Online Supplementary File

Online Supplementary file

Variants in KIF1A gene in dominant and sporadic forms of hereditary spastic paraparesis

1Andrea Citterio, 1Alessia Arnoldi, 1Elena Panzeri, 2Luciano Merlini, 3Maria Grazia D’Angelo,4Olimpia Musumeci, 4Antonio Toscano, 5Alice Bondi, 6Andrea Martinuzzi, 1,7 Nereo Bresolin,1Maria Teresa Bassi.

1Scientific Institute IRCCS E. Medea, Laboratory of Molecular Biology, 23842 Bosisio Parini, Lecco, Italy; 2SC Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, IRCCS, Bologna, Italy; 3Scientific Institute IRCCS E. Medea, Neuromuscular Disorders Unit, 23842 Bosisio Parini, Lecco, Italy; 4Department of Neurosciences, University of Messina, Messina, Italy; 5Dept. Rizzoli-Sicilia, Istituto Ortopedico Rizzoli, 90011 Palermo, Italy; 6Scientific Institute IRCCS E. Medea, Conegliano Research Center, Conegliano, Italy;7Dino Ferrari Centre, Neurology Unit, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Foundation, Dept. of Physiopathology and Transplantation, Universita’ di Milano, Italy.

Key words: NGS targeted resequencing, spastic paraparesis, KIF1A, dominant inheritance.

Corresponding author: Maria Teresa Bassi PhD, E. Medea Scientific Institute, Laboratory of Molecular Biology, Via D. L. Monza 20, 23842 Bosisio Parini, Lecco, Italy; phone 0039031877111, fax 0039031877499; email .

Online resource Patient and Methods

Clinical description of the patients carrying KIF1A variants.

Family P7814

The index case (V-7) was a 10-year-old child in which the very first step was obvious spasticity of the lower limbs. At school it became evident a mild psychomotor retardation (Brunet Lézin QS = 68). He had a spastic gait requiring unilateral support, minimal stiffness in the upper limbs, lumbar hyperlordosis, foot cables. He had a positive Babinski, clonus of the feet, no ataxia, no tremor and urinary urgency.

His 31-year-old mother (IV-4) had a minimal disability with slight stiffness of the legs, cavus of the feet and positive Babinski sign at age 12 yrs; she displayed no intellectual disability, no ataxia, tremor nor any other cerebellar sign.

His 60-year-old maternal grandfather (III-8) showed the first disease symptoms (leg stiffnes and gait instability) in the childhood. However he walked with bilateral support from age 55; had marked lower legs spasticity, pes cavus, mild hand amyotrophy, superficial and deep sensory abnormalities in the feet, urinary urgency, no intellectual disability.

The grandfather cousin (III-6) displayed a clinical picture overlapping the one of subject III-8 with lower severity. Subject III-1 was healthy.

P67809

This patient is a 24 year old girls, born to non-consanguineous parents, after regular pregnancy and podalic natural delivery. Low weight at birth was associated to normal Apgar values.

Mild motor developmental delay was described together with very mild language delay. Unsteadiness during walking and mild spasticity guided a neurological evaluation by the age of 7ys. Brain and spine imaging were normal, as Somatosensory evoked potentials. IQ level was within the normal range.

Spasticity of lower limbs, affecting ambulation has been increasing with time.

At the last neurological evaluation (24 yrs) the patient showed pes cavus and spastic paraparesis with brisk –policynetic lower limb reflexes with Babinski sign, with mild proximal lower limb weakness; brisk reflexes were present also at the upper limb. Ambulation required bilateral support. Alteration at motor and somatosensory evoked potentials together with axonal neuropathy have been recorded at electrophysiological studies. Brain imaging is normal.

Family P22413

The proband is a 68 year old woman with family history of neurological disorders. She reported that her father experienced gait disturbances in his sixties with progressive loss of ambulation in 3 to 4 years. He died wheelchair-bound at 87 year old. He suffered of blood hypertension. She started complaining of imbalance problems with frequent falls since age 63 (yrs) with writing difficulties and urinary urgency.

Neurological examination revealed: spastic ataxic gait, increased deep tendon reflexes at four limbs, mild bilateral cerebellar dysmetria

Brain MRI showed multiple subcortical, and periventricular hyperintensities at TR sequences, in the fronto-parietal regions and pons

Muscle biopsy, EMG and NCVs were normal. Sensory and motor evoked potentials at 4 limbs were normal whereas visual evoked potentials revealed a mild increase of P100 latency

P116909

This patient is 52 year old man who started showing gait instability at age 22. After 30 years of disease duration he showed a moderate disability (he could still walk without support) with frequent falls. Neurological examination revealed spastic gait with Babinski sign and increased deep tendon reflexes at the lower limbs. Urinary urgency was present. No cerebellar signs were noted. Brain and spinal cord images were normal. EMG and NCVs were normal. IQ level was normal.

NGS Sequencing

For gene screening, a targeted next generation sequencing (NGS) method was used that included 84 genes linked to HSP, neuropathy or related diseases (Supplementary Table 1). Target enrichment and amplification were done with the HaloPlex kit (Agilent Technologies, Santa Clara, CA, USA), and sequencing on MiSeq (Illumina, San Diego, CA, USA).

