Supplementary methods

Protein structural modelingHomology modeling was carried out on the basis of the crystallographic structure of E. coli SufA (sequence similarity of 37.61% to the human ISCA1) that is involved in biosynthesis of Fe-S clusters (PDB ID: 2D2A).1, 2 The modeled structures were further validated using Protein Structure Evaluation Suite & Server or PROSESS (http://www.prosess.ca ) that utilizes Volume, Area, Dihedral Angle Reporter and VADAR (http://vadar.wishartlab.com/).3-5 Multiple sequence alignment was performed using Clustal Omega.6 Amino acid conservation was also evaluated using the ConSurf server (http://consurf.tau.ac.il/2016/ ) .7 The structural effect of the missense substitution Glu87Lys was predicted using STRUM8, I-Mutant2.09 (http://folding.biofold.org/i-mutant/i-mutant2.0.html) and Eris10 (http://dokhlab.unc.edu/tools/eris/) web-servers. Additionally HOPE11 (http://www.cmbi.ru.nl/hope/ ) was used to predict the consequences of the ISCA1 p.E87K variant. The protein structure was visualized using YASARA View simulation software.12 Using PROSESS torsion angles for wild-type Glu87 were estimated to be −62.3φ, 138.6ψ and that for mutant Lys87 were −67.8φ, 136.9ψ. The p.E87K alteration has drastic effects on Val50 and Ala90 residues that are hydrogen (H) bonding partners of the native Glu87. As a result, in the mutant, the φ angle of Ala90 potentially increases from −101.9φ to −106.2φ, whereas ψ angle decreases from −15.3ψ to −15.1ψ, similarly the φ angle of Val50 decreases from −95.8φ to −95.6φ and ψ angle increases from −125.6ψ to −138.6ψ. The observed p.E87K variation also seems to affect the main chain residue accessible surface area (ASA) that increases from 41.4 Å2 to 79.9 Å2 and the main-chain residue volume increases from 145.7 Å3 to 178 Å3. Moreover, the side-chain residue ASA increases from 41.4 Å2 to 79.9 Å2 and the solvation energy of the mutant Lys87 decreases from -1.3 kcal/mol to -3.5 kcal/mol.

Supplementary Figure S1: (A) Working model of mitochondrial Fe-S biogenesis Adapted from Cameron et. al. (2011).13 Sulfide (S2-) is derived from cysteine via the cysteine desulfurase NFS1 in complex with LYM4. The S2- is transiently bound to Nfs1 as a persulfide, transferred to the scaffold protein ISCU, and reduced to sulfide by electrons (e-) that originate from NADH and pass through ferredoxin reductase (FdxR) and a [2Fe–2S] ferredoxin (Fdx) electron transport system. Fe2+ is provided by the chaperone Frataxin (FXN) through an interaction with LYRM4 and ISCU and [2Fe-2S] clusters are assembled on the ISCU protein monomers. The co-chaperones HSCB, HSPA9, and GLRX5 bind to ISCU and aid in the transfer of [2Fe-2S] clusters into recipient apoproteins. GLRX5 mediates the transfer of [2Fe-2S] clusters to a heteromeric complex of ISCA1 and ISCA2 that act as ‘assembler’ of [4Fe-4S] clusters, assisted by IBA57, together, they facilitate the maturation of [4Fe-4S] mitochondrial proteins, such as mitochondrial aconitase-like and radical S-adenosylmethionine (SAM)-dependent proteins. Human diseases described previously due to deregulation of mitochondrial Fe-S cluster assembly pathway are italicized. (B) Exons and domain structure of ISCA1. (a) The pathogenic sequence variation, c.259G>A occurs in exon 4 of human ISCA1. (b) The ISCA1 gene encodes for a 129 amino acid product with a C-terminal HESB signature that is involved in the biogenesis of iron–sulfur (Fe-S) clusters. The resultant missense substitution p.(Glu87Lys) occurs at a highly conserved residue in the only essential functional domain for iron-sulfur (Fe-S) biogenesis in the ISCA1 protein.

