Comprehensive Bleeding Disorder Panel

References Johnson B, Lowe GC et al. 2016. Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function Andrikovics H, Klein I et al. 2003. Analysis of large structural changes of the factor defects. Haematologica. 101(10):1170-1179. VIII gene, involving intron 1 and 22, in severe hemophilia A. Haematologica. 88:778–84. Kunicki TJ, Williams SA, Nugent DJ. 2012. Genetic variants that affect platelet function. Current Opin Hematol.19:371-9. Comprehensive Bagnall RD, Giannelli F et al. 2006. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their Lancellotti, S, Basso M et al. 2013. Congenital prothrombin deficiency: an update. detection. J Thromb Haemost. 4:591–8. Semin Thromb Hemost. Sep;39(6):596-606. Bagnall RD, Waseem N et al. 2002. Recurrent inversion breaking intron 1 of the Lentaigne C, Freson K et al. 2016. Inherited platelet disorders: toward DNA-based factor VIII gene is a frequent cause of severe hemophilia A. Blood. 99:168–74. diagnosis. Blood. 127(23):2814-2823. Bleeding Disorder Bajaj SP, Thompson AR. 2006. Molecular and structural biology of factor IX. In: Lillicrap D. 2013. Molecular Testing for Disorders of Hemostasis. Int J Lab Hematol. Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN, eds. Hemostasis and Jun; 35(3):290–296. Thrombosis: Basic Principles and Clinical Practice. 5 ed. Philadelphia: Lippincott- Lillicrap D. 2013. von Willebrand disease: advances in pathogenetic understanding, Raven.131-150. diagnosis, and therapy. Blood. Nov 28;122 (23):3735-40. Panel Bolton-Maggs PH et al. 2010. A study of variations in the reported haemophilia A Maclachlan A, Watson SP, Morgan NV. 2017. Inherited platelet disorders: Insight prevalence around the world. Haemophilia.16:20–32. from platelet genomics using next-generation sequencing. Platelets. Jan ;28(1);14- Casini A, Blondon M et al. 2015. Natural history of patients with congenital 19. dysfibrinogenemia. Blood. Jan 15; 125(3): 553-561. Mumford AD, Nisar S et al. 2013. Platelet dysfunction associated with the novel Cox K, Price V, Kahr WHA. 2011. Inherited platelet disorders: a clinical approach to Trp29Cys thromboxane A₂ receptor variant. J Thromb Haemost. Mar;11(3):547-54. BloodCenter of Wisconsin offers a specifically This panel evaluates for single nucleotide variants and small diagnosis and management. Expert Rev Hematol. Aug;4(4):455-72. Neerman-Arbez M, de Moerloose P et al. 2016. Laboratory and Genetic deletions and duplications, which are most commonly de Moerloose P, Schved JF et al. 2016. Rare Coagulation disorders: fibrinogen, factor Investigation of Mutations Accounting for Congenital Fibrinogen Disorders. Semin designed Comprehensive Bleeding Disorder Thromb Hemost. Jun; 42(4):356-365. responsible for genetic disease. However, large deletions and VII and factor XIII. Haemophilia. Jul;22 Suppl5:61-5. Panel (test code 4825) optimized for the duplications, also referred to as copy number variants (CNV), are Faioni EM, Razzari C et al. 2014. Bleeding diathesis and gastro-duodenal ulcers Ng C, Motto DB et al. 2015. Diagnostic approach to von Willebrand disease. Blood. Mar 26;125(13):2029-2037. detection of germline variants in 51 genes a known cause of genetic disorders, but can escape detection in inherited cytosolic phospholipase-A2 alpha deficiency. Thromb Haemost. by next-generation sequence analysis. Further testing with Dec;112(6):1182-9. Nurden AT, Nurden P. 2011. Advances in our understanding of the molecular basis known to cause bleeding due to disorders of of disorders of platelet function. J Thromb Haemost. 9 Suppl 1:76-91. the BloodCenter of Wisconsin custom designed, high density Freson K, Wijgaerts A, Van Geet C. 2014. Update on the causes of platelet disorders coagulation and/or platelet function. gene-focused array, aCGH Deletion/Duplication Analysis, allows and functional consequences. John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 36, Saultier P,Vidal, L.et al. 2017. Macrothrombocytopenia and dense granule 313–325. deficiency associated with FLI1 variants: ultrastructural and pathogenic features. for the possible detection of large deletions and duplications within a single exon of a given gene, encompassing one or Gresele P. 2014. Diagnosis of inherited platelet function disorders: guidance for the Haematologica.102:1006-1016. Coagulation disorders and inherited platelet function disorders more exons, or affecting an entire gene. This testing may be SSC of the ISTH. J Thromb and Haemost. 13:314-322. are a heterogeneous group of inherited bleeding disorders with warranted when results of sequence analysis do not fully explain Hinckley J, Di Paola J. 2014. Genetic basis of congenital platelet disorders. overlapping clinical phenotypes. Bleeding symptoms can include a clinical phenotype, or when a suspected disorder is known to Hematology Am Soc Hematol Educ Program. Dec5;(1): 3337-42. epistaxis, easy bruising, gingival bleeding, prolonged bleeding be caused by deletions or duplications. Specifically, the Quebec Hua B, Fan L et al. 2009. Alpha 1- antitrypsin Pittsburgh in a family with bleeding following an injury, surgery or dental extractions, gastrointestinal Platelet Disorder is associated with a heterozygous 78-kb tandem tendency. Haematologica. June; 94(6): 881-4. or urinary bleeding, hematomas, hemoptysis, intracranial duplication of the PLAU gene, which will be detected by aCGH Israels SJ, El-Ekiaby M, Quiroga T, Mezzano D. 2010. Inherited disorders of platelet bleeding and menorrhagia or postpartum bleeding in women. and not by the Comprehensive Bleeding Disorder Panel. Please function and challenges to diagnosis of mucocutaneous bleeding. Haemophilia. Symptoms can present at any age and range in severity: in mild 16 (Suppl 5):152-9. refer to the aCGH Deletion/Duplication Analysis test description cases, individuals remain asymptomatic until the event of a for more information about specific genes included in this array. trauma or surgery, and in severe cases, patients may present with spontaneous life threatening hemorrhage or bleeding symptoms Analysis of genes included in the Comprehensive Bleeding in the newborn period. Disorder Panel may also be ordered as a stand-alone single gene sequencing test as dictated by the patient’s laboratory phenotype. Although results of functional hemostasis testing often guide Alternatively, custom panels may be ordered if a patient’s history molecular testing for a specific inherited coagulation disorder, suggests a specific bleeding disorder with multiple causative there are situations where functional tests are not definitive. genes, or if functional testing results narrow the diagnosis to Likewise, the diagnosis of a specific platelet