HUMAN MUTATION Mutation and Polymorphism Report #141 (2000) Online Mutation and Polymorphism Report Authors: Doroti Pirulli 1, Silvia Zezlina 1 , Laura Vatta 1, Paola Di Stefano 2 , Michele Boniotto 1, Guido Tarone 2, Tiziana Mongini 3, Isabella Ugo 3, Laura Palmucci 3, Antonio Amoroso 1, and Sergio Crovella 1 Affiliations 1 Medical Genetic Service, IRCCS Burlo Garofolo, Chair of Genetics, University of Trieste, Italy; 2 Department of Genetics, Biology and Biochemistry, University of Turin, Italy; 3 Department of Neuroscience, University of Turin, Italy Corresponding Author Address and E-mail: Sergio Crovella, Chair of Genetics, University of Trieste, Medical Genetic Service, IRCCS Burlo Garofolo,Via dell'Istria 65/1,34137 Trieste, Italy; E-mail: [email protected] Title : A new polymorphism, g119A>G, in the integrin alpha 7 (ITGA7) gene integrin; ITGA7; PCR; myopathy Keywords: Species: Homo sapiens Change is: Polymorphism Gene/Locus Name: integrin, alpha 7 Symbol: ITGA7 Genbank accession number: AJ228850 exon 15 OMIM accession number: 600536 Locus specific database: Chromosomal location: 12q13 Inheritance: Unknown Mutation / polymorphism name Nucleotide change–Systematic name: g119A>G Amino acid change–Trivial name: H651R Mutation / polymorphism type: Missense Polymorphism frequency: 44/56 G/A Detection method: RFLP-PCR, Direct sequencing Detection conditions: RT-PCR Primers F: 5'-TCAAGAGCTTCGGCTACTCC-3' R: 5'-AGTCAGGAAGCATGACCAGG-3' Genomic PCR-Primers F: 5'-GCCCCCTGCCTTGCC-3' R: 5'-AGGGCTTTCTCTCAATCCTTGA-3' RT-PCR conditions RT-PCR was performed on total mRNA of 3 unclassified myopathy patients with the RNA-PCR Core Kit (PE Biosystems, Foster City, CA) HUMAN MUTATION Mutation and Polymorphism Report #141 (2000) Online using the reverse primer as antisense. cDNA was subsequently amplified using the following conditions: initial hold 10 min 95°C, then 35 cycles at 95°C, 30 sec, 50°C, 30 sec, 72°C 30 sec. The PCR reactions were performed on genomic DNA of 50 healthy controls (100 ng) in 50 mL final volume, using 6 pmol of each primer, 2 Genomic PCR conditions: mM MgCl2, 200 mM dNTPs, AmpliTaq GOLD DNA polymerase, 1 unit (PE Biosystems).10 min at 95 °C, 15 cycles of : 30 sec at 95 °C, 30 sec at 61 °C, 30 sec at 72 °C; 25 cycles of: 30 sec at 95 °C, 30 sec at 61 °C (touchdown: -0.3 °C/cycle), 30 sec at 72 °C; 7 min at 72 °C. The direct sequencing was performed with BigDye Terminators Sequencing Ready Reaction Kit (PE Biosystems) on both DNA strands. ABI PRISM 310 Genetic Analyzer (PE Biosystems) was used to obtain DNA sequences which were handled with Navigator 2.0 and Factura 2.1 Software (PE Biosystems). The substitution of A119 with G create a restriction site for Hha I (Celbio, Milano, Italy) in the 231 bp PCR amplicon. The digestions Direct Sequencing were detected by electrophoretic run on 3% agarose gel and ethidium bromide staining under UV lights. RFLP-PCR Diagnosis method developed: RFLP-PCR Evidence for existence and effect of mutation: Yes No Don’t know 1. Base change found on repeat PCR sample X 2. Base change segregates or appears with trait X 3. Base change affects conserved residue X 4. Expression analysis supports hypothesis for causation X 5. Normals tested (50 required) X Ancillary data 1. Haplotype association : 2. Ethnic background/Population association : Caucasian 3. Geographic association : Italians 4. Frequency (of polymorphism) in population: 0.44 5. Clinical phenotype of proband : Congenital Myopathy 6. Homologous allele (if recessive trait): 7. PIC: (if microsatellite) 8. Other: Yes: No: X 9. Present in HGMD listing: (http://www.cf.ac.uk/uwcm/mg/hgmd0.html) HUMAN MUTATION Mutation and Polymorphism Report #141 (2000) Online Comments In order to determine the role of alpha-7 muscle specific integrin in these pathologies, we performed a retrospective study on muscle biopsies, stored in liquid nitrogen, of unclassified myopathies (Gu et al., 1994; Hayashi et al., 1998; Mayer et al., 1997; Tomatis et al., 1999). In one out of 89 cases, we found a significant decrease of immunohistochemical staining confirmed by two different integrin specific antibodies. The patient was a 12-year-old girl born with joint contractures and diffuse muscle hypotrophy, who walked at 30 months and showed a mild mental retardation. Neurological examination demonstrated elongated face, high arched palate and diffuse muscle hypotrophy with normal muscle strength, marked scoliosis. Muscle biopsy displayed global fiber atrophy, with normal connective tissue. No structural abnormalities were observed; dystrophin, sarcoglycan and merosin were normally distributed. Her clinical diagnosis was unclassified congenital myopathy. We hypothesized that a mutation in the ITGA7 gene sequence was responsible for the significant decrease of integrin specific immunohistochemical staining of the patient with congenital unknown myopathy. We amplified the ITGA7 cDNA of this patient, three other myopathic patients (characterized by normal levels of integrin, after immunohistochemical studies) and 50 healthy controls. We have found a new polymorphism in the exon 15 of the ITGA7 gene, not related to the unknown myopathy patient showing decrease of integrin specific immunohistochemical staining. In a healthy Italian population, the frequencies of the two alleles (G and A at position 119) were 0.44 and 0.56, respectively. The frequencies of the genotypes homozygous G/G, heterozygous G/A and homozygous A/A were 0.14, 0.6 and 0.26, respectively. This distribution was in Hardy- Weinberg equilibrium. Finally although the described G/A polymorphism does not seem to be in relation with pathological conditions, it could be used as intragenic marker for linkage studies on informative families where muscle diseases are characterized by a decrease of alpha-7 integrin. Acknowledgments This work was supported by grants from the Italian Ministries of Research (MURST) and of Health (RC18/99). Doroti Pirulli is recipient of a long term fellowship from University of Trieste, Michele Boniotto and Silvia Zezlina are recipients of fellowship from IRCCS Burlo Garofolo, Trieste. References Gu M, Wang W, Song WK, Cooper DNW and Kaufman SJ: Selecrive modulation of the interaction of a7b1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. Journal of Cell Science 107:175- 181, 1994. Hayashi YK, Chou FL, Engvall E, Ogawa M, Matsuda C, Hirabayashi S, Yokochi K, Ziober BL, Kramer RH, Kaufman SJ, Ozawa E, Goto Y, Nonaka I, Tsukahara T, Wang J, Hoffman EP and Arahata K: Mutations in the integrin a7 gene cause congenital myopathy. Nature Genetics 19: 94-97, 1998. Mayer U, Saher G, Fassler R, Bornemann A, Echtermeyer F, von der Mark H, Miosge N, Poschl E & von der Mark K: Absence of integrin a7 causes a novel form of muscular dystrophy. Nature Genetics 17:318-323, 1997. Tomatis D, Echtermayer F, Schober S, Balzac F, Retta SF, Silengo L and Tarone G: The muscle-specific laminin receptor a7b1 integrin negatively regulates a5b1 fibronectin receptor function.Experimental Cell Research 246:421- 432, 1999..