Exp. Anim. 52(5), 391Ð396, 2003

Analyses of Beta-1 , Syndecan 2 and Gem GTPase as Candidates for Chicken Muscular Dystrophy

Kanako YOSHIZAWA1), Kyoko INABA1), Hideyuki MANNEN2), Tateki KIKUCHI3), Makoto MIZUTANI4), and Soichi TSUJI2)

1)Graduate School of Science and Technology, Kobe University, Kobe 657-8501, 2)Laboratory of Animal Breeding and Genetics, Faculty of Agriculture, Kobe University, Kobe 657-8501, 3)Department of Animal Models for Human Disease, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, 182-8502, and 4)Nippon Institute of Biological Science, Kobuchizawa, 408-0041, Japan

Abstract: Despite intensive studies of muscular dystrophy of chicken, the responsible has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11Ð24.3, where the beta- 1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease. Key words: beta-1 syntrophin, syndecan2, Gem GTPase, muscular dystrophy, linkage map

Introduction movement [8]. The diseases are associated with muta- tions in the genes encoding several classes of muscle Muscular dystrophy refers to a group of inherited , whose purpose is to maintain the normal func- diseases marked by progressive weakness and degen- tion of the internal face of the membrane of muscle eration of the skeletal, or voluntary, muscles that control cells [3].

(Received 3 April 2003 / Accepted 18 June 2003) Address corresponding: H. Mannen, Laboratory of Animal Breeding and Genetics, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan 392 K. YOSHIZAWA, ET AL.

Animal models for the etiological and pathological lated from the leg muscle and liver of a normal male studies of human muscular dystrophies have been es- White Leghorn chicken, and cloned into the ZAP Ex- tablished in mice, dogs and cats [7, 8]. The disease press vector (Stratagene, CA). Reverse transcription of symptoms and conditions are not identical among spe- mRNA from leg muscle and liver of mouse (500 ng) cies because of different pathological manifestations of were performed using a RT-PCR system (Life Tech- similar genetic defects. Therefore, establishing addi- nologies, Inc., MD). The mouse muscle cDNA was tional animal models will contribute to a better used for PCR amplification of mouse Sntb1 and Gem, understanding of the mechanisms of muscular dystro- and the mouse liver cDNA was used for PCR amplifi- phies and provide a basis and strategy for clinical cation of mouse Sdc2. Primers used were therapy trials for such incurable diseases. 5’-AAGGAAGTGCTGCTGGAAGTGAAGTATA-3’ Chicken muscular dystrophy with abnormal muscle (Sntb1-F), 5’-AGACAGAAAGGAGTGGATGATGAA (AM) was first reported in 1956 [2]. The disorder is AACG-3’ (Sntb1-R), 5’-AGAGACGAGAACAGAGCT transmitted codominantly by a single gene, AM, whose GACATCCG-3’ (Sdc2-F), 5’-GTCGTAGCTTCCTTCA phenotype is modified by other “background” genes [2, TCTTTCTTCC-3’ (Sdc2-R), 5’-GACTCTGAATAATG 4, 12]. In our previous study, we found the AM locus TCACCATGCGCC-3’ (Gem-F for first round of PCR), was mapped on the ‘q’ arm of chromosome 2 [5]. Re- 5’-GCCACAATTTTGCCCCAGAAGCGCCT-3’ cently, Schmid et al. [10] showed synteny between (Gem-R for first round of PCR), 5’-CCACCCCTGCAA chicken chromosome 2q and human 8q CCTCCGAAACCGCC-3’ (Gem-F for second round of and 18p. The region including the AM locus shows PCR) and 5’-GAATGCTCTCCCGCCTCTTCTGG synteny with human chromosome 8q11Ð24.3 [5]. Since TAG-3’ (Gem-R for second round of PCR). The PCR the genes located on human chromosome 8q11Ð24.3 was done for 30 cycles at 94°C for 1 min, 60°C for 1 are possible candidates responsible for this disease, in min, 72°C for 3 min for Sntb1; for 30 cycles at 94°C this study we focused on three genes located on human for 1 min, 60°C for 1 min, 72°C for 90 s for Sdc2; for chromosome 8q22Ð24: beta-1 syntrophin (SNTB1), 30 cycles at 94°C for 30 s, 63°C for 30 s, 72°C for 1 syndecan 2 (SDC2) and Gem GTPase (GEM). min for the first round of Gem amplification; and for Chicken SNTB1, SDC2 and GEM genes were cloned 30 cycles at 94°C for 30 s, 67°C for 30 s, 72°C for 1 and sequenced, then subsequently mapped to chicken min for the second round of Gem amplification. The chromosomes in order to investigate whether they are Sntb1 PCR product with 1,032 bp length, Sdc2 PCR related to chicken muscular dystrophy. product with 490 bp length and Gem PCR product with 677 bp length were labeled with digoxigenin (Roche, Materials and Methods Swiss) and were used as probes. The isolated clones were sequenced with SequiTherm Resource family EXCELª II Long-Readª DNA Sequencing Kits-LC The Kobe University (KU) resource family was es- (Epicentre Technologies, Madison, WI) on an automated tablished in a previous study [5], and was used for DNA sequencer (model 4200L, LI-COR, NE), and with linkage analysis in this study. This family is a back- the SILVER SEQUENCEª DNA Sequencing System cross pedigree with 55 offspring produced from the (Promega, Madison, WI) on a manual sequencing ap- mating of a normal male (White Leghorn: strain WL-F) paratus. and a hybrid female produced from a cross between the WL-F male and a muscular dystrophy female (Fayoumi: Identification of polymorphisms strain OPN) homozygous for the AM gene [5]. Ge- PCR primers were designed to amplify the intron nomic DNA was prepared from blood of 55 backcross region by use of exon sequences. Intron amplification chickens and their parents. was performed, using Ex Taqª (Takara, Tokyo, Ja- pan), by the following program: 1 min at 94°C, followed cDNA cloning and sequencing of SNTB1 and GEM cDNA by 35 cycles of 30 s at 94°C, 30 s at 65°C, 1 min at In order to isolate the SNTB1, SDC2 and GEM genes, 72°C. The amplification products were sequenced. The two cDNA libraries were generated using mRNA iso- sequences were compared between WL-F male and CANDIDATE GENES FOR MUSCULAR DYSTROPHY 393

