SUPPLEMENTARY NOTES, FIGURES AND TABLES Clinical features · cerebellar vermis (arrow). (c) Patient#4: Coronal T2-weighted FLAIR image showing symmetric lesions of the putamina and

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SUPPLEMENTARY NOTES, FIGURES AND TABLES

Clinical features

Patients #1 and #2 are siblings from reportedly non-consanguineous, healthy parents, both

originating from a small village in the alpine region in North-Eastern Italy. Patient #1 is now a 37

year-old lady that was first examined at 5 years of age because of mild mental retardation and an

ataxic gait. These signs progressed over several years, and were accompanied by

hypodiadokokinesis, nystagmus on lateral gaze, diplopia, dysphagia, and dysphonia. At 24 years

she was unable to walk unaided because of ataxia and a mild right hemiparesis. She had rest and

intentional tremor and developed severe incoordination of both hands, with bilateral dysmetria, and

dysarthria, increased deep tendon reflexes and a bilateral Babinski sign. A series of brain MRIs

showed progression of necrotic lesions from an initial bilateral involvement of the caudate nuclei (at

18 years of age) to the present condition characterized by severe cerebellar atrophy with

leukodystrophic abnormalities, bilateral involvement of the inferior olives, substantia nigra, and

periaqueductal grey of the mesencephalon, external segment of the left putamen and both caudate

nuclei (Figure 1A). Although blood lactate was referred as normal, proton-specific mass

spectroscopy (1H-MRS) showed a lactate peak in both putamina. Axonal degeneration of the

second-motor neurons was documented by EMG. Because the brain MRI exams suggested a

metabolic, possibly mitochondrial, disease, we obtained a muscle biopsy from the left quadriceps at

30 years of age. Muscle histology and histochemistry, including reactions for COX and succinate

dehydrogenase (SDH, part of cII), were normal (Supplementary Figure 2), but analysis of the MRC

activities in muscle homogenate revealed the presence of an isolated, severe defect of cIII (Table 1).

Other MRC activities, several Krebs cycle enzymatic activities, as well as pyruvate dehydrogenase

Nature Genetics: doi:10.1038/ng.761

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(PDHC) activity were all normal. The patient is now wheelchair bound, with a stable IQ of 77,

severe dysarthria and upper limb ataxia.

Patient #2, the younger brother of patient #1, is now 26 yo. His disease onset was at 10 years with

mild gait ataxia and poor scholastic performance mainly due to difficulty in verbal and written

language skills. He then suffered of a rapid downhill course characterized by progressive

incoordination of the upper and lower limbs, dysarthria, dysphonia, severe cognitive regression,

bilateral hearing loss, spontaneous nystagmus, and dystonia of both hands and right foot. Weakness

of the ocular muscles ensued in the left eye, due to a pontine-mesencephalic lesion involving the

nuclei of the VI and IV left cranial nerves. The patient is now bedridden, in a fluctuating comatose

status, under permanent mechanical ventilation through tracheostomy, and fed by a percutaneous

endoscopic gastrostomy (PEG) tube. A muscle biopsy performed at age 20 years was

morphologically normal, but again revealed a severe isolated defect of cIII by biochemical assay

(Table 1).

Myoblasts obtained from the muscle biopsy of patient #2 showed an isolated, 50% reduction of cIII

activity, whereas skin fibroblasts from both patients were biochemically normal in standard

conditions. However, a partial cIII defect was detected when fibroblasts were forced to rely on

OXPHOS rather than glycolysis, by replacing glucose with galactose in the culturing medium

(Table 1). In addition, using a micro-scale oxygraphic method, the oxygen consumption in intact

fibroblasts from both patients #1 and #2, and myoblasts from patient #2, all cultured in either

glucose (Figure 1B of main text) or galactose (not shown) medium, was significantly decreased

compared to normal control fibroblasts, clearly indicating impairment of overall respiratory activity.

