Whole Exome Sequencing (WES): Questions and Answers for Providers

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1 Whole Exome Sequencing (WES): Questions and Answers for Providers 1. What is Whole Exome Sequencing? What is the difference between Whole Exome Sequencing (WES) and Whole Exome Sequencing Plus Pharmacogenomics (WESPP)? What paperwork and clinical information is requested for WES? Is it required? What sample types are accepted? Are parental samples required for WES? What if both of the patient s biological parents are not available for testing? If the patient s parents are not available for testing, can I send samples from other family members to complete the required trio? If other family members share the same phenotype as the proband, can they also be tested? Which variants will be reported and how will they be categorized on the report? What is the policy for reporting secondary findings? Are parental results reported? Are mitochondrial variants assessed? Is carrier status reported for conditions recommended by ACMG or ACOG for reproductive carrier screening? How are results reported? What is the turnaround time? What types of variants are not detected by this test? Are there genes that are not evaluated by WES? What is the depth of coverage for WES? What is the sensitivity of WES for detecting single nucleotide variants? What size insertions/deletions can the lab reliably detect? How are variants classified? How are variants filtered? What method is being used for WES testing? Do you perform Sanger verification of detected variants? Can WES reveal that 1 of the patient s parents is not related to the patient (ie,nonpaternity, nonmaternity, or adoption)? Can you perform reanalysis of a patient s exome? Can I have a copy of the patient s raw data? Can I order targeted testing for the relative of a patient whose variant was identified by WES? What is the cost of the test? What are the CPT codes for WES?... 4 References... 5 Table 1. Genes analyzed in WES for secondary findings Mayo Foundation for Medical Education and Research Page 1 of 6

2 1. What is Whole Exome Sequencing? Whole Exome Sequencing is a next-generation sequencing test that evaluates patients with suspected genetic disorders for germline mutations within the coding regions (exons) of approximately 23,000 genes. 2. What is the difference between Whole Exome Sequencing (WES) and Whole Exome Sequencing Plus Pharmacogenomics (WESPP)? Pharmacogenomic variants are not evaluated when Whole Exome Sequencing (WES) is ordered. WESPP includes evaluation of pharmacogenomic variants in 11 genes: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, HLA-A, HLA-B, SLCO1B1, UGT1A1, and VKORC1. For additional information about WESPP, please refer to the LTC or the Whole Exome Sequencing Plus Pharmacogenomics (WESPP): Questions and Answers for Providers document 3. What paperwork and clinical information is requested for WES? Is it required?.thorough clinical information is required for the most accurate interpretation of variants identified by whole exome sequencing. In order to ensure appropriate interpretation of your patient s test results, the Patient Information and Informed Consent forms are required. This paperwork is available as a fillable PDF that consists of: Ordering Checklist (page 1) Patient Information (pages 2-4) Informed Consent Form (pages 5-7) Please also submit the following: Clinic notes from specialists relevant to the patient s clinical features A 3-generation pedigree.please send all paperwork with the samples. Although not required, a faxed copy of the paperwork can also be sent directly to the Molecular Genetics Laboratory, Attn: WES Genetic Counselors at What sample types are accepted? WES is currently validated on peripheral whole blood samples only..if insurance preauthorization is being requested prior to testing, CRYO / Cryopreservation for Molecular Genetic Studies can also be ordered. Please contact the Molecular Genetics laboratory to request WES testing on a cryopreserved sample. 5. Are parental samples required for WES? Yes, samples from the patient and both biological parents (trio) are required. Whole exome sequencing is performed on all members of the trio in order to determine the inheritance pattern of all variants. Interpretation of variants is more accurate when the laboratory is able to compare the results between the parents and their child. 6. What if both of the patient s biological parents are not available for testing? Because a trio including the patient and both biological parents is required for WES, testing cannot be completed without both parental samples. 7. If the patient s parents are not available for testing, can I send samples from other family members to complete the required trio? No, the trio must include the patient and both biological parents in order to proceed with WES. 8. If other family members share the same phenotype as the proband, can they also be tested? Samples from additional affected family members outside of the trio may be accepted on a case-by-case basis. Although whole exome sequencing will not be performed on samples from additional affected family members outside of the trio, these samples may be tested for variants of interest identified in the proband to assess segregation. For more information, call Mayo Clinic Laboratories at and ask to speak with a WES genetic counselor. 9. Which variants will be reported and how will they be categorized on the report? Variants will be reported in the following categories: Likely Causative Variants Possibly Relevant Variants Variants in Genes of Uncertain Significance (GUS) Note: Variants in GUS will only be reported if there is a high degree of suspicion that they may be causative of a patient s reported clinical features. Secondary Findings (unless the patient opts-out of receiving these results on the informed consent form) Note: Secondary findings will only be reported if they are in 1 or more of the 59 genes recommended by the American College of Medical Genetics and Genomics (ACMG). Refer to question 10 and Table 1 for more details. Page 2 of 6