function disorder specific phenotypes that can be due to different underlying may be difficult to establish based solely on functional studies, genetic conditions. Targeted familial variant testing can also be especially in patients with milder disorders, as these assays are performed on any gene in the panel when the specific genetic often technically challenging and typically require immediate variant is known in a family. testing on fresh patient platelets due to limited sample stability. In both instances, results of functional tests may be difficult to interpret. For cases in which the laboratory phenotype is not fully Refer to the table inside for further information about each consistent with clinical symptoms, or the specific bleeding disorder gene in the Comprehensive Bleeding Disorder Panel, including is unclear, the Comprehensive Bleeding Disorder Panel offers an the clinical phenotype, OMIM numbers and inheritance pattern. efficient and cost-effective means of diagnostic genetic evaluation. Accurate diagnosis provides information about phenotype and prognosis, guides medical management decisions, assists with the identification of affected family members, and allows for accurate genetic recurrence risk assessment. Variants in several different genes known to cause syndromic or non-syndromic bleeding disorders may be inherited in an autosomal recessive, autosomal dominant or X-linked manner.

© Copyright 2017 r0818 BloodCenter of Wisconsin, Inc. , Part of Versiti. All rights reserved. Indications for testing Assay sensitivity and limitations Shipping requirements CPT Codes/Billing/Turnaround time Comprehensive Bleeding Disorder Panel: The analytical sensitivity of this test is >99% for single nucleotide Ship on an ice pack or at room Test Code: 4825 • Clarification and/or confirmation of diagnosis in a patient with changes and insertions and deletions of less than 20 bp. This temperature. Protect from freezing. CPT codes: 81404, 81238, 81407, 81408, 81479 an unspecified bleeding disorder assay does not detect large deletions or duplications (>20 bp) Place the specimen and the requisition or deletions, duplications or variants that are outside the regions into plastic bags and seal. Insert into Turnaround time: 21 days • Identification of carriers with family history of an unspecified sequenced. To order analysis of copy number variants at the exon a Styrofoam container, seal and place The CPT codes provided are subject to change as more bleeding disorder to provide accurate reproductive risk or gene level, please refer to the aCGH Deletion/Duplication into a sturdy cardboard box, and information becomes available. CPT codes are provided only as assessment and genetic counseling Analysis test, if available, or contact Client Services before placing tape securely. Ship the package in guidance to assist clients with billing. your order. compliance with your overnight carrier Single gene sequencing or custom gene panel: D P For additional information related to shipping, billing or pricing, guidelines. Label with the following • Analysis of genes included in the Comprehensive Bleeding please contact, BloodCenter Client Services: (414) 937-6396 or address: Disorder Panel may also be ordered as a stand-alone single 800-245-3117, Option 1, or [email protected]. Reporting of results Client Services/Diagnostic Laboratory gene sequencing test or custom panel (2-10 genes) as dictated BloodCenter of Wisconsin by the patient’s clinical and laboratory phenotype While this assay is designed to detect germline genetic variants associated with bleeding disorders, variants unrelated to the 638 N. 18th St. Targeted familial variant analysis: indication for testing, but with other clinical and/or reproductive Milwaukee, WI 53233 • Targeted variant analysis for clinical diagnosis, carrier implications, may also be detected. A comprehensive database of identification or prenatal diagnosis can also be performed on gene-phenotype relationships listed by gene name can be found any gene in the panel when the pathogenic variant(s) is known at http://www.omim.org. Required forms in the family (test code: 4970) Results are classified and reported in accordance with ACMG Please complete all pages of the For clinical questions about laboratory tests and test utilization next-generation sequencing standards. Variants predicted to requisition form. Clinical history support, contact BloodCenter Client Services: (414) 937-6396 or be pathogenic, likely pathogenic, and of uncertain significance (including patient’s ethnicity, clinical 800-245-3117, Option 1, to be directed to our genetic counselors will be reported; variants classified as likely benign or benign are diagnosis, family history and relevant and clinical support team. typically not reported but such data are available upon request. laboratory findings) is necessary for optimal interpretation of genetic test Sequence variants are described using standard Human Genome D results and recommendations. ClinicalP Test method Variation Society (HGVS) nomenclature (http://hgvs.org). and laboratory history can either be recorded on the requisition form or This next-generation sequencing assay analyzes 51 genes, clinical and laboratory reports can be spanning the full coding regions plus a minimum 30bp of non- Specimen requirements submitted with the sample. coding DNA including intron-exon junctions, in addition to 67 Parental/Patient/Pediatric: 3-5 mL Whole Blood (EDTA tube, bp upstream of F9 exon 1 to cover F9 Leyden variants, the 3’ lavender top), 2-5 mL Bone Marrow (EDTA tube, lavender top), 3-4 UTR of F2, the 5’ UTR of F8, VWF, as well as targeted sequencing Buccal Swabs, or ≥1ug of DNA at ≥50ng/uL of High Quality DNA. of SERPINA1 c.1145T. These targeted regions are captured by hybridization, amplified and sequenced by massively parallel Fetal: 7-15 mL Amniotic fluid, 5-10 mg Chorionic villi; back up sequencing. Regions will have a minimum coverage of 50x culture of amniocytes or chorionic villi is highly recommended. and those regions with less than 50 sequencing reads or low Cultured: Two T25 flasks cultured amniocytes or chorionic villi quality coverage are supplemented with Sanger sequencing. (2x106 minimum). Maternal blood sample of 3-5 mL Whole Blood All regions are covered by bi-directional analysis. Variants are (EDTA tube, lavender top) is requested for all prenatal samples for identified by a customized bioinformatics pipeline, analyzed maternal cell contamination studies. and comprehensively interpreted by our team of directors, If questions please contact the laboratory to discuss sample scientists, and genetic counselors. All reported variants, including requirements. pathogenic, likely pathogenic, and variants of uncertain significance, are confirmed by Sanger sequencing. For prenatal testing, analysis of variable number tandem repeats (VNTR) is used to confirm results are not affected by maternal cell contamination. Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Gene Clinical Phenotype Phenotype/Gene Inheritance Gene Clinical Phenotype Phenotype/Gene Inheritance OMIM number OMIM number VWF von Willebrand disease (VWD): variable bleeding due to quantitative or qualitative ANO6 Scott syndrome: moderate bleeding disorder due to abnormality of platelet 262890/608663 Autosomal Recessive defects of von Willebrand factor (VWF). There are three types of VWD: procoagulation activity with normal platelet count. Impaired exposure of phosphatidylserine on the outer leaflet of platelet membrane. Type 1 VWD: accounts for more than 80% of cases; typically associated with mild 193400/613160 Autosomal Dominant mucocutaneous bleeding due to reduction in quantity of normal functioning VWF. AP3B1 (HPS2) Hermansky-Pudlak syndrome (HPS): multisystem disorder characterized by 608233/603401 Autosomal Dominant Type VWD 1C: associated with significant increase of VWF clearance and higher than BLOC1S3 oculocutaneous albinism and platelet defects with reduced or absent delta granules on 614077/609762 Autosomal Recessive normal response to DDAVP, but has a shortened half-life. (HPS8) electron microscopy of platelets. Subtypes are caused by recessive pathogenic variants in nine different genes and variable additional features including neutropenia and/or Type 2 VWD: qualitative defects of VWF, accounting for up to 10-15% of all cases of VWD, 613554/613160 Autosomal Dominant BLOC1S6 immune defects (HPS2), pulmonary fibrosis (HPS1, HPS2 and HPS4), or granulomatous 614171/604310 Autosomal Recessive with four primary subtypes: (type 2B, 2M, and (HPS9) colitis (HPS1 and HPS4). HPS3 is associated with milder bleeding symptoms and minimal the majority of 2A); • 2A: associated with mild-moderate mucocutaneous bleeding due to decreased hypopigmentation. Autosomal Recessive DTNBP1 614076/607145 Autosomal Recessive VWF-dependent platelet adhesion with loss of high molecular weight multimers. (HPS7) (type 2N, some cases • 2B: associated with mild-moderate mucocutaneous bleeding with similar laboratory of type 2A) F2 Prothrombin deficiency: rare bleeding disorder with variable disease severity correlating 613679/176930 Autosomal Recessive findings to type 2A but also increased VWF-platelet GP1b binding and often with level of prothrombin (a vitamin K dependent glycoprotein) activity.1 thrombocytopenia. F5 Factor V deficiency: rare bleeding disorder with variable disease severity and age of 227400/612309 Autosomal Recessive • 2M: associated with mild-moderate mucocutaneous bleeding due to decreased VWF- onset.2 dependent platelet adhesion with normal distribution of multimers; a rare form of this type is characterized by decreased interaction with collagen. F7 Factor VII deficiency: rare bleeding disorder with the most severe cases apparent in 227500/613878 Autosomal Recessive infancy. Up to one-third of people with factor VII deficiency never have any bleeding • 2N: characterized by a reduced factor VIII levels due to impaired ability of VWF to bind problems. Thrombotic episodes can occur in a minority of patients, usually with other factor VIII; associated with excessive bleeding following surgery and can resemble a mild form of hemophilia A. thrombotic risk factors. F8 Factor VIII deficiency (Hemophilia A): severe, moderate or mild bleeding disorder 306700/300841 X-linked Recessive Type 3 VWD: accounts for <5% of VWD and is associated with severe bleeding 277480/613160 Autosomal Recessive symptoms due to essentially absent VWF and decreased factor VIII activity. that primarily affects males. Female carriers may show varying degrees of factor VIII deficiency and related bleeding symptoms. F9 Factor IX deficiency (Hemophilia B): severe, moderate or mild bleeding disorder primarily 306900/300746 X-linked Recessive The PLAU gene is not included in this panel. For analysis of the Quebec Platelet Disorder associated with a heterozygous 78-kb tandem affecting males. Female carriers may show varying degrees of factor IX deficiency and duplication of the PLAU gene, please order aCGH. related bleeding symptoms. Hemophilia B Leyden (caused by specific F9 variants): associated with very low levels of functional factor IX at birth, but hormonal changes cause factor IX levels to increase gradually during puberty. Bleeding problems in adulthood not typically seen. F10 Factor X deficiency: rare bleeding disorder with variable disease severity associated with 227600/613872 Autosomal Recessive reduced (type 1) or impaired (type II) factor X. F11 Factor XI deficiency: typically presents with bleeding after trauma or surgery; 612416/264900 Autosomal Recessive homozygotes are more severely affected; there are variable bleeding problems in heterozygotes. F13A1 Factor XIII deficiency: rare bleeding disorder with symptoms shortly after birth. Without 613225/134570 Autosomal Recessive F13B treatment, life-threatening intracranial hemorrhage may occur. Pathogenic variants in 613235/134580 Autosomal Recessive F13A1 and F13B severely reduce the amount or activity of the factor XIII A subunit or B subunit respectively. FERMT3 Leukocyte integrity adhesion deficiency type III: increased bleeding symptoms, poor 612840/607901 Autosomal Recessive wound healing, normal/reduced platelets, bacterial infections and neutrophilia. FGA Pathogenic variants in FGA, FGB and FGG result in quantitative and/or qualitative 202400/134820 Autosomal Recessive changes in the fibrinogen alpha, beta or gamma subunit chains respectively. Congenital afibrinogenemia: rare bleeding disorder with excessive bleeding often in the newborn period. Congenital hypofibrinogenemia results in decreased amounts of these subunit chains and may lead to varying bleeding symptoms from mild-severe. Congenital dysfibrinogenemia and congenital hypodysfibrinogenemia may result in 616004/134820 Autosomal Dominant/ bleeding symptoms, thromboembolic complications or both. Autosomal Recessive FGB 202400/134830 Autosomal Recessive 616004/134830 Autosomal Dominant/ Autosomal Recessive FGG 202400/134850 Autosomal Recessive 616004/134850 Autosomal Dominant/ Autosomal Recessive 1. The thrombophilia-related prothrombin variant F2 20210G>A in the 3’ 2. Factor V Leiden, the most common inherited form of thrombophilia will be untranslated region, which results in enhanced prothrombin production, will be detected with this assay. Individuals homozygous have an increased risk over detected with this assay. heterozygotes.

