Congenital Neutropenia Panel 3 Weeks Showing Some Symptoms at ANC Nadir but Generally Milder Infectious Complications

Specimen requirements CPT Codes/Billing/Turnaround time Parental/Patient/Pediatric: 3-5 mL Whole Blood (EDTA tube, Test Code: 4845 lavender top), 2-5 mL Bone Marrow (EDTA tube, lavender top), 3-4 CPT codes: 81250, 81406x4, 81479 Buccal swabs, or ≥1ug of DNA at ≥50ng/uL of High Quality DNA. Congenital Turnaround time: 21 days Fetal: 7-15 mL Amniotic Fluid, 5-10 mg Chorionic Villi; back up culture of Amniocytes or Chorionic Villi is highly recommended. The CPT codes provided are subject to change. CPT codes are Cultured: Two T25 flasks cultured amniocytes or Chorionic Villi provided only as guidance to assist clients with billing. (2x106 minimum). Maternal Blood sample of 3-5 mL Whole Blood Neutropenia For additional information related to shipping, billing or pricing, (EDTA tube, lavender top) is requested for all prenatal samples for please contact, BloodCenter Client Services: (414) 937-6396 or maternal cell contamination studies. 800-245-3117, Option 1, or [email protected]. If questions, please contact the laboratory to discuss sample requirements. Panel References Ancliff, PJ, Blundell, MP et al. 2006. Two novel activating mutations in the Wiskott- Shipping requirements Aldrich syndrome protein result in congenital neutropenia. Blood 108: 2182-2189. Beel K, Cotter MM et al. 2008. A large kindred with X-linked neutropenia with an of the ANC, typically with periods of severe neutropenia every Ship on an ice pack or at room I294T mutation of the Wiskott-Aldrich syndrome gene. Br J Haematol. 144:120– BloodCenter of Wisconsin offers a specifically temperature. Protect from freezing. 126. designed Congenital Neutropenia Panel 3 weeks showing some symptoms at ANC nadir but generally milder infectious complications. Cyclic neutropenia is typically Place the specimen and the requisition Boztug K, Klein C. 2013. Genetics and pathophysiology of severe congenital (test code: 4845) optimized for detection of into plastic bags and seal. Insert into neutropenia syndromes unrelated to neutrophil elastase. Hematolo Oncol Clin N inherited in an autosomal dominant manner caused by a Styrofoam container, seal and place Am. 27:43-60. germline variants in 23 genes known to cause heterozygous pathogenic variants in ELANE. Additional genes in into a sturdy cardboard box, and Dale DC, Link DC. 2009. The many causes of severe congenital neutropenia. N Engl severe congenital neutropenia (SCN), cyclic this panel are associated with congenital syndromes that have tape securely. Ship the package in J Med. Jan 1; 360(1):3-5. neutropenia as a common finding among other non-hematologic Donadieu J, Feneteau O et. al. 2011. Congenital neutropenia: diagnosis, molecular neutropenia, and syndromic neutropenia with features. P compliance with your overnight carrier guidelines. Label with the following bases and patient management. Orphanet J Rare Dis. 6: 26. non-hematological manifestations. This panel evaluates for single nucleotide variants and small address: Germeshausen M, Deerberg S et al. 2013. The spectrum of ELANE mutation and deletions and duplications, which are most commonly Client Services/Diagnostic Laboratory their implications in severe congenital and cyclic neutropenia. Hum Mutat. June; 34(6): 905-14. Severe congenital neutropenia (SCN) is a heterogeneous responsible for genetic disease. However, large deletions and BloodCenter of Wisconsin group of disorders characterized by reduction in the absolute duplications, also referred to as copy number variants (CNV), are 638 N. 18th St. Horwitz, MS, Corey, SJ et al. 2013. ELANE mutations in cyclic and severe congenital neutropenia: genetics and pathophysiology. Hematol Oncol Clin North Am. Feb; neutrophil count (ANC) of less than 500/uL (<0.5x10 9/L) and a known cause of genetic disorders, but can escape detection Milwaukee, WI 53233 27(1):19-41. recurrent severe infections, fever, and inflammation of the skin by next-generation sequence analysis. Further testing with Klein C, Grudzien M et al. 2007. HAX1 deficiency causes autosomal recessive severe and mucous membranes. Another hallmark of certain disorders the BloodCenter of