Table 1. PCR primers to amplify introns and restriction enzymes to detect the mutations of chicken SNTB1, SDC2 and GEM genes

Locus PCR Restriction Amolified Genotype of parental line Primer sequencea) symbol product (bp) enzyme resion WL-F OPN

SNTB1 F: 5’-ACGAGCAGAGACCTCTCACTGTGGAC-3’ 1.5 kbp Eco72I Intron4 Ð/Ðb) +/+b) R: 5’-GCAATTGTCTTAGAGAAGGCACCGTC-3’ SDC2 F: 5’-AAGAGCAATGAGCCAGGGGATGACAC-3’ 1 kbp HinfI Intron4 +/+ Ð/Ð R: 5’-TCCTTCTTTCTCATGCGATATACCAG-3’ GEM F: 5’-atccacaaaatacttacaaacgcacc-3’ 156 bp Tsp509I Intron1 Ð/Ð +/+ R: 5’-cctctgactcactggctagaaatatt-3’ a) Exon and intron sequences are denoted by uppercase and lowercase letters,respectively. b) -/-: homozygous for the uncut PCR product, +/+: homozygous for the cut PCR product.

OPN female to find nucleotide substitutions.

PCR-RFLP analysis In order to perform genotyping for the resource fam- ily, the PCR-RFLP method was applied and the primers used are shown in Table 1. The intron amplification products were digested by restriction enzyme and were analyzed by electrophoresis in a 1% agarose gel.

Linkage analysis Intron polymorphisms of SNTB1, SDC2, and GEM were used in the linkage analysis. These polymorphisms were analyzed in conjunction with previously published microsatellite and AFLP (Amplified Fragment Length Polymorphism) markers of the Kobe University link- age map [5]. Linkage groups were determined by two-point analysis under linkage criterion of P<0.05 as a G-statistic for independence and Kosambi function, using Map Manager QTXPb12 [6].