Comprehensive Bleeding Disorder Panel, Insert Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Gene Clinical Phenotype Phenotype/Gene Inheritance Gene Clinical Phenotype Phenotype/Gene Inheritance MIM number MIM number FLI1 Paris-Trousseau thrombocytopenia (TCPT): mild bleeding tendency with variable 188025/193067 Deletion syndrome ITGA2B Glanzmann thrombasthenia: mild to severe bleeding disorder with platelet aggregation 273800/607759 Autosomal Recessive thrombocytopenia, dysmorphic facies, abnormal giant alpha-granules in platelets and abnormalities due to quantitative or qualitative defects of platelet glycoproteins IIb dysmegakaryopoiesis as part of contiguous gene deletion syndrome of 11q (Jacobsen and/or IIIa. Caused by homozygous or compound heterozygous recessive pathogenic syndrome). variants in ITGA2B or ITGB3. Bleeding disorder, platelet-type, 21 (BDPLT21): moderate macrothrombocytopenia, 617443/ 193067 Autosomal Recessive/ Bleeding disorder, platelet-type 16 (BDPLT16): congenital macrothrombocytopenia 187800/607759 Autosomal Dominant abnormal giant alpha-granules in platelets, functional platelet disorder. Phenotype Autosomal Dominant associated with platelet anisocytosis with mild or absent symptoms due to specific caused by heterozygous and homozygous pathogenic variants.. heterozygous dominant activating mutations in ITGA2B or ITGB3. ITGB3 273800/173470 Autosomal Recessive GATA1 GATA1-related cytopenia: thrombocytopenia and/or anemia ranging from mild 187800/173470 Autosomal Dominant to severe; may be associated with platelet dysfunction, mild β-thalassemia, LMAN1 Factor V and factor VIII combined deficiency 1 (F5F8D1) and 2 (F5F8D2) are caused by 227300/601567 Autosomal Recessive neutropenia, and congenital erythropoietic porphyria (CEP). Thrombocytopenia present in infancy and anemia may range from mild to severe hydrops fetalis. Carrier MCFD2 pathogenic variants in LMAN1 and MCFD2 respectively: Symptoms are usually mild and 613625/607788 Autosomal Recessive females may have mild to moderate symptoms. may include epistaxis, menorrhagia, and excessive bleeding during or after trauma. X-linked dyserythropoietic anemia and thrombocytopenia (XLTDA): variable severity of 300367/305371 LYST Chediak-Higashi syndrome: multisystem disorder characterized by partial 214500/606897 Autosomal Recessive thrombocytopenia and dyserythropoietic anemia may be present. X-linked Recessive oculocutaneous albinism, immunodeficiency with neutropenia and giant neutrophil granules, bleeding tendency due to absent, reduced or abnormal platelet-dense bodies, Thrombocytopenia with beta-thalassemia, X-linked (XLTT): variable thrombocytopenia, 314050/305371 malignant lymphoma and varying neurologic problems. splenomegaly, and unbalanced hemoglobin chain synthesis resembling that of beta- thalassemia minor. NBEAL2 Gray platelet syndrome (GPS): large platelets that lack α-granules leading to mild 139090/614169 Autosomal Recessive to moderate bleeding tendency and moderate thrombocytopenia with increased GGCX Combined deficiency of vitamin K dependent clotting factors-1 (VKCFD1): mild to 277450/137167 Autosomal Recessive development of myelofibrosis and splenomegaly. severe bleeding symptoms including risk of episodes of perinatal/neonatal intracranial hemorrhage due to deficiency of factors II, VII, IX, and X. P2RY12 Bleeding disorder, platelet-type 8 (BDPLT8): mild to moderate mucocutaneous bleeding 609821/600515 Autosomal Recessive and excessive bleeding after surgery or trauma due to ADP receptor defect. Pseudoxanthoma elasticum-like disorder multiple coagulation factor deficiency: 610842/137167 Autosomal Recessive hyperlaxity of the skin involving the entire body and deficiency of factors II, VII, IX and X. PLA2G4A Recurrent episodes of multiple complicated ulcers of the small intestine, platelet See reference/ Autosomal Recessive dysfunction and globally decreased eicosanoid synthesis due to phospholipase A2 600522 GFI1B Bleeding disorder, platelet-type 17 (Gray Platelet-like syndrome): variable 187900/604383 Autosomal Dominant deficiency (Faioni EM et al. 2014). bleeding symptoms due to disorder of platelet alpha granules with moderate PRKACG Bleeding disorder platelet-type 19 (BDPLT19): severe macrothrombocytopenia with 616176/176893 Autosomal Recessive macrothrombocytopenia and red cell anisopoikilocytosis. moderate-severe bleeding tendency due to defects in megakaryocyte proplatelet GP1BA Bernard-Soulier syndrome (BSS): mild to severe bleeding disorder due to absence 231200/606672 Autosomal Recessive formation, platelet activation and cytoskeleton reorganization. or dysfunction of the platelet glycoprotein receptor Ib/V/IX complex with mild to RASGRP2 Bleeding disorder, platelet-type 18 (BDPLT18): mild bleeding with increased bleeding 615888/605577 Autosomal Recessive moderate thrombocytopenia, unusually large platelets and abnormal platelet function times and platelets showed reduced aggregation in response to ADP or epinephrine. with absent or markedly reduced aggregation response to ristocetin. caused by homozygous or compound heterozygous pathogenic variants in GP1BA (606672), GP1BB RUNX1 Familial platelet disorder with associated myeloid malignancy (FPDMM): mild to 601399/151385 Autosomal Dominant (138720), or GP9 (173515). moderate thrombocytopenia, qualitative platelet defects and a predisposition to development of myeloid malignancies. Bernard-Soulier syndrome (BSSA2): autosomal dominant (mono-allelic) form of BSS with 153670/606672 Autosomal Dominant mild thrombocytopenia, variable large platelets, and mild or no bleeding tendency, due SERPINA1 Bleeding disorder of variable severity caused by specific variant SERPINA1 c.1145T>G 613490/107400 Autosomal Dominant to specific heterozygous variants in GP1BA gene or rarely in GP1BB or GP9. (p.Met358Arg) “Pittsburgh allele” resulting in an altered protein that inhibits thrombin, fXa and protein C. Sequencing of the full coding region of SERPINA1 gene is not Platelet-type von Willebrand disease (also known as pseudo-von Willebrand disease): 177820/606672 Autosomal Dominant available. thrombocytopenia and mucosal bleeding due to dominant pathogenic variants in GP1BA that cause excessive binding of the GPIb-IX-V complex to von Willebrand factor. SERPINE1 Plasminogen activator inhibitor-1deficiency (PAI-1D): rare bleeding disorder associated 613329/173360 Autosomal Recessive/ with increased bleeding after trauma, injury or surgery due to increased fibrinolysis of Autosomal Dominant fibrin blood clots. GP1BB 231200/138720 Autosomal Recessive 153670/138720 Autosomal Dominant SERPINF2 Alpha-2-plasmin inhibitor deficiency (APLID): severe increased susceptibility to bleeding. 262850/613168 Autosomal Recessive GP9 231200/173515 Autosomal Recessive STIM1 Stormorken syndrome: multisystem disorder characterized by mild bleeding tendency 185070/ 605921 Autosomal Dominant 153670/173515 Autosomal Dominant due to platelet dysfunction, thrombocytopenia, anemia, asplenia, tubular aggregate myopathy, congenital miosis, and ichthyosis, with variable headache or recurrent stroke- GP6 Bleeding disorder, platelet-type 11 (BDPLT11): mild to moderate bleeding disorder 614201/605546 Autosomal Recessive like episodes. due to glycoprotein VI deficiency with defective platelet activation and aggregation in response to collagen. TBXA2R Susceptibility to bleeding disorder platelet-type 13 (BDPLT13): susceptibility to mild 614009/188070 Autosomal Dominant mucocutaneous bleeding due to defective platelet thromboxane A2 receptor, with HPS1 Hermansky-Pudlak syndrome (HPS): multisystem disorder characterized by 203300/604982 Autosomal Recessive bleeding phenotype occurring only in the presence of a second variant in this gene or oculocutaneous albinism and platelet defects with reduced or absent delta granules on HPS3 614072/606118 Autosomal Recessive another gene affecting platelet function. electron microscopy of platelets. Subtypes are caused by recessive pathogenic variants HPS4 in nine different genes and variable additional features including neutropenia and/or 614073/606682 Autosomal Recessive VKORC1 Combined deficiency of vitamin K dependent clotting factors-2 (VKCFD2): mild to severe 607473/608547 Autosomal Recessive HPS5 immune defects (HPS2), pulmonary fibrosis HPS1,( HPS2 and HPS4), or granulomatous 614074/607521 Autosomal Recessive bleeding symptoms including risk of perinatal/neonatal intracranial hemorrhage due to colitis (HPS1 and HPS4). HPS3 is associated with milder bleeding symptoms and minimal deficiency of factors II, VII, IX, and X. HPS6 614075/607522 Autosomal Recessive hypopigmentation. VIPAS39 Arthrogryposis, renal dysfunction and cholestasis syndrome (ARCS1 and 2): multisystem 613404/613401 Autosomal Recessive (VIPAR) disorder characterized by early onset and limited survival with arthrogryposis, renal dysfunction and cholestasis with additional symptoms of ichthyosis, abnormal platelet VPS33B count and function, secondary infection, and cardiovascular anomalies. 208085/608552 Autosomal Recessive

Comprehensive Bleeding Disorder Panel, Insert Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Comprehensive Bleeding Disorder Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Gene Clinical Phenotype Phenotype/Gene Inheritance Gene Clinical Phenotype Phenotype/Gene Inheritance OMIM number OMIM number VWF von Willebrand disease (VWD): variable bleeding due to quantitative or qualitative ANO6 Scott syndrome: moderate bleeding disorder due to abnormality of platelet 262890/608663 Autosomal Recessive defects of von Willebrand factor (VWF). There are three types of VWD: procoagulation activity with normal platelet count. Impaired exposure of phosphatidylserine on the outer leaflet of platelet membrane. Type 1 VWD: accounts for more than 80% of cases; typically associated with mild 193400/613160 Autosomal Dominant mucocutaneous bleeding due to reduction in quantity of normal functioning VWF. AP3B1 (HPS2) Hermansky-Pudlak syndrome (HPS): multisystem disorder characterized by 608233/603401 Autosomal Dominant Type VWD 1C: associated with significant increase of VWF clearance and higher than BLOC1S3 oculocutaneous albinism and platelet defects with reduced or absent delta granules on 614077/609762 Autosomal Recessive normal response to DDAVP, but has a shortened half-life. (HPS8) electron microscopy of platelets. Subtypes are caused by recessive pathogenic variants in