Patient #3, a young lady now 19 years old, is the only child from reportedly non-consanguineous

healthy parents, originating from the same area of Central Italy. She had a clinical course very

similar to that of patient #2, with onset at 5 years of an ataxic gait, severe regression of language

skills, and mental impairment characterized by lack of interest, and poor social interaction. The


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disease progressed up to the present condition characterized by severe ataxia, scoliosis, dystonia of

the left foot and both hands, and cognitive impairment, worsened by a recent episode of

cardiorespiratory arrest due to prolonged central apnea, requiring heroic resuscitation in an ICU.

Lactate was slightly elevated in blood. She is now bedridden, in a fluctuating comatose status, under

permanently assisted ventilation and PEG feeding. Several brain MRIs performed over time

documented the development of necrotic lesions scattered from the medulla oblongata, including

both inferior olives, through the pons and cerebellum, and up to the mesencephalon, including

cerebral peduncles and periaqueductal grey matter. She later developed multiple bilateral lesions in

the thalami, cerebellar atrophy and severe axonal degeneration of motor peripheral nerves,

indicating second-motor neuron involvement (Supplementary Figure 1). A muscle biopsy was

morphologically normal but biochemical assay of MRC complexes showed marked isolated

reduction of cIII activity (Table 1).

Patient #4, born from reportedly non-consanguineous healthy parents from Southern Italy, had a

normal psychomotor development with no clinically relevant complaints until the age of 42 years,

when he was first examined for a subacute, rapidly progressive neurological syndrome

characterized by weakness of the four limbs with fasciculations, initially suggesting a motor-neuron

disease, followed by apraxia of gait and hand movements, dysarthria, bradykinesia, dystonia and

paraparesis. Lack of initiative and avoidance behavior rapidly ensued with marked slowness in

ideation, but relative conservation of cognition and critical judgment. A brain MRI showed diffuse

cortical atrophy and necrotic lesions in the right caudate nucleus and both putamina (Supplementary

Figure 1). In these regions a 1H-MRS revealed the presence of a lactate peak. A brain SPECT

showed reduced perfusion of the frontal and parietal lobes. Axonal degeneration of the peripheral

motor nerves was documented by EMG. Jacob-Creutzfeld and Wilson disease, chorea-

acanthocytosis, and several other neurodegenerative conditions were all excluded. A CT scan

showed marked muscle atrophy in the lower limbs and upper limb girdle, with fat substitution. A


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muscle biopsy showed mild anisometry, mild lipid accumulation and a few scattered COX-negative

fibers (Supplementary Figure 2). The measurement of the MRC complex activities revealed

marked, isolated cIII deficiency (Table 1). The patient died at 45 years, two years after the disease

onset.


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Supplementary Figures and Legends

Supplementary Figure 1

Brain MRI of patients #3 (P3) and #4 (P4)


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(a) Patient#3: Transverse T2-weighted image of cerebellum and medulla oblongata. The arrows

indicate bilateral hyperintense signals in the inferior olives.

(b) Patient#3: Median sagittal T1-weighted image showing atrophy of the cerebral cortex and

cerebellar vermis (arrow).

(c) Patient#4: Coronal T2-weighted FLAIR image showing symmetric lesions of the putamina and

caudate nuclei, and severe atrophy of the brain cortex.

(d) Patient#4: The same features as in (c) are shown in a T2-weighted FLAIR transverse sequence.

The lesions are very similar among patients (see also Figure 1 of main text).


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Morphological analysis of muscle biopsies of patients #1 (P1) and #4 (P4)

(A) (B) Gomori trichrome showing hardly any alteration in P1 and P4.

(C) (D) Cytochrome c oxidase (COX) staining of P1 and P4. An asterisk indicate partial COX-

depletion in a muscle fiber of P4.

(E) (F) Oil red O (ORO) staining for lipids, showing accumulation in P4.


