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Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia

SUPPLEMENTARY METHODS

Ascertainment, consent and clinical characteristics of research subjects

Maritime Canadian patients with sideroblastic anemia were identified in the course of routine

clinical care in the Hematology/Oncology Clinic at the IWK Health Centre in Halifax, Nova

Scotia. All individuals were of Acadian descent. Patients (2 males, 1 female) presented at less

than 4 months of age with a severe anemia (hemoglobin 5.1-6.8 g/dL, normal for age is 9.5-

13.5 g/dL), moderate microcytosis (MCV = 60-67 fl, normal for age is 74-108 fl) and an

elevated serum ferritin (513-551ng/mL, normal for age is 40-200 ng/mL). Hemoglobin

electrophoresis and cytogenetics were normal in all of the probands. Bone marrow aspirates

showed ringed sideroblasts on iron stain. None had response to a trial of pyridoxine and all

were transfusion dependent and receiving iron chelation therapy. A fourth male patient was

ascertained, however, he was deceased prior to the study due to cardiac complications of

transfusional iron overload. Other congenital defects were observed in two of the patients;

bilateral club feet and hypospadias in one and atrial and ventricular septal defects in the other;

the remaining two were without other developmental anomalies. Approval for the research study

was obtained from the IWK Research Ethics Board. All sampled family members or parents

provided written informed consent to participate in the study. DNA was obtained from living

patients’ and relatives’ blood samples using routine extraction methods.

Additional clinical data and DNA samples were obtained from a repository of patients referred to

one of us (S.S.B.) for evaluation of SA and/or ALAS2 sequencing under a human subjects

research protocol approved by the University of Oklahoma Health Sciences Center Institutional

Review Board. Samples included in the cohort for SLC25A38 analysis were all negative for

mutations in ALAS2 by intron/exon sequencing. In the SLC25A38 mutation positive group, the

anemia was generally characterized by marked microcytosis and hypochromia, with ringed

sideroblasts in the bone marrow, and an erythrocyte protoporphyrin in the normal range (see

Table 1). Iron overload was evident in all cases, even before transfusion. In most cases the HFE

C282Y and H63D hemochromatosis genotypes were assessed with no significant findings.

Other than the anemia and iron overload, all mutation-positive patients were developmentally

normal. Pyridoxine supplements were of no benefit, and most patients were transfusion

dependent. Four patients (D1, D2, 1A, and 20A) underwent marrow/stem cell transplantation,

which was successful in two cases (1A, 20A). To our knowledge, none of these have been

reported previously in the literature.

1 Nature Genetics: doi:10.1038/ng.359


Genotyping and sequencing

Whole genome SNP scanning was performed on the Maritime kindreds at the McGill University

and Genome Quebec Centre for Innovation, using the Illumina HumanHap300v2_A panel. Data

were scanned using the Bead Array Reader, plate Crane Ex, and Illumina BeadLab software, on

Infinium II fast scan setting. Allele calls were generated using Beadstudio version 3.1 with

genotyping module. Homozygosity was assessed by computational inspection for long runs of

consecutive homozygous SNPs identical by state in the three Maritime Canadian affected

patients, followed by visual inspection of the longest runs for informativeness in unaffected

family members.

For mutation detection, annotated coding exons were amplified from genomic patient DNA by

PCR using standard methods, and sequenced at the McGill University and the Genome Quebec

Centre for Innovation, or at Dalhousie University, using Sanger fluorescent sequencing and

capillary electrophoresis. Sequence traces were analyzed using MutationSurveyor (Soft

Genetics, Inc.) Specific primers and PCR conditions for amplification of SLC25A38 exons and

intraon/exon boundaries are provided in Supplementary Table 2. Short tandem repeat markers

were amplified radioactively and analyzed on 6% denaturing polyacrylamide gels using standard

methodologies.

