MFN2 Transcripts Escaping from Nonsense-Mediated Mrna Decay Pathway Cause Charcot-Marie-Tooth Disease Type 2A2

jnnp-2015-312646.R1

SUPPLEMENTARY DATA

MFN2 transcripts escaping from nonsense-mediated mRNA decay pathway cause Charcot-Marie-Tooth disease type 2A2

Authors: Toshitaka Kawarai, Kanto Yamasaki, Atsuko Mori, Naoko Takamatsu , Yusuke

Osaki, Chimeglkham Banzrai, Ryosuke Miyamoto, Ryosuke Oki, Lucia Pedace, Antonio

Orlacchio, Hiroyuki Nodera, Akihiro Hashiguchi, Yujiro Higuchi, Hiroshi Takashima,

Yoshihiko Nishida, Yuishin Izumi and Ryuji Kaji

#Correspondence to: Dr. Toshitaka Kawarai Department of Clinical Neuroscience, Institute of Biomedical Sciences, Tokushima University Graduate School. Postal number 770-0042 Phone + 81-88-633-7207 Fax + 81-88-633-7208 E-mail: [email protected]

jnnp-2015-312646.R1

Supplementary materials caption:

Supplementary Table S1. Summary of clinical manifestations of affected members

Supplementary Table S2. Electrophysiological studies in the two patients (II-5 and

III-3) with CMT2A2

Supplementary Figure S1. Ultrasonographic images of nerves in the proband (II-5)

Supplementary Figure S2. Foot of the affected members.

Supplementary Figure S3. Measurement of MFN2, OPA1 and MFN1 transcripts

Supplementary Figure S4. Nucleotide variations in MFN2 found in asymptomatic carriers

Supplementary Figure S5. Truncation mutations and age-at-onset reported in

MFN2-CMT2A2

jnnp-2015-312646.R1

Methods

Clinical Investigation

Electrophysiologic and ultrasonographic analyses of peripheral nerve were carried out as previously described.S1,S2 We also reviewed the published reports of

MFN2-CMT2A2 and compared our clinico-genetic data with the reports from the literature review.

Molecular analyses

Extraction of genomic DNA and RNA from blood, and synthesis of complementary DNA

Genomic DNA extraction was done using the RBC Genomic DNA Extraction

Kit Maxi (SciTrove, Inc. Tokyo, Japan) following the manufacturer's instructions.

Lymphocytes were isolated from whole blood using Lymphoprep (Cosmobio, Inc.

Tokyo, Japan). Total RNA was extracted from lymphocytes using RNAzol RTR

(Cosmobio, Inc. Tokyo, Japan). Complementary DNA (cDNA) was prepared from 5 mg of total RNA, with random hexamer primers and the PrimeScript IIR 1st strand cDNA synthesis Kit (TaKaRa Bio, Inc. Kyoto, Japan).

Targeted resequencing of CMT genes, Sanger sequencing and PCR-RFLP analysis

The purpose-built GeneChip CustomSeq Resequencing Array (Affymetrix, Inc.,

Santa Clara, CA) was designed to screen for CMT and related diseases, in which 64

jnnp-2015-312646.R1

genes were analyzed. Genomic DNA extracted from the proband (II-5) was amplified by multiplex PCR. Amplified DNA fragments were labelled and subjected to chip hybridization. The analysis of microarray data was performed using GeneChip

Sequence Analysis Software, version 4.0 (Affymetrix, Inc.). In addition, Optic atrophy 1

(OPA1) and Mitofusin 1 (MFN1) genes were also sequenced as previously reportedS3 or using a pair of specific PCR primers (primer sequence available upon request).