3 10. What is the policy for reporting secondary findings? Secondary findings will be assessed and reported in accordance with the American College of Medical Genetics and Genomics (ACMG) Recommendations for Reporting of Secondary Findings in Clinical Exome and Genome Sequencing, 2016 Update(1), unless the patient opts-out of receiving these results on the informed consent form. ACMG s recommendations include the analysis of variants in 59 genes associated with cardiac conditions, cancer predisposition, and other genetic syndromes. Please see Table 1 at the end of this document for a list of the genes and associated syndromes included in ACMG s recommendations. Only variants identified in these 59 genes will be reported as secondary findings. In accordance with ACMG recommendations, only variants that are known or expected to be pathogenic will be reported and novel missense variants will not be further evaluated as secondary findings. Variants of uncertain significance in these genes will not be reported (unless they are in a gene associated with the patient s reported clinical features). 11. Are parental results reported? The parent from whom a reported variant was inherited will be stated on the proband s report. Parents will not receive a separate interpretative report. 12. Are mitochondrial variants assessed? Variants in nuclear encoded mitochondrial genes will be assessed. However, mitochondrial DNA is not sequenced as part of WES and therefore variants in these genes are not reported. If analysis of mitochondrial DNA is desired, consider ordering a mitochondrial DNA sequencing test. 13. Is carrier status reported for conditions recommended by ACMG or ACOG for reproductive carrier screening? No, carrier status for conditions recommended by ACMG or ACOG for reproductive carrier screening is not specifically evaluated or reported unless the patient is found to have a variant in a gene for a condition that overlaps with the patient s reported clinical features. 14. How are results reported? WES results will be reported directly to the client laboratory and will be incorporated into the patient s medical record in accordance with individual institution processes. 15. What is the turnaround time? The turnaround time for WES is approximately 12 weeks. 16. What types of variants are not detected by this test? Due to the limitations of next-generation sequencing, the following variant types may not be detectable by this test: some insertions and deletions, structural chromosome rearrangements, polyploidy, intronic variants outside of the consensus splice site, variants in repetitive regions, variants occurring in regions with pseudogenes, mitochondrial variants, low-level mosaic variants, and other unknown abnormalities. 17. Are there genes that are not evaluated by WES? While WES targets the entire exome (coding areas of the genome), some genes may not be adequately evaluated due to poor coverage of a region in which a specific gene is located. At this time, greater than 95% of the exome can be sequenced well enough to be interpreted. Variants in genes lacking evidence of clinical significance and variants in genes unrelated to the patient s reported clinical features will not be evaluated (unless present in genes evaluated for medically actionable secondary findings in accordance with ACMG recommendations). Pharmacogenomic variants are not evaluated by WES. If pharmacogenomic results are desired, consider ordering WESPP / Whole Exome Sequencing Plus Pharmacogenomics. 18. What is the depth of coverage for WES? For each patient, 98.3% of the exome is covered at >10X, and 97% of the exome is covered at >20X. 19. What is the sensitivity of WES for detecting single nucleotide variants? With the depth of coverage metrics described above, this assay detects >99% of single nucleotide variants in the exome. 20. What size insertions/deletions can the lab reliably detect? With the depth of coverage metrics described above, this assay detects 95% of small insertions and deletions up to 30 bases in the exome. Genomic copy number variants are not assessed or reliably detected. CMACB / Chromosomal Microarray, Congenital, Blood should be ordered for formal evaluation of clinically relevant copy number variants. Page 3 of 6