Phylogenetic analysis and multiple sequence alignments The sequences of 46 human SLC25 proteins and the Yeast orthologues of proteins closely

related to SLC25A38 (Pet8p, Crc1p, YDL119cp) were used in the analyses. The sequences

were aligned with MUSCLE1. Phylogenetic trees were calculated using both maximum likelihood

(Supplementary Figure 2) and neighbor joining methods (data not shown). The results of the

two methods were not substantially different and supported the same conclusion. Maximum

likelihood trees were constructed using PhyML v2.4.42, employing a Jones-Taylor-Thornton

amino acid substitution model, 1000 bootstrap data sets and a relative substitution rate of 4.

Phylogenetic trees were plotted using TreeView3. Multiple sequence alignments were created

with CLUSTAL 2.0.104.

Pathogenicity of missense variants The effects of amino acid substitutions on protein function were predicted with SIFT, PolyPhen,

PANTHER, and Align-GVGD using the protein sequence of human SLC25A38 as the input.



Query options used for PolyPhen prediction are Structural database: PQS; Sort hits by: Identity;

Map to mismatch: No; Calculate structural parameters: For all hits; Calculate contacts: For all

hits; Minimal alignment length: 100; Minimal identity in alignment: 0.1; Maximal gap length in

alignment: 50; Threshold for contacts: 6Å. Multiple sequence alignment (MSA) of protein

orthologues of SLC25A38 MSA1 was used as the input MSA for Align-GVGD. Homologous

protein sequences of human SLC25A38 gene were retrieved from NCBI genome database with

BLASTP.

Zebrafish Slc25a38 orthologue analysis and fish methods

Teleost fish underwent a whole genome duplication following divergence from other vertebrates,

with duplicate genes in the process of being eliminated, thus some human genes retain two

separate active orthologues in fish whereas others have only one per haploid gene. Two

potentially active zebrafish SLC25A38 orthologues were identified by BLAST analysis of the D.

rerio genome and EST clones in public databases. Putative orthologues were found on fish

chromosomes 3 (drSLC25A38a) and 6 (drSLC25A38b). For orthologue a,

ENSDARESTG00000010168 appears to represent a full length cDNA clone; for orthologue b,

no full length clone was identified, but the structure could be assembled from three partial

cDNA clones and the genomic sequence and was consistent with functionality.

Wild-type zebrafish (Danio rerio) were raised and mated using routine procedures. Antisense

morpholinos (AMO) were designed and synthesized by the supplier (Gene Tools, Corvallis, OR,

USA). AMOs targeted to the region immediately at the translational start site, with sequences 5′-

CCGGATGAGCCACAGAGAACTCCAT-3′ for drSLC25A38a and 5′-

CAGGATGAGCCAGGGCAACTTCCAT-3′ for drSLC25A38b. ~1.5nl of AMOs were injected by

using a sharp electrode mounted on a pipette holder hooked to a picospritzer at serial

concentrations (1mM, 0.5mM, 0.25mM and 0.125mM) to determine the maximum dose that did

not result in overt developmental abnormalities during the first several days of embryonic

development. An AMO blocking the zebrafish aminolevulinic acid synthase 2 gene (drALAS2, 5′-

CAGTGATGCAGAAAAGCAGACATGA-3′ was used as a positive control.

Fish were injected at 1 hour post-fertilization (hpf). At 20 to 22 hpf, the water was supplemented

with 0.002% PTU (1-phenyl-2-thiourea) to prevent background melanin pigment synthesis

(reducing the background for heme staining). At 48 hpf, the dechorionated embryos were

stained in the dark for 15 min in o-dianisidine solution (0.6 mg/ml, Sigma D9143), 10mM Sodium



Acetate pH 4.5, 0,65% H2O2 and 40% ethanol), and then fixed in 4% parafomaldehyde (in

PBS) overnight at 4°C. Embryos were dehydrated in ethanol (70% for 30min; 95% for 60min

and 100% for 60min) and cleared in Benzylbenzoate/Benzyl Alcohol (2:1) until they sank, and

were finally mounted with Permount.

