Huichin Pan

Long Tract of Untranslated CAG Repeats Is Deleterious in Transgenic Mice

The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3′ untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats.

Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans

We have utilized Caenorhabditis elegans as a model to investigate the toxicity and underlying mechanism of untranslated CAG repeats in comparison to CUG repeats. Our results indicate that CAG repeats can be toxic at the RNA level in a length-dependent manner, similar to that of CUG repeats. Both CAG and CUG repeats of toxic length form nuclear foci and co-localize with C. elegans muscleblind (CeMBL), implying that CeMBL may play a role in repeat RNA toxicity. Consistently, the phenotypes of worms expressing toxic CAG and CUG repeats, including shortened life span and reduced motility rate, were partially reversed by CeMbl over-expression. These results provide the first experimental evidence to show that the RNA toxicity induced by expanded CAG and CUG repeats can be mediated, at least in part, through the functional alteration of muscleblind in worms.

Application of FTA® sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR‐based southern blotting

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme‐digested genomic DNA. The development of PCR‐based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7‐deaza‐dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7‐deaza‐dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM. J. Clin. Lab. Anal. 13:188–193, 1999.

Haplotype analysis of the myotonic dystrophy type 1 (DM1) locus in Taiwan: implications for low prevalence and founder mutations of Taiwanese myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder caused by a CTG trinucleotide expansion at the DM1 locus. In this study, we investigated the frequency distribution of various CTG repeats in normal alleles and haplotyped the normal and expanded DM1 locus in a group of Taiwanese people. In the 496 normal chromosomes examined, up to 18 alleles with different CTG lengths from 5 to 30 repeats were found and the frequency of (CTG)>18 alleles was only 1.4% (7/496), predicting a low prevalence of DM1. In addition, there is no absolute association between (CTG)5–19 alleles and Alu insertion/deletion polymorphism observed on normal chromosomes. All DM1 alleles examined, however, were found to be associated with the Alu insertion. Further detailed genetic analysis demonstrated that at least eight haplotypes, including a new haplotype (L), were present in the Taiwanese population and that all DM1 alleles were with the same haplotype (haplotype A) as that identified in Canadian and Japanese DM1 populations. These findings support the notion that the out-of-Africa DM1 alleles were originated by stepwise expansion from a pool of large-sized normal chromosomes with haplotype A.

The critical role of protein arginine methyltransferase prmt8 in zebrafish embryonic and neural development is non-redundant with its paralogue prmt1.

Protein arginine methyltransferase (PRMT) 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio) as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount in situ hybridization revealed ubiquitous prmt8 expression pattern during early embryonic stages, similar to that of prmt1. Knockdown of prmt8 with antisense morpholino oligonucleotide phenocopied prmt1-knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive prmt8 failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length prmt8 but not prmt1 cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the prmt8 morphants. In contrast, N-terminus- deleted but not full-length prmt8 cRNA can rescue the prmt1 morphants as efficiently as prmt1 cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon prmt8 knockdown confirm the critical roles of prmt8 in neural development. In summery, our study is the first report showing the expression and function of prmt8 in early zebrafish embryogenesis. Our results indicate that prmt8 may play important roles non-overlapping with prmt1 in embryonic and neural development depending on its specific N-terminus.

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones

BACKGROUND/OBJECTIVES Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. METHODS Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. RESULTS Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. CONCLUSION From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO(3) from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate.

The predominant protein arginine methyltransferase PRMT1 is critical for zebrafish convergence and extension during gastrulation

Protein arginine methyltransferase (PRMT)1 is the predominant type I methyltransferase in mammals. In the present study, we used zebrafish (Danio rerio) as the model system to elucidate PRMT1 expression and function during embryogenesis. Zebrafish prmt1 transcripts were detected from the zygote period to the early larva stage. Knockdown of prmt1 by antisense morpholino oligo (AMO) resulted in delayed growth, shortened body‐length, curled tails and cardiac edema. PRMT1 protein level, type I protein arginine methyltransferase activity, specific asymmetric protein arginine methylation and histone H4 R3 methylation all decreased in the AMO‐injected morphants. The morphants showed defective convergence and extension and the abnormalities were more severe at the posterior than the anterior parts. Cell migration defects suggested by the phenotypes were not only observed in the morphant embryos, but also in a cellular prmt1 small‐interfering RNA knockdown model. Rescue of the phenotypes by co‐injection of wild‐type but not catalytic defective prmt1 mRNA confirmed the specificity of the AMO and the requirement of methyltransferase activity in early development. The results obtained in the present study demonstrate a direct link of early development with protein arginine methylation catalyzed by PRMT1.

