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The American Journal of Human Genetics, 91

Supplemental Data

Mutations in DDHD2, Encoding an Intracellular

Phospholipase A1, Cause a Recessive Form

of Complex Hereditary Spastic Paraplegia

Janneke H.M. Schuurs-Hoeijmakers, Michael T. Geraghty, Erik-Jan Kamsteeg, Salma Ben-Salem, Susanne T.

de Bot, Bonnie Nijhof, Ilse I.G.M. van de Vondervoort, Marinette van der Graaf, Anna Castells Nobau, Irene

Otte-Höller, Sascha Vermeer, Amanda C. Smith, Peter Humphreys, Jeremy Schwartzentruber, FORGE Canada

Consortium, Bassam R. Ali, Saeed A. Al-Yahyaee, Said Tariq, Thachillath Pramathan, Riad Bayoumi, Hubertus

P.H. Kremer, Bart P. van de Warrenburg, Willem M.R. van den Akker, Christian Gilissen, Joris A. Veltman,

Irene M. Janssen, Anneke T. Vulto-van Silfhout, Saskia van der Velde-Visser, Dirk J. Lefeber, Adinda Diekstra,

Corrie E. Erasmus, Michèl A. Willemsen, Lisenka E.L.M. Vissers, Martin Lammens, Hans van Bokhoven, Han

G. Brunner, Ron A. Wevers, Annette Schenck, Lihadh Al-Gazali, Bert B.A. de Vries, and Arjan P.M. de

Brouwer

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Figure S1. DDHD2 Exome Experiment

Raw sequencing reads of mutations in DDHD2 identified by exome sequencing and visualized with

Integrative Genomics Viewer (http://www.broadinstitute.org/igv/home).

(A) Individual II-2 of family 1, left panel: 19 of 71 reads (26%) showed an deletion of A at

chr8(hg19):g.38117560; right panel: 36 of 66 reads (55%) showed an insertion of T at

chr8(hg19):g.38110558-38110559.

(B) Both affected siblings (II-1 and II-2) of family 2 show on the left panel a heterozygous insertion

of C at chr8(hg19):g.38109471 and on the right panel a heterozygous base-pair substitution G to C at

chr8(hg19):g.38111160. The father (I-1) shows the heterozygous insertion (left panel), but not the

substitution (right panel).

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Figure S2. DDHD Domain Conservation

(A) Alignment of the DDHD domain is shown for all three mammalian iPLAs1: DDHD2

(NP_056029.2), DDHD1 (NP_001153620.1) and SEC23IP (NP_009121.1).

(B) Cross-species alignment of part of the human DDHD domain for human (Homo sapiens;

ENSP00000380352), mouse (Mus musculus; ENSMUSP00000096459), zebrafish (Danio rerio;

ENSDARP00000103209) and fruitfly (Drosophila melanogaster; FBpp0292462). The box indicates

the conserved RIDYXL sequence motif. The position of Asp660, which is substituted in family 2, is

highlighted in black. The box with the dotted line indicates the last 14 amino acids, that are altered by

the p.Glu686Glyfs*35 change in family 1.

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Figure S3. Cerebral Imaging of Family 1

Upper panels: Mid-sagittal T1-weighted MRI of the brain showing a marked thin corpus callosum in

both affected siblings (white arrow).

Middle panels: Transverse T2-weighted MRI of the brain showing subtle white matter

hyperintensities in the same individuals.

Bottom panels: Proton MR spectra obtained at a magnetic field of 1.5 Tesla. Voxel was fixed just

cranial to the basal ganglia/ thalamus area, from which a proton MR spectrum long echo time (144

ms) was obtained showing the prominent pathologic lipid peak at 1.3 ppm, apart from the common

spectral peaks of choline (Cho), creatine (Cr), and N-acetylaspartate (NAA).

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Figure S4. Cerebral Imaging of Family 2

Upper panels: Mid-sagittal T1-weighted MRI of the brain showing a marked thin corpus callosum in

both affected siblings (white arrow). Middle panels: Transverse T2Flair-weighted MRI of the brain

showing subtle white matter hyperintensities in the same individuals.

Bottom panels: Proton MR spectra obtained at a magnetic field of 1.5 Tesla. Voxel was fixed at the

basal ganglia area, from which a proton MR spectrum at short echo time (35 ms) was obtained

showing the prominent pathologic lipid peak at 1.3 ppm, apart from the common spectral peaks of

choline (Cho), creatine (Cr), and N-acetylaspartate (NAA).