Yeast methods

Yeast strains and growth conditions. The parental yeast strain BY4741 MATa his3Δ1 leu2Δ0

met15Δ0 ura3Δ0 and the isogenic deletion strain ydl119cΔ::MX4 were obtained from Open

Biosystems (Huntsville, AL). Yeast were maintained on enriched yeast-peptone medium with

2% glucose (YPD) or in synthetic defined medium with yeast nitrogen base, essential amino

acids, and supplemented with 2% glucose (SD). Where indicated, 3% glycerol was substituted

for glucose in the synthetic defined medium (SG), and sodium nitroprusside (35 mM), 5-

Aminolevulinic Acid (ALA, 50mg/L) and Glycine (5 mM) were added as supplements.

Plasmids. The CEN expression vector pPJS209 was constructed by liberating the

phosphoglycerate kinase (PGK) promoter sequence from pSM703 (gift of Dr. Val Culotta, Johns

Hopkins School of Public Health) at the HindIII and BamHI sites and integrated into the same

sites in vector pRS416. The YDL119c expressing vector pPJS211 was constructed by

amplifying the entire coding sequence of YDL119c using primers engineered with an EcoRI site

immediately upstream of the start codon and a BamHI site immediately past the stop codon.

This fragment was integrated at these same sites in pPJS209.

Sample Preparation for Metabolite Analysis. Metabolite extractions were prepared through a

slight modification of a previous method described by Villas-Boas5. Triplicate yeast cultures

were grown overnight shaking in SD media at 300 C to OD600 = 3.5, and sample cultures were

quenched by quickly adding 10ml of overnight cultures to 40ml of methanol-water solution (60%

v/v) chilled in an ethanol-dry ice bath. Cells were harvested (-100C) for 5 min at 1540 x g, and

the pellets were re-suspended in 3ml chilled 100% methanol. A 1ml aliquot of this suspension

was snap frozen in liquid nitrogen and subsequently thawed in an ice-bath. The suspension was

then centrifuged at 770 x g (-200C) for 20 minutes and the supernatant was collected. An

additional 0.5 ml of chilled 100% methanol was added to the pellet and vortexed for 30 seconds.

This suspension was again harvested at 770 x g (-200C) for 20 minutes and both supernatants



were pooled. Samples were transferred to glass tubes and dried under nitrogen gas for 60

minutes at 300C.

ALA and glycine mass spectrometry Dried yeast metabolites were reconstituted in mobile phase A (see below) and quantitated using

LC/MSMS (Quattro Premier, Waters Inc, MA). An internal standard (D3-arginine) was added to

the samples to normalize for recovery and samples are ionized using an electrospray source in

positive mode. The Q1/Q3 transition for aminolevulinic acid (ALA), glycine were monitored at

m/z 131.97/113.8 and 75.77/29.9, respectively. The two transitions for the internal standard

were monitored at 181.33/46 and 181.33/74.

Liquid chromatography Instrumentation :

LC Instrumentation Waters Acquity UPLC Column ACQUITY UPLC™ BEH C18 Column, 2.1 x 50 mm, 1.7 μm Column Temp 45˚C Sample Temp 5˚C Flow rate 0.8 mL/min Mobile Phase A 99.5%/0.5% water/acetonitrile

0.1% formic acid 0.1% pentadecafluorooctanoic acid

Mobile Phase B 10%/90% water/acetonitrile 0.1% formic acid 0.1% pentadecafluorooctanoic acid

Gradient Table

Time(min) Flow Rate %A %B Curve Initial 0.800 100.0 0.0 - 0.50 0.800 98.0 2.0 6 2.00 0.800 80.0 20.0 6 4.00 0.800 60.0 40.0 6 4.50 0.800 0.0 100.0 6 6.50 0.800 100.0 0.0 1

Weak Wash: Same as MP A, 600μL Strong Wash: Same as MP B, 200μL Injection Volume: 5μL

Mass spectrometry Instrumentation:



MS Instrument Waters Quattro Premier Capillary voltage: 0.8kV LM 1 Resolution 13.0

