“Genetic testing in 2014: How do you choose?”

1. “Genetic testing in 2014: How do you choose?” Chanika Phornphutkul, MD Associate Professor of Pediatrics Director, Division of Human Genetics Department of Pediatrics Warren Alpert Medical School of Brown University

2. The Father of Down Syndrome • In 1866 the physical characteristics of people with Down Syndrome were linked with decreased intellectual ability and grouped as one syndrome. The English physician, John Langdon Down, was the first to publish these findings. Due to the fact that the facial features of people with Down Syndrome were similar to those of people from Mongolia, he used the term mongoloid to refer to a person with (what is now known as) Down Syndrome

4. Genetics Background: The DNA era of 1950 1953: James Watson and Francis Crick - DNA structure as the “double-helix” 1956: Human cells contain 46 chromosomes 1959: Jerome Lejeune- Down syndrome children had an extra copy of chromosome 21

5. The Traditional Karyotypes

6. Genetics in the 60’s-80’s • Astute clinical description of various syndromes associated with cardiac malformation- DiGeorge Syndrome, William Syndrome, Turner Syndrome, Noonan Syndrome. • Explanations- • Chromosome abnormalities- Cytogenic study • Single Gene Disorders- Molecular &Biochem study • Environmental-drug, toxin • Association- VATER

7. FISH (Fluorescence In situ Hybridization)

8. N England J Med 359;16. 2008.

9. Basics of Array Comparative Genomic Hybridization • A microarray has thousands of spots (“array elements”) • Each spot has millions of copies of a particular strand of DNA representing groups of genes. • In the case of array comparative genomic hybridization, small areas of the chromosome are each assigned a unique spot.

10. Interpreting Spots: Red versus Green Equal amounts of Patient and Control DNA are co-hybridized onto a chip and then analyzed! Control DNA Patient DNA Control DNA Patient DNA Patient Duplication in This Spot Patient Deletion In These Spots

16. Common Chromosome Abnormality • William syndrome • 22q11 deletion • 4p-, 9p-,4q-, 11q-, 13q-, 18q- • 45, X • Trisomy 13,18,21

20. Chromosome abnormalities did not explain all the cardiac malformation • Noonan syndrome- Turner-like….

22. Genomics begins here.. 1970: Fred Sanger sequences DNA

23. Genomics in 1980’s-Gene by gene discovery 1983: polymerase chain reaction enabling the easy amplification of DNA 1989: Francis Collins and Lap-Chee Tsui sequence the human gene that encodes the CFTR protein (defects in CFTR cause cystic fibrosis)

24. The Human Genome Project The $3-billion project was formally founded in 1990 by the United States Department of Energy and the U.S. National Institutes of Health, and was expected to take 15 years. A 'rough draft' of the genome was finished in 2000 (announced jointly by then US president Bill Clinton and British Prime Minister Tony Blair on June 26, 2000). Ongoing sequencing led to the announcement of the essentially complete genome in April 2003, 2 years earlier than planned.

25. Incidence ~1:1000-2,500 Autosomal Dominant

26. Facial Characteristics • Triangular facies • Ear abnormalities in 44-90%: low-set posteriorly rotated ears and thick helix. • Ptosis, hypertolorism in 95% • Deeply grooved philtrum • Short or webbed neck in 95% • Malocclusion • Low posterior hairline • Micrognathia

28. Other phenotypic abnormalities:- • Short stature (post natal onset) in 50-80%. • FTT (40%) • Cardiac: pulmonic valve stenosis (50%). Hypertrophic cardiomyopathy (10-20%) • Thoracic abnormalities (53-70%) • Cryptorchidism (60%) • Neurologic: developmental delay, learning disability • Hematologic: bleeding (20%), juvenile myelomonocytic leukemia.

29. 1994:- Linkage analysis linked to 12q • 2001:- Gain of function mutations in PTPN11 found in 50% of NS (Tartaglia et al, Nature Genetics 29:465-468) Explains, AD inheritance! • PTPN11 encodes protein tyrosine phosphatase SHP-2 which is a cytoplasmic protein, function as an adaptor protein as well. • SHP-2 has been implicated in GH,IGF-I, insulin and leptin signaling.

31. From single gene disease to pathway disease • Noonan syndrome- Rasopathy • Marfan syndrome- Aortopathy

32. Along came more cardiac technology-Echo/ECG machineMore findings • Hypertrophic cardiomyopathy • Dilated cardiomyopathy • Long QT syndrome, Arrhythmogenic right ventricular dysplasia

33. PKP2 • Mutations in PKP2 cause arrhythmogenic right ventricular dysplasia • Account for 25-40% of positive ARVC cases • 15/133 mutations listed in HGMD are splicing mutations • 6/133 are large deletions

37. Multi-gene Panel

38. NGS Technology • Noonan syndrome- Rasopathy panel • Marfan syndrome- Aortopathy panel • Arrhythmia panel • HCM/DCM panel

39. Whole Exome Sequencing (WES) • Term coined in 2008 • Look at coding region (Exon) -1% of genome • Fall 2011, WES was available as a clinical test. • NEJM- 70 articles • Nature Genetics- 80 articles • Pubmed

40. July 1, 2014 Pubmed articles

41. Global view Whole Exome 50 million bp 25,000 genes Whole Genome 3 billion bp HGMD Genes 6 million bp 2000 genes Panel testing 500,000 bp 50 genes Slide from Samantha Baxter, MS, CGC

43. Targeted Panels WES

45. The Revolution of Genomics: 2001-2014

46. The Decreasing Cost of Genotype Information. Lu JT et al. N Engl J Med 2014;371:593-596.

47. Sounds promising..But we need to be thoughtful • Variants • Unintended information • How do we address “possible/potential/likely disease causing”? • Is WES better than WGS or we are just buying time?

48. Available Genetic Testing • Karyotype • FISH- the fish is almost dead??? • Microarray- Microarray is dying??? • Single Gene Testing- Sanger seq- Do we still need you????? • Multi-gene Panels- Next Generation seq • Biochemical testing- Barth syndrome, Pompe, mitochondrial disease • Whole ExomeSeq • Whole Genome Seq

49. Case 1- symptomatic child • Dysmorphic child, developmental delay • Abnormal Echo- pulmonic stenosis • Microarray revealed 7q11 deletion • AAP recommendation for William syndrome

50. From Bedside to Bench and Back to Bedside • RASopathy- Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of MEK162 in Noonan Syndrome Hypertrophic Cardiomyopathy • Marfan- Micro RNAs as a Marker of Aortic Aneurysm in Hereditary Aortopathy Syndromes