SUPPLEMENTARY INFORMATION
NBEAL2 is mutated in Gray Platelet Syndrome and required
for biogenesis of platelet alpha-granules.
Meral Gunay- Aygun 1,2*, Tzipora C Falik-Zaccai 3,4*, Thierry Vilboux1, Yifat Zivony-Elboum3, Fatma Gumruk5, Mualla Cetin5, Morad Khayat3, Cornelius F Boerkoel1, Nehama Kfir3 Yan Huang1, Dawn Maynard1, Heidi Dorward1, Katie Berger1, Robert Kleta1, Yair Anikster 6,7, Mutlu Arat8, Andrew S Freiberg9, Beate E Kehrel10, Kerstin Jurk 10, Pedro Cruz 11, Jim C Mullikin11, James G White12, Marjan Huizing 1, William A Gahl1,2
1 Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute National Institutes of Health, Bethesda, MD;
2 Office of Rare Diseases Research, Office of the Director, National Institutes of Health, Bethesda, MD;
3 Institute of Human Genetics, Western Galilee Hospital, Naharia, Israel;
4 Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel;
5 Pediatric Hematology Unit, Hacettepe University Children’s Hospital, Ankara, Turkey;
6 Metabolic Disease Unit, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Aviv, Israel
7 Sackler Medical School, Tel Aviv, Israel
8Department of Hematology, Ankara University Faculty of Medicine, Ankara, Turkey;
9Division of Pediatric Hematology/Oncology, PennState Hershey Children’s Hospital, Hershey, PA;
10Department of Anaesthesiology and Intensive Care, Experimental and Clinical Haemostasis, University Hospital Münster, Münster, Germany;
11NIH Intramural Sequencing Center, NIH, Bethesda, MD;
12Department of Laboratory Medicine, University of Minnesota, Minneapolis, MN;
Supplementary Figure 1: Mutation analysis results of NBEAL2 in 15 GPS patients.
(p) Pathogenicity prediction of NBEAL2 missense variants
Patient / Nucleotide change / Amino acid change / Polyphen / Panther / SiftPSIC / subPSEC / Pdel / Effect/score
GPS-3 / c.1163T>C / p.Leu388Pro / 1.575 / -4.98068 / 0.87875 / Affect/0
GPS-5 / c.5515C>T / p.Arg1839Cys / 2.257 / -6.72405 / 0.97643 / Affect/0
GPS-10 / c.2029T>A / p.Trp677Arg / 3.150 / -6.37715 / 0.96698 / Affect/0
GPS-13 / c.6787C>T / p.His2263Tyr / 1.834 / -6.75049 / 0.97703 / Affect/0.01
GPS-14 / c.5497G>A / p.Glu1833Lys / 1.336 / -3.63828 / 0.65436 / Affect/0
Predicted effect on the protein
(color classifications are arbitrary)
benign / ambiguous / deleteriousLegend to Supplementary Figure 1:
(a) Patient GPS-1 is homozygous for the nonsense mutation c.2701C>T; p.Arg901X (exon 19).
(b) Patient GPS-2 is homozygous for the nonsense mutation c.881C>G; p.Ser294X (exon 8).
(c) Patient GPS-3 is homozygous for the missense mutation c.1163T>C; p.Leu388Pro (exon 11), which has a deleterious missense pathogenicity prediction (see (p)).
(d) Patient GPS-4 is homozygous for the splice site variant c.5720+5G>A (intron 35), predicted to result in an alternative splice donor site 148-bp downstream, confirmed by cDNA sequencing.
(e) Patient GPS-5 is homozygous for the missense variant c.5515C>T; p.Arg1839Cys (exon 34), which has a deleterious missense pathogenicity prediction (see (p)).
(f) Patient GPS-6 is homozygous for the splice site variant c.1296+5G>C (intron 12), predicted to result in an alternative splice donor site 68-bp downstream, confirmed by cDNA sequencing of a different patient with the same variant (GPS-8, see (h)).
(g) Patient GPS-7 is homozygous for a 4-bp frame-shifting deletion c.2257_2260delGCCC; p.Ala753SerfsX65 (exon 16).
