Carrier Screening in ART 2014 The Value of Next-Generation DNA Sequencing Stephanie Hallam, PhD, FACMG, MBA VP Laboratory Operations & Medical Director, Good Start Genetics

Agenda Need for Carrier Screening Responsible Approach Clinically relevant tests Best in class technologies The Need for Accurate Screening Value of Next-Generation Sequencing Technology Clinical Experience Clinical benefits of sequencing-based assay

The Need for Carrier Screening

Importance of Carrier Screening Recommended by leading medical societies ACOG ASRM ACMG Jewish advocacy organizations (e.g., NTSAD)  Disorders should be prevalent, have severe forms, and be costly to treat For all women for all women of reproductive age

The Need for Better Screening 1/100 babies are born with an inherited disease1 20-30% of all infant deaths are due to genetic disorders2 11.1% of pediatric hospital admissions are for children with genetic disorders3 Preconception screening for disease-causing mutations and genetic counseling for carriers can reduce the incidence of these diseases Incidence of Tay Sachs disease was reduced by 90% in AJ due to awareness and screening4 1 Monogenic disorders, World Health Organization 2 Berry, et al, 1987 3 Scriver, et al, 1973 4 Kaback, et al, 2000

Cystic Fibrosis (CF) CF is the most common life- threatening, genetic disorder in Caucasians Despite widespread availability of carrier screening, more than 2,500 babies are born with cystic fibrosis each year The average survival age is late 30’s¹ Individuals with CF endure chronic symptoms with lifetime treatment costs estimated over $2,000,000¹ 1 in 25 Caucasians is a carrier of CF ¹CF foundation website

Spinal Muscular Atrophy (SMA) SMA is the leading genetic cause of death in infants and toddlers. 1/6,000 to 1/10,000 children are born with the disease The most severe form (Type I) manifests before 6 months of age and generally results in death before age two One in 40 to one in 50 people (approximately 6 million Americans) are carriers of the SMA gene. Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disease characterized by muscle atrophy and weakness. Data from SMA foundation website

Carrier Screening: Assessing Inherited Disease in Couples Helps determine risk for conceiving a child with an inherited disease About 1 in 16 Caucasians is a carrier for cystic fibrosis or spinal muscular atrophy, the most common autosomal recessive diseases. Preconception carrier screening for pathogenic gene mutations and genetic counseling can reduce the incidence of disease Fragile x syndrome does not follow this inheritance pattern.

Pre-Pregnancy Screening Provides Valuable Early Information Multiple options Proceed at-risk Prenatal testing IVF with pre-implantation genetic diagnosis (PGD) of embryos Sperm or egg donation Adoption Prenatal Screening Preparation, genetic counseling, support groups Newborn Screening Early detection enabling early treatment, genetic counselor, support groups

Responsible Approach

A Balanced Approach to Genetic Screening Testing for society guideline recommended disorders using a comprehensive set of disease-causing mutations Society Guideline Recommended Disorders Disease- Causing Mutations Balance drives clinical value and sets the new standard for excellence Prevalent & Severe Validated Pathogenicity

Guideline and Society-Recommended Disorders Focus on society-recommended disorders: Provides clinicians confidence that they are screening for relevant diseases Assures payers that testing is justifiable Jewish Societies Every test that GSG offers is recommended by either ACOG and/or ACMG and/or societies supportive of the Ashkenazi Jewish population. GSGs advisors and other opinion-leaders in the field have advised GSG to concentrate on the society-recommended disorders. Concentrating on these 22 disorders has allowed GSG to develop tests that are both highly accurate and clinically actionable. * Recommended if indicated by family history ** Recommended for individuals of French Canadian or Cajun descent AJ – Tests recommended by national Jewish advocacy societies.