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Comparative protein sequence analysis of TTC19

Homo MAQPHTTSVPYFARSPAPPPPSRSGAPPQPPATLRPSRRRTRPPRPADRRDAPADCAYLW 60 Pan MAQPHTTSVPYFARSPAPPPPSRSGAPPQPPATLRPSRRRTRPPRLADRRDAPADCAYLW 60 Pongo ------------------------------------------------------------ Mus ------------------------------------------------------------ Bos ------------------------------------------------------------ Gallus ------------------------------------------------------------ Danio ------------------------------------------------------------ Drosophila ------------------------------------------------------------ Homo RILTPRRGRARRSDVGARHRACGRRDVLLSRQGPANPEGARRVVGGQERVWPAVRRGRGG 120 Pan RILTPRRGRARRSDVGARHRACGRKDVLLSRQGPANPEGARRVVGGQDRVWPEVRRGRGG 120 Pongo ------------------------------------------------------------ Mus ------------------------------------------------------------ Bos ------------------------------------------------------------ Gallus ------------------------------------------------------------ Danio --------------------------------------------------------MALR 4 Drosophila ---------------------------------------------------------MLV 3 Homo SMFRLLSWSLGRGFLRAAG----RRCRGCSARLLPGLAGGPGPEVQVPPSRVAPHGRGPG 176 Pan SMYRLLSWSLGRGFLRAAG----RRCRGCSARLLPGLAGGPGPEVQVPPSRVAPHGRGPG 176 Pongo -MYRLLSRSLGRGLLRAAG----RRYGGCSARLLPGLAGGPGPEVQVPPSRVAPHGRGPG 55 Mus -MFRLLRWRLGRTLLRAAG----RRCGGCTARLLPERTGDAGTGAERLRTRGAP-ARGHG 54 Bos -MYRLLAVGLARGLLRVAG----RRGRCHPVCLLPGPPG-----ARAPPCRGAPRGRGSG 50 Gallus -----MAAALPRALCRLG-----RRCPAGLARPRPAWHGGARRERGGTGTSTDTKSAGRR 50 Danio SYCRQLAAQVFR--LKLCGNECPRNAPGCSFSLAGIKSLHEPTGVLARSSRCVKRWHRRS 62 Drosophila RNICKFTQVMGRFRVVVNP------RDYCHLAPLKRSRYHQRSEFGCRPLCSNAAGYEVS 57 : : * Homo LL-PLLAALAWFSRPAAAEEEE------------QQGADGAAAEDGADEAEAEIIQLLKR 223 Pan LL-PLLAALAWFSRPAAAEEEE------------QQGADGAAAEDGADEAEAEIIQLLKR 223 Pongo LL-PLLAALAWFSRPAAAEEEEE-----------QQGADGAAAEDGADEAEAEIIQLLKR 103 Mus VL-PLLAALAWFSRPAATAEQP--------------------GEDASDEAEAEIIQLLKQ 93 Bos LV-PLLAALAWSSRPAAAEQER-----------------RGAGGAAAGDAEAEIIQLLKR 92 Gallus WARPGGAALGFLGAFSLSPR-----------------------DDEEEDGEDAIILLLKK 87 Danio WH--RCASLQMPSQKSHSDTTDFSRWTLAWGAVAFSLFGGSDEKTEEQKLEDELILLLKK 120 Drosophila WAAPPASGSSGGMFWAFSAAFTLN--------------LFGGADEKEETPEEKLIKTIKR 103 :. : : * :* :*: Homo AKLSIMKDEPEEAELILHDALRLAYQTDNKKAITYTYDLMANLAFIRGQLENAEQLFKAT 283 Pan AKLSIMKDEPEEAELILHDALRLAYQTDNKKAITYTYDLMANLAFIRGQLENAEQLFKAT 283 Pongo AKLSIMKDEPEEAELILHDALRLAYQTDNKKAITYTYDLMANLAFIRGQLENAEQLFKAT 163 Mus AKLSIMKDEPEAAELILHDALRLAYESDNRKAITYTYDLMANLAFIRGQLENAEQLFKAT 153 Bos AKFCIMKDEPEEAELILHDALHLAHKSHNTKAIAYTYDLMANLAFIRGQLENAEKLFKAT 152 Gallus AKLSVMKGELGEAERLLHQALRLAHQADNRRAVIYTYSLMANVAFMQGQLDNAEKLYKAT 147 Danio AKYSMMIGELDAADGFLHRAVRLAHQMHNNDAIIYTYSLMANLAFVRGQLDNAEKLFKAA 180 Drosophila SILCIQREQYDKAEQMLHLALRMAQDIQSKDGITYVFDLMANLAMEREQFKKAEKIFTDV 163 : .: : *: :** *:::* . .. .: *.:.****:*: : *:.:**:::. . Homo MSYLLGGGMKQEDNAIIEISLKLASIYAAQNRQEFAVAGYEFCISTLEEKIEREKELAED 343 Pan MSYLLGGGMKQEDNAIIEISLKLASIYAAQNRQEFAVAGYEFCISTLEEKIEREKELAED 343 Pongo MSYLLGGGMKQEDNAIIEISLKLASIYAAQNRQEFAIAGYEFCISTLEEKIEREKELAED 223 Mus MSYLLGGGMKQEDNAIIEISLKLANIYAAQNKQEFALAGYEFCISTLEGKIEREKELAED 213 Bos MSYLLGGGMQQEDNAIIEISLKLATIYAAQSRQELALAGYEFCISTLEEKIERERELSED 212 Gallus MSSMLAGDTKEDDNAILEMSLKLASIYAAQKQHKLALAGYEFCILTLEEKIAKEKDLPED 207 Danio MSFMLSGGTPQDDNALIEMSLKLASIYATQNKNELAEHGFQFCTDSLEAKMDKQKDLPPE 240 Drosophila MKRLFAEGHTEESPKILHISSKIAHMSQLQGDLEKSFQGFTWTLQQLAKLLEKMP----- 218 *. ::. . ::. ::.:* *:* : * : : *: : * : :