(h) Patient GPS-8 is homozygous for the splice site variant c.1296+5G>C (intron 12), resulting in an alternative splice donor site 68-bp downstream, confirmed by cDNA sequencing, as shown.
(i) Patient GPS-9 is homozygous for a 356-bp frame-shifting deletion c.3819_4174del; p.Val1274GlyfsX32 (exon 27).
(j) Patient GPS-10 is homozygous for the missense mutation c.2029T>A; p.Trp677Arg (exon 14), which has a deleterious missense pathogenicity prediction (see (p)). This variant occurs at a splice junction, however it does not seem to affect the splice site prediction score significantly (0.81 wild type sequence versus 0.98 mutant sequence) (http://www.fruitfly.org/cgi-bin/seq_tools/splice.pl). We have no material available of this patient to test this NBEAL2 defect on cDNA.
(k)Patient GPS-11 is homozygous for the 1-bp frame-shifting mutation c.7604delG; p.Gly2535ValfsX5 (exon 50).
(l) Patient GPS-12 is heterozygous for the nonsense mutation c.5505T>G; p.Tyr1835X (exon 34). A mutation in the coding exons and intronic junctions on the other allele could not be identified. No material available of this patient as she passed away, to test NBEAL2 mRNA expression.
(m)Patient GPS-13 is compound heterozygous for the nonsense mutation c.2701C>T; p.Arg901X (exon 19) and the missense mutation c.6787C>T; p.His2263Tyr (exon 42), which has a deleterious missense pathogenicity prediction (see (p)).
(n) Patient GPS-14 is compound heterozygous for the 1-bp frame-shifting deletion c.2156delT; p.Phe719SerfsX100 (exon 16) and the missense mutation c.5497G>A; p.Glu1833Lys (exon 34), which has an overall deleterious missense pathogenicity prediction (see (p)).
(o) Patient GPS-15 is homozygous for the splice site variant c.5301+1G>A (intron 32), resulting in an alternative splice donor site 14-bp downstream, confirmed by cDNA sequencing, as shown.
(p) Pathogenicity prediction results of identified NBEAL2 missense variants using the prediction programs Polyphen, Panther and PMut.
Supplementary Figure 2: SNP-microarray analysis of patient GPS-15.
Supplementary Figure 2: SNP-microarray analysis of patient GPS-15.
Genomic DNA from patient GPS-15 was analyzed on a human 1M-Duo DNA analysis BeadChip (Illumina). Data were analyzed using the GenomeStudio software and showed regions of homozygosity. Shown is the “B allele frequency plot” of chromosome 3, indicating two regions of homozygosity (shown within red accolades), including the 3p21.1-22.1 interval, to which we previously mapped GPSS1.
13
Supplementary Figure 3: NBEAL2 isoform studies.
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Legend to Supplementary Figure 3: NBEAL2 isoform studies.
(a) Schematic rendering of NBEAL2 mRNA transcripts, representing 9 predicted isoforms, as currently published in Ensembl:
(http://uswest.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000160796;r=3:47021173-47051193). Predicted protein length of each isoform (AA, amino acids) is indicated. cDNA amplicons were designed (AMP A-F, indicated as rectangles in top row), which were employed for tissue-specific expression studies.
(b) Table of predicted amplicon size (bp) after cDNA PCR for each NBEAL2 mRNA transcript. Neg = not amplified.
(c) Representative gel images of NBEAL2 tissue-specific expression studies. Shown are gel images of cDNA amplifications of AMP D and AMP F for mRNA extracted from a panel of human hematopoietic cells (indicated above gels). All 3 expected amplicon sizes for AMP D are indicated (152, 276 and 637-bp). Interestingly, isoform transcripts 008 and 009 are described in Ensembl as predicted to undergo nonsense-mediated decay. However, our cDNA experiments showed expression of these transcripts (152, 637-bp bands in selected tissues). Both expected amplicon sizes for AMP F are indicated (177 and 88-bp). Results of all cDNA amplifications are summarized in Supplementary Table 1.