HM 1 Resolution 14.0 Ion Energy 1 0.1

Cone: 20.00V Extractor: 3.00V Entrance 0 -22

Collision 8 -22 Exit 1 -24

RF Lens: 0.3V LM 2 Resolution 12.5 HM 2 Resolution 15.0 Ion Energy 2 1.0

Source Temperature: 140°C Multiplier (V) 650 Cone Gas Flow: 45 L/Hr Syringe Pump Flow (uL/min): 10.0 Desolvation Temperature: 400°C Gas Cell Pirani Pressure(mbar): 6.01e-3 Desolvation Gas Flow: 1002 L/Hr

Metabolite-specific data acquisition:

Arginine (internal standard)

Scans in function 652 Cycle time (secs) 0.525 Inter Scan Delay (secs) 0.00 Span (Da) 46.00 Retention window (mins) 0.000 to 7.000 Ionization mode ES+ Data type SIR or MRM data Function type: MRM of 2 channels Chan Reaction 1) 181.33 > 46.00

2) 181.33 > 74.00 Dwell(secs) 1) 0.250

2) 0.250 Cone Volt. 1) 25.0

2) 25.0 Col.Energy 1) 35.0

2) 35.0 Delay(secs) 1) NA

2) NA

Aminolevulinic acid (ALA)

Scans in function 651 Cycle time (secs) 0.055 Inter Scan Delay (secs) 0.00 Span (Da) 138.00 Retention window (mins) 0.000 to 7.000 Ionization mode ES+ Data type SIR or MRM data Function type: MRM of 1 channel Chan Reaction 131.90 > 113.80 Dwell(secs) 0.050 Cone Volt. 17.0

Col.Energy 13 Delay(secs) NA

Glycine

Scans in function 651 Cycle time (secs) 0.055



Inter Scan Delay (secs) 0.00 Span (Da) 29.00 Retention window (mins) 0.000 to 7.000 Ionization mode ES+ Data type SIR or MRM data Function type: MRM of 1 channel Chan Reaction 75.70 > 29.90 Dwell(secs) 0.050 Cone Volt. 24.0

Col.Energy 7.0 Delay(secs) NA



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Supplementary Figure 1A. Congenital sideroblastic anemia pedigrees and local haplotypes

surrounding SLC25A38.Short tandem repeats markers spanning an ~12 Mb interval (36.14 Mb-

48.57 Mb, according to the Ensembl human sequence Release 50, www.ensembl.org) on

chromosome 3 are shown in the left-most column. For each family, the SLC25A38 mutated and

unmutated maternal haplotypes are in pink and yellow, respectively. The SLC25A38 mutated

and unmutated paternal haplotypes are in green and blue, respectively. Colors do not

automatically denote shared haplotypes between families. The allele sizes of single probands

for whom parental DNA was unavailable are shown, without implying a haplotype, in lilac. The

three families in the upper left are from the Canadian Maritimes and were used for initial

chromosomal mapping studies.



L108fs (exon 4): 2 base pair deletion (CT) at (Ch3:39,407,983 – 39,407,984) DNA sequence: TCC ATT GTG AGA TGT GTC CCT GGC GTT GGA ATC TAC TTT GGC ACT CTC TAC TCT TTG AAG CAG TAT TTC TTG CGA GGC CAT CCC CCA ACC GCC CTG GAG TCA GTC ATG CTG GGG GTG GGC TCT CGC TCT GTT GCA GGG GTC TGT ATG TCA CCT ATC ACT GTA ATC AAG ACG CGC TAT GAG Reference amino acid sequence: SIVRCVPGVGIYFGTL YSLKQYFLRGHPPTALESVMLGVGSRSVAGVCMSPITVIKTRYE Mutant amino acid sequence: SIVRCVPGVGIYFGTL(del) LFEAVFLARPSPNRPGVSHAGGGLSLCCRGLYVTYHCNQDAL*



R117X (exon 4): 8667 C>T, 117R>X at (Ch3: 39,408,008) TCC ATT GTG AGA TGT GTC CCT GGC GTT GGA ATC TAC TTT GGC ACT CTC TAC TCT TTG AAG CAG TAT TTC TTG CGA GGC CAT CCC CCA ACC GCC CTG GAG TCA GTC ATG CTG GGG GTG GGC TCT CGC TCT GTT GCA GGG GTC TGT ATG TCA CCT ATC ACT GTA ATC AAG ACG CGC TAT GAG



ivs3-1G>A: 8593 A>G at (Ch3: 39,407,934; Just before the Exon 4; Consensus splice site mutation)