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Homo IMSVEEKANTHLLLGMCLDACARYLLFSKQPSQAQRMYEKALQISEEIQGERHPQTIVLM 403 Pan IMSVEEKANTHLLLGMCLDACARYLLFSKQPSQAQRMYEKALQISEEIQGERHPQTIVLM 403 Pongo IMSVEEKANTHLLLGMCLDACARYLLFSKQPSQAQRMYEKALQISEEIQGERHPQTIVLM 283 Mus IMS-EETANTYLLLGMCLDSCARYLLFSKQLSQAQRMYEKALQICQEIQGERHPQTIVLM 272 Bos VLSADDKANTQLLLGMCLDTYARYLLFSQQPSQAQRMYEKALRISEEILGERHPQTIVLL 272 Gallus VLPAEEKANTRLLLGMSLDSYARYLLDINQLSVAQKMYEKALQISNDVQGETHPQTVVLM 267 Danio SLSDEERKDTRLLLGLSLDARARYLAANHRFIGACRDYRHALQICQEEQGESHPQTLVLM 300 Drosophila -----DDKDILELYGLTKNWFGQLLMKQGKYLEAKNLFKEAFDTLINVYGAVNDASVTIL 273 : : * *: : .: * : * . :..*: : * : ::.:: Homo SDLATTLDAQGRFDEAYIYMQRASDLARQINHPELH-MVLSNLAAVLMHRERYTQAKEIY 462 Pan SDLATTLDAQGRFDEAYIYMQRASDLARQINHPELH-MVLSNLAAVLMHRERYTQAKEIY 462 Pongo SDLATTLDAQGRFDEAYIYMQRASDLARQINHPELH-MVLSNLAAVLMHRERYTQAKEIY 342 Mus SDLATTLDAQGHFDDAYIYMQRASDLAREINHPELH-MVLSNLAAILIHRERYTQAKEIY 331 Bos NDLATTLDTQGRTDEACVHAQRASDLARQVEHPELH-VLLSNLAAVLTHRELYAQAEETY 331 Gallus NDLATVLDAQGHYDEAYSHVKRAAELAKVTQHPEEH-MVLNNLAAILMHKKDFLQAKQVY 326 Danio SDLATVLDLQGKHDEALVYVKKAVELGQAAGHPEQH-VLLGNMAGILMHNGEFEESAKLY 359 Drosophila NNISVAYVNLEKYAEARETLLEAMELTKELKDATQEGILQANLGLVYLREGLMSQAENAC 333 .:::.. : :* .* :* : .. . :: *:. : :. :: : Homo QEALKQAKLKKDEISVQHIREELAELSKKSRPLTNSVKL----- 501 Pan QEALKQAKLKKDEISVQHIREELAELSKKSRPLT---------- 496 Pongo QEALKQAKLKKDEISVQHIREELAELSKKSRPLN-SVKL----- 380 Mus QEALKRAELKRDEVSVQHIREELAELSRKSRRLT---------- 365 Bos REALRQAELKRDEASVQHIREELAELARKSRPLS---------- 365 Gallus KEALKQAEQKGDADSVRHIQEELAELAKRRKGSK---------- 360 Danio QEALALAHTAGDAEAIEQLQEGLKELDNRRNAKDNSKVEDELKE 403 Drosophila RLAWKLGKQHQNPDAVEQAEYCLNEIKTTLNGEKRQ-------- 369 : * .. : ::.: . * *: .