Supplementary Figure 4: Identification of NBEAL2 protein fragments in normal platelets by mass spectrometry.
Legend to Supplementary Figure 4: Identification of NBEAL2 (NBEL2) protein fragments in normal platelets by mass spectrometry.
(a) Sucrose gradient separation of control platelet organelles. Fraction 4 proteins were separated on a 4-12% Tris/glycine gel and stained with Coomassie R-250 stain. Protein bands were excised, reduced, alkylated, and digested with trypsin. Resulting peptides were separated and analyzed by HPLC-MS/MS.
(b) Peptides identified from NBEAL2 (NBEL2) by a Mascot search of Fraction 4 proteins.
(c) MS/MS fragmentation pattern of peptide “AFFAEVVSDGVPLVLALVPHR” from NBEAL2 (NBEL2) (B in Fig. 1g).
Supplementary Table 1: cDNA expression pattern of NBEAL2 isoform transcripts in hematopoietic cells and other tissues
NBEAL2-001 / NBEAL2-201/003 / NBEAL2-203/004 / NBEAL2-202 / NBEAL2-008 / NBEAL2-009 / NBEAL2-011Platelets / + / + / + / + / - / - / -
Megakaryocytes / + / + / + / + / - / - / -
B lymphocytes / + / - / + / + / - / + / +
T lymphocytes / + / + / + / + / + / - / +
Neutrophils / + / + / + / + / + / - / +
Natural Killer Cells / + / + / + / + / + / - / +
Monocytes / + / + / + / + / - / + / +
Liver / + / - / - / + / - / + / -
Fibroblasts / + / - / ND / + / - / - / +
Heart / + / - / + / + / - / + / -
Brain / + / - / - / + / + / + / -
Placenta / + / - / + / + / - / + / +
Lung / - / - / + / + / - / + / +
Skeletal Muscle / + / - / - / + / - / + / -
Kidney / + / - / - / - / - / + / -
ND: No data.
Supplementary Methods
Patients
Between January 2000 and January 2011, a total of 117 individuals (26 patients) from 15 unrelated families were evaluated. The inclusion criterion was platelet EM characteristic for GPS. All patients or their parents gave written informed consent. Sixty individuals from 15 unrelated GPS families were enrolled in the protocol, “The Genetic Analysis of Gray Platelet Syndrome” (www.clinicaltrials.gov, NCT00069680), approved by the NHGRI Institutional Review Board. Sixty individuals from the Bedouin family (GPS-1) were enrolled in the “Clinical and Genetic Analysis of Gray Platelet Syndrome” study approved by the Israeli Supreme Helsinki Committee of the Israeli Ministry of Health. In addition, 25 individuals including, 10 patients from 7 families (Table 1, GPS-1, 2, 4, 5, 11, 13 and 14) who were clinically evaluated at the NIH Clinical Center, were also enrolled in the protocol “Diagnosis and Treatment of Patients with Inborn Errors of Metabolism and other Genetic Disorders.” (www.clinicaltrials.gov, NCT00369421). Detailed clinical data of patients GPS-1-GPS-14 were recently reportedS1. Patient GPS-15 was of Hispanic descent and recently recruited to our protocol. This patient was shown to be homozygous in the 3p21.1-22.1 region (Supplementary Fig. 2).
Bone Marrow Analysis
Posterior iliac crest bone marrow biopsy sections were stained with Reticulin silver stain (Fig. 1e, f). Images were obtained via Olympus BX-51 digital microscope (Olympus America, Melville, NY) equipped with a UPlanFL 20X/0.50 numeric aperture objective and Olympus DP70 digital camera system. Imaging software was Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).
Megakaryocyte Cultures
Under clinical trial NCT00086476 (Investigations of Megakaryocytes from Patients with Abnormal Platelet Vesicles), control megakaryocytes were cultured from CD 34+ cells in serum free liquid medium (X-vivo 20, Cambrex, East Rutherford, NJ) supplemented with recombinant human thrombopoietin (R&D Systems, Inc., Minneapolis, MN) as previously describedS2, and harvested on day 14 and purified using CD61 microbeads and the AutoMacs magnetic cell separator positive selection program (Miltenyi Biotec Inc., Auburn, CA).