Wisconsin custom designed, high density congenital neutropenia (Kostmann Disease). Nat Genet. Jan;39: 86-92. gene-focused array, aCGH Deletion/Duplication Analysis, allows Required forms in this group is predisposition to myelodysplastic syndrome and Klein C, Welte K. 2010. Genetic insights into congenital neutropenia. Clin Rev acute myeloblastic leukemia (AML). Diagnosis is based on clinical for the possible detection of large deletions and duplications Please complete all pages of the Allergy Immunol. Feb;38(1): 68-74. findings and serial measurement of the ANC. within a single exon of a given gene, encompassing one or more requisition form. Clinical history Rehm HL, Bale SJ et al. 2013. Working Group of the American College of Medical exons, or affecting an entire gene. This testing may be warranted The diagnosis of a specific congenital neutropenia disorder may (including patient’s ethnicity, clinical Genetics and Genomics Laboratory Quality Assurance Committee. ACMG clinical when results of sequence analysis do not fully explain a clinical be difficult to establish solely on functional studies or clinical diagnosis, family history and relevant laboratory standards for next-generation sequencing. Genet Med.15:733-747. phenotype, or when a suspected disorder is known to be caused history. Advances in genetic testing through next-generation laboratory findings) is necessary for Richards S, Aziz N A et al.2015. Standards and guidelines for the interpretation of by deletions or duplications. Please refer to the aCGH Deletion/ sequencing allow for the identification of underlying genetic optimal interpretation of genetic test sequence variants: a joint consensus recommendation of the American College Duplication Analysis test description for more information about of Medical Genetics and Genomics and the Association for Molecular Pathology. defects associated with neutropenia that have overlapping results and recommendations. Clinical specific genes included in this array. Genet Med.17:405-424. clinical and laboratory findings. Accurate diagnosis provides and laboratory history can either be P Xia J, Bolyard AA et al. 2009. Prevalence of mutations in ELANE, GFI1, HAX1, information about phenotype and prognosis, guides medical Refer to the table inside for further information about each recorded on the requisition form or SBDS, WAS and G6PC3 in patients with severe congenital neutropenia. Br J management decisions, assists with the identification of affected gene in the Congenital Neutropenia Panel, including the clinical and laboratory reports can be Haematol.147:535–42. family members, and allows for accurate genetic recurrence risk clinical phenotype, OMIM numbers and inheritance pattern. submitted with the sample. assessment. Variants in several different genes are known to cause syndromic or non-syndromic congenital neutropenia, which may be inherited in an autosomal dominant, autosomal recessive or X-linked recessive manner. Most common causes of severe congenital neutropenia include autosomal dominant pathogenic variants in ELANE, (38-80% of SCN cases, varying with ethnicity and other factors) and autosomal recessive pathogenic variants in HAX1 (up to 15-20% of cases of SCN). Genes responsible for rare cases of non-syndromic SCN analyzed by this panel include CSF3R, G6PC3, GFI1, JAGN1, VPS45 and WAS. Cyclic neutropenia is distinguished from congenital neutropenia by regular oscillations

1 Congenital Neutropenia Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Gene Clinical Phenotype Phenotype/Gene Inheritance OMIM number AP3B1 Hermansky Pudlak syndrome, type 2: oculocutaneous albinism, platelet defects and 608233/603401 Autosomal Recessive congenital neutropenia. CSF3R Severe congenital neutropenia- 7(SCN7): isolated severe congenital neutropenia with 617014/138971 Autosomal Recessive recurrent infections. CXCR4 WHIM syndrome: neutropenia, hypogammaglobulinemia- related recurrent infections, 193670/162643 Autosomal Dominant extensive human papillomavirus (HPV) infection, and myeloid hyperplasia and apoptosis. ELANE Severe congenital neutropenia-1 (SCN1): isolated severe congenital neutropenia with 202700/130130 recurrent infections. Autosomal Dominant

