ONLINE RESOURCES

Mutation screening.

The eleven coding exons of the MLC1 gene were PCR amplified in a total volume of 50 µl using 30 ng of genomic DNA, 144 µM dNTPs, 2 mM MgCl2, 1 U of Taq Gold (Applied Biosystems, Foster City, CA, USA) and 200 nM of forward and reverse primers reported in supplementary table S1. a final concentration of 1 M betaine (B0300, Sigma-Aldrich, St.Louis, MO, USA) was added. Thermal cycling conditions were: 7 min at 95°C, followed by 14 cycles of 30 sec at 95°C, 30 sec at annealing temperature (Ta)+7°C-0.5°C/cycle (see supplementary table 1) and 1 min at 72°C; then 25 cycles consisting of 30 sec at 95°C, 30 sec at annealing temperature (see supplementary table S1), and 1 min at 72°C. End step of 10 minutes at 72°C for final extension.

cDNA analysis.

MLC1 cDNA was divided in five partially overlapping fragments: PCR was performed in a total volume of 25 µl using 50 ng of cDNA, 144 µM dNTPs, 2 mM MgCl2, 1 U of KAPA2G Fast Polymerase (KAPA Biosystems) and 100 nM of forward and reverse primers reported in supplementary table S1. Thermal cycling conditions were: 1 min at 95°C, followed by 14 cycles of 10 sec at 95°C, 10 sec at annealing temperature (Ta)+7°C-0.5°C/cycle (see supplementary table 1) and 15 sec at 72°C; then 25 cycles consisting of 10 sec at 95°C, 10 sec at annealing temperature (see supplementary table S1), and 15 sec at 72°C. End step of 1 minute at 72°C for final extension.

Supp. Table S1. Primers, PCR conditions for MLC1 mutation screening
Exon (size)
/ Primers (Forward/Reverse) / PCR Ta (°C)
2
(301bp) / 2F: 5’- CCAGATAAAGAACTGGTGACACG
2R:5’- TCCGTCACCAGAGGGACC / 58
3
(244bp) / 3F: 5’-GAAGTTGAAGGGTCAGGGTCC
3R: 5’-ACCTGCACATCTCAGAACAAAG / 58
4
(635bp) / 4F: 5’-TCTGGAAGCGCAAATGTTAGAG
4R: 5’-ATTTGCTGACACCATTCGTGG / 58
5
(250bp) / 5F: 5’-CCTGAAGTGTGGTTTATCTGTTACCTT
5R: 5’-ATTTGCTGACACCATTCGTGG / 61
6
(261bp) / 6F: 5’-GTCCGGTGGACGCTGAAG
6R: 5’-CCGAGGGTGACGCTGAGAC / 58
7
(207bp) /

7F: 5’-CAGCGGCAGTGCTGAGTC

7R: 5’-CGTGACGTTTAATCCAGCCTC / 58
8
(274bp) /

8F: 5’-GCACAGTGCATTCTGGGAATC

8R: 5’-ACCAAGACTGAGCTCGGGG / 58
9
(214bp) /

9F: 5’-TGTTTGGAATTCGACTTCTTCG

9R: 5’-CTGGAGCCTGGTCACATGG / 58
10
(361bp) / 10F: 5’-CCAGCTTGGGACTATGCC
10R: 5’-CCTCCTGGAGCTGTCCCTG / 58
11
(779bp) /
11F: 5’-CAGTGGCTCGGTCACTTTTA
11R: 5’-CACCCCTGAAACCCACAG / 61
12
(229bp) / 12F: 5’-GAGTGGCGTGTCCACCTTC
12R: 5’-AGTAGCTCAGGGCGATTAGGG / 58
cDNA analysis
cDNA Fragment (size)
/ Name: Primer sequence (Forward/Reverse) / PCR Ta (°C)
A
(416 bp)
/
c2F: 5’-AGCTGTGGTGTCCCCAAGT
c5R: 5’-TGCATCCAAACCAAATTAAA
/ 57
B
(254 bp)
/
c4F: 5’-GCCAATGTGATTCCCAACTT
c7R: 5’-GGCACTTCGTCCAGAATGTT
/ 57
C
(408 bp)
/
c.5F: 5’-GTCCTGAACCCATCAGCAAT
c10R: 5’-AGACGTGAGGCTGCTTATGG
/ 57
D
(322 bp)
/

c10F: 5’-CCATAAGCAGCCTCACGTCT

c12R: 5’-GCTCTCCAGGCTTTCTCCTT

/ 57


Minigene assay.

A minigene assay was set up to test the effect of MLC1 c.895-226T>G sequence variant on splicing. We amplified a 469 bp fragment from subject II-8 carrying the variant G in homozygousity, and her healthy sibling II-4, wild-type T. The forward primer (5’-gtgctccgtggtcactctct) was located 84 bp upstream the acceptor splice site, and the reverse primer (5’-cgagtcggagccccagtaac) 99 bp downstream the donor splice site. The PCR products were cloned into a pGEM®-T Easy Vector (Promega) and transformed in DH5α bacterial cells following manufacturer’s recommendations (RBC Bioscience, Chung Ho City, Taiwan).

Plasmids containing the wild-type or the variant sequence were extracted using PureYield™ PlasmidMiniprepSystem(Promega) according to manufacturer protocol, checked by PCR and sequencing, and digested with EcoRI restriction endonucleases. Inserts were gel-purified and sub-cloned into a New Exon Trapping System pSPL3 vector (Life sciences-Invitrogen).

pSPL3 plasmids containing the wild-type (pSPL3_T) or the variant (pSPL3_G) were extracted using PureYield™ PlasmidMidiprepSystem(Promega) according to manufacturer protocol, checked by PCR for the correct orientation and transfected in Hela cells using the TurboFect kit (Fermentas), according to protocols. After 36h, RNA was extracted and retrotranscribed with the Cell-To-CT kit (Life sciences).

The cDNA was amplified with vector primers pSPL3_F (5’- tctgagtcacctggacaacc) and pSPL3_R (5’- atctcagtggtatttgtgagc).

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