Interspecies alignment of TTC19 protein sequences, obtained by ClustalW software online.

Predicted sequences corresponding to the translatable ORF of human TTC19 protein sequence from

the first predicted methionine (M1) to the second methionine (M122). The M1 is consistently absent

in non-human species, with the exception of chimp (Pan) and gorilla (not shown).

Homo: Homo sapiens, Pan: Pan troglodytes, Pongo: Pongo pygmaeus, Bos: Bos Taurus, Mus: Mus

musculus,Gallus: Gallus gallus, Danio: Danio rerio, Drosophila: Drosophila melanogaster.


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Mutation analysis of h.TTC19 gene in patient #1 (top panel) and #4 (bottom panel).

The wild-type sequences are displayed above the mutant sequences in each panel. The stop codons

TAG and TGA are underlined. The mutant nucleotides are highlighted in yellow in each

electropherogram.


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Quantitative PCR of h.TTC19 on patient #4 genomic DNA

0

0,2

0,4

0,6

0,8

1

1,2

1,4

ex1 ex5 ex10

ctP4

Values reported on y-axis are the relative amount of TTC19 amplicons in comparison to a refence

gene (RNAseP), obtained using specific primers for exons 1, 5 or 10 (ex1, ex5, ex10) of TTC19

gene. Patient#4 (P4) and control (ct) DNA samples were used. Error bars indicate the standard

deviation.


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Subcellular localization of h.TTC19HA in Hela cells

In both fields the HA-specific CRM (in green) does not co-localize with Aurora B (in red, see

arrows), a midbody protein marker.


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Cyt b expression and synthesis in mutant TTC19 myoblasts

(A) Quantitative real-time PCR of mt-cyt b and CoxI mRNA relative to GAPDH mRNA in patient

#2 (P2) and control (Ctrl) myoblasts.

(B) In vitro mitochondrial synthesis and Coomassie staining of control (Ctrl) and patient #2 (P2)

myoblasts.

(C) 2D-BNGE of radiolabeled newly synthesized mitochondrial proteins in control (Ctrl) and

patient #2 (P2) myoblasts.


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Analysis of the insertion of the PiggyBac element within the cg15173 gene (A) and transcript (B).

