RR-MADD in mainland Chinese patients

Supporting information

Riboflavin responsive multiple Acyl-CoA dehydrogenation deficiency in 13 cases, and literature review in mainland Chinese patients

Min Zhu 1, Xuan Zhu 1, Xueliang Qi 2, Weijiang Ding 2, Yajing Yu 1, Hui Wan 1, Daojun Hong 1

1: Department of Neurology, The first affiliated hospital of Nanchang University, China

2: Department of Neurology, The second affiliated hospital of Nanchang University, China

Corresponding author:

Daojun Hong MD, PhD

Department of Neurology

The first affiliated hospital of NanchangUniversity

Yong Wai Zheng Street 17#, Nanchang, 330006, P.R.China

Telephone: 86-791-8869-2511

Fax number: 86-791-8869-2511

E-mail:

Figure S1. Myopathological characteristics in patient 11. HE showed many small round or irregular vacuoles in fibers (A), the vacuoles were filled with lipid droplets on oil red O stain (B), the vacuole fibers mainly involved in type I fibers on ATPase PH 4.3 stain (C).

Figure S2. the sequence of 5 novel ETFDH mutation. c.295C>T mutation (A), control (B), c.303T>A mutation (C), control (D), c.518T>G mutation (E), control (F), c.1586A>G mutation (G), control (H), c.1810G>T mutation (I), control (L).

Figure S3. Result of multiplex PCR to detect exon deletion/duplication of the ETFDH gene. A: The multiplex PCR products were divided into two groups: group 1 including exon 3, 6, 7, 8, 11/12, 13, and exon1 of GAPDH as reference gene; group 2 including exon 1, 2, 4/5, 9, 10, and exon 7 of GAPDH as reference gene. B: Exon deletion/duplication of the ETFDH gene was not found in patients with single heterozygous ETFDH mutation.

Figure S4.In RT-PCR assays,cDNAfragments of the expected size (1974bp) were observedboth in controls and patients.No additional bands were detected in theaffected individuals. M: marker, N: normal controls.

Figure S5.The sequence of cDNA of ETFDH in 3 patients with single heterozygous mutations. In case 7, c.3G>C mutation is a heterozygous mutation (A), control (B); in case 10, c.1399G>A is a homozygous mutation (C), control (D); in case 2, c. 1773-1774delAT is a homozygous mutation (E), control (F).

Table S1: Primers and annealing temperature for amplification of exons in the ETFDH, ETFA, and ETFB gene.

PCR target / forward primer 5’-3’ / reverse primer 5’-3’ / annealing temperature
ETFDH exon1 / tgcagcagagttcttgcttt / gtcccttttgccctaactcc / 57°C
ETFDH exon2 / gtgttcacctaggaaagatt / tgacttggtaaagccacaaaa / 57°C
ETFDH exon3 / tgtgcaaaacacagggagaa / aaaaagatagagttacatgaaaagg / 57°C
ETFDH exon4 / atgccaaaagaaaattaggg / tgtctgtaaaaggaggaaat / 57°C
ETFDH exon5 / aaagtgtgaccatcaatgtagca / agatactttaaagattccttctaatc / 57°C
ETFDH exon6 / cctgaacattttaaactctgtatttc / ataccacgaagcccagctaa / 57°C
ETFDH exon7 / ttgcactgtctcttctacatctga / tgaacatttcaagacaatgaaaaa / 57°C
ETFDH exon8 / tcacagtttttcacttgtaattttcat / cagtcttcagattcaaaaccctta / 57°C
ETFDH exon9 / tgagttccagagcacaagga / ccaggcagactacccaactc / 59°C
ETFDH exon10 / tcctttgctcaattcattttca / tgacagatgcaatacaaatc / 57°C
ETFDH exon11 / gagcaagaccttgtctcgaa / ttgaaaacattaaaacaatcactttc / 57°C
ETFDH exon12 / gggctagtcatatttctttggtg / ggaagctggatgaagtcattt / 57°C
ETFDH exon13 / agcaatgcaccaaggtcttc / tctgttcagtaatatgcagaaagaca / 57°C
ETFA exon1 / cctagggcgtcttatctcgttc / gaatctgagggggccagtgat / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon2 / aaaaaaaaaccctgtgacaacat / gattagagcccagtttgggttac / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon3 / taactggcccttttgctaataag / gagttgccaaaatcaatggtgtc / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon4 / aagttctggtggctcagcatgat / ttcagataaatccagattggctac / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon5 / tatttaaaaagctgtgcttgaagtt / acttaaagcaattgtatggtttgat / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon6 / tttcttcaccgttatggttcattt / attagaaatgtaggcagaggtcatc / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon7 / aatgtggcaaaggctggaatct / aatatcccaaacttcccaacttct / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon8 / tcagaggccagaaaaggtttat / aaaaactgaaaaatccggaaaagt / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon9 / ttaagcccaaattaaaatctgaaat / tattcaccatggtactggctaact / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon10 / tttaagcaatacattttgcctctatt / tcagttcccagatctacgttagtg / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon11 / ttgacacagatgcctcaaatcttat / catttctgagagtttctcggatgac / 56.0°C,-0.5°C/cycle, to 52°C
ETFA exon12 / gaagagagactaccggagaacag / ataaacaagcattctggcatctg / 56.0°C,-0.5°C/cycle, to 52°C
ETFB exon1 / ccttccttccggcctctcgattg / gatttttgccctcaggtgacttt / 65.0°C,-0.5°C/cycle, to 59°C
ETFB exon2 / agttctgtccctggagctgtgt / ccctccccaagaccttcat / 65.0°C,-0.5°C/cycle, to 59°C
ETFB exon3 / tcctaggtccccctgaggatag / ctcagcctggagtggtgaatg / 65.0°C,-0.5°C/cycle, to 59°C
ETFB exon4 / gaggttgcagtgagccaagatt / cagtgttccaggaggagagaac / 65.0°C,-0.5°C/cycle, to 59°C
ETFB exon5 / attgttcgctccctcattcat / ctgtgcacgagacctcctttg / 65.0°C,-0.5°C/cycle, to 59°C
ETFB exon6 / aggtgtggccctgtgagaagt / catcaatacagaggctcctcat / 65.0°C,-0.5°C/cycle, to 59°C