4 21. How are variants classified? Variants are evaluated and classified as either benign, likely benign, variant of uncertain significance (VUS), likely pathogenic, or pathogenic in accordance with ACMG recommendations.(2) Variants classified as benign or likely benign are not reported. Variant classification may change over time if additional information becomes available. At this time, it is not standard practice for the laboratory to systematically review likely pathogenic variants and variants of uncertain significance that are detected and reported. Health care providers are encouraged to contact the laboratory to learn how the classification of a particular variant may have changed over time. 22. How are variants filtered? Inheritance pattern filtering is performed based upon the affected status of the proband s parents. Variants fitting any biologically plausible mechanism are evaluated further. Variants with a minor allele frequency greater than or equal to 1% are not typically evaluated. Unless previously reported as pathogenic or disease-causing in ClinVar or HGMD, the following variants may not be evaluated: low impact variants (synonymous, deep intronic, 5 /3 UTR, etc), variants in genes lacking evidence of clinical significance, and variants in genes unrelated to the patient s reported clinical features (unless present in genes evaluated for medically actionable secondary findings in accordance with ACMG recommendations). 23. What method is being used for WES testing? Whole exome sequencing is performed on genomic DNA extracted from all samples submitted. The exome is captured utilizing a custom reagent developed by Mayo Clinic and Agilent Technologies. Sequencing is performed on an Illumina HiSeq 2500 Next-generation sequencing instrument, using HapMap Sample NA12878 as an internal control. Paired-end 101 base-pair reads are aligned to a modified human reference genome (GRCh37/hg19) using Novoalign (Novocraft Technologies, Malaysia). Sequencing quality is evaluated using FastQC.(3) All germline variants are jointly called through GATK Haplotype Caller and GenotypeGVCF.(4) Each variant is annotated using the BioR Toolkit(5), and subsequently evaluated for clinical relevance by a team of scientists, genetic counselors, and laboratory directors. 24. Do you perform Sanger verification of detected variants? Variants of interest in the patient are confirmed by automated fluorescence dideoxy sequencing (aka Sanger sequencing), if technically necessary. 25. Can WES reveal that 1 of the patient s parents is not related to the patient (ie, nonpaternity, nonmaternity, or adoption)? WES requires samples from the patient and both biological parents in order to be able to compare variants found in the patient to those found in the parents. Thus, with this sequencing data it is possible to discover that a parent is unrelated to the patient (misattributed maternity or paternity). In this situation, no results will be generated. The ordering provider will be contacted by the laboratory to discuss available options. 26. Can you perform reanalysis of a patient s exome? A health care provider can request reanalysis of the patient s reported variants. Contact the laboratory and ask to speak with a WES genetic counselor for more information. 27. Can I have a copy of the patient s raw data? A health care provider can request the patient s raw data. A fee will be charged for the cost of the storage device. Contact the laboratory and ask to speak with a WES genetic counselor for more information. 28. Can I order targeted testing for the relative of a patient whose variant was identified by WES? Yes, FMTT / Familial Mutation Targeted Testing can be ordered for variants identified by WES. Documentation of the specific variants is required and must be provided with the sample in order to perform this test. Contact the laboratory and ask to speak with a WES genetic counselor for more information. 29. What is the cost of the test? Contact Mayo Clinic Laboratories ( ) for current pricing information. 30. What are the CPT codes for WES? The following CPT codes will be applied to the proband only: and x 2. Page 4 of 6