Supplementary Figure S2. Integrated Genomics Viewer14 of the exome sequencing of family 1, P2 (A) and family 2, P4 (B) aligned to the human genome assembly (hg19/ GRCh37) showed homozygous variation, c.259G>A (p.E87K).

BA

Supplementary Figure S3. Autozygosity mapping was performed from the VCF files of family 1, P2 and family 2, P4, using the AutEx algorithm in FILTUS15. The plot displays the posterior autozygosity probability for each variant against its position (red curve). The autozygous segments are indicated in green color, the homozygous variants are displayed in the upper region and the heterozygous variants in the lower region. Out of the two autozygous segments in family 1, P2 (A) the genomic coordinate of the longest region (6.14 Mb) in chromosome 9, was found to range from 84205860 to 90343780 and in family 2, P4 (B) the 3.3 Mb segment ranges from 85613354 to 88925774.

B

A

Supplementary Table S1. Variant filtering strategy implemented in the study.

Family 1, P2 Family 2, P4Total called variants 74,557 79,702Rare variants (<1%)* 10,170 11,193Read number (>20) 6,468 6,580PHRED quality score (>20) 6,034 6,075Variants less than 20bp from exon 3,835 3,894Excluding synonymous variants 3,300 3,350Homozygous variants 332 248Shared by both the families 1*Variant frequency equal to or less than 1% in population databases (1000 Genomes Project, ExAC and ESP)

Supplementary Table S2: Comparison of salient features of known multiple mitochondrial dysfunctions syndromes with the phenotypes in our patients.

Clinical features MMDS 1 MMDS 2 MMDS 3 MMDS 4 Family 1 Family 2

Intellectual disability + + + + + +

Feeding difficulty + + NA NA + +

Failure to thrive + + + - + +

Neurologic regression + + + + + +

Lactate peak on MRS/lactic acidosis + + + - + +

Leukodystrophy + + + + + +

Pachygyria - - - - + NA

Seizures + + - - + +

Tone Hypotonia Spasticity Hypotonia Spasticity Spasticity Spasticity

Optic atrophy + + NA + - NA

Respiratory insufficiency + + + - + NA

Gene NFU1 BOLA3 IBA57 ISCA2 ISCA1 ISCA1

MMDS: Multiple mitochondrial dysfunctions syndrome; + present; - absent; NA not available; MRS Magnetic Resonance spectroscopy

Supplementary Table S3. Regions of homozygosity observed in the two affected probands. The shared region harboring the ISCA1 variant is highlighted.

Family 1, P2 Family 2, P4

Chromosome From To Mb From To Mb

1 34158472 38450367 4.29

1 152281948 161022380 8.74

1 193051685 196461513 3.41

1 201181223 206239574 5.06

1 206589233 233515102 26.93

2 192214833 197065964 4.85

4 10090178 13615293 3.53 128544514 135121601 6.58

4 90169925 100871360 10.7

9 27455095 32542204 5.09

9 84205860 90343780 6.14 85613354 88925774 3.3

11 14991539 22881002 7.8912 31132479 42503505 11.3712 42554505 52307308 9.7512 54367061 58204283 3.8412 62696599 65856899 3.1612 82751062 88177791 5.4318 14752957 20564800 5.8119 23837145 29704010 5.8720 76962 17605240 17.5320 17705677 21186161 3.48

Supplementary Table S4. Homozygous block (red) around the ISCA1 locus, using the exome sequencing data from family 1, P1 and family 2, P4, demonstrating the shared haplotype block between the two affected probands with the ISCA1 mutation (green) suggesting a founder effect.