nine different genes and variable additional features including neutropenia and/or Type 2 VWD: qualitative defects of VWF, accounting for up to 10-15% of all cases of VWD, 613554/613160 Autosomal Dominant BLOC1S6 immune defects (HPS2), pulmonary fibrosis (HPS1, HPS2 and HPS4), or granulomatous 614171/604310 Autosomal Recessive with four primary subtypes: (type 2B, 2M, and (HPS9) colitis (HPS1 and HPS4). HPS3 is associated with milder bleeding symptoms and minimal the majority of 2A); • 2A: associated with mild-moderate mucocutaneous bleeding due to decreased hypopigmentation. Autosomal Recessive DTNBP1 614076/607145 Autosomal Recessive VWF-dependent platelet adhesion with loss of high molecular weight multimers. (HPS7) (type 2N, some cases • 2B: associated with mild-moderate mucocutaneous bleeding with similar laboratory of type 2A) F2 Prothrombin deficiency: rare bleeding disorder with variable disease severity correlating 613679/176930 Autosomal Recessive findings to type 2A but also increased VWF-platelet GP1b binding and often with level of prothrombin (a vitamin K dependent glycoprotein) activity.1 thrombocytopenia. F5 Factor V deficiency: rare bleeding disorder with variable disease severity and age of 227400/612309 Autosomal Recessive • 2M: associated with mild-moderate mucocutaneous bleeding due to decreased VWF- onset.2 dependent platelet adhesion with normal distribution of multimers; a rare form of this type is characterized by decreased interaction with collagen. F7 Factor VII deficiency: rare bleeding disorder with the most severe cases apparent in 227500/613878 Autosomal Recessive infancy. Up to one-third of people with factor VII deficiency never have any bleeding • 2N: characterized by a reduced factor VIII levels due to impaired ability of VWF to bind problems. Thrombotic episodes can occur in a minority of patients, usually with other factor VIII; associated with excessive bleeding following surgery and can resemble a mild form of hemophilia A. thrombotic risk factors. F8 Factor VIII deficiency (Hemophilia A): severe, moderate or mild bleeding disorder 306700/300841 X-linked Recessive Type 3 VWD: accounts for <5% of VWD and is associated with severe bleeding 277480/613160 Autosomal Recessive symptoms due to essentially absent VWF and decreased factor VIII activity. that primarily affects males. Female carriers may show varying degrees of factor VIII deficiency and related bleeding symptoms. F9 Factor IX deficiency (Hemophilia B): severe, moderate or mild bleeding disorder primarily 306900/300746 X-linked Recessive The PLAU gene is not included in this panel. For analysis of the Quebec Platelet Disorder associated with a heterozygous 78-kb tandem affecting males. Female carriers may show varying degrees of factor IX deficiency and duplication of the PLAU gene, please order aCGH. related bleeding symptoms. Hemophilia B Leyden (caused by specific F9 variants): associated with very low levels of functional factor IX at birth, but hormonal changes cause factor IX levels to increase gradually during puberty. Bleeding problems in adulthood not typically seen. F10 Factor X deficiency: rare bleeding disorder with variable disease severity associated with 227600/613872 Autosomal Recessive reduced (type 1) or impaired (type II) factor X. F11 Factor XI deficiency: typically presents with bleeding after trauma or surgery; 612416/264900 Autosomal Recessive homozygotes are more severely affected; there are variable bleeding problems in heterozygotes. F13A1 Factor XIII deficiency: rare bleeding disorder with symptoms shortly after birth. Without 613225/134570 Autosomal Recessive F13B treatment, life-threatening intracranial hemorrhage may occur. Pathogenic variants in 613235/134580 Autosomal Recessive F13A1 and F13B severely reduce the amount or activity of the factor XIII A subunit or B subunit respectively. FERMT3 Leukocyte integrity adhesion deficiency type III: increased bleeding symptoms, poor 612840/607901 Autosomal Recessive wound healing, normal/reduced platelets, bacterial infections and neutrophilia. FGA Pathogenic variants in FGA, FGB and FGG result in quantitative and/or qualitative 202400/134820 Autosomal Recessive changes in the fibrinogen alpha, beta or gamma subunit chains respectively. Congenital afibrinogenemia: rare bleeding disorder with excessive bleeding often in the newborn period. Congenital hypofibrinogenemia results in decreased amounts of these subunit chains and may lead to varying bleeding symptoms from mild-severe. Congenital dysfibrinogenemia and congenital hypodysfibrinogenemia may result in 616004/134820 Autosomal Dominant/ bleeding symptoms, thromboembolic complications or both. Autosomal Recessive FGB 202400/134830 Autosomal Recessive 616004/134830 Autosomal Dominant/ Autosomal Recessive FGG 202400/134850 Autosomal Recessive 616004/134850 Autosomal Dominant/ Autosomal Recessive 1. The thrombophilia-related prothrombin variant F2 20210G>A in the 3’ 2. Factor V Leiden, the most common inherited form of thrombophilia will be untranslated region, which results in enhanced prothrombin production, will be detected with this assay. Individuals homozygous have an increased risk over detected with this assay. heterozygotes.

