Subjects, sequencing, and genotyping

Whole-exome sequence data were generated for 457 AIS European American cases and 987 European American controls for the discovery cohort. For all AIS cases, spinal radiographs were used for diagnosis or Cobb angle was abstracted from clinical records, if unavailable. Exome sequenced cases were included only if Cobb angle > 20. The mean Cobb angle of cases was 52. Controls were not evaluated for scoliosis, and were therefore unselected for presence or absence of scoliosis. Informed consent was obtained for all subjects. IRB approval was obtained for this study by each contributing institution. Exome capture was performed using Agilent All-exome capture kits. Sequencing was performed using paired-end Illumina sequencing to a minimum depth of 30x with >90% of the captured regions covered with at least 8× read depth. Sequencing was performed at the McDonnell Genome Institute at Washington University in St. Louis. Alignment was performed using Novoalign and variant called using SAMTOOLS. For the expanded control cohort, all non-Finnish European individuals from the Exome Aggregation Consortium (ExAC) were used as control samples (N = 34,057). All genotypes, including ExAC genotyping counts, were processed excluding SNPs with call rates < 0.95, minor allele frequency < 0.01 or Hardy–Weinberg equilibrium P-values < 10−5. As a replication cohort, 857 AIS cases and 1095 controls from seven sites (Washington University in St. Louis, Shriners Hospital for Children in St. Louis, University of Iowa, Hospital for Special Surgery, University of Wisconsin, Texas Scottish Rite Hospital and University of Colorado) and were genotyped for rs13107325 using either Affymetrix 6.0 genotyping array or KASPAR genotyping (Supplementary Table 1). A subsample was checked for consistency (N = 100) and there was complete concordance between genotyping methods. This was also done for exome sequenced individuals to ensure accuracy and we see 100% concordance in this group as well. (Supplementary Data 1). We performed power calculations using the genetic power calculator available here: [http://zzz.bwh.harvard.edu/gpc/]. Given our sample sizes, this study had >80% power to detect associations with OR > 1.5 and MAF > 0.07.

Statistical analysis

For the discovery exome association analysis, expanded exome analysis, replication analysis and combined analysis, associations between genotype and case/control status were assessed using logistic regression in PLINK37. Regional association plots were generated using SNAP [https://www.broadinstitute.org/mpg/snap/]. Association with curvature severity was assessed using linear regression with allele count in R. For associations with height, body-mass index, plasma manganese and plasma iron concentrations, t-tests were performed in R. For height, the difference of measured height from CDC reported averages for age-matched and sex-matched individuals was reported [http://www.cdc.gov/growthcharts/]. Ancestry was verified for all AIS cases and in-house controls using EIGENSTRAT38 (Supplementary Fig. 6) and individuals were confirmed to be unrelated using IBD estimation in PLINK37.

In vitro metal ion uptake assays

Human SLC39A8 cDNA was obtained from GE Dharmacon (GenBank® accession number BC012125; Catalog no. MHS6278-202832132) and subcloned into a pCMV-Sport6 vector. To generate the A391T (rs13107325) SLC39A8, the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies) was used to mutate the alanine residue at position 391 to threonine. The mutant construct was subsequently verified by Sanger sequencing. HEK 293T cells (ATCC) were transiently transfected with human SLC39A8, the A391T SLC39A8 variant or empty vector pCMV-Sport6 (Effectene; Qiagen) and incubated for 48 h. Prior to uptake, the cells were washed twice with warm serum-free DMEM medium (for iron uptake) or HBSS medium (for zinc/manganese uptake) and incubated for 1 h in serum-free medium (SFM) containing 2% (w/v) BSA to deplete cells of transferrin and to block the nonspecific binding at 37 °C. For iron uptake, the cells were incubated with 2 µM [59Fe]ferric citrate in SFM in the presence of 1 mM l-ascorbate for 2 h at 37 °C followed by three washes of iron chelator solution (1 mM bathophenanthroline sulfonate and 1 mM diethylenetriaminepentaacetic acid) to remove any surface-bound iron. For zinc and manganese uptake, cells were incubated with either 2 µM [65Zn]ZnCl 2 or 2 µM [54Mn]MnCl 2 in SFM for 2 h at 37 °C followed by three washes of chelator solution (10 mM ethylenediaminetetraacetic acid). The cells were lysed in buffer containing 0.2 N NaOH and 0.2% (w/v) SDS. Radioactivity (as counts per minute; CPM) was determined by γ-counting, and protein concentration was determined colorimetrically by using the RC DC protein assay (Bio-Rad). Transport was normalized to SLC39A8 protein level as measured by western blot. Data are expressed as CPM per mg protein and represent mean ± SE of three independent experiments.