Cyclic Neutropenia: regular oscillations of neutropenia and generally milder infectious 162800/130130 complications. G6PC3 Severe congenital neutropenia-4, (SCN4) (includes Dursun syndrome): congenital 612541/611045 Autosomal Recessive neutropenia, recurrent infections, prominent super!cial venous pattern; variable pulmonary hypertension, cardiac defects, thrombocytopenia, urogenital abnormalities. GATA1 X-linked anemia with or without neutropenia and /or platelet abnormalities (XLANP): 300835/305371 variable severity of early-onset anemia with erythroid hypoplasia; variable neutropenia, X-linked Recessive variable thrombocytopenia or platelet dysfunction. Thrombocytopenia, abnormal platelet morphology, variable dyserythropoietic anemia. 300367/305371 GATA2 Primary lymphedema with myelodysplasia (Emberger syndrome). 614038/137295 Immunode!ciency 21 (IMD21, DCML, or MONOMAC): primary immunode!ciency with 614172/137295 monocytopenia, B-cell de!ciency without hypogammaglobulinemia, NK-cell de!ciency, Autosomal Dominant but generally normal T-cell numbers. Susceptibility to myelodysplastic syndrome. 614286/137295 Susceptibility to the development of acute myeloid leukemia. 601626/137295 GFI1 Severe congenital neutropenia-2 (SCN2): isolated severe congenital neutropenia with 613107/600871 Autosomal Dominant recurrent infections. HAX1 Severe congenital neutropenia-3 (SCN3; Kostmann disease): neutropenia, recurrent 610738/605998 Autosomal Recessive infections; with or without neurologic features. JAGN1 Severe congenital neutropenia-6 (SCN6): isolated severe congenital neutropenia with 616022/616012 Autosomal Recessive recurrent infections. LAMTOR2 Immunode!ciency syndrome: congenital neutropenia, partial albinism, short stature and 610798/610389 Autosomal Recessive B-cell and cytotoxic T-cell de!ciency. LYST Chediak-Higashi syndrome: multisystem disorder characterized by partial 214500/ 606897 Autosomal Recessive oculocutaneous albinism, immunode!ciency with neutropenia and giant neutrophil granules, bleeding tendency due to absent, reduced or abnormal platelet-dense bodies, malignant lymphoma and varying neurologic problems. RAB27A Griscelli syndrome, type 2: hypomelanosis of skin and hair with immunologic 607624/603868 Autosomal Recessive abnormalities (variable hypogammaglobulinemia and leukopenia, hemophagocytosis) with or without neurologic involvement. RAC2 Neutrophil immunode!ciency syndrome (NEUID): leukocytosis, neutrophilia, severe 608203/602049 Autosomal Dominant recurrent bacterial infections, poor wound healing. SBDS Shwachman-Diamond syndrome: neutropenia, variable anemia or thrombocytopenia, 260400/607444 Autosomal Recessive pancreatic insu"ciency, skeletal abnormalities, susceptibility to myelodysplastic syndrome (MDS), aplastic anemia or acute myeloid leukemia (AML); possible hepatic and/or renal involvement, variable developmental delay. SLC37A4 Glycogen storage disease type 1b (GSD1B): congenital metabolic disorder with 232220/602671 Autosomal Recessive hypoglycemia, lactic acidosis, hyperuricemia, and hyperlipidemia, short stature and hepatomegaly; variable neutropenia, recurrent infections, in#ammatory bowel disease. TAZ Barth syndrome: dilated cardiomyopathy, skeletal myopathy, short stature, neutropenia. 302060/300384 X-linked TCIRG1 Osteopetrosis 1: life-threatening disorder of subnormal osteoclast function, defective 259700/604592 Autosomal Recessive bone remodeling, bone marrow insu"ciency. Severe congenital neutropenia and/or associated with lower absolute neutrophil count See Autosomal Dominant (Rosenthal et al. 2016) Reference /604592 USB1 Poikiloderma with neutropenia: hypo-/hyperpigmentation, telangiectasias and skin 604173/613276 Autosomal Recessive atrophy; nail dystrophy, neutropenia, and recurrent pulmonary infections. Congenital Neutropenia Panel: gene, clinical phenotype, OMIM number and inheritance pattern. Gene Clinical Phenotype Phenotype/Gene Inheritance OMIM number VPS13B Cohen syndrome: developmental delay, distinctive facial features, microcephaly, 216550/ 607817 Autosomal Recessive hypotonia, truncal obesity, progressive retinopathy, intermittent congenital neutropenia. VPS45 Severe congenital neutropenia-5 (SCN5): primary immunode!ciency with