Table e-1: Phylogenetic alignment of the AIF orthologs

Species / Amino acid numbering and sequence
H.sapiens / 249-298 / SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRSLEK--I
P.trogloodytes / 213-262 / SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRSLEK--I
M.mulatta / 245-294 / SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRSLEK--I
C.lupus / 249-298 / SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRTLEK--I
B.taurus / 249-298 / SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIEDFRTLEK--I
M.musculus / 248-297 / SQITFEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRALEK--I
R.norvegicus / 248-297 / SQITFEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRALEK--I
G.gallus / 227-276 / TQISYDKCLIATGGSPRNLPAIERAGKEVQQRLTLFRKIEDFKNLEK--I
D.rerio / 251-300 / SEISYEKCLIATGGVPRNLQVIDRAGEEVIKRTTLFRKIEDFRSLEK--I
X.tropicalis / 271-320 / TQISYEKCLIATGGVPRSLPAIERAGEEVTKRTTLFRRISDFRTLEA--I
D.melanogaster / 373-422 / YEISYDECLIATGCAPKNLPMLRDAPPSVLEKVMVYRTPDDFDRLRK--L

Alignment of AIF orthologs, using the following Refseqs: NP_004199.1, XP_003317723.1, XP_001092025.1, XP_538170.2, NP_001179913.1, NP_036149.1, NP_112646.1, NP_001007491.1, NP_956396.2, NP_001017244.1, NP_001259907.1, NP_499564.2. The amino acids corresponding to the human Gly262, mutated in our patient, are in red.

Table e-2: In silico prediction of pathogenicity for published AIFM1missense mutations

Amino acid Change / Polyphen2 / SIFT / Mutationtaster / Panther / ExACfrequencya / Reference
p.[Gly262Ser] / Possiblydamaging / Tolerated / Diseasecausing / Deleterious / Ø / Presentpaper
p.[Gly308Glu] / Probablydamaging / Affectproteinfunction / Diseasecausing / Deleterious / Ø / Berger et al. 2011
p.[Glu493Val] / Probably damaging / Affect protein function / Disease causing / Not predictable / Ø / Rinaldi et al. 2012

aAllele frequency reported in the Exome Aggregation Consortium(ExAC) browser ( containing >120000 alleles, sequenced as part of various disease-specific and population genetic studies.Ø, not found.

Table e-3: Clinical, instrumental and laboratory findings in patients carrying AIFM1 mutations

Patients / Mutation / Onset / Clinicalfeatures / Brain MRI / Hearingloss / Neuropathy / Lactate-pyruvate / Musclebiopsy / Deficits in ETCcomplexes / Outcome
Age / Symptoms/signs
pt1 a / c.601_603del;p.Arg201del / Earlymo / Psychomotor delay, reduced spontaneus motility, involuntary movements / Psychomotor delay, seizures and psychomotor regression(15 mo); transient improvement, tracheotomy (2 yrs) / N (5 mo);
T2 hyperintensities in neostriatum (1yr) / n.i. / axonalsensorymotorneuropathy / ↑(plasma and CSF) / Largelysubstituted by fat / Fibroblasts: III (65%), IV (54%) / Tetraplegic, unable to comunicate, artificialventilation (5yr)
pt2 a / 11 mo / Psychomotorregression / Hypotonia, reduced spontaneus motility, irritabiliy / T2 hyperintensities in neostriatum / n.i. / n.i. / ↑ (plasma) / Dystrophic alterations, several ragged red fibers, COX deficiency / Muscle: multiple defects, mainly IV (11%) / Suddendeath(16 mo)
pt 3b / c.923G>A;p.Gly308Glu / Prenatal / Bilateral ventriculomegaly, choroid plexus cysts (brain US) / hypotonia, reduced spontaneus motility, swallowing difficulties, neonatal seizures / T2 hyper-intensities in basal ganglia / n.i. / n.i. / N / n.d. / Muscle: IV (12%),I (21%), / Cardiopulmonaryfailure,death(4 yrs)
pt 4 b / Prenatal / Bilateral ventriculomegaly, choroid plexus cysts (brain US) / hypotonia, reduced spontaneus motility, swallowing difficulties, neonatal seizures / n.d. / n.i. / n.i. / N / Dystrophicalterations, COX deficiency / Muscle: IV (4%), I (19%), / Cardiopulmonaryfailure, death(3 mo),
pts 5-12 c / c.1478A>T; p.Glu493Val / Childhood / Weakness, cognitive impairment / Slow progressive axonal neuropathy (7/7) associated with cognitive impairment / T2 hyper-intensities in supraventricular white matter (2/7) / Bilateralhearingloss (3/7) / Axonalmotorsensoryneuropathy (7/7) / n.i. / Neurogenic atrophy-COX staining N (1/7) / Muscle: N / Stable
Presentpatient / c.784G>A; p.Gly262Ser / 1 year / Walking difficulties / Sensory and cerebellar ataxia, hearing and visual loss, muscle wasting and mild weakness, cognitive impairment / Cerebellar, cortical, thalamic atrophy; dentate, olivar, posterior column T2 hyper-intensities / Bilateralhearingloss / Axonal sensory motor neuropathy / Inconstant ↑ (plasma) / Chronic denervation, ragged-red fibers,COX deficiency / Muscle: IV (19%), I (57%) / Slow progression

n.i. not investigated, n.d. not done ; mo: months; yr: year. N: normal; COX: cytochrome c oxidase. a Ghezzi et al. 2010; b Berger et al. 2011; c Rinaldi et al. 2012