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Neurodevelopment Next-Generation Sequencing Panels

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NEURODEVELOPMENT NEXT-GENERATION SEQUENCING PANELS 62 Southfield Avenue, Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Stamford CT Lic#: CL-1016 Page 1 of 125 www.sema4.com 62 Southfield Avenue, Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Page 2 of 125 Stamford CT Lic#: CL-1016 www.sema4.com 62 Southfield Avenue, Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Page 3 of 125 Stamford CT Lic#: CL-1016 www.sema4.com TABLE OF CONTENTS GENETIC TESTING FOR NEURODEVELOPMENTAL DISEASE 5 GENETICS 5 INDICATIONS 5 TESTING METHODS, SENSITIVITY, AND LIMITATIONS 6 TURNAROUND TIME 8 SPECIMEN AND SHIPPING REQUIREMENTS 8 CUSTOMER SERVICES AND GENETIC COUNSELING 9 THE COMPREHENSIVE EPILEPSY AND AUTISM PANEL 10 THE COMPREHENSIVE EPILEPSY PANEL 32 FOCAL, GENERALIZED, AND MYOCLONIC EPILEPSY SUBPANEL 48 INFANTILE EPILEPSY SUBPANEL 52 MIGRAINE SUBPANEL 56 NEURONAL CEROID LIPOFUSCINOSES SUBPANEL 57 NEURONAL MIGRATION SUBPANEL 58 SYNDROMIC EPILEPSY AND INTELLECTUAL DISABILITY SUBPANEL 61 THE COMPREHENSIVE AUTISM SPECTRUM DISORDER PANEL 67 STAT AUTISM SPECTRUM DISORDER SUBPANEL 80 MICROCEPHALY PANEL 84 REFERENCES 89 DISCLAIMER 108 62 Southfield Avenue, Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Page 4 of 125 Stamford CT Lic#: CL-1016 www.sema4.com Genetic Testing for NEURODEVELOPMENTAL DISEASE Sema4 offers targeted next-generation sequencing (NGS) for neurodevelopmental conditions such as autism spectrum disorder, Intellectual disability, epilepsy, seizures, brain malformations, and microcephaly. NGS technology is ideal for diagnostic testing of these disorders due to the extreme locus heterogeneity and phenotypic overlap of the genes involved. Our customizable targeted NGS panel uses Agilent SureSelectTM QXT target enrichment and Illumina NovaSeq sequencing to detect pathogenic variants in genes involved in neurodevelopmental disease. Sema4 offers three primary neurodevelopmental panels: 1) the Comprehensive Epilepsy and Autism Panel (401 genes); and 2) the Microcephaly Panel (78). The Comprehensive Epilepsy and Autism Panel includes the Comprehensive Autism Spectrum Disorder (ASD) Panel (228) and the Comprehensive Epilepsy (226) panels. The Comprehensive Autism Spectrum Disorder Panel includes the STAT Autism Panel (30 genes). The Comprehensive Epilepsy Panel includes the Focal, Generalized, and Myoclonic Epilepsy (52), Infantile Epilepsy (58), Migraine (7), Neuronal Migration (22), Neuronal Ceroid Lipofuscinoses (9), and the Syndromic Epilepsy and Intellectual Disability (ID; 93) Subpanels. The Microcephaly Panel includes conditions such as microcephaly, holoprosencephaly, lisencephaly, and other brain malformations for differential diagnosis. These genes were selected for inclusion based on literature review, clinical actionability scores, and comparison with commercially available assays. Further information about these genes and panels is available in the following pages. Customizable testing is available for ordering a neurodevelopment disease group-specific gene panel. Targeted familial testing for a specific variant is also available. Parental testing is recommended. Sema4 offers whole exome sequencing if needed. Genetic testing may clarify the cause of an individual’s neurodevelopmental disease, provide information on the likelihood of related health issues, and also establish risk to other family members and future generations. The American College of Medical Genetics (ACMG) recommends that every person with developmental delay, ID, or ASD should receive a chromosomal microarray (CMA) to identify copy number variants (CNVs) that contribute to risk in 10-15% of individuals. Sema4 offers CMA testing as a first-tier test and an ultra-high resolution medical exome array for exon-level CNV detection as an add-on to NGS. Genetics The disorders included in this panel may be inherited in an autosomal dominant (AD), autosomal recessive (AR), -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 neurodevelopmental 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. 62 Southfield Avenue, Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Page 5 of 125 Stamford CT Lic#: CL-1016 www.sema4.com 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 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: ALDH7A1 chr5:125907050-125907056, ARX chrX:25031027-25031926, ATP13A2 chr1:17338212-17338244, ATP2A2 chr12:110718408-110718414, ATP6AP2 chrX:40440306-40440365, ATP7A chrX:77269723-77269729, ATP7A chrX:77278955-77279156, ATR chr3:142184699- 142184705, CACNA1H chr16:1203726-1204047, CACNA2D2 chr3:50540637-50540865, CDKN1C chr11:2905888- 2906730, CHRNA7 chr15:32460129-32460670, CHRNA7 chr15:32446096-32446198, COL18A1 chr21:46825134- 46825167, COL18A1 chr21:46825282-46825399, DMD chrX:31897426-31897627, DMD chrX:32668999-32669253, DMD chrX:32644476-32644680, DMD chrX:31627637-31627838, DMD chrX:32460213-32460334, DMD chrX:31219361- 31219367, DMD chrX:31219126-31219287, DMD chrX:32644160-32644321, EHMT1 chr9:140513469-140513512, ELP4 chr11:31703323-31703634, EPM2A chr6:146056322-146056645, FKTN chr9:108368751-108368962, FMR1 chrX:147018844-147019305, GABRD chr1:1950851-1950941, GK chrX:30687487-30687493, HGSNAT chr8:42995524- 42995881, HPRT1 chrX:133625461-133625467, HTRA1 chr10:124221157-124221651, IDS chrX:148584720-148585050, IQSEC2 chrX:53263389-53264377, KRIT1 chr7:91829915-91829921, LAMP2 chrX:119604075-119604081, MAGI2 chr7:77648620-77649304, NFIX chr19:13106640-13106689, NHS chrX:17753589-17753595, NOTCH3 chr19:15311587- 15311727, OTC chrX:38269298-38269509, SCN1B chr19:35521713-35521775, SCN3A chr2:165986438-165986817, SGSH chr17:78193967-78194168, ST3GAL5 chr2:86115935-86116039, SYNGAP1 chr6:33388030-33388119, TBX1 chr22:19748416-19748814, TMLHE chrX:154721184-154721338, TMLHE chrX:154722214-154722372, TMLHE chrX:154722006-154722167, TRIO chr5:14143823-14144002, TUBB2A chr6:3154085-3155190, TUBB2A chr6:3156266- 3156397, TUBB2B chr6:3224973-3226056, VPS13A chr9:79829178-79829419, and ZIC2 chr13:100637565-100637947. The
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  • Serum Albumin OS=Homo Sapiens