A

PCR amplification of the cg15173 gene from genomic DNA obtained from wild-type (WT) and

knock-out (KO) flies. Numbers refer to the amplicons reported in Supp Table 5. M: molecular

weight markers ΦX174 (HaeIII). Amplicons #4 and #5 are present only in the KO DNA, since they

contain the 5’ and 3’ insertion points of the PiggyBac element respectively, being obtained using

primer pairs corresponding to a region of the cg15173 gene and of the PiggyBac transposable

element. Amplicon #2 is present only in WT DNA, being obtained by a primer pair corresponding

to 5’ and 3’ regions of the cg15173 gene, flanking the transposable element, which is too big to

produce a detectable fragment from the KO DNA.


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B

PCR amplification of the cg15173 transcript encoding dm.TTC19 from the corresponding cDNA

obtained from wild-type (WT) and knock-out (KO) flies. Numbers refer to the amplicons reported

in Supp Table 5. M: molecular weight markers ΦX174 (HaeIII). Results are identical to those

shown in A and indicate the absence of the normal transcript in the KO fly, which is replaced by an

aberrant transcript that includes the transposable element.


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Subcellular localization of dm.TTC19 protein.

Dm.TTC19 HA Mitotracker MergeDm.TTC19 HA Mitotracker Merge

Confocal IF images of COS7 cells transfected with dm.TTC19HA. The IF signal specific to

dm.TTC19HA is in green, whereas the red signal corresponds to MitoTracker, a specific

mitochondrial marker (Mitotracker Red, Invitrogen).


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Morphology of male spermatids in WT and dm.TTC19 mutant KO flies.

A Bdm wt dm.TTC19 mutant

A Bdm wt dm.TTC19 mutant

Phase-contrast 40x images of 4 spermatids from testes of control and dm.TTC19 null mutant flies.

The two sections are morphologically normal (see main text).


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Electroretinogram in wild-type and dm.TTC19 KO adult aged flies

Electroretinograms recorded extracellularly from 27 day-old adult wild type (A) and dm.TTC19-KO

(B) flies, following white light stimulation. After the onset of a light stimulus, a brief ON transient

positive deflection of the trace, corresponding to lamina neurons activity, is followed by a negative

sustained deflection of the trace, determined by photoreceptor response. At the cessation of the light

stimulus, an OFF transient, expressed as a negative deflection, is followed by a slow recovery to the

pre-stimulus condition. The original responses (3s duration) have been magnified to show both ON

and OFF transients. In both wild-type (A) and dm.TTC19-KO (B) electroretinograms, ON and OFF

transients are equally present. In conclusion, the electroretinogram of the TTC19 KO individual is

comparable to that of the wild-type individual, indicating the integrity of the photoreceptive organ

in both animals.


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Characterization of dm.TTC19 knock-down flies

(a) Levels of dm.TTC19 transcript in control (CTRL) and RNAi knock-down (RNAi) flies. Bars

indicate SD.

(b) Specific activities of MRC complexes I, III and IV (cI, cIII, cIV) in controls (CTRL1= CantonS

and CTRL2= WTALA) and dm.TTC19 RNAi knock-down (TTC19 RNAi) flies, normalized to that

of citrate synthase (CS).


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Supplementary Tables

Supplementary Table 1. Gene prioritization

Symbol HUGO nomenclature Protein accession number AKAP A kinase (PRKA) anchor protein 1 NP_003479.1 C17orf45 chromosome 17 open reading frame 45 Q8N1F1 C17orf76 chromosome 17 open reading frame 76 NP_001107039.1 CORO6 coronin 6 NP_116243.2

DHRS7B dehydrogenase/reductase (SDR family) member 7B NP_056325.2

DRG2 developmentally regulated GTP binding protein 2 NP_001379.1

ERAL1 Era G-protein-like 1 NP_005693.1

SGK494 uncharacterized serine/threonine-protein kinase SgK494 NP_001167574.1

Loc201164 phospholipase D family, member 6 NP_849158.2 Loc201229 chromosome 17 open reading frame 108 NP_001070148.1 NT5M 5',3'-nucleotidase, mitochondrial NP_064586.1