Family 1, P2 Family 2, P4

ChromosomeGenomic Position

Reference Variant Zygosity

Index Frequency

Index Read Number Chromosome

Genomic Position

Reference Variant Zygosity

Index Frequency

Index Read Number

chr9 82267732 C G Hom 98.9 94

chr9 82286286 C G Hom 100 36 chr9 82320887 A T Hom 95.5 66 chr9 82320887 A T Hom 100 106 chr9 84205860 G A Hom 99 97 chr9 84205860 G A Hom 100 137

chr9 84214974 T G Hom 100 68 chr9 84267147 T C Hom 100 92 chr9 84267147 T C Hom 100 62

chr9 84268878 A G Hom 100 82 chr9 85613354 A G Hom 98.5 134 chr9 85613354 A G Hom 96.9 128 chr9 85677562 G A Hom 99.4 164 chr9 85677562 G A Hom 100 113 chr9 85964563 C T Hom 97 66 chr9 85964563 C T Hom 100 56 chr9 85987880 A - Hom 91.1 56 chr9 85987880 A - Hom 89.5 38 chr9 86153159 T G Hom 100 47 chr9 86153159 T G Hom 98.3 60 chr9 86238086 T C Hom 100 41 chr9 86238086 T C Hom 100 23 chr9 86241489 A G Hom 100 28 chr9 86241489 A G Hom 100 32 chr9 86278817 C A Hom 100 79 chr9 86278817 C A Hom 97.3 74 chr9 86284178 C A Hom 100 108 chr9 86284178 C A Hom 97.5 120 chr9 86468715 A T Hom 100 209 chr9 86468715 A T Hom 100 179 chr9 86504005 C T Hom 100 60 chr9 86504005 C T Hom 93.8 64 chr9 86592026 A G Hom 100 92 chr9 86592026 A G Hom 100 83 chr9 87285915 C T Hom 100 294 chr9 87285915 C T Hom 100 147 chr9 88694179 G T Hom 96.8 63 chr9 88694179 G T Hom 100 88 chr9 88881089 C T Hom 98.5 66 chr9 88881089 C T Hom 98.4 63 chr9 88924057 A C Hom 100 35 chr9 88924057 A C Hom 100 62 chr9 88925774 A G Hom 100 80 chr9 88925774 A G Hom 100 54

chr9 90258248 C T Hom 100 38

chr9 90258413 TTT C Hom 100 46

chr9 90258413 TT - Hom 100 54

chr9 90258415 T C Hom 100 56

chr9 94518328 C T Hom 100 117 chr9 95219597 G A Hom 100 118 chr9 95219597 G A Hom 100 108 chr9 95738781 C A Hom 100 88 chr9 95766471 A T Hom 100 9 chr9 95780465 A G Hom 100 75 chr9 95780465 A G Hom 100 149

chr9 95838070 G C Hom 100 52

chr9 95840077 A G Hom 100 14

chr9 95840256 A G Hom 100 68

Supplementary Table S5. MIM genes within the shared region of homozygosity in chromosome 9.

HGNC Gene symbol

Gene name Gene/LocusMIMnumber

Phenotype PhenotypeMIMnumber

HCHGQ2 Hematocrit/hemoglobin quantitative trait locus 2

609320 Hematocrit/hemoglobin quantitative trait locus 2

609320

SPG19 Spastic paraplegia-19 607152 Spastic paraplegia 19, autosomal dominant

607152

CTRCT26 Cataract 26, multiple types 605749 Cataract 26, multiple types 605749

GSM1 Geniospasm 1 190100 Geniospasm 190100

ETL4 Epilepsy, familial temporal lobe, 4

611631 Epilepsy, familial temporal lobe, 4

611631

PVOP2 Pelvic organ prolapse, susceptibility to, 2

613088 Pelvic organ prolapse, susceptibility to, 2

613088

FHL1 Hemophagocytic lymphohistiocytosis, familial, 1

267700 Hemophagocytic lymphohistiocytosis, familial, 1

267700

AGTPBP1 ATP/GTP-binging protein 1 606830 None

GOLM1 Golgi membrane protein 1 606804 None

ISCA1 Iron-sulfur cluster assembly 1, S. cerevisiae, homolog of

611006 None

HGNC: HUGO Gene Nomenclature Committee; MIM: Mendelian Inheritance in Man16

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