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Figure S5. Cerebral Imaging of Family 3

Left panel: Mid-sagittal T1-weighted MRI of the brain showing a marked thin corpus callosum in

affected individual IV-3 (white arrow).

Right panel: Transverse T2-weighted MRI of the brain showing subtle white matter hyperintensities

in the same individual.

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Figure S6. Cerebral Imaging of Family 4

Upper left panel: Mid-sagittal T1-weighted MRI of the brain showing a marked thin corpus callosum

(white arrow).

Upper right panel: Transverse T2-weighted MRI of the brain showing subtle white matter

hyperintensities.

Bottom panel: Proton MR spectrum obtained at a magnetic field of 3 Tesla. Voxel was fixed at the

thalamus area, from which a proton MR spectrum at short echo time (30 ms) was obtained showing

the prominent pathologic lipid peak at 1.3 ppm, apart from the common spectral peaks of choline

(Cho), creatine (Cr), and N-acetylaspartate (NAA).

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Figure S7. Spinal Imaging of Families 1 and 2

Sagittal T2-weighted MRI of the spine of affected individual II-2 of family 1, and II-2 of family 2,

both showing a spinal syrinx (white arrow).

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Figure S8. Effect of Mutations in Families 1 and 4 on DDHD2 Expression

Effect of the compound heterozygous mutations, c.1804_1805insT and c.2057delA, from family 1,

and c.859C>T from family 4 on DDHD2 expression in Epstein Barr Virus transformed

Lymphoblastoid cells (EBV-LCLs). Shown is the mean expression of DDHD2 in EBV-LCLs of

affected individuals of family 1 (n=2), family 4 (n=1) and controls (n=8), treated without (-CH) and

with cycloheximide (+CH). Quantifications were performed in duplicate and normalized against

GUSB and PPIB. Differences in expression between samples of the affected individuals and eight

controls were calculated by the comparative Ct or 2ΔΔCt

method 1; 2

. The p-value was derived from the

standard score (Z-value) calculated for each individual as compared to the normal distribution of the

eight controls. Since we assume a lower expression level as a result of nonsense-mediated decay

(NMD), a one-sided test was enough to reject the null hypothesis, i.e., no statistically significant

difference between the expression of DDHD2 in EBV-LCLs of an affected individual and that in

EBV-LCLs of controls. We used an alpha level of 0.05, because only one gene was assessed.

Inhibition of translation was obtained by treatment of EBV-LCLs with 20 µl cycloheximide

(concentration: 100 mg/ml DMSO) that was added to the medium and incubated for 4 hours at 37°C.

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Figure S9. Expression of DDHD2 in Different Human Tissues

Expression of DDHD2 by mRNA expression analysis in human fetal and adult tissues. Relative

expression levels are given as the fold change in comparison to the tissue with the lowest expression

level. Quantifications were performed in duplicate and normalized against GUSB and PPIB.

Differences in expression between tissues were calculated by the comparative Ct or 2ΔΔCt

method 1; 2

.

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Figure S10. Oil Red O Staining of Cultured Fibroblasts of Controls and Affected Individuals

(A–E) Representative fibroblast cells of individuals II-1 of family 1 (A), II-2 of family 2 (C) and II-1

of family 4 (E) and two control individuals (B and D).

(F) Positive control.

Cells were stained with Oil Red O to visualize lipid droplets. There was no difference observed in the

number and appearance of lipid droplets between cells of affected individuals (n=3) and controls

(n=3). Cells were seeded and cultured overnight on glass slides, fixed in 3,7% formalin for 10

minutes, rinsed in demineralized water and stained for 30 minutes in filtered Oil Red O solution

dissolved in isopropanol and rinsed in demineralized water followed by a brief counterstaining in

hematein. After rinsing in tap water for 10 minutes the slides were sealed by using Xylol-based

mounting medium.

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Figure S11. Electron Micrographs of Fibroblasts of Affected Individuals

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Representative electron micrographs of two control individuals (A–F) and individuals II-1 of family 1

(G–I), IV-10 of family 3 (J–L) and II-1 of family 4 (M–O).

Left panels: 1.2K (A, D, G, and M) and 2K (J) images.

Middle and right panels: 6K images.