Results cDNA cloning and sequencing of SNTB1, SDC2 and GEM genes The cDNA library from the chicken leg muscle was screened using mouse Sntb1 PCR product as a probe. The longest positive clone included approximately 3 kbp of the 3’ untranslated region and the partial coding sequence. There were 382 amino acid residues Fig. 1. Deduced amino acid sequence of chicken SNTB1 gene in estimated from the determined coding sequence. Hu- comparison with corresponding human and mouse amino man and mouse SNTB1 have 481 and 480 amino acid acid sequences. Dots and hyphens indicate no change in residues, respectively. Figure 1 shows the alignment amino acid residue and a gap among chicken, human and mouse genes. of the deduced amino acid sequences of human/mouse/ 394 K. YOSHIZAWA, ET AL.

Fig. 3. Deduced amino acid sequence of chicken GEM gene in comparison with corresponding human and mouse amino acid sequences. Dots and hyphens indicate no change in amino acid residue and a gap among chicken, human and Fig. 2. Deduced amino acid sequence of chicken SDC2 gene in mouse genes. comparison with corresponding human, mouse, frog and fish amino acid sequences. Dots and hyphens indicate no change in amino acid residue and a gap among chicken, human and mouse genes. and GEM genes were recorded in the DDBJ databases under accession numbers AB105810- AB105812. chicken SNTB1. Their alignment revealed that chicken PCR-RFLP analyses SNTB1 is highly identical to human (94.53%) and Exon-based primers were designed to amplify across mouse (94.31%) SNTB1. introns. The exon-intron structure was estimated from We used mouse Sdc2 PCR product as a probe and human orthologues. Introns were amplified using PCR. isolated a cDNA clone containing an approximately 2- Sequence analyses of PCR products from the resource kb insert from chicken liver cDNA library. The clone family parents were conducted to identify base substi- contained a complete open reading frame. Two hun- tutions in either parent. Sequence analyses of SNTB1/ dred and two amino acid residues were composed of intron4, SDC2/intron4 and GEM/intron1 between the chicken SDC2. The amino acid sequences of chicken parents identified mutations. Since these introns create SDC2 cDNA were 73.8% identical to the deduced hu- differences of restriction enzyme sites between WL-F man, 72.4% identical to the deduced mouse, 66.2% and Fayoumi OPN (Table 1), PCR-RFLP analyses to identical to the deduced frog and 55.7% identical to the detect these mutations were performed for the KU re- deduced fish SDC2 (Fig. 2). source family. Screening of the chicken leg muscle cDNA library using mouse Gem as a probe yielded five positive Linkage analysis clones. All inserts had an open reading frame. The Linkage analysis was performed by analyzing the seg- first methionine codon was in a context favoring aligns regation of alleles of SNTB1, SDC2, GEM and AM loci with that described for the human cDNA (Fig. 3). The in conjunction with 12 microsatellite and 16 AFLP mark- deduced protein contained 241 amino acids. Its amino ers [5]. As a result of linkage analysis, all markers were acid sequences were 90.9% and 90.3% identical to hu- located on chromosome 2q. Figure 4 indicates the link- man GEM and mouse Gem sequences, respectively. age map analyzed in this study as well as the cytogenetic The nucleotide sequences of chicken SNTB1, SDC2 map [10]. The AM gene was mapped 130 cM from the CANDIDATE GENES FOR MUSCULAR DYSTROPHY 395

