Introducing high-throughput sequencing into mainstream of genetic diagnosis practice in inherited platelet disorders

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Published Ahead of Print on October 5, 2017, as doi:10.3324/haematol.2017.171132. Copyright 2017 Ferrata Storti Foundation.

1 Published Ahead of Print on October 5, 2017, as doi: /haematol Copyright 2017 Ferrata Storti Foundation. Introducing high-throughput sequencing into mainstream of genetic diagnosis practice in inherited platelet disorders by José M. Bastida, María L. Lozano, Rocío Benito, Kamila Janusz, Verónica Palma-Barqueros, Mónica Del Rey, Jesús M. Hernández-Sánchez, Susana Riesco, Nuria Bermejo, Hermenegildo González-García, Agustín Rodriguez-Alén, Carlos Aguilar, Teresa Sevivas, María F. López-Fernández, Anna E. Marneth, Bert A. van der Reijden, Neil V. Morgan, Steve P. Watson, Vicente Vicente, Jesús M. Hernández-Rivas, José Rivera, and José R. González-Porras. Collaborative Groups: On behalf of the Project (Jose Maria Bastida, Jose Rivera, Maria L. Lozano), On behalf of the Group of the Castilla y Leon Society of Thrombosis and Haemostasis (Jose Ramon Gonzalez-Porras) Haematologica 2017 [Epub ahead of print] Citation: Bastida JM, Lozano ML, Benito R, Janusz K, Palma-Barqueros V, Del Rey M, Hernández-Sánchez JM, Riesco S, Bermejo N, González-García H, Rodriguez-Alén A, Aguilar C, Sevivas T, López-Fernández MF, Marneth AE, van der Reijden BA, Morgan NV, Watson SP, Vicente V, Hernández-Rivas JM, Rivera J, and González-Porras JR. Collaborative Groups: On behalf of the Project (Jose Maria Bastida, Jose Rivera, Maria L Lozano), On behalf of the Group of the Castilla y Leon Society of Thrombosis and Haemostasis (Jose Ramon Gonzalez-Porras). Introducing high-throughput sequencing into mainstream of genetic diagnosis practice in inherited platelet disorders. Haematologica. 2017; 102:xxx doi: /haematol Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.

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39 Introducing high-throughput sequencing into mainstream of genetic diagnosis practice in inherited platelet disorders. Bastida et al. Supplemental Methods Blood sampling, platelet phenotyping and DNA isolation Venous blood was drawn from each patient into commercial 7.5% K3 EDTA tubes (for complete blood count [CBC], and DNA isolation), and into buffered 3.2% sodium citrate for platelet studies. Platelet function testing was performed mainly in citrated plateletrich-plasma. Assays included, as appropriate, blood smears, PFA-100, light transmission aggregometry, flow cytometric evaluation of platelet glycoprotein s and agonist-induced platelet activation, alpha and dense granule secretion, clot retraction, immunoblotting analysis of selected proteins, immunofluorescence and electron microscopy. 1-3 Genomic DNA from peripheral blood samples was isolated using a DNeasy blood and tissue kit, following the manufacturer s protocol (Qiagen, Hilden, Germany). DNA concentration was measured using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA). High-throughput sequencing (HTS) The HTS test was performed on a MiSeq Instrument, running MiSeq Control Software and applying a Nextera sequencing design (Illumina). Fifty ng of patient s DNA was sequenced following Illumina s standardized protocol. DNA libraries were normalized to 4 nm and pooled in equal volumes. We used the 300-cycle reagent kit. Specifically, samples were sequenced using paired 150 nt reads, multiplexing twelve, dual-indexed samples per run. Twenty-four samples were included in each run. Sanger sequencing To verify candidate variants found using the HTS multigenic platform and to examine their segregation among available family members, we performed standard Sanger sequencing. Briefly, specific forward and reverse primers were designed using Primer3 ( (sequences available on request). Genomic DNA was amplified with the Fast Start High Fidelity PCR System (Roche, Basel,

40 Switzerland) following the manufacturer s instructions, with some variations in the annealing temperature. DNA sequences were evaluated using Chromas Lite v2.1.1 (Technelysium, South Brisbane, Australia) and DS Gene v1.5 (Accerlys, San Diego, CA) software. Data were analyzed using annotations of genome version hg19/grch37.