Table S2: Primers and products size formultiplex PCRof the ETFDHgene.

PCR target / forward primer 5’-3’ / reverse primer 5’-3’ / fragment size
exon1 / tggctgtttcaccgctccc / atccccaccgcccgtttc / 355bp
exon2 / gccaaaagctattcaaagtgttc / cgtgggtagtaattcgaggc / 249bp
exon3 / ggaactctaaagcacagtggata / cggtttaaaagcacctggatc / 298bp
exon4/5 / ggtgagacccagatatttgaagg / gcactaaatgtcccaagcgta / 387bp
exon6 / ggatgatggtagtgtaaaaggaat / gagatggggtttcactttgatg / 150bp
exon7 / ggatttgagctatacagtgacttg / ggattacagaaacaaaccaggata / 403bp
exon8 / gccccttggacagacatacc / ggcacagcagccataagcac / 234bp
exon9 / attactgcacatagtgctccaa / aagagttacctgaaagccacc / 292bp
exon10 / cgaatctttgtttcctcagtctat / gctttgattggagattttcactag / 182bp
exon11/12 / ggagccgtggactctgaaacata / gggttttggatactcaataggtg / 345bp
exon13 / ggtgatggatttcggttacagataa / gaactgaagaggtaggaagatgctg / 360bp
GAPDH exon1 / ccatgttgcaaccgggaag / ttaccagagaataatctagga / 178bp
GAPDH exon7 / tgacctgccgtctagaaaaacctg / aggaaatgagcttgacaaagtggt / 204bp

Table S3: Summary of the ETFDH mutations in previously reported patients with RR-MADD.