Discussion

The inherited nature of the chicken muscular dystro- phy has been previously well-described [2, 14]. However the pathogenesis of the progressive loss of muscle func- tion is unclear. To elucidate this pathogenesis, it is essential to identify the gene responsible for this disor- der. Recently, our studies have revealed that the AM locus is mapped on the ‘q’ arm of chromosome 2, and the region including the AM locus shows synteny with human chromosome 8q11Ð24.3 [5]. However, the num- ber of available genetic markers, especially those of functional genes, is limited in chicken, and a compre- hensive comparative map between chicken and human has not been constructed. Therefore, in order to identify the responsible genes for chicken muscular dystrophy, a positional candidate approach or development of func- tional gene markers in this region is required. It should be noted that SNTB1, SDC2 and GEM, which were mapped on human chromosome 8q11Ð24.3, were thought to be candidate genes of chicken muscular dystrophy, or at least to be useful markers for constructing a compre- hensive comparative map between human and chicken. The are a multigene family expressing intracellular -associated proteins (DAP) [1, 9]. It has been reported that defects in the DAP com- Fig. 4. Linkage and cytogenetic map of the chicken chromosome plex often cause muscular dystrophy [11]. Syntrophin 2q. Linkage map produced using the Kobe University re- is thought to link signaling proteins (ion channels, ki- source family is shown on the right. The map corresponds to the region from approximately 380 cM to 520 cM of nases, nitric oxide producing enzymes, etc.) to chicken chromosome 2 in the Kobe University linkage dystrophin/utrophin complexes and thus target these sig- map [5]. Cytogenetic map of chicken chromosome 2 is naling systems for membrane specializations. Any of shown on the left [10]. Thirty-two loci include four func- tional genes (GEM, SDC2, SNTB1 and CALB1), 11 the dystrophin-associated proteins are candidates for the microsatellite markers, 16 AFLP markers and abnormal site of mutation in neuromuscular disease. To date, muscle (AM). Four functional genes are shown in italic however, the map location of the SNTB1 gene appears and boldface type. Microsatellite markers are underlined. to exclude it as responsible for any of the human neu- KUA indicates Kobe University AFLP markers. Dis- tances between adjacent markers are given in cM romuscular diseases with known map location. (Kosambi map function). However, due to the wide tissue distribution of the SNTB1 transcript, it is possible that a defect of this gene would result in a non-muscular disorder. distal end of chromosome 2q [5], and was closely linked Syndecans are transmembrane heparan sulfate to calbindin 1 (CALB1), while MCW0166 was 9.5 cM proteoglycans (HSPGs) that are present in most cell distal and KUA610 was 7.6 cM proximal from the AM types. HSPGs have been known for some time to regu- locus. SNTB1 was mapped 88.5 cM distal from the AM late a variety of biological processes, ranging from locus. SDC2 was located 27.6 cM distal from AM. GEM coagulation cascades, growth factor signaling, cell ad- was mapped 18.5 cM distal from AM and 9.1 cM proxi- hesion to extracellular matrix and subsequent mal from SDC2. Orthologues of SNTB1, SDC2 and cytoskeletal organization, to infection of cells with mi- GEM were syntenic to human chromosome 8q. croorganisms [15]. The pathological roles of syndecan 396 K. YOSHIZAWA, ET AL. have not been extensively studied, but the effects of chromosomal locations, and each bind to dystrophin and SDC2 on matrix assembly may well contribute to many its relatives. J. Biol. Chem. 271: 2724Ð2730. 2. Asmundson, V.S. and Julian, L.M. 1956. Inherited muscle diseases involving a fibrotic response. abnormality in the domestic fowl. J. Hered. 47: 248Ð252. GEM is a member of a small GTP binding family of 3. Imamura, M., Araishi, K., Noguchi, S., and Ebashi, S. 2000. proteins within the Ras superfamily, sometimes referred A - complex anchors Dp116 and to as the Rad/Gem/Kir (RGK) subfamily of Ras-related utrophin in the peripheral nervous system. Hum. Mol. Genet. 