The variants identified in the Resequencing Array were validated by direct sequencing, which was also applied for surveillance of variant in 400 control chromosomes. The primer pair used for sequencing MFN2 genomic DNA (gDNA) was:

MFN2-exon19F, 5'-GCCTCCTTGTCCCCATCTGTG-3' and MFN2-exon19R,

5'-GTGCTGGAGGGCTCAAAATGG-3'. The primer pair used for complementary

DNA (cDNA) was: MFN2-cDNA-2421F, 5'-CAAGGAGAGGGCCTTCAAGCG-3' and

MFN2-cDNA-2763R, 5'-GCACTTAGGGCTGGCAGCACC-3' that span exons 18-19 of MFN2 cDNA. Sequence read was aligned to the genome reference sequences using

Sequencher, version 5.2 (Gene Codes, Ann Arbor, MI USA). Nucleotide variations were identified comparing the reported reference sequence (MFN2; NG_007945 for gDNA, and NM_014874.3 for cDNA, OPA1; AC048351 for gDNA and NM_015560.2 for cDNA, MFN1; NC_018914 for gDNA, XM_005247596.2 for cDNA) and further

jnnp-2015-312646.R1

analyzed in control individuals. The mutation c.2251C>T in MFN2 was also confirmed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis with the restriction enzyme SpeI. This assay was also used for surveillance of the mutation in genomic DNA samples from 109 Japanese (female 56.0%, 61.8 ± 7.8 years (range 44 - 79)) and 200 Italian (female 52.5%, 46.2 ± 8.7 years (range 30 - 62)) control subjects.

Measurement of MFN2, OPA1 and MFN1 transcripts and Nonsense pre-mRNA mediated decay analysis

The amount of MFN2, OPA1 and MFN1 transcripts in lymphocytes was evaluated with semi-quantitative reverse-transcription (RT)-PCR. MFN2 transcript levels in response to nonsense-mediated RNA decay (NMD) inhibitor were also evaluated using the same technique. T cells in whole blood withdrawn from the patient

(II-1) were cultured using TLY CULTURER kit (Cosmobio, Inc. Tokyo, Japan).

Exponentially growing (60% confluence) cells were treated with 100 μg/ml of emetine dihydrochloride hydrate (Sigma-Aldrich, St. Louis, USA) at 37°C for 6 h before preparation of total RNA in order to evaluate the effects of NMD. RNAiso PlusR reagent (TaKaRa Bio, Inc. Kyoto, Japan) was used for the extraction of total RNA. All

RNA samples were treated with RNase-free DNase I (Takara Bio, Inc. Kyoto, Japan).

jnnp-2015-312646.R1

cDNA was prepared from 1 ug of total RNA, with random hexamer primers and

PrimeScript IIR 1st strand cDNA synthesis Kit (TaKaRa Bio, Inc. Kyoto, Japan). The synthesized cDNA was diluted; 1/500th of the first-strand cDNA, equivalent to the cDNA reverse-transcribed from 2 ng of total starting RNA, was used for real-time quantitative PCR. SYBR quantitative real-time PCR analysis was performed using specific oligonucleotide primers MFN2-cDNA-2593F

(5'-ATTGCCGCCATGAACAAGAAAATT-3') and MFN2-cDNA-2763R

(5'-GCACTTAGGGCTGGCAGCACC-3') that span exons 18-19 of MFN2 cDNA,

OPA1-cDNA-F (5'-ATCTGTGGATGCTGAACGCA-3') and OPA1-cDNA-R

(5'-GAATCCTGCTTGGACTGGCT-3') that span exons 14-15 of OPA1 cDNA, and

MFN1-cDNA-F (5'-TGTTTTGGTCGCAAACTCTG-3') and MFN1-cDNA-R

(5'-CTGTCTGCGTACGTCTTCCA-3') that span exons 6-8 of MFN1 cDNA. One housekeeping gene, beta-2-microglobulin (B2M) was selected as internal control gene to normalize the PCR.S4 Relative expression levels were analyzed with the 2−ΔΔCP method.S5 Real-time quantitative reverse transcription-PCR was performed in quadruplicate and analyzed using real-time quantitative reverse transcription-PCR

(StepOnePlus; Life Technologies) with StepOneSoftware, version 2.3 (Life

Technologies). The Mann-Whitney test was used to compare the 2 groups: patient’s

jnnp-2015-312646.R1

cultured T cells treated with emetine and untreated cultured T cells.

