Supplementary information
Novel mutatons in genes causing hereditary spastic paraplegia and Charcot-Marie-Tooth neuropathy identified by an optimized protocol for homozygosity mapping based on whole exome sequencing
Daliya Kancheva MSc 1,2,3 *, Derek Atkinson MSc 1,2, *, Peter De Rijk PhD 4 , Magdalena Zimon PhD 1,2,12 , Teodora
Chamova MD, PhD 5 , Vanyo Mitev MD, DSc 3 , Ahmet Yaramis MD 6 , Gian Maria Fabrizi MD, PhD 7 , Haluk
Topaloglu MD, PhD 8 , Ivailo Tournev MD, DSc 5,9 , Yesim Parman MD, PhD 10 , Esra Battaloglu PhD 11 , Alejandro
Estrada-Cuzcano PhD 1,2,& and Albena Jordanova PhD 1,2,3,&
1.
Molecular Neurogenomics Group, Department of Molecular Genetics, VIB, University of Antwerp, Antwerp
2610, Belgium
2.
Neurogenetics Laboratory, Institute Born-Bunge, University of Antwerp, Antwerp 2610, Belgium
3.
Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Medical University-Sofia,
Sofia 1431, Bulgaria
4.
Department of Molecular Genetics, VIB, University of Antwerp, Antwerp 2610, Belgium
5.
Department of Neurology, Medical University-Sofia, Sofia 1000, Bulgaria
6.
Department of Pediatrics, Dicle University School of Medicine, Diyarbakir 21280, Turkey
7.
Department of Neurological, Neuropsychological, Morphological and Motor Sciences, University of Verona,
Verona 37134, Italy
8.
Division of Pediatric Neurology, Department of Pediatrics, Faculty of Medicine, Hacettepe University,
Ankara 06100, Turkey
9.
Department of Cognitive Science and Psychology, New Bulgarian University, Sofia 1618, Bulgaria
10.
Department of Neurology, Istanbul Medical Faculty, Istanbul University, Capa 34390, Istanbul, Turkey
11.
Department of Molecular Biology and Genetics, Bogazici University, Istanbul 34342, Turkey
12.
Present affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL),
Meyerhofstraße 1, Heidelberg 69117, Germany
* These authors contributed equally to this study
& Shared last co-authors

Figure S1. Pedigree structures of the nine families with both SNP and WES data available

A.
Families included in the training set; B.
Families included in the test set.
The individuals who were genotyped with a SNP array are indicated with the “S” symbol, and those on whom
WES was performed – with the “E” symbol. The individuals with DNA material available are labelled with a number. Segregation analysis is presented for the families where a pathogenic variant was detected in a known
CMT or HSP gene. The mutant allele is shown in red. The declared consanguinity is denoted by a double line between the parents. Square – male; circle – female; empty symbol – unaffected individual; filed black symbol – affected individual.

Figure S2. Pedigrees of the three families with only WES data available and haplotype sharing analysis for the ZFYVE26 c.2639T>C mutation
A. Pedigrees of families HSP-2, HSP-3 and HSP-4 with only WES data available. The intermittent line indicates relatedness between HSP-3 and HSP-4. The individuals on whom WES was performed are indicated with the
“E” symbol. The individuals with DNA material available are labelled with a number. The genotype for the identified mutation is shown under each tested individual, the mutant allele is highlighted in red. Square – male, circle – female, empty symbol – unaffected individual, filed black symbol – affected individual.
B.
Haplotype sharing analysis for the ZFYVE26 c.2639T>C mutation. Informative SNP markers flanking the
ZFYVE26 gene were chosen from the WES data. Green shading indicates shared disease haplotype; the core haplotype is delineated by a thick line. The c.2639T>C mutation is shown in red. MAF (1000G) – minor allele frequency of the respective SNP based on the 1000Genome database.

Figure S3. Specificity and sensitivity of ROH detection using different quality filtering strategies after
SNP extraction from WES data

Table S1. Mutations identified using HOMWES in known HSP and CMT genes
Family
CMT-2
CMT-3
CMT-8
HSP-2
Gene
HSPB1
PRX
GAN
GBA2
Nucleotide change c.250G>A c.2098delG c.140T>C c.451+2 T>C
Mutation present in: Conservation
-
In silico predictions
Protein change
Matched controls
NHLBI /
1000Genome
GERP PhyloP Condel Meta-Snp NetGene2 p.G84R p.A700P fs*17 p.I47T
-
0/190
0/190
0/190
0/50
-
-
-
-
4.94
1.39
3.94
3.97
5.82
-0.51
6.88
8.79
D(0.62) D(RI=2)
-
-
-
D(0.58) D(RI=4)
- -
-
Donor splicesite broken
ACMG
Classification
P
P
LP
P
HSP-3 ZFYVE26 c.2639T>C
HSP-4 ZFYVE26 c.2639T>C p.L880P p.L880P
0/50 - 5.95 8.81 N(0.50) D(RI=3) - LP
ACMG - classification of the mutations according to the ACMG standards and guidelines; the Condel score is a value between 0 and 1 (values > 0.5 are predicted to be disease-causing); RI - Reliability Index between 0 and 10 (higher values indicating higher reliability of prediction); D- disease causing; N – neutral; P – pathogenic; LP - likely pathogenic.