neutropenia 615285/610035 Autosomal Recessive and neutrophil dysfunction, a lack of response to G-CSF, recurrent infections, bone marrow !brosis, and renal extramedullary hematopoiesis. WAS WAS related disorders comprise a spectrum of disorders, not distinct entities, as clinical manifestations can vary, even within the same family: Wiskott-Aldrich syndrome (WAS): profound thrombocytopenia, small platelet size, 301000/300392 eczema and recurrent infections; increased risk for autoimmune disorders and lymphoma; absent or decreased intracellular WAS protein (WASP) detection in hematopoietic cells by #ow cytometry or western blotting. X-linked Recessive X-linked neutropenia: congenital neutropenia; variable severity, infectious history, 300299/300392

myelodysplasia and increased risk for MDS and AML. Normal WAS protein (WASP) expression by #ow cytometry or western blotting. X-linked thrombocytopenia: thrombocytopenia, possibly intermittent, with small platelet 313900/300392 volume; variable severity of bleeding, autoimmune disease and malignancies, variable WASP expression by #ow cytometry or western blotting. WIPF1 Wiskott-Aldrich syndrome-2 (WAS2): thrombocytopenia, recurrent infections, eczema, 614493/602357 Autosomal Recessive B-cell and T-cell de!ciency, impaired NK cell function; normal WASP sequence and mRNA; defective WASP expression.

Indications for testing coverage of 50x and those regions with less than 50 sequencing reads or low quality coverage are supplemented with Sanger Congenital Neutropenia Panel: sequencing. All regions are covered by bi-directional analysis. • Clari!cation and/or con!rmation of diagnosis in a patient Variants are identi!ed by a customized bioinformatics pipeline, with clinical !ndings of neutropenia or an associated genetic analyzed and comprehensively interpreted by our team of syndrome when patient’s history suggests multiple congenital directors, scientists, and genetic counselors. All reported variants, neutropenia disorders including pathogenic, likely pathogenic, and variants of uncertain • Identi!cation of carriers with family history of an unspeci!ed signi!cance, are con!rmed by Sanger sequencing. neutropenia disorder to provide accurate reproductive risk For prenatal testing, analysis of variable number tandem repeats assessment and genetic counseling (VNTR) is used to con!rm results are not a$ected by maternal cell Single gene sequencing or custom gene panel: contamination. • Analysis of genes included in the Congenital Neutropenia Panel may also be ordered as a stand-alone single gene sequencing Assay sensitivity and limitations test(s) or custom panel (2-10 genes) as dictated by the patient’s clinical and laboratory phenotype The analytical sensitivity of this test is >99% for single nucleotide changes and insertions and deletions of less than 20 bp. This Targeted familial variant analysis: assay does not detect large deletions or duplications (>20 bp), • Targeted variant analysis for clinical diagnosis, carrier or deletions, duplications or variants that are outside the regions identi!cation or prenatal diagnosis can also be performed on sequenced. To order analysis of copy number variants at the exon any gene in the panel when the pathogenic variant(s) is known or gene level, please refer to the aCGH Deletion/Duplication in the family (test code: 4970 ) Analysis test or contact Client Services before placing your order. For clinical questions about laboratory tests and test utilization support, contact BloodCenter Client Services: (414) 937-6396 or Reporting of results 800-245-3117, Option 1, to be directed to our genetic counselors and clinical support team. While this assay is designed to detect germline genetic variants associated with neutropenia, variants unrelated to the indication for testing but with other clinical and/or reproductive implications Test method may also be detected. A comprehensive database of gene- phenotype relationships listed by gene name can be found at This next-generation sequencing assay analyzes 23 genes, http://www.omim.org . spanning the full coding regions plus a minimum 30bp of non- coding DNA including intron-exon junctions. These targeted Results are classi!ed and reported in accordance