5 References 1. Kalia SS, Adelman K, Bale SJ, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med. 2016; Epub ahead of print 2. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5): McKenna A, Hanna M, Banks E, et al: The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010;20(9): Kocher JP, Quest DJ, Duffy P, et al: The Biological Reference Repository (BioR): a rapid and flexible system for genomics annotation. Bioinformatics 2014;30(13): Table 1. Genes analyzed in WES for secondary findings In accordance with ACMG recommendations for reporting of secondary findings in clinical exome and genome sequencing.(1) Gene Conditions Inheritance Pattern Variants Reported ACTA2 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD ACTC1 Hypertrophic or dilated cardiomyopathy AD KP APC Familial Adenomatous Polyposis AD APOB Familial Hypercholesterolemia SD KP ATP7B Wilson disease AR* BMPR1A Juvenile polyposis syndrome AD BRCA1 Hereditary Breast and Ovarian Cancer AD BRCA2 Hereditary Breast and Ovarian Cancer AD CACNA1S Malignant Hyperthermia Susceptibility AD KP COL3A1 Ehlers-Danlos Syndrome, Vascular Type AD DSC2 Arrhythmogenic Right-Ventricular Cardiomyopathy AD DSG2 Arrhythmogenic Right-Ventricular Cardiomyopathy AD DSP Arrhythmogenic Right-Ventricular Cardiomyopathy AD FBN1 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD GLA Hypertrophic or dilated cardiomyopathy XL KCNH2 Romano-Ward Long QT syndrome types 1, 2, and 3, Brugada Syndrome AD KCNQ1 Romano-Ward Long QT syndrome types 1, 2, and 3, Brugada Syndrome AD LDLR Familial Hypercholesterolemia SD LMNA Hypertrophic or dilated cardiomyopathy AD MEN1 Multiple Endocrine Neoplasia type 1 AD MLH1 Lynch syndrome AD MSH2 Lynch syndrome AD MSH6 Lynch syndrome AD MUTYH MYH-associated polyposis AR* MYBPC3 Hypertrophic or dilated cardiomyopathy AD MYH11 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD MYH7 Hypertrophic or dilated cardiomyopathy AD KP MYL2 Hypertrophic or dilated cardiomyopathy AD KP MYL3 Hypertrophic or dilated cardiomyopathy AD KP NF2 Neurofibromatosis type 2 AD OTC Ornithine transcarbamylase deficiency XL PCSK9 Familial Hypercholesterolemia AD KP PKP2 Arrhythmogenic Right-Ventricular Cardiomyopathy AD PMS2 Lynch syndrome AD PRKAG2 Hypertrophic or dilated cardiomyopathy AD KP PTEN PTEN Hamartoma Tumor Syndrome AD RB1 Retinoblastoma AD Page 5 of 6 Continued on next page

6 Continued Gene Conditions Inheritance Pattern Variants Reported RET Multiple Endocrine Neoplasia type 2; Familial Medullary Thyroid Cancer AD KP RYR1 Malignant Hyperthermia Susceptibility AD KP RYR2 Catecholaminergic Polymorphic Ventricular Tachycardia AD KP SCN5A Romano-Ward Long QT syndrome types 1, 2, and 3, Brugada Syndrome AD SDHAF2 Hereditary Paraganglioma-Pheochromocytoma syndrome AD KP SDHB Hereditary Paraganglioma-Pheochromocytoma syndrome AD SDHC Hereditary Paraganglioma-Pheochromocytoma syndrome AD SDHD Hereditary Paraganglioma-Pheochromocytoma syndrome AD SMAD3 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD SMAD4 Juvenile polyposis syndrome AD STK11 Peutz-Jeghers AD TGFBR1 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD TGFBR2 Marfan syndrome, Loeys Dietz syndromes, and familial thoracic aortic AD TMEM43 Arrhythmogenic Right-Ventricular Cardiomyopathy AD KP TNNI3 Hypertrophic or dilated cardiomyopathy AD KP TNNT2 Hypertrophic or dilated cardiomyopathy AD TP53 Li-Fraumeni syndrome AD TPM1 Hypertrophic or dilated cardiomyopathy AD KP TSC1 Tuberous sclerosis complex AD TSC2 Tuberous sclerosis complex AD VHL Von Hippel-Lindau syndrome AD WT1 WT1-Related Wilms Tumor AD *Homozygous and biallelic mutations will be reported only (carrier status will not be reported). AD = Autosomal dominant AR = Autosomal recessive EP = Expected pathogenic KP = Known pathogenic SD = Semidominant XL = X-linked Page 6 of 6

Informed Consent Columbia Whole Genome or Whole Exome Sequencing

Informed Consent Columbia Whole Genome or Whole Exome Sequencing Please read the following form carefully and discuss with your ordering physician before signing consent. This consent is intended for the