IMMUNODEFICIENCY NEXT-GENERATION SEQUENCING PANELS Mail: One Gustave L. Levy Place, Box 1497 CLIA #: 33D2097541 Specimens: 1428 Madison Ave, Atran Bldg, Rm 2-25 T: 800-298-6470 1 New YorK, NY 10029 F: 212-241-0139 www.sema4genomics.com Mail: One Gustave L. Levy Place, Box 1497 CLIA #: 33D2097541 Specimens: 1428 Madison Ave, Atran Bldg, Rm 2-25 T: 800-298-6470 2 New YorK, NY 10029 F: 212-241-0139 www.sema4genomics.com Table of Contents GENETIC TESTING FOR IMMUNODEFICIENCY 4 GENETICS 4 INDICATIONS 4 TESTING METHODS, SENSITIVITY, AND LIMITATIONS 4 TURNAROUND TIME 7 SPECIMEN AND SHIPPING REQUIREMENTS 7 CUSTOMER SERVICES AND GENETIC COUNSELING 8 THE COMPREHENSIVE IMMUNODEFIENCY PANEL 9 PRIMARY IMMUNODEFIENCY (PID) SUBPANEL 22 INFLAMMATORY BOWEL DISEASE (IBD) SUBPANEL 32 SEVERE COMBINED IMMUNODEFICIENCY (SCID) SUBPANEL 36 REFERENCES 38 DISCLAIMER 46 Mail: One Gustave L. Levy Place, Box 1497 CLIA #: 33D2097541 Specimens: 1428 Madison Ave, Atran Bldg, Rm 2-25 T: 800-298-6470 3 New YorK, NY 10029 F: 212-241-0139 www.sema4genomics.com Genetic Testing for IMMUNODEFICIENCY Inherited disorders of the immune system include over 300 Known diseases in the world with prevalence rates of more than 1 in 1,000 in the US (Joshi, et al 2009). Genetic defects constitute the vast majority and are the main driver of immunological diseases with diagnosable causes (Zhernakova, et al 2009). Symptoms and severity range from mild increases in susceptibility to infection and gastrointestinal disorders to the inability to survive in non-sterile environments. Sema4 utilizes Agilent SureSelectQXT target enrichment library prep with Illumina NovaSeq NGS platform to detect clinically significant variants in immunodeficiency-related genes selected based on available evidence to date. The genes in this panel and subpanels were curated based on careful consideration of literature review, active physician input, and comparison against existing tests. The Comprehensive Panel includes 250 genes with the following subpanels: Primary Immunodeficiency (PID; 206), Inflammatory Bowel Disease (IBD; 59), and Severe Combined Immunodeficiency (SCID; 26). Our results aim to pinpoint the genetic basis of a patient’s immunological disease thereby aiding physicians when developing healthcare plans and allowing genetic counselors to advise the patient in family planning and carrier status relative risK for family members. Genetics The disorders included in this panel may be inherited in an autosomal dominant (AD), autosomal recessive (AR), X-linKed (XL), or isolated cases (IC) manner. For genes displaying an AD mode of inheritance, an affected parent carrying the mutated gene has a 50% chance of passing the variant on to an offspring, regardless of gender. Some of these genes are not fully penetrant, meaning that an individual may have a mutated gene but not display any of the signs/symptoms of the disorder. Additionally, these disorders may have variable expressivity indicating that individuals carrying the same pathogenic variant may display differing features and/or differing severity. For diseases with AR inheritance, the risk for a couple who are both carriers to have a child affected with the disease is 25% for each pregnancy. The parents of an affected child are most often obligate carriers (heterozygotes) and each carry one mutant allele (unless a de novo mutation occurs). An X-linKed inheritance means that the risK of a male offspring with the disorder will be 50% if the mother carries an XL mutation. Depending on the X-inactivation pattern of the gene, a mother and her daughters may rarely be affected. Although X-linKed diseases are normally transmitted from mother to son, transmission of an X-linKed mutation will occur from an affected father to each daughter, but will not occur from father to son. An IC mode of inheritance indicates no prior family history. Indications 1. Clinical status: to confirm a clinical diagnosis in an affected patient, in an individual with unKnown status (no screening/evaluation), or in unaffected relatives of an affected patient (all screening/evaluations(s) normal). The purpose of the test may be diagnostic, carrier testing, familial follow-up on a known family variant, or prenatal testing for known variant(s). 2. Treatment: to clarify the cause of an individual’s immunodeficiency and/or inflammatory bowel disease, provide information on the likelihood of related health issues, and guide treatment. 