K112fs (exon 4): 11 base pair deletion (GCAGTATTTCT) at (Ch3:39,407,995 – 39,408,005) DNA sequence: TCC ATT GTG AGA TGT GTC CCT GGC GTT GGA ATC TAC TTT GGC ACT CTC TAC TCT TTG AAG CAG TAT TTC TTG CGA GGC CAT CCC CCA ACC GCC CTG GAG TCA GTC ATG CTG GGG GTG GGC TCT CGC TCT GTT GCA GGG GTC TGT ATG TCA CCT ATC ACT GTA ATC AAG ACG CGC TAT GAG Reference amino acid sequence: SIVRCVPGVGIYFGTLYSL KQYFLRGHPPTALESVMLGVGSRSVAGVCMSPITVIKTRYE Mutant amino acid sequence: SIVRCVPGVGIYFGTLYSL(del) NARPSPNRPGVSHAGGGLSLCCRGLYVTYHCNQDAL*



G130E (exon 4): 8707 G>GA, 130 G>G/E at (Ch3: 39,408,048)



R134H (exon 4): 8719 G>GA, 134 R>R/H at (Ch3: 39,408,060)



R187P (exon 5): 9110 G>GC, 187 R>R/P at (Ch3: 39,408,451)



D209H (exon 5): 9175 G>GC, 209 D>D/H at (Ch3: 39,408,516)



K264X (exon 6): 11728 A>T, 264 K>X at (Ch3: 39,411,069)



Y293X (exon 7): 13638 T>TG, 293 Y>Y/X at (Ch3: 39,412,979)



X305R (exon 7): 13672 T>TC, 305 X>X/R at (Ch3: 39,413,013)

Supplementary Figure 1B. Mutations in CSA patients. Ideogram depicting the domain

structure of human SLC25A38 and the mutations identified in CSA patients in this

cohort. MLS, mitochondrial localization signal; MCF, mitochondrial carrier family

domain. *Denotes a recurrent mutation found in more than one family or individual

patient. Screenshots from MutationSurveyor with nucleotide and predicted translation

mutations.



Supplementary Figure 2. Tissue specific expression profile of SLC25A38 according to GNF

BioBGPS (http://symatlas.gnf.org/SymAtlas/ ).


http://symatlas.gnf.org/SymAtlas/


Supplementary Figure 3A. Multiple sequence alignment of putative SLC25A38

orthologues.Alignment performed using MSA1. Amino acids are shaded according to sequence

conservation.



Supplementary Figure 3B. SLC25A38 comparative genomic alignment and structure

annotated with CSA missense mutations. In this comparative genomic alignments, created with

WebLogo 3 (weblogo.berkeley.edu) the height of the residue reflects greater evolutionary

conservation. The four missense mutations found in CSA patients in this cohort are indicated in

red above the sequence. The tandem repeated hinge motifs (H1~H3) of mitochondrial carriers

are indicated with black lines. The proposed ligand binding sites or “contact points” (CPI, CPII,

and CPIII) are indicated with red lines.



Supplementary Figure 3C. Potential model of three-dimensional structure of human

SLC25A38 and evolutionary conservation of the residues with missense mutations. The

color scale range from red to blue represent the conservation scores from 1-most

variable to 9-most conserved. The color code for residues with missense mutations

G130, R134, R187, and D209 are 9, 9, 9, and 1, respectively. Surface-mapping of

phylogenetic information was done with ConSurf; view is from the cytosolic side.