9: 3091Ð3100. GTPases whose biochemical functions are largely un- 4. Kikuchi, T., Ishiura, S., Nonaka, I., and Ebashi, S. 1981. known. Recently, it has been shown that GEM Genetic heterozygous carriers in hereditary muscular interfaces with the Rho pathway through association dystrophy of chickens. Tohoku J. Agr. Res. 32: 14Ð26. with the Rho effectors, Rho kinase (ROK) alpha and 5. Lee, E.J., Yoshizawa, K., Mannen, H., Kikuchi, H., Kikuchi, beta, and the physical role for GEM in cytoskeltal regu- T., Mizutani, M., and Tsuji, S. 2002. Localization of the muscular dystrophy AM locus using a chicken linkage map lation mediated by ROK has been identified [13]. constructed with the Kobe University resource family. Anim. These probable functions and the locations of SNTB1, Genet. 33: 42Ð48. SDC2, and GEM make them candidates for this muscu- 6. Manly, K.F., Cudmore, R.H. Jr., and Meer JM. 2001. Map lar dystrophy. Linkage analysis mapped SNTB1, SDC2 Manager QTX, cross-platform software for genetic mapping. Mamm. Genome 12: 930Ð932. and GEM genes to chicken chromosome 2q and indi- 7. Nonaka, I. 1998. Animal models of muscular dystrophies. cated a high level of conserved synteny between chicken Lab. Anim. Sci. 48: 8Ð17. chromosome 2q and human chromosome 8q21Ð24. 8. Partridge, T. 1991. Animal models of muscular dystrophy— However, the three genes do not overlap with the AM what can they teach us? Neuropathol. Appl. Neurobiol. 17: locus. Therefore, it is unlikely that SNTB1, SDC2 and 353Ð363. 9. Peters, M.F., Adams, M.E., and Froehner, S.C. 1997. GEM genes are responsible for chicken muscular dys- Differential association of syntrophin pairs with dystrophin trophy. The AM gene locus was closely linked to complex. J Cell Biol. 138: 81Ð93. CALB1 and MCW0166 (9.5 cM distal), KUA610 (7.6 10. Schmid, M., Nanda, I., Guttenbach, M., Steinlein, C., cM proximal), and GEM (18.5 cM distal). The genes Hoehn, M., Schartl, M., Haaf, T., Weigend, S., Fries, R., Buerstedde, J.M., Wimmers, K., Burt, D.W., Smith, J., located in the proximal region of the CALB1 gene on A’Hara, S., Law, A., Griffin, D.K., Bumstead, N., Kaufman, human chromosome 8q are therefore possible candi- J., Thomson, P.A., Burke, T., Groenen, M.A., Crooijmans, dates for this disease. Additional development of R.P., Vignal, A., Fillon, V., Morisson, M., Pitel, F., Tixier- genetic markers based on functional genes will be re- Boichard, M., Ladjali-Mohammedi, K., Hillel, J., Maki-Tanila, A., Cheng, H.H., Delany, M.E., Burnside, J., quired to determine the correct location of the AM locus and Mizuno, S. 2000. First report on chicken genes and where the responsible gene exists. chromosomes 2000. Cytogenet Cell Genet. 90: 169Ð218. 11. Terri, G.T. and Kunkel, L.M. 2000. Advances in muscular Acknowledgments dystrophy: Exiciting new projects for millennium. Neuro. Sci. News 3: 4Ð12. 12. Wagner, W.D. and Peterson, R.A. 1970. Muscular We thank Dr. S. Takeda in the Department of Mo- dystrophy syndrome in the Cornish chicken. Am. J. Vet. lecular Therapy, National Institute of Neuroscience, Res. 31: 331Ð338. NCNP, Tokyo, Japan for encouraging and supporting 13. Ward, Y., Yap, S.F., Ravichandran, V., Matsumura, F., our study. This work was supported in part by the Ito, M., Spinelli, B., and Kelly, K. 2002. The GTP binding proteins Gem and Rad are negative regulators of the Rho- Research Grant for Nervous and Mental disorders from Rho kinase pathway. J. Cell Biol. 157: 291Ð302. the Ministry of Health, Labour and Welfare, Japan. 14. Wilson, B.W., Randall, W.R., Patterson, G.T., and Entrikin, R.K. 1979. Major physiologic and histochemical References characteristics of inherited dystrophy of the chicken. Ann. N.Y. Acad. Sci. 317: 224Ð246. 1. Ahn, A.H., Freener, C.A., Gussoni, E., Yoshida, M., Ozawa, 15. Woods, A. 2001. Syndecans: transmembrane modulators E., and Kunkel, L.M. 1996. The three human syntrophin of adhesion and matrix assembly. J. Clin. Invest. 107: 935Ð genes are expressed in diverse tissues, have distinct 941.