HEARING AND VISION LOSS NEXT- GENERATION SEQUENCING PANEL 62 Southfield Ave. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 F: 646-859-6870 Stamford CT Lic#: CL-1016 1 www.sema4.com 62 Southfield Avenue. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 2 F: 646-859-6870 Stamford CT Lic#: CL-1016 www.sema4.com 62 Southfield Avenue. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 3 F: 646-859-6870 Stamford CT Lic#: CL-1016 www.sema4.com Table of Contents GENETIC TESTING FOR HEARING AND VISION LOSS 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 VISION LOSS PANEL 9 RETINAL DISEASE SUBPANEL 14 ALBINISM, HERMANSKY-PUDLAK SYNDROME, AND WAARDENBURG SYNDROME SUBPANEL 24 DEVELOPMENTAL EYE SUBPANEL (ANOPHTHALMIA/MICROPHTHALMIA/ANIRIDIA) 26 STICKLER & CATARACT SUBPANEL 28 THE COMPREHENSIVE HEARING LOSS PANEL 32 BRANCHIO-OTO-RENAL SYNDROME SUBPANEL 37 USHER SYNDROME SUBPANEL 38 ZELLWEGER SYNDROME SUBPANEL 39 REFERENCES 41 DISCLAIMER 41 62 Southfield Avenue. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 4 F: 646-859-6870 Stamford CT Lic#: CL-1016 www.sema4.com Genetic Testing For HEARING AND VISION LOSS Hearing and/or vision loss can result from both genetic and non-genetic etiologies. In general, up to 50% of prelingual hearing loss has a genetic basis, as does up to 60% of congenital blindness among infants. Next generation sequencing (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 XT target enrichment and Illumina HiSeq sequencing to detect pathogenic variants in genes involved in hearing and or vision loss. These genes were selected for inclusion based on literature review, clinical actionablity scores, and comparison with commercially available assays. The Hearing and Vision Loss Panel (308 genes) includes the Comprehensive Hearing Loss (92) and Comprehensive Vision Loss (250) panels. Hearing loss subpanels include Usher Syndrome (11 genes), Zellweger Spectrum Disorder (9), and Branchio-Oto-Renal Syndrome (3). Vision loss subpanels include Retinal Disease (154 genes), Albinism, Hermansky-Pudlak Syndrome, and Waardenburg Syndrome (18), Developmental Eye Disorders (21), and Stickler Disease and Cataracts (41). Customizable testing is available for ordering a hearing-specific or vision-specific gene panel. Targeted familial testing is also available. Genetic testing may clarify the cause of an individual’s deafness and/or vision loss, provide information on the likelihood of related health issues, and also establish risk to other family members and future generations. Genetics The disorders included in this panel may be inherited in an autosomal dominant (AD), autosomal recessive (AR), or X-linked manner (XL). 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. 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). 62 Southfield Avenue. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 5 F: 646-859-6870 Stamford CT Lic#: CL-1016 www.sema4.com 2. Treatment: to clarify the cause of an individual’s hearing and/or vision loss, 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 individual gene sequencing or syndromic subpanels prior to ordering the full panel. Testing Methods, Sensitivity, and Limitations Next Generation Sequencing (NGS) Agilent SureSelectTM XT technology uses a custom capture library to target the exonic regions of the genes listed below. These targeted regions are sequenced using the Illumina HiSeq2500 system with 100 bp paired-end reads. The DNA sequences are mapped to and analyzed in comparison with the published human genome build UCSC hg19 reference sequence. The targeted coding exons and splice junctions of the known protein-coding RefSeq genes are assessed for the average depth of coverage and data quality threshold values. In our validation, average coverage was greater than 500X per sample with 99% of regions covered at greater than 20X. Sanger sequencing, as indicated, is performed in both directions using BigDye Terminator chemistry on the ABI 3730 DNA Analyzer with target specific amplicons as a confirmatory method for NGS positive results. Sanger sequencing may also be used to supplement targets for which NGS returned a low depth of coverage (<20 reads) or poor mapping scores. False negative results may occur if rare variants interfere with amplification or annealing. In addition, SALSA® MLPA® P163-D1 probemix is used to test the copy number of GJB2 and GJB6 where all testing is approximately 99% accurate. This MLPA test will detect the two most common GJB6 deletions, del(GJB6-D13S18830) and del(GJB6-D13S1854). SALSA® MLPA® P461-A1 probemix is used to test the copy number of OTOA and STRC where all testing is approximately 99% accurate. The sensitivity of this NGS panel is estimated at 99% for single base substitutions. This NGS technology may not detect all small insertions/deletions and is not diagnostic for large duplications/deletions, repeat expansions, and structural genomic variation. Therefore, oligonucleotide array CGH is available for this test for deletion duplication analysis (please see details below). Variant interpretation and classification was performed based on the American College of Medical Genetics Standards and guidelines for the interpretation of sequence variants (Richards et al, 2015). Frequency in control populations were evaluated based on the Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/) and Genome Aggregation Database (http://gnomad.broadinstitute.org/). All potentially pathogenic variants may have been confirmed by either a specific genotyping assay or Sanger sequencing as indicated. Variants classified as likely benign in the proband and any further familial testing of such variants will only be confirmed by Sanger sequencing if indicated. Any benign polymorphisms identified during this analysis were not reported. Variant interpretations, based on current knowledge, may change over time as more information arises. Sanger Sequencing 62 Southfield Avenue. Stamford, CT 06902 P: 800-298-6470 Branford CT Lic#: CL-0830 6 F: 646-859-6870 Stamford CT Lic#: CL-1016 www.sema4.com Sanger sequencing, as indicated, was performed in both directions using BigDye Terminator chemistry with the ABI 3730 DNA analyzer with target specific amplicons. It also may be used to supplement specific guaranteed target regions that fail NGS sequencing due to poor quality or low depth of coverage <20 reads or as a confirmatory method for NGS positive results. False negative results may occur if rare variants interfere with amplification or annealing. Oligonucleotide array Comparative Genomic Hybridization (Optional Add-on) The customized oligonucleotide microarray is a highly-targeted exon-focused array capable of detecting medically relevant microdeletions and microduplications at a much higher resolution than traditional aCGH methods. Each array matrix has approximately one hundred and eighty thousand 60-mer oligonucleotide probes that cover the entire gene panel. This platform is designed based on human genome NCBI Build 37 (hg19) and the CGH probes are selected to target the exonic regions of 304 genes. This test does not include analysis of MT-RNR1, P2RX2, STRC, and RAB28. For the majority of genes there are a minimum of 4 probes per exon. For very large exons, probes are distributed evenly along the exon with 1 probe every 125 bp. In the untargeted backbone regions, this array has one probe every 42kb. All genomic coordinates are reported using human genome NCBI Build 37 (hg19). Copy number aberrations are identified using the Aberration Detection Method-2 (ADM2) algorithm with a sensitivity threshold of 6.0 (Agilent Technologies). The log2 ratio threshold values to detect aberrations are < -0.25 for copy number losses and > 0.25 for copy number gains. Please note that any inconsistencies in the reported biological familial relationships could significantly change the interpretation of these results. For reported CNVs with uncertain clinical significance, continued surveillance of the medical literature for new information is recommended. The sensitivity of this assay is estimated to be greater than 99% for microdeletions and microduplications in the exonic regions of 304 medically-relevant genes.