Measurment of mutant MFN2 transcripts escaping NMD

The presence or absence of mutation in RT-PCR products was determined by the signal ratio of wild type to mutant using a MassARRAY system with technology based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (Agena Bioscience, San Diego, CA). The DNA fragments containing the mutation, c.2251C>T mutation was amplified by a pair of oligonucleotide primers,

MFN2_Q751X_W1_F, 5’-ACGTTGGATGTCAGGTGCCCACTATCTGC-3’and

MFN2_Q751X_W1_R, 5’-ACGTTGGATGATAAAGCCGGTTGGTTGGAC-3’. Ratios of mutant to wild-type DNA were determined with allele-specific extended primer,

MFN2_Q751X_W1_E, 5’-GCTGGGCTGCAGGTACT-3’. Data were analyzed with the

MassARRAY Typer software module (Agena Bioscience, San Diego, CA). Furthermore,

Percentage of wild MFN2 peaks that were higher than the corresponding mutant peak in the sequencing chromatogram was calculated in independent RT-PCR amplicons using

Minor Variant Finder Software (Thermo Fisher Scientific Inc. South San Francisco,

CA.).

Bioinformatic analysis

The 3 dimensional protein structures of wild and mutant MFN2 were predicted

jnnp-2015-312646.R1

by SWISS-MODEL (http://swissmodel.expasy.org).S6 The effect on the coiled-coil domain lacking the last 7 amino acids of the C-terminus of the MFN2 protein was investigated using web-based prediction program COILS under default settings

(http://www.ch.embnet.org/software/COILS_form.html).S7

Results

Clinical investigation

Clinical features of the nine affected members were summarized in supplementary table

S1. No symptom related to cerebral involvement was observed, and no abnormal finding, including white matter change, was detected in brain MRI. Electrophysiological analyses demonstrated axonal degeneration, leading to the diagnosis of CMT2

(supplementary table S2). Ultrasonographic analysis in the proband (II-5 in Fig. 1A)) demonstrated cervical nerve root atrophy at C5 (supplementary figure S2). Literature review demonstrated that a total of 12 mutations have been reported in asymptomatic carriers (supplementary figure S3)

Molecular analyses

The PCR-RFLP analysis demonstrated that the mutation co-segregates with the disease in the family. This mutation was not found in 122 Japanese and 200 Italian control chromosomes (data available upon request).

jnnp-2015-312646.R1

No mutation was found in ganglioside-induced differentiation-associated protein 1

(GDAP1) gene by GeneChip CustomSeq Resequencing Array system. PCR-direct sequencing showed no mutation in OPA1 and MFN1 in the two affected members, II-1 and III-3. Measurement of MFN2, OPA1 and MFN1 transcripts in lymphocytes demonstrated no significant difference between affected and unaffected family members and their expression levels were not influenced by inhibition of nonsense-mediated mRNA decay in emetine-treated T cells (supplementary figure S5). No significant difference of peaks of wild and mutant MFN2 allele was observed in the MassARRAY system and sequencing chromatogram. Peaks of mutant MFN2 allele ranged from 110% to 90% of wild allele.

Bioinformatic analysis

Comparison of the predicted structure between wild and mutant MFN2 proteins showed no drastic conformational change. However, each value for scoring function in

SWISS-MODEL was changed by the mutation (data available upon request). No structural alteration or disruption of the coiled-coil domain at C-terminus in the truncated MFN2 protein was shown with the prediction program COILS (data available upon request).

jnnp-2015-312646.R1

Discussion

It has been reported that Mfn1 complements with Mfn2 CMT2A alleles through the formation of heterooligomeric complexes.S8 The results of MFN1, OPA1 and MFN2 expression were obtained using the family members’ lymphocytes or cultured T cells.