    Serum Albumin OS=Homo Sapiens

    Protein Name Cluster of Glial fibrillary acidic protein OS=Homo sapiens GN=GFAP PE=1 SV=1 (P14136) Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 Cluster of Isoform 3 of Plectin OS=Homo sapiens GN=PLEC (Q15149-3) Cluster of Hemoglobin subunit beta OS=Homo sapiens GN=HBB PE=1 SV=2 (P68871) Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4 Cluster of Tubulin beta-3 chain OS=Homo sapiens GN=TUBB3 PE=1 SV=2 (Q13509) Cluster of Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 (P60709) Cluster of Tubulin alpha-1B chain OS=Homo sapiens GN=TUBA1B PE=1 SV=1 (P68363) Cluster of Isoform 2 of Spectrin alpha chain, non-erythrocytic 1 OS=Homo sapiens GN=SPTAN1 (Q13813-2) Hemoglobin subunit alpha OS=Homo sapiens GN=HBA1 PE=1 SV=2 Cluster of Spectrin beta chain, non-erythrocytic 1 OS=Homo sapiens GN=SPTBN1 PE=1 SV=2 (Q01082) Cluster of Pyruvate kinase isozymes M1/M2 OS=Homo sapiens GN=PKM PE=1 SV=4 (P14618) Glyceraldehyde-3-phosphate dehydrogenase OS=Homo sapiens GN=GAPDH PE=1 SV=3 Clathrin heavy chain 1 OS=Homo sapiens GN=CLTC PE=1 SV=5 Filamin-A OS=Homo sapiens GN=FLNA PE=1 SV=4 Cytoplasmic dynein 1 heavy chain 1 OS=Homo sapiens GN=DYNC1H1 PE=1 SV=5 Cluster of ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide OS=Homo sapiens GN=ATP1A2 PE=3 SV=1 (B1AKY9) Fibrinogen beta chain OS=Homo sapiens GN=FGB PE=1 SV=2 Fibrinogen alpha chain OS=Homo sapiens GN=FGA PE=1 SV=2 Dihydropyrimidinase-related protein 2 OS=Homo sapiens GN=DPYSL2 PE=1 SV=1 Cluster of Alpha-actinin-1 OS=Homo sapiens GN=ACTN1 PE=1 SV=2 (P12814) 60 kDa heat shock protein, mitochondrial OS=Homo
  • Supplementary Figure S4