Comprehensive Bleeding Disorder Panel, Insert Indications for testing Assay sensitivity and limitations Shipping requirements CPT Codes/Billing/Turnaround time Comprehensive Bleeding Disorder Panel: The analytical sensitivity of this test is >99% for single nucleotide Ship on an ice pack or at room Test Code: 4825 • Clarification and/or confirmation of diagnosis in a patient with changes and insertions and deletions of less than 20 bp. This temperature. Protect from freezing. CPT codes: 81404, 81238, 81407, 81408, 81479 an unspecified bleeding disorder assay does not detect large deletions or duplications (>20 bp) Place the specimen and the requisition or deletions, duplications or variants that are outside the regions into plastic bags and seal. Insert into Turnaround time: 21 days • Identification of carriers with family history of an unspecified sequenced. To order analysis of copy number variants at the exon a Styrofoam container, seal and place The CPT codes provided are subject to change as more bleeding disorder to provide accurate reproductive risk or gene level, please refer to the aCGH Deletion/Duplication into a sturdy cardboard box, and information becomes available. CPT codes are provided only as assessment and genetic counseling Analysis test, if available, or contact Client Services before placing tape securely. Ship the package in guidance to assist clients with billing. your order. compliance with your overnight carrier Single gene sequencing or custom gene panel: D P For additional information related to shipping, billing or pricing, guidelines. Label with the following • Analysis of genes included in the Comprehensive Bleeding please contact, BloodCenter Client Services: (414) 937-6396 or address: Disorder Panel may also be ordered as a stand-alone single 800-245-3117, Option 1, or [email protected]. Reporting of results Client Services/Diagnostic Laboratory gene sequencing test or custom panel (2-10 genes) as dictated BloodCenter of Wisconsin by the patient’s clinical and laboratory phenotype While this assay is designed to detect germline genetic variants associated with bleeding disorders, variants unrelated to the 638 N. 18th St. Targeted familial variant analysis: indication for testing, but with other clinical and/or reproductive Milwaukee, WI 53233 • Targeted variant analysis for clinical diagnosis, carrier implications, may also be detected. A comprehensive database of identification or prenatal diagnosis can also be performed on gene-phenotype relationships listed by gene name can be found any gene in the panel when the pathogenic variant(s) is known at http://www.omim.org. Required forms in the family (test code: 4970) Results are classified and reported in accordance with ACMG Please complete all pages of the For clinical questions about laboratory tests and test utilization next-generation sequencing standards. Variants predicted to requisition form. Clinical history support, contact BloodCenter Client Services: (414) 937-6396 or be pathogenic, likely pathogenic, and of uncertain significance (including patient’s ethnicity, clinical 800-245-3117, Option 1, to be directed to our genetic counselors will be reported; variants classified as likely benign or benign are diagnosis, family history and relevant and clinical support team. typically not reported but such data are available upon request. laboratory findings) is necessary for optimal interpretation of genetic test Sequence variants are described using standard Human Genome D results and recommendations. ClinicalP Test method Variation Society (HGVS) nomenclature (http://hgvs.org). and laboratory history can either be recorded on the requisition form or This next-generation sequencing assay analyzes 51 genes, clinical and laboratory reports can be spanning the full coding regions plus a minimum 30bp of non- Specimen requirements submitted with the sample. coding DNA including intron-exon junctions, in addition to 67 Parental/Patient/Pediatric: 3-5 mL Whole Blood (EDTA tube, bp upstream of F9 exon 1 to cover F9 Leyden variants, the 3’ lavender top), 2-5 mL Bone Marrow (EDTA tube, lavender top), 3-4 UTR of F2, the 5’ UTR of F8, VWF, as well as targeted sequencing Buccal Swabs, or ≥1ug of DNA at ≥50ng/uL of High Quality DNA. of SERPINA1 c.1145T. These targeted regions are captured by hybridization, amplified and sequenced by massively parallel Fetal: 7-15 mL Amniotic fluid, 5-10 mg Chorionic villi; back up sequencing. Regions will have a minimum coverage of 50x culture of amniocytes or chorionic villi is highly recommended. and those regions with less than 50 sequencing reads or low Cultured: Two T25 flasks cultured amniocytes or chorionic villi quality coverage are supplemented with Sanger sequencing. (2x106 minimum). Maternal blood sample of 3-5 mL Whole Blood All regions are covered by bi-directional analysis. Variants are (EDTA tube, lavender top) is requested for all prenatal samples for identified by a customized bioinformatics pipeline, analyzed maternal cell contamination studies. and comprehensively interpreted by our team of directors, If questions please contact the laboratory to discuss sample scientists, and genetic counselors. All reported variants, including requirements. pathogenic, likely pathogenic, and variants of uncertain significance, are confirmed by Sanger sequencing. For prenatal testing, analysis of variable number tandem repeats (VNTR) is used to confirm results are not affected by maternal cell contamination. References Johnson B, Lowe GC et al. 2016. Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function Andrikovics H, Klein I et al. 2003. Analysis of large structural changes of the factor defects. Haematologica. 101(10):1170-1179. VIII gene, involving intron 1 and 22, in severe hemophilia A. Haematologica. 88:778–84. Kunicki TJ, Williams SA, Nugent DJ. 2012. Genetic variants that affect platelet function. Current Opin Hematol.19:371-9. Comprehensive Bagnall RD, Giannelli F et al. 2006. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their Lancellotti, S, Basso M et al. 2013. Congenital prothrombin deficiency: an update. detection. J Thromb Haemost. 4:591–8. Semin Thromb Hemost. Sep;39(6):596-606. Bagnall RD, Waseem N et al. 2002. Recurrent inversion breaking intron 1 of the Lentaigne C, Freson K et al. 2016. Inherited platelet disorders: toward DNA-based factor VIII gene is a frequent cause of severe hemophilia A. Blood. 99:168–74. diagnosis. Blood. 127(23):2814-2823. Bleeding Disorder Bajaj SP, Thompson AR. 2006. Molecular and structural biology of factor IX. In: Lillicrap D. 2013. Molecular Testing for Disorders of Hemostasis. Int J Lab Hematol. Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN, eds. Hemostasis and Jun; 35(3):290–296. Thrombosis: Basic Principles and Clinical Practice. 