Table S2. Examples of fragmented WES-derived homozygous regions compared to the regions found with
SNP array in family CMT-2.
Chromosome
4
7
11
SNP array
Begin
71 567
61 074 195
54 794 238
End
6 868 155
93 959 918
73 004 053
WES
Size
(Mb)
Chromosome
6.8
32.9
18.2
7
11
11
11
4
4
7
7
Begin End
68 007 3 578 183
4 239 586 6 925 008
63 033 331 65 048 118
66 194 437 76 069 901
76 116 130 94 027 171
55 086 759 56 143 157
56 507 879 59 710 427
59 710 483 73 020 026
Size
(Mb)
17.9
1.1
3.2
13.3
3.5
2.7
2.0
9.9
The genomic coordinates of the start and end and the total size of each region are shown. In gray is indicated the region containing the disease-causing mutation in HSPB1 for this family.

Table S3. Clinical characteristics of the HSP patients with newly identified mutations
Patient
Gene/
Mutation
HSP-2.1 GBA2/ c.451+2 T>C
HSP-2.2 GBA2/ c.451+2 T>C
Sex/age at onset (y)
Disease duration (y)
Spasticity
(Ashworth scale)
LL/UL
Reflexes
Mental
Retardation
Cognitive
Deterioration
Cerebellar
Signs
Visual
Function
M/16
M/17
24
27
3
3
3/4
3/4
-
-
-
-
+
+
Normal
ND
NCS
Mild axonal neuropathy
ND
Brain
MRI
Cerebellar vermian atrophy
ND
HSP-3.1 ZFYVE26/ p.L880P
M/19 14 4 2/5 - - + Normal Mild axonal neuropathy
Normal Normal
Periventricular leukoencephalopathy and thin corpus callosum
Normal HSP-4.1 ZFYVE26/ p.L880P
HSP-4.2 ZFYVE26/ p.L880P
M/15
M/14
4
6
2
2
3/4
3/4
-
-
-
-
-
- Normal Normal y- years; UL/LL – upper/lower limbs; ND – no data; NCS – nerve conduction studies; MRI – magnetic resonance imaging; (-) – not present, (+) – present
Normal

Table S4. Clinical characteristics of the CMT patients with newly identified GAN mutation
Muscle weakness (MRC) NCS
Patient
Gene /
Mutation
UL proximal
UL distal
LL proximal
LL distal n. medianus motor n. medianus sensory n. tibialis motor n.tibialis sensory
CV A CV A CV A CV A
CMT-8.1 GAN/ p.I47T M/2 8 + 4+/5 4/5 4/5 3/5 distal Feet + - 43 2.49 37.1 4.4 NR NR 40 11.8
CMT-8.4 GAN/ p.I47T M/2 14 - 4/5 3/5 2/5 0/5 distal
Feet, vertebral column, thorax
+ + 43 2.7 NR NR NR NR y- years; UL/LL – upper/lower limbs; MRC – Medical Research Council; NCS – nerve conduction studies; (-) – not; present, (+) – present; CV - conduction velocity (in meter per second); A - amplitude (motor: in milivolt; sensory: in microvolt); NR – no response
NR NR

Table S5. Proportion of identical-by-descent alleles between HSP-3.1 and HSP-4.1
Individual 1 Individual 2
Estimated proportion 0, 1 or 2 alleles shared IBD
IBD=0
(Z0)
IBD=1
(Z1)
IBD=2
(Z2) Relatedness (r)
HSP-3.1 HSP-4.1 0.7712 0.1448 0.084 0.1564
Z0, Z1 and Z2 – estimated proportion of 0, 1 or 2 alleles shared IBD between the pairs of individuals; relatedness (r) – overall proportion of alleles shared IBD between the two individuals (r= Z1/2+Z2). Expected values of r for double first cousins, first cousins, first cousins once removed and second cousins are respectively
0.25, 0.125, 0.0625 and 0.03125. The calculated relatedness between HSP-3.1 and HSP-4.1 corresponds to approximately a first cousins relationship.

Table S6. Impact of HOMWES for variant filtering of WES data
Number of variants after WES filtering
Individual
HSP-1.1
CMT-1.1
CMT-1.2
CMT-2.1
CMT-3.1
CMT-4.1
CMT-5.1
CMT-6.1
CMT-7.1
CMT-8.1
No HOMWES
440
429
445
414
428
439
486
379
448
406
HOMWES
71
90
117
116
88
79
130
111
135
113
No HOMWES: includes homozygous coding and splicing variants with <5% 1000Genome frequency.
HOMWES: includes homozygous coding and splicing variants with <5% 1000Genome frequency, which are located within the identified ROH regions. The candidate variant list decreases considerably after applying a filter based on the homozygosity mapping results.