3. Family risK: to establish risK to other family members and future generations. For patients with a suspected syndrome or disorder, please consider single gene sequencing or associated subpanels prior to ordering the coMprehensive panel. Testing Methods, Sensitivity, and LiMitations Next Generation Sequencing (NGS) (Analytical Detection Rate >95%) Agilent SureSelectTM QXT technology is used with a custom capture library to target the exonic regions and intron/exon splice junctions of the relevant genes, as well as a number of UTR, intronic or promoter regions that Mail: One Gustave L. Levy Place, Box 1497 CLIA #: 33D2097541 Specimens: 1428 Madison Ave, Atran Bldg, Rm 2-25 T: 800-298-6470 4 New YorK, NY 10029 F: 212-241-0139 www.sema4genomics.com contain previously reported mutations. Samples are pooled and sequenced on the Illumina NovaSeq platform in the Xp workflow, using 100 bp paired-end reads. The sequencing data are analyzed using a custom bioinformatics algorithm designed and validated in-house. In our validation, average coverage was greater than 200X per sample with >99.9% of regions covered at greater than 20X. The coding exons and splice junctions of the Known protein-coding RefSeq genes are assessed for the average depth of coverage (minimum of 20X) and data quality threshold values. Most exons not meeting a minimum of >20X read depth across the exon are further analyzed by Sanger sequencing. Please note that several genomic regions present difficulties in mapping or obtaining read depth >20X. These regions include, but are not limited to, UTRs, promoters, and deep intronic areas. In addition, a mutation(s) in a gene not included on the panel could be present in this patient. The following regions (hg19 coordinates) have been excluded due to lacK of amenability to NGS or Sanger sequencing, high GC content, high homology, lacK of Known clinically significant variants, or overlap with repetitive regions: C4A chr6:31959812-31960020, C4A chr6:31962010-31962172, C4A chr6:31968818-31968943, C4A chr6:31964594-31964849, C4A chr6:31961479-31961613, C4A chr6:31961681- 31961774, C4A chr6:31962717-31962791, C4A chr6:31962870-31962982, C4A chr6:31963095-31963327, C4A chr6:31949873-31949960, C4A chr6:31950070-31950291, C4A chr6:31950483-31950707, C4A chr6:31950894- 31950987, C4A chr6:31951043-31951154, C4A chr6:31951350-31951455, C4A chr6:31951603-31951722, C4A chr6:31951838-31951966, C4A chr6:31952041-31952196, C4A chr6:31958957-31959095, C4A chr6:31959172- 31959374, C4A chr6:31959497-31959702, C4A chr6:31960151-31960332, C4A chr6:31960561-31960710, C4A chr6:31960855-31960952, C4A chr6:31961190-31961410, C4A chr6:31965470-31965552, C4A chr6:31966579- 31966695, C4A chr6:31966770-31966979, C4A chr6:31967071-31967184, C4A chr6:31967252-31967349, C4A chr6:31969086-31969198, C4A chr6:31969261-31969382, C4A chr6:31969542-31969648, C4A chr6:31969888- 31970043, C4A chr6:31970164-31970328, C4A chr6:31962263-31962495, C4A chr6:31963484-31963582, C4A chr6:31963720-31963899, C4A chr6:31963972-31964111, C4A chr6:31964909-31965099, C4B chr6:31992550- 31992758, C4B chr6:32001555-32001680, C4B chr6:31997331-31997586, C4B chr6:31992235-31992440, C4B chr6:31996458-31996637, C4B chr6:31994217-31994351, C4B chr6:31994419-31994512, C4B chr6:31995455- 31995529, C4B chr6:31995608-31995720, C4B chr6:31995833-31996065, C4B chr6:31982611-31982698, C4B chr6:31982808-31983029, C4B chr6:31983221-31983445, C4B chr6:31983632-31983725, C4B chr6:31983781- 31983892, C4B chr6:31984088-31984193, C4B chr6:31984341-31984460, C4B chr6:31984576-31984704, C4B chr6:31984779-31984934, C4B chr6:31991695-31991833, C4B chr6:31991910-31992112, C4B chr6:31992889- 31993070, C4B chr6:31993299-31993448, C4B chr6:31993593-31993690, C4B chr6:31993928-31994148, C4B chr6:31998207-31998289, C4B chr6:31999316-31999432, C4B chr6:31999507-31999716, C4B chr6:31999808- 31999921, C4B chr6:31999989-32000086, C4B chr6:32001823-32001935, C4B chr6:32001998-32002119, C4B chr6:32002279-32002385, C4B chr6:32002625-32002780, C4B chr6:32002901-32003065, C4B chr6:31995001- 31995233, C4B chr6:31994748-31994910, C4B chr6:31996222-31996320, C4B chr6:31996710-31996849, C4B chr6:31997646-31997836, CCDC40 chr17:78010450-78010501, CFHR1 chr1:196788963-196789043, CFHR1 chr1:196799618-196799823, CFHR3 chr1:196759163-196759368, CORO1A chr16:30200169-30200296, CSF2RA chrX:1422142-1422266, CYBB chrX:37656945-37657156, DCLRE1C chr10:14966805-14967016, DUOX2 chr15:45403297-45403486, IKBKG chrX:153792522-153792687, IKBKG chrX:153784368-153784602, IKBKG chrX:153786735-153786876, IKBKG chrX:153788610-153788785, IKBKG chrX:153791013-153791179, IKBKG chrX:153792162-153792246, IKBKG chrX:153791762-153791927, IKBKG chrX:153789891-153790010, IRAK4 chr12:44178044-44178050, NCF1 chr7:74203371-74203515, NCF1 chr7:74202316-74202443, NCF1 chr7:74199516-74199656, NCF1 chr7:74197270-74197415, NCF1 chr7:74191601-74191704, NCF1 chr7:74197856-74197986, NCF1 chr7:74193591-74193779, NCF1 chr7:74202891-74203059, NCF1 chr7:74193416-74193514, NCF1 chr7:74195114-74195192, NCF1 chr7:74188367-74188461, PMS2 chr7:6026378-6027262, PMS2 chr7:6017207-6017432,