41 Supplemental Tables Table S1. Diagnosis of inherited platelet disorder and underlying causative variants previously identified by Sanger sequencing, in the 10 patients of the validation group. Case Diagnosis Gene Status cdna Sanger Sequencing Protein change Ref 1 CAMT MPL Hom c.304c>t p.arg102cys 2 CHS LYST Hom c.11173g>a p.gly3725arg CHS LYST Hom c.772t>c p.cys258arg c.2944g>a p.val982met 4 GT ITGA2B C. Het c.2965g>a p.ala989thr 1 c.1612g>t p.glu538* 5 GT ITGB3 C. Het c.647a>g c.665t>c p.tyr216cys p.leu222pro 1 6 BSS GP1BA Hom c.673t>a p.cys225ser 1 7 BSS GP9 Hom c.212t>c p.phe71ser 8 BSS GP1BA Het c.268_276del GGGACGCTG p.gln90_leu92del 9 HPS HPS1 Hom c.610g>t p.glu204* 10 GPS GFI1B Het c.859c>t p.gln287* 1 1,8 9 Abbreviations: CAMT: Congenital amegakaryocytic thrombocytopenia; CHS: Chediak-Higashi Syndrome; GT: Glanzmann thrombasthenia; BSS: Bernard Soulier Syndrome, HPS: Hermansky- Pudlak Syndrome; GPS: Gray Platelet Syndrome; Hom: Homozygous; Het: Heterozygous; *: stop codon.

42 Table S2. Target regions in the 72-gene HTS platform covered with a depth read lower than 20X. Genes Region Exon Mean coverage A2M chr12: ,6 ABCA1 chr9: chr10: ,6 ANKRD26 chr10: ,2 chr10: chr10: ,8 ANO6 chr12: AP3B1 chr5: ,6 BLOC1S6 chr15: DHCR24 chr1: ,3 chr1: FERMT3 chr11: ,2 FLI1 chr11: ,3 GNAI3 chr1: GNAQ chr9: ,5 HPS3 chr3: ,2 ITGB3 chr17: ,9 LYST chr1: ,8 chr1: MYH9 chr22: MYO5A chr15: ,4 chr15: ,9 PLCB2 chr15: ,2 RAB27A chr15: ,9 RASGRP2 chr11: ,2 RUNX1 chr21: ,7 TBXAS1 chr7:

43 Table S3. Genetic variants identified with the HTS platform, which we have recently reported in detail elsewhere. Case Clinical and biological phenotype Gen Variants IPD Lifelong macrothrombocytopenia ABCG5 28 Presence of stomatocytes in blood smear c.1890delt Sitosterolemia 10 Xanthelasmas c.914c>g 66 Lifelong macrothrombocytopenia Lack of response to previous ITP treatments WAS c.802delc Wiskott-Aldrich syndrome 11 Markedly reduced platelet aggregation with ADP, epinephrine and low-dose collagen 90 Mildly reduced aggregation with other agonists PFA-100 >300s; normal clot retraction Normal expression of GPIIb/IIIa, GPIb/IX, GPIa and GPVI RASGRP2 c.1142c>t IIb 3 activation defect 2 Severely reduced fibrinogen binding with all agonists but PMA Markedly reduced platelet aggregation with ADP, epinephrine, low-dose collagen and CRP Mildly reduced aggregation with other agonists 91 PFA-100 >300s; normal clot retraction Normal expression of GPIIb/IIIa, GPIb/IX, GPIa and GPVI RASGRP2 c.887g>a IIb 3 activation defect 12 Reduced fibrinogen binding with all agonists but PMA Mildly reduced platelet secretion Markedly reduced platelet aggregation with all agonists 92 PFA-100 >300s; normal clot retraction Normal expression of GPIIb/IIIa, GPIb/IX, GPIa and GPVI Reduced fibrinogen binding with all agonist but RASGRP2 c.706c>t IIb 3 activation defect 12 PMA Mildly reduced platelet secretion