    Supplementary Figure S4

    18DCIS 18IDC Supplementary FigureS4 22DCIS 22IDC C D B A E (0.77) (0.78) 16DCIS 14DCIS 28DCIS 16IDC 28IDC (0.43) (0.49) 0 ADAMTS12 (p.E1469K) 14IDC ERBB2, LASP1,CDK12( CCNE1 ( NUTM2B SDHC,FCGR2B,PBX1,TPR( CD1D, B4GALT3, BCL9, FLG,NUP21OL,TPM3,TDRD10,RIT1,LMNA,PRCC,NTRK1 0 ADAMTS16 (p.E67K) (0.67) (0.89) (0.54) 0 ARHGEF38 (p.P179Hfs*29) 0 ATG9B (p.P823S) (0.68) (1.0) ARID5B, CCDC6 CCNE1, TSHZ3,CEP89 CREB3L2,TRIM24 BRAF, EGFR (7p11); 0 ABRACL (p.R35H) 0 CATSPER1 (p.P152H) 0 ADAMTS18 (p.Y799C) 19q12 0 CCDC88C (p.X1371_splice) (0) 0 ADRA1A (p.P327L) (10q22.3) 0 CCNF (p.D637N) −4 −2 −4 −2 0 AKAP4 (p.G454A) 0 CDYL (p.Y353Lfs*5) −4 −2 Log2 Ratio Log2 Ratio −4 −2 Log2 Ratio Log2 Ratio 0 2 4 0 2 4 0 ARID2 (p.R1068H) 0 COL27A1 (p.G646E) 0 2 4 0 2 4 2 EDRF1 (p.E521K) 0 ARPP21 (p.P791L) ) 0 DDX11 (p.E78K) 2 GPR101, p.A174V 0 ARPP21 (p.P791T) 0 DMGDH (p.W606C) 5 ANP32B, p.G237S 16IDC (Ploidy:2.01) 16DCIS (Ploidy:2.02) 14IDC (Ploidy:2.01) 14DCIS (Ploidy:2.9) -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 Log Ratio Log Ratio Log Ratio Log Ratio 12DCIS 0 ASPM (p.S222T) Log Ratio Log Ratio 0 FMN2 (p.G941A) 20 1 2 3 2 0 1 2 3 2 ERBB3 (p.D297Y) 2 0 1 2 3 20 1 2 3 0 ATRX (p.L1276I) 20 1 2 3 2 0 1 2 3 0 GALNT18 (p.F92L) 2 MAPK4, p.H147Y 0 GALNTL6 (p.E236K) 5 C11orf1, p.Y53C (10q21.2); 0 ATRX (p.R1401W) PIK3CA, p.H1047R 28IDC (Ploidy:2.0) 28DCIS (Ploidy:2.0) 22IDC (Ploidy:3.7) 22DCIS (Ploidy:4.1) 18IDC (Ploidy:3.9) 18DCIS (Ploidy:2.3) 17q12 0 HCFC1 (p.S2025C) 2 LCMT1 (p.S34A) 0 ATXN7L2 (p.X453_splice) SPEN, p.P677Lfs*13 CBFB 1 2 3 4 5 6 7 8 9 10 11