H.s. SLC25A38 MIQNSRPSLLQPQDVGDTVETLMLHPVIKAFLCGSISGTCSTLLFQPLDLLKTRLQTLQP 60 S.c. YDL119c -----------------MTEQATKPRNSSHLIGGFFGGLTSAVALQPLDLLKTRIQQDK- 42 D.r. slc25a38a ------------------MEFSVAHPAVKAFMCGSLSGTCSTLLFQPLDLVKTRLQTLHS 42 D.r. slc25a38b ------------------MEVALAHPALKAFMCGSLSGTCSTLLFQPLDLVKTRLQTLQN 42 * . :: * :.* *:: :*****:***:* : H.s. SLC25A38 SDH-GSRRVGMLAVLLKVVRTESLLGLWKGMSPSIVRCVPGVGIYFGTLYSLKQYFLRGH 119 S.c. YDL119c ----------KATLWKNLKEIDSPLQLWRGTLPSALRTSIGSALYLSCLNLMRSSLAKRR 92 D.r. slc25a38a GVQPGTGRVGMVTVFVNVLRTEKLLGLWRGVSPSFVRCIPGVGIYFSTYFTLKQHYFSSG 102 D.r. slc25a38b NMHPGAPKVGMITVLFNVIRTEKLLGLWKGVSPSFMRCIPGVGIYLSTFYSLKQHYFQEG 102 :: :: . :. * **:* ** :* * .:*:. ::. H.s. SLC25A38 ---------------------PPTALESVMLGVGSRSVAGVCMSPITVIKTRYESGKYGY 158 S.c. YDL119c NAVPSLTNDSNIVYNKSSSLPRLTMYENLLTGAFARGLVGYITMPITVIKVRYESTLYNY 152 D.r. slc25a38a ---------------------APGPLQAVLLGAGARCVAGVFMLPVTVIKTRFESGRYRY 141 D.r. slc25a38b ---------------------SPSAGEAVLLGAGARCVAGVAMLPFTVIKTRFESGRYNY 141 : :: *. :* :.* *.****.*:** * * H.s. SLC25A38 ESIYAALRSIYHSEGHRGLFSGLTATLLRDAPFSGIYLMFYNQTKNIVPHDQVD------ 212 S.c. YDL119c SSLKEAITHIYTKEGLFGFFRGFGATCLRDAPYAGLYVLLYEKSKQLLPMVLPSRFIHYN 212 D.r. slc25a38a SGVFGALRSVCQTEGPKALFSGLMATLLRDAPFSGIYVMIYSQTKNLLPPEISQ------ 195 D.r. slc25a38b ISVAGALKSVCQNEGPKALYSGLTATLLRDAPFSGIYVMFYSQAKKALPQEISS------ 195 .: *: : .** .:: *: ** *****::*:*:::*.::*: :* . H.s. SLC25A38 ------ATLIPITNFSCGIFAGILASLVTQPADVIKTHMQLYPLKFQWIGQAVTLIFKDY 266 S.c. YDL119c PEGGFTTYTSTTVNTTSAVLSASLATTVTAPFDTIKTRMQLEPSKFTNSFNTFTSIVKNE 272 D.r. slc25a38a ------SSYAPVANFSCGVLAGVLASVLTQPADVVKTHIQVSPDVFSRTSDVVRYIYKEH 249 D.r. slc25a38b ------SSIAPLVNFGCGVVAGILASLATQPADVIKTHMQVSPALYPKTSDAMRHVYVKH 249 : . .* ..:.:. **: * * *.:**::*: * : :.. : . H.s. SLC25A38 GLRGFFQGGIPRALRRTLMAAMAWTVYEEMMAKMGLKS 304 S.c. YDL119c NVLKLFSGLSMRLARKAFSAGIAWGIYEELVKRFM--- 307 D.r. slc25a38a GLVGFFRGAVPRSLRRTMMAAMAWTVYEQLMAQIGLKS 287 D.r. slc25a38b GLSGFFRGAVPRSLRRTLMAAMAWTVYEQLMARMGLKS 287 .: :* * * *::: *.:** :**::: :: ***

Supplementary Figure 4. CLUSTAL 2.0.10 multiple sequence alignment

(http://www.ebi.ac.uk/Tools/clustalw2/index.html) of the human (H.s.) SLC25A38 protein with its

putative yeast (S.c. YDL119C.) and zebrafish (D.r. slc25a38a and D.r. slc25a38b) orthologues.