case number[ref] / ETFDH gene mutations feature / mutation remarks
mutation / protein / domain / mutation / protein / domain
18[2] / c.242T>G / p.L81P / FAD / c.993T>G / p.N331K / UQ / c.1227A>C in 7 HeZ
c.770A>G in 1 HoZand 4 HeZ
c.389A>T in 1 HeZ
c.380T>G / p.L127R / FAD / c.1084G>A / p.G362R / UQ
c.389A>T / p.D130V / FAD / c.1211T>C / p.M404T / FAD
c.393G>C / p.W131C / FAD / c.1227A>C / p.L409F / FAD
c.770A>G / p.Y257C / FAD / c.1395T>G / p.Y465X / UQ
c.715G>A / p.A239T / FAD / c.1399G>C / p.G467R / UQ
c.872T>G / p.V291G / UQ / c.1436G>C / p.R479T / UQ
c.973del312 / 325del48 / FAD / c.1531G>A / p.D511N / UQ
IVS3+1G>A / splice site / - / IVS4-9T>C / splice site / -
IVS12-3C>G / splice site / -
21[3] / c.-75A>G / ? / c.835T>C / p.W279R / UQ / c.250G>A in 4 HeZ
c.210_212del / p.C71del / FAD / c.872T>G / p.V291G / UQ / c.770A>G in 5 HeZ
c.236C>G / p.A79G / FAD / c.1027T>C / p.W343R / UQ / c.1227A>C in 4 HeZ
c.242T>C / p.L81P / FAD / c.1227A>C / p.L409F / FAD / c.389A>T in 6 HeZ
c.250G>A / p.A84T / FAD / c.1281_1282del / p.433* / FAD
c.251CT / p.A84V / FAD / c.1295T>A / p.V432G / FAD
c.256C>T / p.R86C / FAD / c.1372_1375del / p.467* / UQ
c.360_361insT / p.125* / FAD / c.1395T>G / p.Y465X / UQ
c.389A>T / p.D130V / FAD / c.1528C>T / p.P510S / UQ
c.503A>G / p.N168S / FAD / c.1531G>A / p.D511N / UQ
c.524G>A / p.R175L / FAD / IVS5+7A>G / splice site
c.528G>C / p.L176F / FAD / VS2+2T>C / splice site
c.770A>G / p.Y257C / FAD
53[7] / c.250G>A / p.A84T / FAD / c.998A>G / p.Y333C / UQ / c.250G>A in 39 HoZ and 11 HeZ
c.770A>G in 4 HeZ
c.524G>A / p.R175L / FAD / c.1395T>G / p.Y465X / UQ
c.643G>A / p.A215T / FAD / c.821G >A / p.G274E / UQ
c.770A>G / p.Y257C / FAD / c.1254_1257del / p.L418TfsX10 / UQ
23[12] / c.3G>C / p.M1I / MTP / c.998A>G / p.Y333C / UQ / c.770A>G in 1HoZ and 8 HeZ
c.1227A>C in 6 HeZ
c.389A>T in 3 HeZ
c.250G>A in 1 HeZ
c.250G>A / p.A84T / FAD / c.1227A>C / p.L409F / FAD
c.251CT / p.A84V / FAD / c.1450T>C / p.W484R / UQ
c.389A>T / p.D130V / FAD / c.1675 C>T / p.R559X / 4Fe4S
c.643G>A / p.A215T / FAD / c.1703T>C / p.F568S / 4Fe4S
c.770A>G / p.Y257C / FAD / c.1717 C>T / p.Q573X / 4Fe4S
c.993T>G / p.N331K / UQ / c.1763A>T / p.H588L / 4Fe4S
IVS6+1G>A / splice site / - / IVS7+2T>C / splice site / -
2[13] / c.65A>G / p.K22R / MTP / c.242T>C / p.L81P / FAD
1[14] / c.1522C>A / p.P508T / 4Fe4S / c.1583-1584insA / p.N528TfsX3 / 4Fe4S
30[15] / c.3G>C / p.M1I / MTP / c.949C>A / p.P317T / UQ / c.250G>A in 5 HoZ and 4 HeZ
c.770A>G in 6 HeZ
c.1227A>C in 1 HoZ and 4 HeZ
c.389A>T in 3 HeZ
c.152G>A / p.R51Q / FAD / c.998A>G / p.Y333C / UQ
c.172G>T / p.E58X / PTC / c.1084G>A / p.G362R / UQ
c.191G>A / p.R64K / FAD / c.1211T>C / p.M404T / FAD
c.250G>A / p.A84T / FAD / c.1227A>C / p.L409F / FAD
c.349G>C / p.A117P / FAD / c.1282-1283del / p.I428X / FAD
c.389A>T / p.D130V / FAD / c.1395T>G / p.Y465X / UQ
c.409C>T / p.P137S / FAD / c.1448C>G / p.P483 L / UQ
c.433G>C / p.D145H / UQ / c.1454C>G / p.T485S / UQ
c.524G>A / p.R175 L / FAD / c.1774A>T / p.N582Y / 4Fe4S
c.770A>G / p.Y257C / FAD / c.1763A>G / p.H588L / 4Fe4S
c.872T>G / p.V291G / UQ / c.1773-1774del / p.T591TfsX2 / 4Fe4S
IVS3+1G>T / splice site / - / IVS12-3C>G / splice site / -

FAD: flavin adenine dinucleotide, UQ: ubiquinone, MTP: mitochondrial targeting peptide, HeZ: heterozygote, Hoz: homozygote.

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