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
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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
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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.
2 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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
3 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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
4 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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
Weak Wash: Same as MP A, 600μL Strong Wash: Same as MP B, 200μL Injection Volume: 5μL
Mass spectrometry Instrumentation:
5 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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
6 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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
7 Nature Genetics: doi:10.1038/ng.359
Guernsey et al. Mutations in SLC25A38 cause sideroblastic anemia
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