The Best Technology for Each Test Test Methodology Disorder(s) Next-Generation DNA Sequencing (comprehensive) Bloom’s syndrome Glycogen storage disease 1a Canavan disease Maple syrup urine disease 1A/1B Cystic fibrosis Mucolipidosis type IV DLD deficiency Niemann-Pick type A/B Familial dysautonomia Tay-Sachs disease Familial hyperinsulinism Usher syndrome type 1F Fanconi anemia group C Usher syndrome type III Next-Generation DNA Sequencing Joubert syndrome 2 (targeted) Walker-Warburg syndrome Multiplex Ligation-Dependent Probe Amplification (MLPA) Alpha-thalassemia Spinal muscular atrophy (SMA) Tri-Nucleotide Repeat PCR & Fragile X syndrome Methylation Analysis Enzyme Analysis Tay-Sachs disease (Hex A) Hemoglobin Capillary Electrophoresis Beta-thalassemia Sickle cell disease Genotyping Gaucher disease Nemaline myopathy

The Need for Accurate Screening

Why is Accurate Screening Important? Screen with ~100 mutations Result = Negative Not tested because partner is negative

Why is Accurate Screening Important? Child affected with cystic fibrosis

Why is Accurate Screening Important? Carrier of a rare mutation, not detected by test that was used Carrier of a common mutation, but not tested

Why is Accurate Screening Important? Carrier identified by test with higher detection rate Carrier identified because his partner screened positive Embryo 1 in 4 chance for each pregnancy to be affected Reproductive Options Available

Next-Generation DNA Sequencing

DNA Sequencing: The “Old” Way First human genome ~13 years ~ $3B At completion, cost to repeat was $300M Routine clinical sequencing was unaffordable

Next-Generation DNA Sequencing Massively parallel chemistry and detection Faster Cheaper Revolutionizing Clinical Molecular Testing HS2500 rapid run dual flowcell 2x100, 27 hrs, reagent cost 2550+3520

Next-Generation Sequencing: Technology Matters Genotyping Used by many companies for routine carrier screening Tests for a limited set of only common mutations Provides limited utility beyond Caucasian and Jewish ethnicities Next-Generation Sequencing Comprehensively evaluates the gene Detects all known common and rare disease-causing mutations Delivers higher accuracy across ethnicities Why what using isn’t “good enough”

Delivering Clinically Actionable Results Comprehensive Set of Known Disease-Causing Mutations Rigorous, multi-year process to catalogue and evaluate each gene for all documented disease-causing variants Accurate, actionable results that do not include variants of unknown significance (VUS)

Evaluating Variants: Not all are pathogenic Some have no detrimental effect Some have a mild effect Some cause severe disease

Mutation Database: Validating the Variants Literature (PubMed) Locus Specific Databases Genetic Evidence Experimental Evidence Sequence Based Evidence Focus on mutations that cause disease and clinically important. Not just what easy to detect with technology! ~ 1,700 variants ~1,000 variants classified as disease-causing

NGS Versus Genotyping: Mutations in the CFTR Gene Each dot represents a disease-causing mutation. (illustrative purposes only) ACMG 23 Standard genotype screening panels (≈100) Previously publicly reported pathogenic mutations (550) Novel truncating mutations detectable by next-generation DNA sequencing (unknown) GoodStart Select™ detects all of the disease-causing mutations above

The Power of Sequencing NGS Tests for 5-10 Times More Pathogenic Mutations Per Disease than traditional genotyping DISEASE CARRIER FREQUENCY* LEADING COMPETITORS GOOD START GENETICS Bloom's Syndrome 1 in 134 1-2 51 Canavan Disease 1 in 55 4 44 Cystic Fibrosis 1 in 23 ~ 100 560 Dihydrolipoamide Dehydrogenase Deficiency 1 in 107 0 - 2 3 Familial Dysautonomia 1 in 31 2 - 32 2 Familial Hyperinsulinism 1 in 68 0 - 3 65 Fanconi Anemia Group C 1 in 100 2 - 4 26 Glycogen Storage Disease Type 1A 1 in 64 2 - 10 69 Maple Syrup Urine Disease Type 1A/1B 1 in 97 3 - 4 40 Mucolipidosis Type IV 1 in 89 9 Niemann-Pick Disease Type A/B 1 in 115 45 Tay-Sachs Disease 1 in 27 7 - 11 73 Usher Syndrome 1F 1 in 147 0 - 1 16 Usher Syndrome III 1 in 120 5 1 3 1 Data on file as of 12/2013. 2 Third mutation reported by one competitor not confirmed by GSG to be of clinical significance. 3 According to most-recently updated version of the GSG mutation list (9/13/2013). * Based on Ashkenazi Jewish population (population selected because figures are available for all disorders). Updated 5/2014

