Polymerase Chain Reaction The sequences of all candidate genes were downloaded from web interface Genome Browser Santa Cruz (http://genome.ucsc.edu/cgi-bin/hgGateway). All coding exons and intron-flanking regions were amplified by PCR from genomic DNA using primers pairs available on request. Primers were compared with results of the web-based program Primer3 (PRIMER3; primer3_www.cgi, v 0.2; http://frodo.wi.mit.edu). Each oligonucleotide was also checked by Blastn

against the NCBI data bank genome for specificity (BLAST, www.ncbi.nlm.nih.gov/BLAST; Inhibitors,research,lifescience,medical NCBI, www.ncbi.nlm.nih.gov). For PCR analysis, 60 ng of genomic DNA was amplified with a DNA Thermocycler System. An initial denaturation step at 95°C for 7 min was set, followed by 34 cycles (95°C for 30 s, 60–61°C for 1 min and 30 s, and 68°C for 1 min) followed by 95°C and a final extension at 68°C for 10 min. DHPLC Analysis We

performed comparative mutation scanning to select amplicons for aberrant DHPLC profiles not shared Inhibitors,research,lifescience,medical by normal controls. Primers were longer than 25 nucleotides to reduce the allele preference determined by sequence differences located in the region of annealing. DHPLC was performed on a WAVE DNA fragment analysis system (Transgenomic Inc.) equipped with a DNASep column (3,500 High Throughput [HT]) employing a UV-C scanner Inhibitors,research,lifescience,medical to detect eluted DNA (23). Based on DHPLC requirements, Inhibitors,research,lifescience,medical special buffer formulations and primer design were used to improve sensitivity and specificity (22, 23). Genomic DNA Sequence Analysis Both strands were sequenced using BigDyes Terminator sequencing chemistry (Applied Biosystems). An ABI3130XL automatic DNA sequencer (Applied Biosystems) was used to analyze the product of the sequence reaction. We verified each nucleotide change by direct sequencing of a second amplified PCR product obtained with different primers. Mutations were numbered based on proteins and cDNA sequences in GenBank (Table ​(Table1).1). Nucleotides were numbered according to international recommendation Inhibitors,research,lifescience,medical (24). Table 1 Genes and relative nucleotide and protein accession numbers. Results Selection of candidate genes

We have selected a pool of EVP4593 nmr eleven candidate genes with different methodology: yeast-two hybrid and bioinformatics approach. It consists in selecting genes however with a combination of interesting characteristics: muscle specific expression or localization (sarcomeric or sarcoplasmatic); function (known or hypothetic for muscle); structure (similarity with other LGMD proteins). Myozenins (1, 2 and 3) These three genes codify for three small Z-disk proteins which specifically binds calcineurin. Transgenic mice that overexpress the calcineurin develop a progressive cardiac hypertrophy, which causes stroke and death (25). Myozenin 1 is also known as FATZ for the interaction with three sarcomeric proteins: alpha-actinin, filamin C and telethonin. Gontier et al.