MS#200201004 Péréon et al, 1

Paramyotonia congenita with an SCN4A mutation

affecting cardiac repolarization

Yann Péréon, MD, PhD, 1,2 Gilles Lande, MD,2

Sophie Demolombe, PhD,2 Sylvie Nguyen The Tich, MD,1

Damien Sternberg, MD,3 Hervé Le Marec, MD, PhD,2 Albert David, MD4

Supplementary material (for Web site only)

SSCP detection and direct sequencing of the R1448C heterozygous mutation in exon 24 of SCN4A

Blood samples were obtained after informed consent of the proband and her eldest child according to the European Union and French bioethics laws and declaration of Helsinki. DNA was extracted from blood samples by a rapid procedure using Qiagen DNA minikit ®. Mutations were searched for in SCN4A coding regions by PCR-SSCP 1. For the part of exon 24 encompassing codon 1448, primers were 5'-CGTCCTCACTAGCTTCTCCC-3' (F) and 5'-CGACTCCTTCTTGACGTAGG-3' (R) (270 bp). PCR were performed in 25 µl of standard PCR buffer containing 2 mM MgCl2, 0.2 mM of each dNTP, 240 µM of each primer, 0.025 U/µl of Taq polymerase Gold® (Perkin-Elmer) and 80 ng of DNA. Steps of amplification were 94°C 15', (94°C 30", 61°C 30", 72°C 1') x 35 cycles, 72°C 3'. PCR products were denatured in two different conditions. In the first one, 3 µl of each PCR were mixed with with 20 µl of saccharose 10%, bromophenol blue 0.05%, xylene cyanol 0.05% (S medium). In the second one, 4 µl of each PCR were mixed with 8 µl of formamide 100%, EDTA 10 mM, bromophenol blue 0.05% and xylene cyanol 0.05% (FE medium). Both kinds of mixes were denatured 5’at 98°C, kept on ice, and 5 µl of each were electrophoresed at two different temperatures (7°C and 25°C) on 10 % polyacrylamide gels (acrylamide/bisacrylamide 37.5/1). Gels were then fixed, silver-stained and dried. Direct sequencing was used to sequence variants responsible for each abnormal SSCP profile using a fluorescent procedure and a DNA sequencer according to the manufacturer protocol (ABI 377, Perkin-Elmer).

RNA isolation

Paravertebral muscle, heart muscle (left and right ventricles, auricles) and aorta samples were quick-frozen in liquid nitrogen and total RNA was isolated separately from each sample. using the classical guanidinium isothiocyanate method and treated with DNase I (Sigma). RNA samples were quantified by spectrophotometric analysis. Absence of genomic DNA contamination was checked by PCR.

SCN4A gene expression

Expression of the SCN4A skeletal and SCN5A and SCN6A cardiac sodium muscle channel a-subunits was investigated using RT-PCR. The primers were 5’-TGTGAACGCATGTGGTAAGG-3’ (foward) and 5’-AGGCACCAAGGAGGGTTTAT-3’ (backward), to yield a 693-bp PCR product in the 3’UTR region of the SCN4A a-subunit; 5’-CCCACTTGACCTGAGATGCT-3’ (foward) and 5’-CCATGGTTCATTCCCCTCTA-3’ (backward), to yield a 713-bp PCR product in the 3’UTR region of the SCN5A a-subunit 5’-TTGAGGCGAAATGACAAAAA-3’ (foward) and 5’-GAATTTGGCATGAAGTCTTGTG-3’ (backward), to yield a 278-bp PCR product in the 3’UTR region of the SCN6A a-subunit. One microgram amounts of DNA free-total RNA from skeletal muscle, heart and aorta were used and RT-PCR was performed with the One-Step RT-PCR kit (Invitrogen) according to the directions of the manufacturer.

Amplifications were performed by an PTC-200 thermocycler (MJ Research). The RT pre-cycle consisted of 30 min at 50°C. The amplifications started with an initial step of 2 min at 94°C, and then 30 cycles each consisting of 15 sec at 94°C, 1 min at 60°C and 1 min at 72°C. PCR products were analyzed by electrophoresis of 10 µl of each sample on a 2% agarose gel and ethidium bromide staining.

The same cardiac total RNA samples used for the non quantitative RT-PCR analysis were employed for the relative quantitative RT-PCR. Two µg of total RNA were reverse transcribed into cDNA by the M-MLV transcriptase (Life Technologies). A dilution range of the produced cDNAs from the control group was added to 50 µl quantitative PCR reactions containing 25 µl of 2x SYBR Green Master Mix (Applied Biosystems) and 300 nmol gene-specific primers or GAPDH primers used as the reference house-keeping gene. GAPDH was chosen because the mRNA level of this gene was not significantly different between the skeletal and cardiac muscles. Amplifications were performed in duplicate with an ABI Prism 7700 Sequence Detection System Perkin-Elmer machine (Applied Biosystems). The PCR started with an initial step of 2 min at 50°C, followed by 10 min at 95°C and then 40 cycles each consisting of 15 sec at 95°C and 1 min at 60°C. The obtained standard curve was necessary to check that PCR efficiency was identical between the genes of interest and GAPDH, our internal standard. Therefore, for data analysis, the fluorescence signals were normalized to GAPDH mRNA (Rh), yielding a normalized value Rh. The threshold cycle (Ct) was set where Rh reached 10 times standard deviation of the baseline. Relative quantification, following the Ct-method2, was applied to compare the amounts of mRNA in skeletal muscle and heart.

References

1.Plassart E, Reboul J, Rime CS, et al. Mutations in the muscle sodium channel gene (SCN4A) in 13 French families with hyperkalemic periodic paralysis and paramyotonia congenita: phenotype to genotype correlations and demonstration of the predominance of two mutations. Eur J Hum Genet 1994;2:110-124.

2.Fink L, Seeger W, Ermert L, et al. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med 1998;4:1329-1333.

Legends

Figure (E)F-1

Pedigree of a family with paramyotonia congenital. Symbol shading indicates the phenotype of each individual ( and , unaffected;  and , affected;  and , affected by history but not examined). The proband is indicated by an arrow.

Figure (E)F-2

R1448C sodium channel (SCN4A) mutation :SSCP detection and direct sequencing of the R1448C heterozygous mutation in exon 24 of SCN4A. A. For this SSCP experiment, a mix of PCR and FE medium was heat at 94°C, ice-cooled, and five µl of this mix was electrophoresed on a 10 % polyacrylamide gel at 7°C. Gels were fixed and silver-stained. 1 : normal profile ; 2 : heterozygous R1448C profile ; 3 : normal profile. B. PCR products was amplified from DNA of the subject presenting with an abnormal SSCP profile, and labeled by using forward primer and Big Dye® (Perkin-Elmer) labeling mix. Electrophoregram shows a double C/T peak at nt 4342 of SCN4A. One allele carries a CGT codon (arginine) and the second allele carries a TGT codon (cysteine).