White paper The Genetic Horizon Improving clinical sensitivity in difficult-to-sequence genes for rare hereditary disorders Meeting clinical needs using custom solutions From Bench to Bedside: The Genetic Horizon Ongoing challenges The long-awaited promise of tailored treatments for individual patients based on their genetic Clinically relevant but highly homologous and · Masked regions in the reference data sets contain makeup is beginning to materialize. Next generation sequencing, along with artificial intelligence, repetitive regions within important genes are an uncertain or ambiguous variant calls that are the source ongoing challenge to analyze: of false positives, false negatives and other genotype have facilitated the rapid, accurate analysis and interpretation of genomic data for disease causing calling errors (e.g., calling homozygous variant at a variants which may be treatable through emerging gene therapy technology. · Certain genomic regions are difficult to analyze due to heterozygous site)1. complex sequence variations therein The number of clinical trials and drug development involves delivering a functional copy of the SMN1 gene in a Blueprint Genetics remains committed to resolving pipelines for rare disease are quickly growing and, one-time intravenous infusion (www.avexis.com). · Not only are there variants in these regions but there difficult-to-sequence regions that are hard to validate, encouragingly, the first approved treatments have been are types of variants that we cannot detect in the clinical interpret and confirm, by developing custom solutions. In successful. As the field of precision medicine is still in its The power of gene therapies is in the specificity of the diagnostic laboratory with current technologies (Table 1) this paper, we share our strategies and what is next in our infancy, increased awareness about the clinical utility of treatments: understanding the genetic mechanism of R&D pipeline. genetic testing is needed to drive the adoption of novel the disease and creating a therapy to counter it. Genetic · We are limited in our ability to validate certain regions/ diagnostic and treatment modalities. testing therefore becomes an integral part of rare disease variants because approximately 12% of the human management as determining the precise gene and variant genome is masked in gold-standard reference data sets At Blueprint Genetics, we feel that being fully transparent responsible for the disease is a prerequisite for targeted about our successes, challenges and limitations is key treatments and gene therapy. to making effective, powerful diagnostic tools available Table 1 Percentage of challenging regions in the human genome. to the rare disease community. By sharing our own troubleshooting strategies, we hope to move genetic The importance of transparency in 1.2% Low-coverage regions diagnostics and the promise of precision medicine forward. clinical genetic testing 6.9% Segmental duplications Actionability for the rare Our ability to accurately detect disease causing variants in the human genome is continuously improving. Single 6.8% Structural variants Rare diseases comprise a heterogeneous group of nucleotide variants, small insertions and deletions and thousands of genetically and clinically distinct disorders. exon-level deletions and duplications can be reproducibly 0.4% Genes with long repeat expansions Molecular diagnosis of these disorders has the potential to detected, almost throughout the entire genome, and their Sub-exon level deletions and duplications allow a patient’s treatment to be tailored and optimized. identification is becoming more important in clinical care. 1.9% Recent advances in gene therapy hold tremendous promise Absolute transparency in genetic diagnostics is essential as a cure for these devastating diseases. so that clinicians understand the quality and limitations of any given test and the suitability of the test for their As an example, retinal gene therapy has demonstrated patient. Blueprint Genetics is committed to providing the potential to cure different forms of inherited vision full disclosure of our analytic validation of testing, the loss. The first FDA-approved gene therapy is a one-time description of our technologies and datasets used in the treatment for individuals with retinal disease such as Leber diagnostic process and to the critical assessment of the congenital amaurosis and retinitis pigmentosa caused by performance metrics. homozygous RPE65 gene variants. The treatment involves delivering a functional copy of the RPE65 gene directly to However, not all patients with rare genetic disease retinal cells (www.luxturna.com). receive a molecular diagnosis. While the industry’s ability to accurately detect clinically actionable variants Similarly, targeted treatments for Spinal Muscular Atrophy is increasing, there are still areas that current next (SMA) have recently entered clinical trials. SMA is a severe generations sequencing (NGS) strategies and standard neuromuscular disease caused by variants in the SMN1 Sanger sequencing do not adequately cover which leads to gene leading to the loss of motor neurons and resulting poor sensitivity and decreased diagnostic yield. in progressive muscle weakness and paralysis. Phase 1 clinical trials have begun investigating the efficacy of gene therapy in the treatment of SMA Type 2. Treatment 1 Zook JM et al. Nat Biotechnol. 