Functional complementation amongst MFN2, OPA1 and MFN1 needs to evaluate it under the same conditions as previously reported.S8

jnnp-2015-312646.R1

Supplementary Table S1. Summary of clinical manifestations of affected members ID II-1 II-3 II-4 II-5 III-2 III-3 III-5 III-6 IV-1 Sex F F F F F F F M M Age at examination 74 71 68 66 49 46 44 41 27 Age at onset a 5 6 6 5 6 6 6 6 6 Disease duration 69 65 62 61 43 40 38 35 21 Hand deformities none none none none none none none none none Foot deformities none none none none none none none none none Lower limb muscle severe mild mild mild severe severe severe severe mild atrophy FDS b 7 7 7 7 4 3 4 4 3 Neuropathy Impairment Score (NIS) c Cranial nerves 0 0 0 0 0 0 0 0 0

Muscle 28 28 26 26 16 16 12 12 10 weakness Reflexes 16 16 16 16 12 12 12 12 10 Sensation 18 18 16 16 10 10 8 10 8 Total NIS score 62 62 58 58 38 38 32 34 28

Charcot-Marie-Tooth disease neuropathy score version 2 (CMTNS2) d Sensory symptoms 2 2 2 2 2 2 2 2 1 Motor symptoms Legs 4 4 4 4 3 2 2 2 2 Arms 1 1 1 1 0 0 0 0 0 Pinprick sensibility 2 2 2 2 2 2 2 2 2 Vibration 2 2 2 2 2 2 2 2 2 Strength Legs 3 3 3 3 2 2 2 2 2 Arms 1 1 1 1 0 0 0 0 0 Ulnar/median 4 n.d. 4 4 n.d. 3 n.d. n.d. n.d CMAP

jnnp-2015-312646.R1

Radial SNAP 3 n.d. 3 3 n.d. 3 n.d. n.d. n.d.

Total CMTNS2 score 22 >15 22 22 >11 16 >10 >10 10

Brain MRI e Diffuse n.p. n.p. n.p. n.p. No n.p. n.p. n.p. brain white atrophy, matter no change white matter change

Foot pictures of the family members are shown in supplementary figure S1.

M = male; F = female; a Age at onset was calculated approximately as the time when difficulty walking or gait instability first appeared in the affected individuals; b FDS (Functional Disability Scale) from 0 to 8 as follows: 0 = normal; 1 = normal, but with cramps and fatigability; 2 = inability to run; 3 = walking difficult, but still possible unaided; 4 = able to walk with a cane; 5 = able to walk with crutches; 6 = able to walk with a walker; 7 = wheelchair bound; 8 = bedridden. c Neuropathy Impairment Score (NIS).S9 d CMTNS2 = CMT neuropathy score version 2 (Maximum: 32).S10 n.d., Ulnar CMAP and radial SNAP amplitude not determined. e Brain MRI. n.p., not performed.

jnnp-2015-312646.R1

Supplementary Table S2. Electrophysiological studies in the two patients (II-5 and III-3) with CMT2A2

Electrophysiological studies in the patient III-3 with MFN2-CMT2A2

NCS DL (ms) MCV (m/s) CMAP (mV) SCV (m/s) SNAP amplitude (mV) Segment, normal value 2.3 - 4.6 Elbow to wrist, 51-65 4.5 - 19.0 Wrist to the 2nd digit, 53.2-71.2 28.7-86.3 Right median 3.6 50 0.18 - not evoked

Segment, normal value 3.1 - 3.2 Elbow to wrist, 50-69 9.0 - 22.0 Wrist to the 4th digit, 59.7-74.9 11.4-89.4 Right ulnar 3.8 50 0.76 - not evoked

Knee to ankle, 41 - Segment, normal value 4.2 - 6.5 5.0 - 21.5 55 Right tibial - - not evoked Left tibial - - not evoked

Fibular head to ankle, Segment, normal value 4.7 - 6.8 1.4 - 9.3 43.1 - 57.9 Right peroneal - - not evoked

Segment, normal value 14 cm, 43.5-61.1 > 7.71 Right sural nerve - not evoked Left sural nerve - not evoked nEMG (right first dorsal interosseous muscle), Chronic denervational changes without positive sharp waves and fibrillation potentials.