5 ed. Philadelphia: Lippincott- Lillicrap D. 2013. von Willebrand disease: advances in pathogenetic understanding, Raven.131-150. diagnosis, and therapy. Blood. Nov 28;122 (23):3735-40. Panel Bolton-Maggs PH et al. 2010. A study of variations in the reported haemophilia A Maclachlan A, Watson SP, Morgan NV. 2017. Inherited platelet disorders: Insight prevalence around the world. Haemophilia.16:20–32. from platelet genomics using next-generation sequencing. Platelets. Jan ;28(1);14- Casini A, Blondon M et al. 2015. Natural history of patients with congenital 19. dysfibrinogenemia. Blood. Jan 15; 125(3): 553-561. Mumford AD, Nisar S et al. 2013. Platelet dysfunction associated with the novel Cox K, Price V, Kahr WHA. 2011. Inherited platelet disorders: a clinical approach to Trp29Cys thromboxane A₂ receptor variant. J Thromb Haemost. Mar;11(3):547-54. BloodCenter of Wisconsin offers a specifically This panel evaluates for single nucleotide variants and small diagnosis and management. Expert Rev Hematol. Aug;4(4):455-72. Neerman-Arbez M, de Moerloose P et al. 2016. Laboratory and Genetic deletions and duplications, which are most commonly de Moerloose P, Schved JF et al. 2016. Rare Coagulation disorders: fibrinogen, factor Investigation of Mutations Accounting for Congenital Fibrinogen Disorders. Semin designed Comprehensive Bleeding Disorder Thromb Hemost. Jun; 42(4):356-365. responsible for genetic disease. However, large deletions and VII and factor XIII. Haemophilia. Jul;22 Suppl5:61-5. Panel (test code 4825) optimized for the duplications, also referred to as copy number variants (CNV), are Faioni EM, Razzari C et al. 2014. Bleeding diathesis and gastro-duodenal ulcers Ng C, Motto DB et al. 2015. Diagnostic approach to von Willebrand disease. Blood. Mar 26;125(13):2029-2037. detection of germline variants in 51 genes a known cause of genetic disorders, but can escape detection in inherited cytosolic phospholipase-A2 alpha deficiency. Thromb Haemost. by next-generation sequence analysis. Further testing with Dec;112(6):1182-9. Nurden AT, Nurden P. 2011. Advances in our understanding of the molecular basis known to cause bleeding due to disorders of of disorders of platelet function. J Thromb Haemost. 9 Suppl 1:76-91. the BloodCenter of Wisconsin custom designed, high density Freson K, Wijgaerts A, Van Geet C. 2014. Update on the causes of platelet disorders coagulation and/or platelet function. gene-focused array, aCGH Deletion/Duplication Analysis, allows and functional consequences. John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 36, Saultier P,Vidal, L.et al. 2017. Macrothrombocytopenia and dense granule 313–325. deficiency associated with FLI1 variants: ultrastructural and pathogenic features. for the possible detection of large deletions and duplications within a single exon of a given gene, encompassing one or Gresele P. 2014. Diagnosis of inherited platelet function disorders: guidance for the Haematologica.102:1006-1016. Coagulation disorders and inherited platelet function disorders more exons, or affecting an entire gene. This testing may be SSC of the ISTH. J Thromb and Haemost. 13:314-322. are a heterogeneous group of inherited bleeding disorders with warranted when results of sequence analysis do not fully explain Hinckley J, Di Paola J. 2014. Genetic basis of congenital platelet disorders. overlapping clinical phenotypes. Bleeding symptoms can include a clinical phenotype, or when a suspected disorder is known to Hematology Am Soc Hematol Educ Program. Dec5;(1): 3337-42. epistaxis, easy bruising, gingival bleeding, prolonged bleeding be caused by deletions or duplications. Specifically, the Quebec Hua B, Fan L et al. 2009. Alpha 1- antitrypsin Pittsburgh in a family with bleeding following an injury, surgery or dental extractions, gastrointestinal Platelet Disorder is associated with a heterozygous 78-kb tandem tendency. Haematologica. June; 94(6): 881-4. or urinary bleeding, hematomas, hemoptysis, intracranial duplication of the PLAU gene, which will be detected by aCGH Israels SJ, El-Ekiaby M, Quiroga T, Mezzano D. 2010. Inherited disorders of platelet bleeding and menorrhagia or postpartum bleeding in women. and not by the Comprehensive Bleeding Disorder Panel. Please function and challenges to diagnosis of mucocutaneous bleeding. Haemophilia. Symptoms can present at any age and range in severity: in mild 16 (Suppl 5):152-9. refer to the aCGH Deletion/Duplication Analysis test description cases, individuals remain asymptomatic until the event of a for more information about specific genes included in this array. trauma or surgery, and in severe cases, patients may present with spontaneous life threatening hemorrhage or bleeding symptoms Analysis of genes included in the Comprehensive Bleeding in the newborn period. Disorder Panel may also be ordered as a stand-alone single gene sequencing test as dictated by the patient’s laboratory phenotype. Although results of functional hemostasis testing often guide Alternatively, custom panels may be ordered if a patient’s history molecular testing for a specific inherited coagulation disorder, suggests a specific bleeding disorder with multiple causative there are situations where functional tests are not definitive. genes, or if functional testing results narrow the diagnosis to Likewise, the diagnosis of a specific platelet function disorder specific phenotypes that can be due to different underlying may be difficult to establish based solely on functional studies, genetic conditions. Targeted familial variant testing can also be especially in patients with milder disorders, as these assays are performed on any gene in the panel when the specific genetic often technically challenging and typically require immediate variant is known in a family. testing on fresh patient platelets due to limited sample stability. In both instances, results of functional tests may be difficult to interpret. For cases in which the laboratory phenotype is not fully Refer to the table inside for further information about each consistent with clinical symptoms, or the specific bleeding disorder gene in the Comprehensive Bleeding Disorder Panel, including is unclear, the Comprehensive Bleeding Disorder Panel offers an the clinical phenotype, OMIM numbers and inheritance pattern. efficient and cost-effective means of diagnostic genetic evaluation. Accurate diagnosis provides information about phenotype and prognosis, guides medical management decisions, assists with the identification of affected family members, and allows for accurate genetic recurrence risk assessment. Variants in several different genes known to cause syndromic or non-syndromic bleeding disorders may be inherited in an autosomal recessive, autosomal dominant or X-linked manner.