PEMT phosphatidylethanolamine N-methyltransferase NP_009100.2

POLDIP2 polymerase (DNA-directed), delta interacting protein 2 NP_056399.1

TLCD1 TLC domain containing 1 NP_612472.1 TMEM11 transmembrane protein 11 NP_003867.1 TMEM199 transmembrane protein 199 NP_689677.1 TMEM97 transmembrane protein 97 NP_055388.2 TTC19 tetratricopeptide repeat domain 19 NP_060245.2


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Supplementary Table 2. SNP-based haplotypization of the h.TTC19 genea dbSNP rs# Position P1 P3 P4 Heterozygosity rs2041460 intron 4 G G A 54,2% rs3826360a intron 7 T T C 54,2% rs9891938a intron 7 A A G 49,0% rs9899002 intron 7 G G A 53,3% rs28663172 intron 5 G G G 86,0% rs2301652 intron 8 T T G 53,4% rs35168566 intron 9 G G A 54,2% rs73276077 5' utr C C C 82,0% rs73276080 5' utr G G G n.a. rs2302414 5' utr C C C n.a. rs11554355 exon 1 C C C n.a. rs11554352 exon 1 G G G n.a. rs11554354 exon 1 C C C n.a. rs11554353 exon 3 C C C n.a. rs58517927 exon 6 G G G 84,0% rs73981411 exon 9 G G G n.a. rs32077384 exon 10 G G G n.a. rs3744328 3' utr T T T 95,0%

a The intragenic haplotypes of P1 and P3 are identical, strongly suggesting that the same mutant allele is shared among the two families b: Hapmap tag SNP; n.a.: not available; P1, P3, P4: patient 1, 3, 4.


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Supplementary Table 3. Primers used for quantitative RT-PCR of exons 1, 5 and 10 of the h.TTC19

gene.

TTC19 ex 1 Fw: CGGGAGCATGTTCCGG

TTC19 ex 1 Rc: GCCCGCAGGAAGCCTC

TTC19 ex 5 Fw: CTTATGCTTTTAGGCTGAACAACTTTT

TTC19 ex 5 Rc: TCATGCCCCCTCCAAGG

TTC19 ex 10Fw: CTACCAGGAAGCACTGAAGCAA

TTC19 ex 10Rc: TCCCTGATGTGTTGTACAGAAATTTC


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Supplementary Table 4. Primers used for quantitative RT-PCR of the h.TTC19 transcript

TTC19 1Fw: CGAGGCAGAGATCATCCAGC

TTC19 1Rc: CTTCTGGCTCATCTTTCATAATGC

TTC19 2Fw: AACAAGAAGGCCATCACTTACACTT

TTC19 2Rc: CAAGCTGACCCCGTATAAATGC


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Supplementary Table 5. Amplicons and primers used for the analysis of the dm.TTC19 gene

Exon and position relative to the PiggyBac insertion

Seq. left primer Seq. right primer size (cDNA)

size (genomic)

1 Exon1, upstream insertion

TTC19_1F GCAAGTTTACGCAAGTTATGGG

TTC19_1R AGTGAAAGCAGCGGAAAATG

218 218

2 Exon1 flanking insertion

TTC19_2F AGGAAACTCCGGAGGAAAAA

TTC19_2R GGCAAAAAGCCGTTTCATAA

239 239

3 Exon2/3, downstream the insertion

TTC19_3F CCCGACGACAAGGACATACT

TTC19_3R ATGGTTACAGAGGCGTCGTT

164 234

4 Exon1 upstream from and within PiggyBac

WH5' plus TCCAAGCGGCGACTGAGATG

TTC19_1F GCAAGTTTACGCAAGTTATGGG

~500 ~500

5 Exon1 downstream from and within PiggyBac

WH3' plus CCTCGATATACAGACCGATAAAAC

TTC19_2R GGCAAAAAGCCGTTTCATAA

~150 ~150


Documents

SUPPLEMENTARY NOTES, FIGURES AND TABLES Clinical features · cerebellar vermis (arrow). (c) Patient#4: Coronal T2-weighted FLAIR image showing symmetric lesions of the putamina and