Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1 Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.

Muscular dystrophies, a group of inherited diseases with the progressive weakness and degeneration of skeletal muscle, contain genetically variable diseases. Though chicken muscular dystrophy with abnormal muscle (AM) has long been known, the gene responsible has not yet been identified. In this study, a resource family for AM was established with 487 F2 individuals and 22 gene markers, including microsatellite and insertion-deletion markers, were developed. The haplotypes were analyzed with these markers for the candidate region of GGA2q described in a previous study. The candidate region was successfully narrowed down to approximately 1Mbp. The region included seven functional genes predicted as the most likely AM candidates.

The WW domain containing E ubiquitin protein ligase (WWP ) is classified into one of ubiquitin ligases which play an important role in ubiquitin-proteasome pathway. Previously, we identified the gene as a candidate gene of chicken muscular dystrophy by linkage analysis and sequence comparison. However, the mechanism causing pathological changes and underlaying gene function remains elucidated. In the present study, we analyzed the gene expression in various muscles and tissues of normal chickens, and compared with those from muscular dystrophic chickens. Two mRNA isoforms were detected in all tissues examined and revealed almost equal expression level. The expression of dystrophic chickens was decreased in almost all skeletal muscles including una ected muscles. These data indicate that there might not be a causal relationship between the alteration of expression level and the severity of muscular dystrophy.: chicken, expression analysis, fast twitch muscle fiber, muscular dystrophy, WWP Poser ; Augustine, ). The WW domain has two conserved tryptophan residues and binds prolineThe WW domain containing E ubiquitin protein ligase rich region (Sudol ). HECT domain, similar to ( ) is classified into an ubiquitin ligase (E ) which E s structurally, has a cysteine residue as an active center plays an important role in ubiquitin-proteasome pathway that transfers the activated Ub from E onto first itself, (UPP) to degrade unneeded or damaged proteins and then onto its substrates (Jackson ). (Sche ner and Staub,). E recognizes and catalyzes The muscular dystrophies are the group of inherited ubiquitin (Ub) conjugation to specific protein substrates diseases with progressive weakness and degeneration of (Liu, ). Comparative genome analysis reveals few skeletal muscle (Partridge, ). It is well known that genes encoding E , tens of E encoding genes and hunabnormalities of muscle proteins linking sarcolemma and dreds of E encoding genes (Semple ). basal lamina lead to cause muscular dystrophies (Lisi and The gene is classified into HECT (homologous Cohn, ), but there are a number of muscular to the E -AP carboxyl terminus)-type E which possesses dystrophies and related diseases of which causes are still one C domain, multiple WW domains and one HECT unknown. We identified gene as a candidate domain (Pirozzi ; Flasza ). The C responsible for the chicken muscular dystrophy by the domain binds to the cellular membranes in a linkage analysis (Matsumoto ) and the seCa -dependent manner (Plant )

SUMMARYMLPA was developed by Schouten JP et al. in 2002, and has become a rapidly growing technique used in clinical diagnosis for genetic diseases, such as Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD), Lynch syndrome and multiple congenital anomalies (MCA)/mental retardation (MR). MLPA is a simple and rapid method for simultaneous quantification of up to 40 nucleic acid sequences in a single reaction. MLPA probe consists of two different oligonucleotides, each containing one of the PCR primer sequences as well as a sequence complementary to the target sequence. The two probe oligonucleotides hybridize to immediately adjacent target sequences. It is only when the two probe parts are both hybridized to their target sequence that they can be ligated to each other by a thermostable ligase. These ligated probes will be amplified exponentially during PCR reaction. The resulting PCR amplification products are subsequently separated by capillary gel electrophoresis. The number of probe ligation products of one probe depends on the number of target sequences in the sample. The DMD gene is located on Xp21 and the gene responsible for DMD/BMD. Large deletions of DMD gene are found in 60%, large duplications are 10%, and small mutations are 30% of the cases. Although the conventional multiplex PCR assay was developed for the genetic testing of the DMD gene, it could not detect large duplication because of its low quantitative performance. The MLPA assay can provide a simple and rapid method for the large deletion and duplication screening done in DMD/BMD patients.

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