2014 Mar; 32(3): 246–51. 2 | The Genetic Horizon | Blueprint Genetics HORA41-02 The Genetic Horizon | Blueprint Genetics | 3 Our Approach to Difficult-to-Sequence Genes — Clinical Research & Development: Opening the Black Box Going the Extra Mile Innovative, customized solutions for Our first step in providing tailored diagnostic services was Our Clinical R&D team’s work is driven by the opportunity to establish a specialized team of dedicated members to to tackle the most challenging parts of our genome and difficult-to-sequence genes troubleshoot and develop custom solutions for challenging by the reward inherent in finding a genetic diagnosis for a genetic regions. Our Clinical Research & Development patient. Developing new methods to analyze challenging genes The need for custom variant confirmation solutions (R&D) team consists of genetics and molecular biology requires innovations in both bioinformatic data analysis may arise when: professionals. The team’s core focus is on genomic regions algorithms as well as laboratory protocols optimized for a · a low-quality variant is detected in a difficult-to-sequence too complex to resolve with standard NGS analysis particular genetic region. or duplicated region, and developing new methods to analyze technically · unusual SNP clusters are observed, or challenging, but clinically important, genes. To date, our key accomplishments with this approach · other quality control steps, such as coverage analysis, include successful sequencing of the highly repetitive detects an event deviating from normal Difficult-to-analyze genomic regions include genes ORF15 region of the RPGR gene, associated with X-linked that have: retinitis pigmentosa, accurate determination of the copy The R&D team then develops custom laboratory · pseudogenes numbers of each of the highly homologous SMN1 and methods that often involve: · other highly homologous regions in the genome SMN2 genes, associated with spinal muscular atrophy and · Specialized primer and probe designs · longer stretches of repetitive sequences a method to identify Alu element insertions and variants in · Long-range PCR and sequencing the polycystic kidney disease associated PKD1 gene. · Quantitative PCR Additionally, inversions, translocations, and other complex · Long-range PCR and sequencing genetic rearrangements are not typically amenable to When a variant is identified in the NGS data that routine NGS methods and require custom methods for successful Sanger sequencing is not able to confirm, a customized Results of a custom confirmation analysis may take up to sequencing and variant confirmation. confirmation assay is needed. Our Clinical R&D team 4–6 weeks depending on the scale of development work develops custom solutions to confirm these variants for required. individual patients after the reporting geneticist refers the case to the Clinical R&D team for evaluation and work-up. 4 | The Genetic Horizon | Blueprint Genetics The Genetic Horizon | Blueprint Genetics | 5 A Clinician–Lab Partnership ClinicalThe importance Information Can of Leadclinical to Finding a Variant Detailed Clinical information can make or break the diagnosis, especially Thatinformation Might Otherwise in genetic Be Missed diagnostics with difficult-to-sequence genes From 20,000–35,000 possible variants explaining the phenotype… Sometimes, when a likely complex disease-causing variant is identified, and a detailed clinical information is provided, the reporting geneticist will refer the case to Clinical R&D to work on customized solutions. The need for customization may arise when a low-quality variant is detected at difficult-to-sequence or duplicated region, when strange SNP clusters are observed or when other quality control steps such as coverage analysis detects any events deviating from normal. ? Clinician Insucient Sucient Did you know? clinical information Patient clinical information Clinical · At the exome level, every patient has approximately 20,000–35,000 variants. The challenge is to Laboratory find the 1 or 2 that are related to the patient’s phenotype Information exchange · Detailed