystrophy type 2 (CCTG) x (CAGG) repeat.

All DNA repeats known to undergo expansion leading to human neurodegenerative disease can form one, or several, alternative conformations, including hairpin, slipped strand, triplex, quadruplex, or unwound DNA structures. These alternative structures may interfere with the normal cellular processes of transcription, DNA repair, replication initiation, or polymerase elongation and thereby contribute to the genetic instability of these repeat tracts. We show that (CCTG) x (CAGG) repeats, in the first intron of the ZNF9 gene associated with myotonic dystrophy type 2, form slipped-strand DNA structures in a length-dependent fashion upon reduplexing. The threshold for structure formation on reduplexing is between 36 and 42 repeats in length. Alternative DNA structures also form in (CCTG)(58) x (CAGG)(58) and larger repeat tracts in plasmids at physiological superhelical densities. This represents an example of a sequence that forms slipped-strand DNA from the energy of DNA supercoiling. Moreover, Z-DNA forms in a (TG) x (CA) tract within the complex repeat sequence 5' of the (CCTG)(n) x (CAGG)(n) repeat in the ZNF9 gene. Upon reduplexing, the presence of the flanking sequence containing the Z-DNA-forming tract reduced the extent of slipped-strand DNA formation by 62% for (CCTG)(57) x (CAGG)(57) compared with 58 pure repeats without the flanking sequence. This finding suggests that the Z-DNA-forming sequence in the DM2 gene locus may have a protective effect of reducing the potential for slipped-strand DNA formation in (CCTG)(n) x (CAGG)(n) repeats.

Differential Gain-of-Function Activity of Three p53 Hotspot Mutants In Vivo.

The majority of TP53 missense mutations identified in cancer patients are in the DNA-binding domain and are characterized as either structural or contact mutations. These missense mutations exhibit inhibitory effects on wild-type p53 activity. More importantly, these mutations also demonstrate gain-of-function (GOF) activities characterized by increased metastasis, poor prognosis, and drug resistance. To better understand the activities by which TP53 mutations, identified in Li-Fraumeni syndrome, contribute to tumorigenesis, we generated mice harboring a novel germline Trp53R245W allele (contact mutation) and compared them with existing models with Trp53R172H (structural mutation) and Trp53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed that all three hotspot mutations exhibited similar inhibitory effects on wild-type p53 transcription in vivo, and tumors from these mice had similar levels of loss of heterozygosity. However, the overall survival of Trp53R245W/+ and Trp53R270H/+ mice, but not Trp53R172H/+ mice, was significantly shorter than that of Trp53+/- mice, providing strong evidence for p53-mutant-specific GOF contributions to tumor development. Furthermore, Trp53R245W/+ and Trp53R270H/+ mice had more osteosarcoma metastases than Trp53R172H/+ mice, suggesting that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Transcriptomic analyses using RNA sequencing data from Trp53R172H/+, Trp53R245W/+, and Trp53R270H/+ primary osteosarcomas in comparison with Trp53+/- indicated that GOF of the three mutants was mediated by distinct pathways. Thus, both the inhibitory effect of mutant over wild-type p53 and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcoma. SIGNIFICANCE: p53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.

The mutation that underlies myotonic dystrophy type 2 (DM2) is a (CCTG)n expansion in intron 1 of zinc finger protein 9 (ZNF9). It has been suggested that ZNF9 is of no consequence for disease pathogenesis. We determined the expression levels of ZNF9 during muscle cell differentiation and in DM2 muscle by microarray profiling, real-time RT-PCR, splice variant analysis, immunofluorescence, and Western blotting. Our results show that in differentiating myoblasts, ZNF9 protein was localized primarily to the nucleus, whereas in mature muscle fibers, it was cytoplasmic and organized in sarcomeric striations at the Z-disk. In patients with DM2, ZNF9 was abnormally expressed. First, there was an overall reduction in both the mRNA and protein levels. Second, the subcellular localization of the ZNF9 protein was somewhat less cytoplasmic and more membrane-bound. Third, our splice variant analysis revealed retention of intron 3 in an aberrant isoform, and fourth quantitative allele-specific expression analysis showed the persistence of intron 1 sequences from the abnormal allele, further suggesting that the mutant allele is incompletely spliced. Thus, the decrease in total expression appears to be due to impaired splicing of the mutant transcript. Our data indicate that ZNF9 expression in DM2 patients is altered at multiple levels. Although toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for the differences in DM1.