44 Supplemental References 1. Sanchez-Guiu I, Anton AI, Padilla J, et al. Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study. Orphanet J Rare Dis. 2014;9: Lozano ML, Cook A, Bastida JM, et al. Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction. Blood. 2016;128(9): Navarro-Nunez L, Teruel R, Anton AI, et al. Rare homozygous status of P43 beta1- tubulin polymorphism causes alterations in platelet ultrastructure. Thromb Haemost. 2011;105(5): Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5): Parrini E, Ramazzotti A, Dobyns WB, et al. Periventricular heterotopia: phenotypic heterogeneity and correlation with Filamin A mutations. Brain. 2006;129(Pt 7): Stritt S, Nurden P, Turro E, et al. A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss. Blood. 2016;127(23): Feng Y, Chen D, Wang GL, Zhang VW, Wong LJ. Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing. Genet Med. 2015;17(2): Sanchez-Guiu I, Torregrosa JM, Velasco F, et al. Hermansky-Pudlak syndrome. Overview of clinical and molecular features and case report of a new HPS-1 variant. Hamostaseologie. 2014;34(4): Monteferrario D, Bolar NA, Marneth AE, et al. A dominant-negative GFI1B mutation in the gray platelet syndrome. N Engl J Med. 2014;370(3): Bastida JM, Benito R, Janusz K, et al. Two novel variants of the ABCG5 gene cause xanthelasmas and macrothrombocytopenia: a brief review of hematological abnormalities of sitosterolemia. J Thromb Haemost. 2017;15(9): Bastida JM, Del Rey M, Revilla N, et al. Wiskott-Aldrich syndrome in a child presenting with macrothrombocytopenia. Platelets. 28(4):

45 12. Sevivas T, Bastida JM, Paul DS, et al. Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis. Platelets. 2017:1-4.

46 Supplemental Figures Figure S1. Filtering strategy of variant called in 82 IPD patients analyzed by the HTS platform.

47 Figure S2. Gene location of variants called in each patient after the filtering procedure. Figure S2 Legend. In each patient the filtering procedure described in Methods, led to calling one o more gene variants. These variants were qualified for contribution to the patient disease using a color code: i) Red: main candidate causal variants, which were classified for pathogenicity according to ACMG guidelines 4 ; Yellow: variants in other genes that could fit with the patient phenotype (for instance, gene already know to be involved in inherited thrombocytopenia; Green: variants in other genes unknown to be involved in phenotype similar with that displayed by the patient.

48 Figure S3. Pedigree of the patient with c.3695c>t variant in FLNA. Figure S3 Legend. HTS test identified in the patient 31 (black symbol) the novel missense variant (c.3695c>t) in exon 22 of the FLNA gene. The patient displayed mild developmental delay and character disorders, congenital right kidney agenesis, corpus callosum hypoplasia and facial abnormalities (small mouth and eyes and bulbar nose), and moderate thrombocytopenia (40 x 10 9 platelets/l) [Table 2]. None of the healthy relatives (white symbols) carried this genetic variant. As occurred in our patient, variants in FLNA have been identified in about 20-30% of cases with sporadic bilateral periventricular nodular heterotopias. 5

Filled black symbols indicate heterozygosity for the R1213* variant in DIAPH1.

49 Figure S4. Family pedigrees in two unrelated families carrying the variant R1213* in DIAPH1. Figure S4 Legend. The index cases in each family are indicated with black arrows. Filled black symbols indicate heterozygosity for the R1213* variant in DIAPH1. Family members in white symbols do not carrier the R1213* variant. They showed not hearing abnormalities and displayed normal blood cell counts.

50 Figure S5. Platelet aggregation and secretion in carriers of the R1213* variant in DIAPH1. Figure S5 Legend. A) Platelet aggregation in response to the indicated platelet agonists was evaluated in unadjusted platelet-rich plasma (PRP), from patients (P1, P2, P3) and two unrelated controls (C1, C2). B) Agonist-induced secretion of α-granules (P-Selectin) and δ-granules (CD63) was evaluated by flow cytometry (FC) in diluted (PRP) from patients (back bars) and two unrelated controls (white bars). Platelets were stimulated (30 min at RT) with the indicated agonist in the presence of CD41*APC and CD62*PE or CD63*PE antibodies. Then, the samples were evaluated by FCusing an Accuri C6 flow cytometer. Bars correspond to the mean, plus standard deviation, of the median fluorescence intensity (MFI) for CD62*PE (P-Selectin) and CD63*PE in the three R1213* carriers from family 18 and two unrelated controls.

Proteins in the lysates were separated by SDS polyacrylamide gel electrophoresis on 4% 20% gradient gels and transferred to polyvinylidene fluoride membranes (Millipore).