The sequencing target was designed with the SureDesign program (Agilent Technologies, Santa Clara, CA, USA) to cover 84 disease genes causative of either HSP or overlapping diseases. Target enrichment and amplification was done with the HaloPlex Target Enrichment Kit (Agilent Technologies), according to the manufacturer’s instructions. The genomic patient DNA was fragmented with restriction enzymes followed by hybridization of the target DNA to a biotinylated probe library and by its capture using streptavidin-coated magnetic beads. The target was PCR amplified to produce a target enriched sample for sequencing. Sequencing was done on an MiSeq sequencer (Illumina, San Diego, CA, USA). Data analysis (alignment and variant calling) was performed by using MiSeq Reporter and Integrative Genomics Viewer (IGV) v. 2.3.20. The created VCF files were then imported and annotated using Illumina Variant Studio version 2.2 (Illumina, San Diego, CA, USA).

At least 98% of the NGS fragments had an average coverage of 150 reads and passed quality control. The remaining 2% had an average coverage of at least 20 reads. All KIF1A exons had an average coverage of 350 reads.

Data filtering

NGS single nucleotide variant (SNV) data were filtered as follows: 1) Exclusion of variants with frequency > 0.01 in the Exome Variant Server and with MAF > 0.01. 2) Exclusion of synonymous or non-splice site changing variants. 3) Exclusion of some recurring false positive calls present in reads using Integrative Genomics Viewer (IGV). Insertion and deletion (indel) data were filtered by: 1) Exclusion of variants outside of genes; 2) Exclusion synonymous or non-splice site changing; 3) Exclusion of variants found in the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). Predictions of variants’ pathogenicity were obtained using SIFT (http://sift.jcvi.org/),

PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and MutationTaster

(http://www.mutationtaster.org/) software.

Online resource Table 1-List of targeted genes for NGS

Disease / Genes
HSP / ABCD1, ALDH18A1, ALS2, AP4B1, AP4E1, AP4M1, AP4S1, AP5Z1, ARL6IP1, ATL1, B4GALNT1, BSCL2, C12ORF65, C19ORF12, CYP2U1, CYP7B1, DARS2, DDHD1, DDHD2, ENTPD1, ERLIN1, ERLIN2, FA2H, FLRT1, GBA2, GJC2, HSPD1, KIAA0415, KIAA0196, KIF1A, KIF1C, KIF5A, L1CAM, MAG, MARS, MARS2, NIPA1, NT5C2, OPA1, PLP1, RAB3GAP2, REEP1, REEP2, RTN2, SLC33A1, SPAST, SPG11, SPG20, SPG21, SPG7, TECPR2,USP8, VPS37A, WDR48, ZFR, ZFYVE26, ZFYVE27
Other related phenotypes (Motor Neuron disease, Familial Cerebral palsy, Ataxia and Neuropathies / VAMP1, VCP, AFG3L2,ADD3, KANK1, GARS, GDAP1, HSPB1, MFN2, TRPV4, YARS, BICD2, SACS, SETX, SIGMAR1, PNPLA6, ATL3, EXOSC3, TFG, PGAP1, MTPAP, GSN, CCDC50, ARSI, AMPD2 DYNC1H1, DCTN1
Total n. of targeted genes 84

Online resource Table 2- List of variants resulting from the filtering process in the patients carrying KIF1A heterozygous variants

P7814
Filtering step
/ Number of variants
Unfiltered- Passing filter
/ 3634 -288
MAF and Exome variant server < 0.01
/ 13
Intragenic variants that alter amino acid sequence or splice site
/ 2
Gene
/ Reference sequence
/ Variant
/ dbSNP
KIF1A / NM_001244008.1 / NM_001244008.1:c.206C>T
NP_001230937.1:p.Ser69Leu (het) / ---
DDHD1 / NM_001160148.1 / NM_001160148.1:c.331_336dupGGCGGC
NP_001153620.1:p.Gly111_Gly112dup (het) / rs140904345
P67809
Filtering step
/ Number of variants
Unfiltered- Passing filter
/ 3775 -288
MAF and Exome variant server < 0.01
/ 14
Intragenic variants that alter amino acid sequence or splice site
/ 3
Gene
/ Reference sequence
/ Variant
/ dbSNP
KIF1A / NM_001244008.1 / NM_001244008.1:c.304G>A
NP_001230937.1:p.Gly102Ser (het) / -----
DDHD1 / NM_001160148.1 / NM_001160148.1:c.331_336dupGGCGGC
NP_001153620.1:p.Gly111_Gly112dup (het) / rs140904345
KIF1C / NM_006612.5 / NM_006612.5:c.884C>T
NP_006603.2:p.Ser295Leu (het) / rs145650252
P116909
Filtering step
/ Number of variants
Unfiltered- Passing filter
/ 777 -60
MAF and Exome variant server < 0.01
/ 4
Intragenic variants that alter amino acid sequence or splice site
/ 3
Gene
/ Reference sequence
/ Variant
/ dbSNP
KIF1A / NM_001244008.1 / NM_001244008.1:c.499C>T
NP_001230937.1:p.Arg167Cys (het) / ---
SACS / NM_014363.4 / NM_014363.4:c.9562T>C
NP_055178.3:p.Phe3188Leu (het) / rs137905181
SACS / NM_014363.4 / NM_014363.4:c.810T>G
NP_055178.3:p.Phe270Leu (het) / rs116907814
P22413
Filtering step
/ Number of variants
Unfiltered- Passing filter
/ 583 -63
MAF and Exome variant server < 0.01
/ 2
Intragenic variants that alter amino acid sequence or splice site
/ 1
Gene
/ Reference sequence
/ Gene
/ Reference sequence
KIF1A / NM_001244008.1 / KIF1A / NM_001244008.1

Online resource Figure 1

Results of the analysis with different prediction software of the novel KIF1A variants identified.