Expression of RNA CCUG repeats dysregulates translation and degradation of proteins in myotonic dystrophy 2 patients.

Myotonic dystrophy 2 (DM2) is a multisystem skeletal muscle disease caused by an expansion of tetranucleotide CCTG repeats, the transcription of which results in the accumulation of untranslated CCUG RNA. In this study, we report that CCUG repeats both bind to and misregulate the biological functions of cytoplasmic multiprotein complexes. Two CCUG-interacting complexes were subsequently purified and analyzed. A major component of one of the complexes was found to be the 20S catalytic core complex of the proteasome. The second complex was found to contain CUG triplet repeat RNA-binding protein 1 (CUGBP1) and the translation initiation factor eIF2. Consistent with the biological functions of the 20S proteasome and the CUGBP1-eIF2 complexes, the stability of short-lived proteins and the levels of the translational targets of CUGBP1 were shown to be elevated in DM2 myoblasts. We found that the overexpression of CCUG repeats in human myoblasts from unaffected patients, in C2C12 myoblasts, and in a DM2 mouse model alters protein translation and degradation, similar to the alterations observed in DM2 patients. Taken together, these findings show that RNA CCUG repeats misregulate protein turnover on both the levels of translation and proteasome-mediated protein degradation.

Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.

Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.

Altered MEF2 isoforms in myotonic dystrophy and other neuromuscular disorders.

Because of their central role in muscle development and maintenance, MEF2 family members represent excellent candidate effectors of the muscle pathology in myotonic dystrophy (DM). We investigated the expression and alternative splicing of all four MEF2 genes in muscle from neuromuscular disorder (NMD) patients, including DM1 and DM2. We observed MEF2A and MEF2C overexpression in all NMD muscle, including 12 MEF2-interacting genes. Exon 4 and 5 usage in MEF2A and MEF2C was different between DM and normal muscle, with DM showing the embryonic isoform. Similar splicing differences were observed in other NMD muscle. For MEF2C, missplicing was more pronounced in DM than in other dystrophies. Our data confirm dysregulation of MEF2A and MEF2C expression and splicing in several NMD, including DM. Our findings demonstrate that aberrant splicing in NMD is independent from expression of mutant repeats, and suggests that some aberrant splicing, even in DM, may be compensatory rather than primary.

(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis.

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening.

Diminished milk synthesis in upstream stimulatory factor 2 null mice is associated with decreased circulating oxytocin and decreased mammary gland expression of eukaryotic initiation factors 4E and 4G.

Previous studies have suggested that upstream stimulatory factors (USFs) regulate genes involved with cell cycle progression. Because of the relationship of USFs to an important oncogene in breast cancer, c-myc, we chose to determine the importance of USF to normal mammary gland development in the mouse. Expression of USF in the mammary gland throughout development demonstrated only modest changes. Mutation of the Usf2 gene was associated with reduced fertility in females, but had no effect on prepartum mammary gland development. However, lactation performance in Usf2-/- females was only half of that observed in Usf2+/+ females, and both lactose and nitrogen were decreased in milk from Usf2-/- dams. This decrease was associated with diminished mammary tissue wet weight and luminal area by d 9 of lactation and with a decreased protein-DNA ratio. This decrease was associated with reduced abundance of the eukaryotic initiation factors eIF4E and eIF4G. Blood oxytocin concentrations on d 9 postpartum were also lower in Usf2-/- mice than Usf2+/+ mice. In contrast, the mutation had no effect on blood prolactin concentrations, mammary cell proliferation or apoptosis, mammary tissue oxytocin receptors, or milk protein gene expression. The mutation had only modest effects on maternal behavior. These data support the idea that USF is important to physiological processes necessary for the establishment and maintenance of normal lactation and suggest that USF-2 may impact lactation through both systemic and mammary cell-specific mechanisms.

Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies.

The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.