51 Figure S6. DIAPH1 protein level in platelet lysates from R1213* carriers and unrelated healthy controls. Figure S6 Legend. Washed platelets and total platelet lysates were prepared from citrated blood from carriers of the R1213* variant from pedigrees 18 and 19, and from two unrelated controls. Proteins in the lysates were separated by SDS polyacrylamide gel electrophoresis on 4% 20% gradient gels and transferred to polyvinylidene fluoride membranes (Millipore). The total platelet content of DIAPH1 protein was assessed by standard immunoblotting, using an anti-diaph1 antibody (ERP7948, Abcam) previously reported, 6 followed by an appropriate peroxidase labeled secondary antibody, and chemiluminiscence detection with ECL kit (GE Healthcare). The level of GPIb (using LJ- Ib10 anti-gpib antibody) was quantified as an internal control of platelet protein loading. As shown, the DIAHP1 protein is expressed in all R1213* carriers from families 18 (Panel A) and 19 (Panel B), although a mild reduction in level is apparent in most of them as compared to controls.

52 Figure S7. Tubulin 1 expression pattern in platelets from a R1213* carrier and an unrelated control. Figure S7 Legend. Detection of β1-tubulin in platelets from the index case 18 and from an unrelated control was performed by standard immunofluorescence microscopy 3. Briefly, diluted platelet-rich-plasma (20.00 platelet/ul) was placed onto(pll)-coated slides, fixed with 4%formaldehyd, permeabilized and blocked with 4% BSA/0.1% Triton X-100 in PBS (90 min, RT). Then, cells were incubated with anti-β1-tubulin antibody (SAB , Sigma-Aldrich) (1:100 in PBS containing 0.5% BSA/0.1% Triton X-100; 90 min, RT), followed by the appropriate Alexa Fluor 546 conjugated secondary antibodies (Thermo Fisher Scientific). Fluorescence was analyzed on a Leica 6000B microscope (Leica Microsystems, Madrid, Spain). As shown, platelets from a R1213* carrier displayed higher labeling for β1-tubulin, both in the periphery and in inside the cells.

Figure S8. Detection of the uncommon rs39428292 variant in RBM8A and a microdeletion in 1q21.

HTS test of DNA from case 30 identified the c.21g>a variant in RBM8A (rs39428292).

53 Figure S8. Detection of the uncommon rs variant in RBM8A and a microdeletion in 1q21.1 in patient 30 confirmed diagnosis of TAR. A Patient Father Mother A G/A G/G B Figure S8 Legend. HTS test of DNA from case 30 identified the c.21g>a variant in RBM8A (rs ). A) Sanger sequencing confirmed the variant in hemizigosity in the patient and in heterozygosity status in his healthy father. B) CGH array analysis of patient DNA revealed a loss of heterozygosity in a 709 kb region in 1q21.1 which involved RBM8A.

54 Figure S9. Detection of 21q deletion in patient 29 by massively parallel sequencing copy number variation analysis. Figure S9 Legend. Whole-genome profiles of log2 ratios of normalized mean coverage of individual exon target of a gene to that of the reference, was plotted against the target. The x-axis shows the targets in the panel plotted by relative genome order. The y-axis corresponds to the log2 ratio of mean coverage of testing to that of reference. CNVs were called using fixed thresholds representing the minimum log2 ratio for gains (0.40) and maximum log2 ratio for losses ( 0.55). An atypical log2 normalized coverage ratio <0.5 suggests a heterozygous deletion of chr21 (21q region, RUNX1). The mean coverage depth of each individual exon target of a sample was first normalized for the total amount of the DNA template loaded onto sequencing flow cells based on the total reads of this sample. The mean coverage of each individual target from the reference samples thus obtained was used as the reference for a specific exon. Reference coverage profile for a normal sample was generated. To detect CNVs, the normalized coverage of each exon of a test sample was compared to the mean coverage of the same target in the reference file generated above 7.

Published Ahead of Print on August 16, 2018, as doi: /haematol Copyright 2018 Ferrata Storti Foundation.

Published Ahead of Print on August 16, 2018, as doi:10.3324/haematol.2018.196121. Copyright 2018 Ferrata Storti Foundation. Computed tomography pulmonary angiography versus ventilation-perfusion lung scan