Fibroblast Cultures
Primary patients’ and control fibroblasts were cultured from forearm skin biopsies. Fibroblasts were grown in high-glucose (4.5 g/l) DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, MEM nonessential amino acid solution and penicillin-streptomycin.
Electron Microscopy
For platelet EM, blood samples were mixed immediately with citrate–dextrose, pH 6.5, in a ratio of 9:1 blood to anticoagulant. Platelet-rich plasma (PRP) was separated by centrifugation at 100 g for 10 minutes at room temperature. Samples of PRP were fixed, embedded, sectioned, and stained for EM as describedS3 (Fig. 1c, d).
SNP Analysis
Whole genome SNP analysis was performed on genomic DNA of patient GPS-15, using an Illumina Human 1M–Duo DNA Analysis BeadChip and Illumina’s standard hybridization protocol. The data were analyzed using the GenomeStudio software (Illumina, San Diego, CA). Regions of homozygosity were identified based on “B allele frequency” plots (Supplementary Fig. 2).
Whole Exome Sequencing and Variant Analysis
Solution hybridization exome capture was carried out using the SureSelect Human 38Mb All Exon System (Agilent Technologies, Santa Clara, CA), which employs biotinylated RNA baits to hybridize to sequences that correspond to exons. The manufacturer’s protocol version 1.0 compatible with Illumina paired end sequencing was used, with the exception that DNA fragment size and quality were measured using a 2% agarose gel stained with SYBR Gold instead of using an Agilent Bioanalyzer. Flowcell preparation and 101-bp paired end read sequencing were carried out as per protocol for the GAIIx sequencer (Illumina Inc, San Diego CA)S4. A single 101-bp paired-end lane on a GAIIx flowcell was used per exome sample to generate sufficient reads to align the sequence. Image analysis and base calling on all lanes of data were performed using Illumina Genome Analyzer Pipeline software (GAPipeline versions 1.4.0 or greater) with default parameters. Reads were aligned to a human reference sequence (UCSC assembly hg18, NCBI build 36) using the package called Efficient Large-scale Alignment of Nucleotide Databases (ELAND). Genotypes were called at all positions where there were high-quality sequence bases using a Bayesian algorithm called the Most Probable Genotype MPGS5. A graphical java tool, VarSifter, was developed by the NIH Intramural Sequencing Center to view, sort, and filter the variants.
Sanger Sequence Analysis
Data regarding position, coding region sequence, and exon-intron boundaries of genes within the 9.4 megabase interval on 3p21.1-22.3 were obtained from Ensembl Genome Browser (http://www.ensembl.org/), National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/mapview/), and UCSC Genome Browser (http://genome.ucsc.edu/), which were used to design primers. Bidirectional sequencing of the PCR-amplified products was analyzed as describedS6. A first-pass ranking of missense variants by sequence conservation, in conjunction with the identification of nonsense, splice-site, and frame-shift variants, was used to create prioritized listings of sequence variants; further analysis established the likelihood of disease relevance and/or correlation with inheritance pattern.
Missense Variant Analysis
The effect of missense variations on protein function was evaluated using a variety of pathogenicity prediction programsS7, including POLYPHEN (http://genetics.bwh.harvard.edu/pph/; POLYmorphism PHENotyping)S8, PANTHER (http://www.pantherdb.org/; Protein ANalysis THrough Evolutionary Relationships)S9, and SIFT (http://sift.jcvi.org/; Sorting Intolerant From Tolerant)S10. Each missense variant was tested on 100 ethnically matched control individuals. The variants p.Leu388Pro and p.His2263Tyr were tested on a Caucasian DNA panel (Coriell Institute of Medical Research). The variants p.Trp677Arg and p.Glu1833Lys were tested on an African American DNA panel (Coriell Institute of Medical Research). The variant p.Arg1839Cys was tested on a Turkish DNA panel (kindly provided by Dr. Ahmet Gul, Istanbul University, Turkey).