The zebrafish homeobox gene irxl1 is required for brain and pharyngeal arch morphogenesis

Iroquois homeobox‐like 1 (irxl1) is a novel member of the TALE superfamily of homeobox genes that is most closely related to the Iroquois class. We have identified the zebrafish irxl1 gene and characterized its structure. The protein contains a homeodomain that shares 100% sequence identity with other vertebrate orthologs. During embryogenesis, irxl1 is expressed from 18 hours postfertilization onward and prominent expression is detected in the pharyngeal arches. Knockdown of irxl1 by morpholinos results in malformed brain and arch structures, which can be partially rescued by cRNA injection. The heads of the morphants become small and flat, and extensions along the anterior–posterior/dorso–ventral axes are reduced without affecting regional specification. Loss of irxl1 function also causes deficit in neural crest cells which consequently results in partial loss of craniofacial muscles and severe deformation of arch cartilages. These observations suggest that irxl1 may regulate factors involved in brain and pharyngeal arch development. Developmental Dynamics 239:639–650, 2010.

Zebrafish muscleblind-like genes: Identification, structural features and expression

Muscleblind-like (MBNL) proteins are a family of RNA-binding proteins that participate in the regulation of tissue-specific alternative splicing. Misregulation of MBNL activity in humans leads to pathogenesis. Here, we report upon the identification and characterization of three muscleblind-like genes in zebrafish (zmbnl1, zmbnl2 and zmbnl3). Alternative splicing of the three zmbnl primary transcripts gives rise to at least four protein isoforms for zmbnl1, four for zmbnl2 and five for zmbnl3, respectively. All of the zmbnl proteins contain the characteristic CCCH zinc fingers required for RNA binding. In addition, several structural motifs, including a C-terminal Ser/Thr-rich region, are conserved among Mbnl orthologs in vertebrates, but not invertebrates. These genes are broadly expressed in most adult tissues. However, the relative expression levels of specific spliceforms vary across different tissues. During embryogenesis, zmbnl1 and zmbnl2 are both maternally and zygotically expressed. In contrast, zmbnl3 transcripts are not detected until the late pharyngula stage. Our results reveal the expression pattern of various mbnl spliceforms for the first time and suggest that they may play specific roles during fish development.

The homeobox transcription factor Irxl1 negatively regulates MyoD expression and myoblast differentiation

Irxl1/Mkx (Iroquois homeobox‐like 1/Mohawk) encodes a member of the TALE subfamily of homeodomain proteins. It is expressed in multiple mesoderm‐derived tissues and has recently been shown to regulate tendon differentiation during mouse embryonic development. Previously we showed that knockdown of Irxl1 in zebrafish caused a deficit in neural crest cells which consequently resulted in deformation of craniofacial muscles and arch cartilages. Here, we further demonstrate that loss of Irxl1 function results in deformed somites with disordered muscle fibers and myotendinous junctions. Because expression of myoD is increased in the somites of Irxl1 knockdown morphants, we test whether Irxl1 negatively regulates myoD expression. When stable C2C12 myoblasts overexpressing Irxl1/Mkx were induced to differentiate, myotube formation was inhibited and protein levels of myoD and myosin heavy chain were decreased accordingly. A series of deletion constructs of myoD promoter fragments were tested by luciferase reporter assays, which identified a promoter fragment that is necessary and sufficient for Irxl1‐mediated repression. Direct interaction of Irxl1 and myoD promoter was subsequently elucidated by yeast one‐hybrid assays, electrophoretic mobility shift assays and chromatin immunoprecipitation analysis. Furthermore, mouse Mkx also binds to and represses myoD promoter. These results indicate that Irxl1/Mkx can repress myoD expression through direct binding to its promoter and may thus play a negative regulatory role in muscle differentiation.