Electrophysiological studies in the patient II-5 with MFN2-CMT2A2

NCS DL (ms) MCV (m/s) CMAP (mV) SCV (m/s) SNAP amplitude (mV) Segment, normal value 2.3 - 4.6 Elbow to wrist, 51-65 4.5 - 19.0 Wrist to the 2nd digit, 53.2-71.2 28.7-86.3 Left median 4.5 26.3 0.15 37.7 1.6

Segment, normal value 3.1 - 3.2 Elbow to wrist, 50-69 9.0 - 22.0 Wrist to the 4th digit, 59.7-74.9 11.4-89.4 Left ulnar 4.4 69.5 0.11 27.7 1.3

Segment, normal value 4.2 - 6.5 Knee to ankle, 41 - 55 5.0 - 21.5 Right tibial - - not evoked Left tibial - - not evoked

Fibular head to ankle, Segment, normal value 4.7 - 6.8 1.4 - 9.3 43.1 - 57.9 Right peroneal - - not evoked

Segment, normal value 14 cm, 43.5-61.1 > 7.71 Right sural nerve - not evoked Left sural nerve - not evoked

NCS, nerve conduction study; CMAP, compound motor action potential; SCV, sensory conduction velocity; DL, distal latency; SNAP, sensory nerve action potential; MCV, motor conduction velocity. Normal values are cited from previous publication.S11,S12

jnnp-2015-312646.R1

Supplementary Figure S1. Ultrasonographic images of nerves in the proband (II-5)

Cervical nerve root Diameter (mmm) Area (mm3) Right Left Standard Right Left Standard C5 1.7 1.9 2.41 ±0.4 C6 3.9 3.3 3.16 ± 0.4 13 8 7.6 ± 1.6 C7 3.6 3.6 3.77 ± 0.5 Area (mm3) Right Standard Median nerve Wrist 6 6.72 ± 1.2 Ulnar nerve Wrist 3 5.34 ± 1.3

(A) Transverse view of cervical nerve root (C5) (B) Axial ultrasonographic image of median nerve at the antecubital fossa. (C) Axial ultrasonographic image of ulnar nerve at the arterial split point. (D) Axial ultrasonographic image of median nerve at the carpal tunnel. Measured areas or diameters are drawn with blue line.

Atrophy of cervical nerve root at the level of C5 was demonstrated without any abnormal echo density. C6 and C7 cervical nerve roots echographically remained intact. Standard values are determined based on the in-house data obtained from 37 Japanese (female 65%, 55.3 ± 8.3 years (range 47-79)) control samples.S13

jnnp-2015-312646.R1

Supplementary Figure S2. Foot of affected members

Distal lower limb muscle atrophy is observed in the affected members, however, foot deformities, such as pes equinus or pes varus, are not evident.

jnnp-2015-312646.R1

Supplementary Figure S3. Measurement of MF2, OPA1 and MFN1 transcripts

No significant differences of expression levels of MFN2, OPA1 and MFN1 transcripts were demonstrated between the unaffected (II-2) and affected member (II-1). Moreover, the expression level in cultured T cells is not increased by treatment with nonsense-mediated decay (NMD) inhibitor, emetine (E).

jnnp-2015-312646.R1

Supplementary Figure S4. Nucleotide variations in MFN2 found in asymptomatic carriers

A total of 12 variations have been literally reported in MFN2-CMT2A2 asymptomatic carriers. Round mark () indicates missense mutation. Diamond mark () indicates nonsense mutation. Triangle mark () indicates in frame deletion mutation.

jnnp-2015-312646.R1

Supplementary Figure S5. Truncation mutations and age-at-onset reported in

MFN2-CMT2A2

Nucleotide numbering (number in parentheses) is based on the human longest transcript of MFN2 (NM_014874.3). Scheme of mRNA with putative regions for NMD-sensitive and -insensitive are also depicted. Star mark () indicates missense mutation. Diamond mark () indicates nonsense mutation. Exclamation mark (!) indicates autosomal dominant inheritance. Percent (%) indicates double mutation in cis. Hash mark (#) indicates inheritance mode not described. Ampersand (&) indicates de novo mutation. Dollars sign ($) indicates no family history.