with ACMG regions are captured by hybridization, ampli!ed and sequenced next-generation sequencing standards. Variants predicted to be by massively parallel sequencing. Regions will have a minimum pathogenic, likely pathogenic, and of uncertain signi!cance will Specimen requirements CPT Codes/Billing/Turnaround time Parental/Patient/Pediatric: 3-5 mL Whole Blood (EDTA tube, Test Code: 4845 lavender top), 2-5 mL Bone Marrow (EDTA tube, lavender top), 3-4 CPT codes: 81406x3, 81408, 81479 Buccal swabs, or ≥1ug of DNA at ≥50ng/uL of High Quality DNA. Turnaround time: 21 days Fetal: 7-15 mL Amniotic Fluid, 5-10 mg Chorionic Villi; back up culture of Amniocytes or Chorionic Villi is highly recommended. The CPT codes provided are subject to change. CPT codes are Cultured: Two T25 "asks cultured amniocytes or Chorionic Villi provided only as guidance to assist clients with billing. (2x106 minimum). Maternal Blood sample of 3-5 mL Whole Blood For additional information related to shipping, billing or pricing, (EDTA tube, lavender top) is requested for all prenatal samples for please contact, BloodCenter Client Services: (414) 937-6396 or maternal cell contamination studies. 800-245-3117, Option 1, or [email protected] . If questions, please contact the laboratory to discuss sample requirements. References Ancli%, PJ, Blundell, MP et al. 2006. Two novel activating mutations in the Wiskott- Shipping requirements Aldrich syndrome protein result in congenital neutropenia. Blood 108: 2182-2189. Beel K, Cotter MM et al. 2008. A large kindred with X-linked neutropenia with an Ship on an ice pack or at room I294T mutation of the Wiskott-Aldrich syndrome gene. Br J Haematol. 144:120– temperature. Protect from freezing. 126. Place the specimen and the requisition Boztug K, Klein C. 2013. Genetics and pathophysiology of severe congenital into plastic bags and seal. Insert into neutropenia syndromes unrelated to neutrophil elastase. Hematolo Oncol Clin N a Styrofoam container, seal and place Am. 27:43-60. into a sturdy cardboard box, and Dale DC, Link DC. 2009. The many causes of severe congenital neutropenia. N Engl tape securely. Ship the package in J Med. Jan 1; 360(1):3-5. Donadieu J, Feneteau O et. al. 2011. Congenital neutropenia: diagnosis, molecular SHIP compliance with your overnight carrier guidelines. Label with the following bases and patient management. Orphanet J Rare Dis. 6: 26. address: Germeshausen M, Deerberg S et al. 2013. The spectrum of ELANE mutation and Client Services/Diagnostic Laboratory their implications in severe congenital and cyclic neutropenia. Hum Mutat. June; 34(6): 905-14. BloodCenter of Wisconsin 638 N. 18th St. Horwitz, MS, Corey, SJ et al. 2013. ELANE mutations in cyclic and severe congenital neutropenia: genetics and pathophysiology. Hematol Oncol Clin North Am. Feb; Milwaukee, WI 53233 27(1):19-41. Klein C, Grudzien M et al. 2007. HAX1 de#ciency causes autosomal recessive severe congenital neutropenia (Kostmann Disease). Nat Genet. Jan;39: 86-92. Required forms Klein C, Welte K. 2010. Genetic insights into congenital neutropenia. Clin Rev Please complete all pages of the Allergy Immunol. Feb;38(1): 68-74. requisition form. Clinical history Rehm HL, Bale SJ et al. 2013. Working Group of the American College of Medical (including patient’s ethnicity, clinical Genetics and Genomics Laboratory Quality Assurance Committee. ACMG clinical diagnosis, family history and relevant laboratory standards for next-generation sequencing. Genet Med.15:733-747. laboratory #ndings) is necessary for Richards S, Aziz N A et al.2015. Standards and guidelines for the interpretation of optimal interpretation of genetic test sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. results and recommendations. Clinical Genet Med.17:405-424. and laboratory history can either be ORDER Rosenthal EA, Makaryan V et al. 2016. Association Between Absolute Neutrophil recorded on the requisition form or Count and Variation at TCIRG1: The NHLBI Exome Sequencing Project. Genet clinical and laboratory reports can be Epidemiol. 2016 Sep;40(6):470-4. submitted with the sample. Xia J, Bolyard AA et al. 2009. Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia. Br J Haematol.147:535–42.