(:) and (*) denote amino acid similarity and identity, respectively. The RD dipeptide in contact

point II (CPII) is highlighted in red.



SLC25A19/dNDPs

SLC25A16/CoA

SLC25A31/ADP

SLC25A4/ADP

1000

680801

SLC25A30/KMCP1

SLC25A27/UCP4

849

SLC25A7/UCP1

SLC25A10/Malate

SLC25A11/Oxoglutarate

825

583

1000

199

SLC25A21/Oxoadipate

SLC25A1/Citrate

346

SLC25A22/Glutamate

SLC25A18/Glutamate

1000SLC25A13/Aspartate GlutamateSLC25A12/Aspartate Glutamate

1000998

152

Ydl119cp

SLC25A38

1000

SLC25A26/S-adenosylmethionine

Pet8p

1000

SLC25A37/Fe2+SLC25A28

1000

624

231

90

SLC25A3/Phosphate

SLC25A17/ATP

285

Crc1p

SLC25A20/Carnitine

507SLC25A2/Ornithine

SLC25A15/Ornithine1000998

Supplementary Figure 5A. Unrooted phylogenetic tree with bootstrap values of the selected

Human SLC25 family members and Yeast orthologs calculated using PhyML. The phylogenetic

tree was plotted using TreeView. These data support the assignment of YDL119C as the yeast

orthologue of human SLC25A38.



Supplementary Figure 5B. The unrooted tree of the 46 human SLC25 family members

calculated using PhyML. Clusters with high confidence are shown in red. The phylogenetic tree

was plotted using MEGA.



#SNPs Chr BeginSNP EndSNP Begin End Size (bp) 299 3 rs7624701 rs1918029 36,801,155 39,823,345 3,022,191 61 3 rs16810 rs3843370 42,546,733 43,195,853 649,121 59 15 rs900543 rs2277598 69,884,515 70,814,531 930,017 51 5 rs981782 rs11948152 45,321,475 45,990,384 668,910 40 5 rs2436396 rs155806 139,513,747 140,330,024 816,278

Supplementary Table 1. Homozygosity mapping of congenital sideroblastic anemia. The 5

longest runs of homozygosity sorted by the number of consecutive homozygous SNPs shared

by the three affected Maritime patients. Positions are based on the March 2006 human genome

assembly, build 36.1.



CSBA (SLC25A38) primers Exon

Forward primer

Reverse primer Product

size Anneal. temp. Mg

E01 TCTACAGAGTTCCTCCGGC AAAGGGTAGCCGAGCCTTAG 520 60 1.5

E02

GCTGGTCAGGTATAGAGAAAGG CATCCAACAGAATGGAAGTTG 289 60 1.5

E03

TTGAGTGGGGAATTGTTTTATG TCTCACATATCCTTAAGAGCTGG 255 60 1.5

E0405 CTTTTGGGAAAACCCAGC TCATATCCCAGAGAAAATGGTG 733 60 1.5

E06 GGAAGAATTGGTGGGCAAC GAGTGAAGGGTAAGAACTACTGCT

C 329 60 1.5

E07 CACCTACTCGGAAGGCTGAG GAAGGCAAAGGCAACACAAT 512 60 1.5

*E04 (F1)

CTTTTGGGAAAACCCAGC (R1) CAGGAGCCCAGTGGCTAAG 314 60 1.5

(F2) CTTGCATGCGAATCATCTTG

(R2) CAGGAGTTGACATCGGTGG 365 60 1.5

*E05

CTGCAGTCTGCTTGTTCAGTG TCATATCCCAGAGAAAATGGTG 315 60 1.5

**E07 (F2) GTTGTCTCCTTGGACCCATT

Supplementary Table 2. Primer sequences used for sequence analysis of SLC25A38. Exon

number corresponds to Ensembl transcript ENST00000273158 and gene ENSG00000144659.

PCR conditions are available upon request.

* Initially, two sets of primers were designed for the exon 4. Because the exons 4 and 5 are

located close together, they were amplified with E04-F1 and E05-R and obtained 733-bp

amplicon.

** E07-F2 was designed inside of T-repeats to obtain better forward sequence.


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