Mutations Reported Uncommon Mutations Common Mutations High frequency Previously reported in the literature In Validated Database

Mutations Reported Traditional genotyping tests for a limited set of mutations. Carrier screening by genotyping Common Mutations Uncommon Mutations

Truncating mutations expected to disrupt protein function Mutations Reported Good Start’s NGS test for 5-10 times more mutations than carrier screening tests performed by genotyping. Carrier screening by next-generation DNA sequencing Uncommon Mutations Novel Mutations Common Mutations Truncating mutations expected to disrupt protein function Any two mutations from the same disorder can cause disease

Detection of New Pathogenic Mutations NGS enables the discovery of rare and novel mutations in a pan-ethnic population Case Study: New CFTR Mutation Found Using NGS Normal Normal working protein Truncating Mutation Definition of Novel: Not yet described in the literature Disrupts protein structure and will cause disease Protein is cut off and does not work properly *Data presented at the 2013 Annual Meeting of the Pacific Coast Reproductive Society

Clinical Experience

Clinical Experience 22,296 patients from infertility centers across the US Screened for up to 14 disorders using next-generation DNA sequencing Carrier status determined by the presence or absence of a pathogenic mutation Analysis of results: Good Start’s NGS versus genotyping-based carrier screening tests Self-reported Ethnicity % of Total Caucasian 44.7% Not Provided 29.0% African American 7.0% Asian 6.3% Hispanic 5.8% Other 4.8% Ashkenazi Jewish 2.5% Not all sequencing is the same: anyone can buy the machine, but it is not as simple as that to have an accurate test. NGS has been used in research much longer than in a clinical setting because it is not easy to obtain the level of accuracy that is necessary for clinical use. It takes time to develop and validate to obtain that level of accuracy. Data up to 1/31/2014 .

Carriers Detected Carriers Detected Gene Genotyping Assays GSG Only Total Carriers Familial Hyperinsulinism 23 7 30 Canavan Disease 36 6 42 Maple Syrup Urine Disease Type 1A/1B 18 14 32 Bloom Syndrome 13 20 33 Cystic Fibrosis 952 51 1003 Usher Syndrome Type III 9 4 Dihydrolipoamide Dehydrogenase Deficiency 2 15 Fanconi Anemia Group C Glycogen Storage Disease Type 1a 5 41 Tay-Sachs Disease 87 105 Familial Dysautonomia 34 43 Mucolipidosis Type 1V 8 Usher Syndrome Type IF 10 19 Niemann-Pick Disease Type A/B 26 Total 1294 169 1463 ~12% of carriers would not have been identified using genotyping-based carrier screening1,2 CF has higher values because (1) more patients are being screened for it (2) it has a high carrier frequency 1 – Results based on analysis of 22,296 patients in IVF centers across the country screened for up to 14 disorders using GoodStart Select. 2 – Analysis was done by comparing the mutations found using GoodStart Select to competitors’ genotyping panels. If all competitors did not test for a particular mutation, then it was marked as GSG only.

Clinical Implications of NGS for Your Patients Reduce the risk of having a child with a common, severe genetic disorder High detection rate, regardless of ethnicity Low residual risk, regardless of ethnicity Confidence in a negative test result

Thank You!