Western blotting of wild-type and A391T SLC39A8

HEK 293T cells were transiently transfected with SLC39A8 constructs as described above. Cells were washed and lysed in RIPA lysis buffer containing 1× protease inhibitor (Roche). Protein concentration was determined by using the RC DC protein assay (Bio-Rad) prior to western blot analysis. Proteins (15 µg) were mixed with Laemmli buffer and electrophoretically separated on a 7.5% polyacrylamide gel, and transferred to a nitrocellulose membrane. The blots were blocked for 1 h in 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST). The blots were then incubated overnight at 4 °C in blocking buffer containing rabbit anti-hZIP8 (0.05 µg/ml; Sigma; Catalog no. HPA038833) or mouse anti-tubulin (1:10,000; Sigma). The membranes were washed four times for 5 min with TBST and incubated with the appropriate HRP-conjugated secondary antibodies. Immunoreactivity was visualized by using enhanced chemiluminescence (SuperSignal West Pico; Pierce) and the FluorChem E digital darkroom (ProteinSimple).

CRISPR-mediated slc39a8 disruption in zebrafish

Briefly, gRNAs designed to target exon 7 of the slc39a8 gene were synthesized in vitro and microinjected with codon-optimized Cas9 mRNA into one-cell-stage embryos of the AB* zebrafish strain. F 0 founders were identified with Illumina sequencing of F 1 embryos. F 1 mutants were confirmed by Sanger sequencing. In one line used in this study, a 17-bp insertion and 2-bp deletion was detected on one allele at the slc39a8 locus. Movement was measured using the Noldus DanioVision and data analyzed using EthoVision XT12 software package. Comparisons of movements were performed using t-tests. Movement was defined as reaching a velocity of 5 mm/s and was measured until fish slowed to 1 mm/s. Approval was obtained for this study by the Washington University institutional review board. For all experiments, both male and female zebrafish were used.

Alizarin Red staining of zebrafish

Zebrafish were first fixed in 4% phosphate buffered formalin for 48 h. After rinsing with deionized water, they were dehydrated by in 50% ethanol for 48 h followed by 95% ethanol for 48 h. Fish were then stained using a filtered solution of 20 mg Alcian blue in 30 ml acetic acid and 70 ml 100% ethanol for 24 h. After staining, fish were neutralized in a saturated sodium borate solution for 12 h and bleached in a solution of 15 ml 3% hydrogen peroxide and 85 ml 1% potassium hydroxide for 3 h, cleared in a solution of 35 ml saturated sodium borate and 65 ml deionized water for 7 days, stained for bone using a solution of 1% w/v Alizarin red in 1% potassium hydroxide for 24 h and cleared again in 3 day intervals using alternating solutions of 35% saturated sodium borate and 1% potassium hydroxide until cleared (>4 weeks). The number of animals stained was determined to ensure adequate power to detect altered rates of skeletal phenotypes in KO vs WT. For each genotype, fish were selected randomly for staining and assessment. Three independent blinded assessors of skeletal phenotypes were used to determine the relationship between genotype and phenotype.

Whole-mount in situ RNA hybridization (WISH)

To synthesize RNA probes for slc39a8 gene, we first amplified 833 bp of slc39a8 cDNA by RT-PCR using 4 dpf embryos (Supplementary Table 2). The slc39a8 cDNA fragment was re-amplified by PCR to insert T7 RNA polymerase promoter and subcloned into the pSMART vector (CloneSmart HCKan Blunt Cloning Kit, Lucigen). After sequence confirmation, two plasmids (one for anti-sense and the other for sense slc39a8 RNA probe) were linearized with NotI. From the linearized DNAs, we synthesized both anti-sense and sense RNA probes using a digoxigenin RNA labeling kit (Roche). WISH was performed as described previously39 with several modifications. We used a higher concentration of Proteinase K (25 μg/ml) and treated embryos for 80 min (from 50 to 80 min PK treatment showed similar results). We used a higher probe concentration (200 μg/ml). Lastly, we added 5% of Dextran sulfate (Sigma-aldrich) to the hybridization mix40.