Supplementary material J Neurol Neurosurg Psychiatry

Supplementary File

METHODS

Whole- (WES)

DNA was isolated from peripheral blood using a DNA Isolation Kit (Bioteke, AU1802). One microgram of genomic DNA was fragmented into 200-300 bp

pieces by the Covaris Acoustic System. The DNA fragments were then processed by end-repairing, A-tailing and adaptor ligation, followed by 4-cycle

pre-capture PCR amplification and targeted sequence capture. Captured DNA fragments were eluted and amplified by 15 cycles of post-capture PCR. The

final products were sequenced as 150-bp paired-end reads on an Illumina HiSeq X platform according to the standard manufacturer’s instructions. The raw

data were filtered and aligned to the human reference (hg19) using the BWA Aligner (http://bio-bwa.sourceforge.net/). The variants were called by

using GATK software (Genome Analysis Toolkit;

Filtering of causative mutations

Variants were filtered for the presence of nonsynonymous heterozygous variants with a minor allele frequency <1% in the Exome Aggregation Consortium

(ExAC) database (http://exac.broadinstitute.org/), the Exome Sequencing Project (ESP) (http://evs.gs.washington.edu), the 1000 Project (1000G)

database (http://www.1000genomes.org/) and the Genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org/). In silico predictive

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483 Supplementary material J Neurol Neurosurg Psychiatry

programs, including PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), SIFT-2 (http://sift.jcvi.org) and Mutation Taster (http://mutationtaster.org), were

used to identify potential functional effects of the mutations. The genomic evolutionary rate profiling (GERP) scores were acquired by the GERP++ program

(http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html).

Sanger sequencing

All suspected variants were validated using Sanger sequencing and interpreted according to the American College of Medical Genetics and

standards and guidelines.

Exons or intron of the KIF5A gene were amplified by polymerase chain reaction (PCR) using the primers listed in table 1. The PCRs were carried out in

a total volume of 30 µl containing 50 ng of genomic DNA, 0.2 mM of each primer and 1×Taq Plus Master Mix II (Vazyme Biotech Co., Nanjing, China).

The cycling protocol consisted of denaturation at 95℃ for 3 min, followed by 30 cycles at 95℃ for 20 s, 50–63 ℃ for 20 s, 72 ℃ for 0.5–1 min and a

final extension at 72 ℃ for 5 min. The amplified products were purified using the gel purification system (Omega Bio-Tek Inc., Norcross, GA, USA) and

sequenced using the ABI Prism BigDye Terminator Cycler Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 3730xl

DNA Analyzer.

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483 Supplementary material J Neurol Neurosurg Psychiatry

Supplementary Table 1. Primers used for human KIF5A gene PCR amplification

Fragment Annealing Region Forward primer (5’-3’) Reverse primer (5’-3’) size temperature

Exon1 AGCCCGAAAGGACCAGAC TCCCGAAGGTAGGAAAGCAG 586 bp 60℃

Exon 2 ACAATCTCACTGTGCGACAC CACCAAAGCCAGAACCCT 802 bp 62℃

Exon 7 CCAGCTACTCTGGAGGCTGA TGATGAGGAAGATGCTGTGG 616bp 59℃

Exon 8 CCTTCTCTCTGGGTGGGC CTTCCACCCACCCCATATCC 261bp 59℃

Exon10 CAAGACTGGAGCAGAGGG GGGAGTATGGGAGGTAGGT 902 bp 63℃

Exon15 AGAAGGTGGTAAGTGGTG TGTATCTTTTCAATTGGG 643 bp 50℃

Intron 17 AAAACTCCTAACACCCACC CAGAGCCTTCTGCAAACA 938 bp 62℃

Exon 27 GCCCTTGCCTTCCAGTTC TGCCCACGTTGTTTTCGT 507 bp 60℃

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483 Supplementary material J Neurol Neurosurg Psychiatry

Supplementary Table 2. KIF5A LOF and rare missense variants found in this study.

Pati Ex cDNA Amino Local Public In silico analysis GERP Age Gen Site survival Phenotype ent on Acid Control Exome f of der of time ID frequency Databa onset onset ia se PolyPhen-2 SIFT-2d Mutation Freque c Tastere ncy b C-1 1 c.35T>C p.V12A 0 0 Probably Damaging Disease 3.62 lower >13years 17 F damaging causing limbs (alive) CMT2 7 c.565 p.S189P 0 0 Probably Damaging Disease 4.48

T>C damaging causing C-2 8 c.610 p.R204 0 0 Probably Damaging Disease 3.60 18 F lower >11years CMT2 C>T W damaging causing limbs (alive) A-1 1 c.86A p.K29R 0 0 Benign Tolerated Disease 3.58 39 M upper 53 ALS >G causing limbs months A-2 2 c.136 p.P46S 0 0 Benign Tolerated Disease 4.59 40 M upper 79 ALS C>T causing limbs months A-3 10 c.890 p.R297Q 0 1/2462 Probably Damaging Disease 4.23 59 M upper 23 ALS G>A 14 damaging causing limbs months (2.031* 10-6) A-4 15 c.1624 p.H542N 0 0 Possibly Tolerated Disease 5.46 26 M lower (lost to ALS C>A damaging causing limbs follow up) A-5 - c.2024 IVS18-6 0 0 - - - - 60 M upper 52 ALS

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483 Supplementary material J Neurol Neurosurg Psychiatry

-6C>T C>T limbs months A-6 27 c.2999 p.T1001 0 0 - - - - 45 M upper 13 ALS delC Qfs limbs months H-1g 23 c.2437 p.A813T ／ 0 Benign Tolerated Disease 5.15 - F - - - G>A causing

H-2 g 6 c.470 p.H157R ／ 0 Possibly Deleterious disease_ 5.28 - F - - - A>G damaging causing

H-3 g 9 c.788 p.G263D ／ 0 Probably Deleterious disease 4.23 - M - - - G>A damaging causing

H-4 g 13 c.1338 p.K446N ／ 0 Benign Deleterious disease_ 4.82 - M - - - G>T causing

H-5 g 28 c.3041 p.Y1014 ／ 0 Benign Tolerated polymorp 4.79 - M - - - A>T F hism

a. Variant allele frequencies in the 1015 controls.

b. Variant allele frequencies in the following databases:

1. ExAC: Exome Aggregation Consortium database (http://exac.broadinstitute.org)

2. ESP: Exome Sequencing Project database (http://evs.gs.washington.edu)

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483 Supplementary material J Neurol Neurosurg Psychiatry

3. 1000G: 1000 Genomes Project database (http://www.1000genomes.org/)

4. gnomAD: Genome Aggregation database (http://gnomad.broadinstitute.org/)

c. Prediction of pathogenicity in PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)

d. Prediction of pathogenicity in SIFT-2 (http://sift.jcvi.org/)

e. Prediction of pathogenicity in Mutation Taster (http://mutationtaster.org)

f. Genomic evolutionary rate profiling scores (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html)

g. These five participants（H-1,H-2,H-3,H-4,H-5）were controls.

- none

M male F female

FIGURE LEGENDS

Supplementary Figure 1. Sequencing chromatograms of KIF5A mutations found in two CMT2 patients and their parents. Patient C-1 carried two mutations,

one inherited from her father and one from her mother.

Supplementary Figure 2. Sequencing chromatograms of KIF5A mutations found in sALS patients.

He J, et al. J Neurol Neurosurg Psychiatry 2019; 0:1–2. doi: 10.1136/jnnp-2019-320483