(A–I and M–O) Spun down pellets of cultured fibroblasts were fixed in 2% glutaraldehyde in 0.1M

Phosphate buffer for 4 hours, rinsed in Phosphate buffer and postfixed for 1 hour in 1% Osmium

containing 1% Kaliumhexacyanoferrat. Semithin (1 m) sections for previewing and ultrathin (70

nm) sections were cut on an ultramicrotome, Leica EM UC6, collected on 200mesh copper grids and

contrasted with uranyl acetate and lead citrate double stain (as previously described by Sjostrand et

al.3 and Reynold et al.

4).

(J and K) Cultured fibroblasts were fixed with formaldehyde-glutaraldehyde method as described by

Karnovsky et al5. Semithin (1,30 m) and ultrathin (95 nm) sections were prepared and stained with

1% aqueous toluidine blue on glass slides and 200mesh Cu grids, respectively, and contrasted with

uranyl acetate and lead citrate double stain3,4

. All sections were examined and images generated in a

Jeol JEM1200 Transmission Electron Microscope (Jeol, Netherlands). Abbreviations: N= Nucleus,

Nuc=nucleolus, ER= endoplasmic reticulum, M= Mitochondria. Red asterisks indicate vacuoles,

arrows indicate glycogen.

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Figure S12. Quantification of NMJ Morphology in Drosophila Lavae

Synapse morphology and organization at the Drosophila neuromuscular junction of control and

CG8552 (Ddhd) knockdown flies. Three different RNAi lines, vdrcGD35956, vdrcGD35957, and

vdrcKK108121, from the Vienna Drosophila Research Center, were used and compared to their

genetic background lines, vdrcGD60000 and vdrcKK60100, respectively. Quantification of NMJ area

(A), branch length (B), longest branch length (C), number of branching points (D), number of

branches (E) and number of island (F). Error bars indicate standard error of the mean.

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Table S1. Raw Sequencing Statistics & Prioritization of Variants of Family 1

Family 1: II-2

Total number of sequenced reads (×106) 189.24

Total number of mapped reads (×106) 144.51

Total number of bases mapped (Gb) 6.7

Total bases mapping to targets (Gb) 5.8

% targets with 10x coverage 88.9%

Mean target coverage (fold) 99.9

Median target coverage (fold) 74

all variants 30,182

QC filtering a 29,117

After exclusion of known variants 568

Affecting protein sequence or canonical splice sites 180

Of which fit a recessive disease modelb 37

Of which fit a recessive disease model after manual

inspection of raw sequence reads 11c

Gene(s) in overlap with family 2 1 (DDHD2)

a>2 unique variation reads and >20% variation reads,

b>1 variation in a gene for compound heterozygous variants and >80% variation reads for candidate

homozygous variants. cFour candidate compound heterozygous mutations and three candidate homozygous mutations. The

candidate mutations in DDHD2 were the only recessive protein truncating mutations.

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Table S2. Raw Sequencing Statistics & Prioritization of Variants of Family 2

Family 2: II-2 Family 2: II-1 Family 2: I-1

Total number of sequenced reads (×106) 152.41 122.98 120.02

Total number of mapped reads (×106) 152.40 122.98 120.02

Total number of bases mapped (Gb) 14.6 11.8 11.5

% of CCDS bases with 10x coverage 92.7 92.2 92.2

Mean target coverage (fold)a 109.5 93.3 94.3

Median target coverage (fold)a 90 77 78

all variants 154,566 143,418 142,473

QC filteringa 110,606 104,530 103,559

After exclusion of known variants 728 671 860

Affecting protein sequence or canonical splice sites 119 142 185

Of which fit a recessive disease model 13 15 NA

Of which fit a recessive disease model after comparison of

brother, sister and father. 1 (DDHD2)

Gene(s) in overlap with family 1 1 (DDHD2)

aAfter duplicate read removal. NA= not analyzed.

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References

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quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25,402-408.

2. Pfaffl M.W. (2001). A new mathematical model for relative quantification in real-time RT-

PCR. Nucleic. Acids Res. 29,e45.

3. Sjostrand F.S. (1956). A method to improve contrast in high resolution electron microscopy

of ultrathin tissue sections. Exp. Cell. Res. 10,657-664.

4. Reynold E.S. (1965). The use of lead nitrate at high pH as an electron opaque stain in electron

microscopy. The Journal of Cell biology 17,208.

5. Karnovsky M.J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in

electron microscopy. The Journal of Cell Biology 27,137A.