jnnp-2015-312646.R1

REFERENCES S1. Nodera H, Takamatsu N, Shimatani Y, et al. Thinning of cervical nerve roots and peripheral nerves in ALS as measured by sonography. Clin Neurophysiol. 2014;125:1906-11. S2. Nodera H, Bostock H, Kuwabara S, et al. Nerve excitability properties in Charcot-Marie-Tooth disease type 1A. Brain. 2004;127:203-11. S3. Zhang L, Shi W, Song L, et al. A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family. Sci Rep. 2014;4:6936. S4. Lupberger J, Kreuzer KA, Baskaynak G, et al. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR. Mol Cell Probes. 2002;16:25-30. S5. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8. S6. Biasini M, Bienert S, Waterhouse A, et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014;42:W252-8. S7. Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Science. 1991;252:1162-4. S8. Detmer SA, Chan DC. Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations. J Cell Biol. 2007;176(4):405-14. S9. Dyck PJ, Davies JL, Litchy WJ, et al. Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort. Neurology. 1997;49:229-39. S10. Murphy SM, Herrmann DN, McDermott MP, et al. Reliability of the CMT neuropathy score (second version) in Charcot-Marie-Tooth disease. J Peripher Nerv Syst. 2011;16:191-8. S11. Kurokawa K, Tanaka E, Yamashita H, et al. Ratios of nerve conduction parameters in proximal to distal limbs remain constant through the second to the eighth decades. Electromyogr Clin Neurophysiol. 1998;38:169-76. S12. Kurokawa K, Tanaka E, Yamashita H, et al. Age-related changes in amplitude ratio, duration ratio and area ratio in nerve conduction studies. Nihon Ronen Igakkai Zasshi. 1995;32:547-52. S13. Nodera H, Takamatsu N, Shimatani Y, et al. Thinning of cervical nerve roots

jnnp-2015-312646.R1

and peripheral nerves in ALS as measured by sonography. Clin Neurophysiol. 2014;125:1906-11. S14. Polke JM, Laura M, Pareyson D, et al. Recessive axonal Charcot-Marie-Tooth disease due to compound heterozygous mitofusin 2 mutations. Neurology. 2011;77:168-73. S15. Nicholson GA, Magdelaine C, Zhu D, et al. Severe early-onset axonal neuropathy with homozygous and compound heterozygous MFN2 mutations. Neurology. 2008;70:1678-81. S16. Piscosquito G, Saveri P, Magri S, et al. Mutational mechanisms in MFN2-related neuropathy: compound heterozygosity for recessive and semidominant mutations. J Peripher Nerv Syst. 2015;20:380-6. S17. Carr AS, Polke JM, Wilson J, et al. MFN2 deletion of exons 7 and 8: founder mutation in the UK population. J Peripher Nerv Syst. 2015;20:67-71. S18. Sivera R, Sevilla T, Vilchez JJ, et al. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series. Neurology. 2013;81:1617-25. S19. Park SY, Kim SY, Hong YH, et al. A novel double mutation in cis in MFN2 causes Charcot-Marie-Tooth neuropathy type 2A. Neurogenetics. 2012;13:275-80. S20. Vital A, Latour P, Sole G, et al. A French family with Charcot-Marie-Tooth disease related to simultaneous heterozygous MFN2 and GDAP1 mutations. Neuromuscul Disord. 2012;22:735-41. S21. Verhoeven K, Claeys KG, Züchner S, et al. MFN2 mutation distribution and genotype/phenotype correlation in Charcot-Marie-Tooth type 2. Brain. 2006;129:2093-102. S22. Feely SM, Laura M, Siskind CE, et al. MFN2 mutations cause severe phenotypes in most patients with CMT2A. Neurology. 2011;76:1690-6. S23. Engelfried K, Vorgerd M, Hagedorn M, et al. Charcot-Marie-Tooth neuropathy type 2A: novel mutations in the mitofusin 2 gene (MFN2). BMC Med Genet. 2006;7:53.