4/24/2017 Personalized Medicine: What This Talk Will Cover Genetic Testing and the Implications for Future Therapies Genetic testing for inherited marrow failure . General background: What are gene mutations? . Risk and severity: Do all people with inherited mutations develop disease? National Patient and Living with . How does medical genetic testing differ from commercially available testing? Aplastic Anemia, Family Conference MDS or PNH Las Vegas, NV April 22, 2017 Cytogenetic testing in Bone Marrow Failure Genomic profiling of bone marrow cells in MDS and AA for acquired mutations Implications for diagnosis, prognosis and therapies Katherine R. Calvo, M.D., Ph.D. Department of Laboratory Medicine, Clinical Center National Institutes of Health Benefits and Limitations of Genetic Testing and Genomic Profiling Bethesda, Maryland Bone marrow failure syndromes: How can genetic testing help to distinguish and guide therapy? AA/PNH Part One: PNH LGL Autoimmune SDS Disease: AA MS, IBD, uveitis, HYPOCELLULAR Genetic Testing for DM type 1, etc. TELOMERE MDS (DKC) GATA2 MDS AML Inherited Marrow Failure FA Acquired AA SDS AA, aplastic anaemia; AID, autoimmune disease; AML, acute myelogenous leukemia; DKC, dyskeratosis congenita; IBD, inflammatory bowel disease; LGL, large granular lymphocyte leukemia; GATA2, Gata2 deficiency; FA, Fanconi anemia; SDS, Shwachman–Diamond syndrome; MDS, myelodysplastic syndrome; MS, multiple sclerosis; DM, diabetes mellitus; PNH, paroxysmal nocturnal hemoglobinuria Modified from Young NS, et al. Blood 2006;108:2509–19 1 4/24/2017 GENETIC TESTING – Background GENETIC TESTING – Background The Basics: GENES are made of DNA The Basics: DNA is made up of Nucleotides (building blocks) Each person has a unique “Genome” made up of DNA is made up of four types of SNPs= Single Nucleotide Polymorphisms DNA that forms individual genes building blocks (called nucleotides) Humans have around 30,000 genes These spell out the genetic “code” Genes encode the directions for making: used in our genes Proteins and everything that is you We all have normal “variations” in Hair color/texture, eye color, skin color our genetic code that make each one All the cells in the blood, bone marrow, and of us different or unique https://neuroendoimmune.wordpress.com/2014/03/27/dna-rna-snp-alphabet-soup-or-an- every organ of the body introduction-to-genetics/ http://prn.fm/wp-content/uploads/2015/05/DNA.jpg A=Adenosine C=Cytosine G=Guanine T=Thymidine GENETIC TESTING – Background What is a mutation? WHAT IS A MUTATION? A mutation is a change in the genetic code that can increase your risk of getting: Disease or Conserved region A major change in whatever the gene is responsible for Mutations usually occur in conserved regions of genome 2 4/24/2017 GENETIC TESTING – Background GENETIC TESTING – Background WHAT ARE CHROMOSOMES? WHAT ARE CHROMOSOMES? DNA CHROMOSOMES FROM ONE - We normally have 23 pairs of BONE MARROW CELL => NORMAL MALE KARYOTYPE In the nucleus of cells DNA is chromosomes tightly wound and forms Chromosomes - Each chromosome contains thousands of genes TELOMERES = protective caps - Chromosomes are numbered at the ends of each chromosome according to size Chromosome Telomeres - Sex Chromosomes: - XX female https://ghr.nlm.nih.gov/primer/basics/howmanychromosomes - XY male http://www.yourgenome.org/facts/what-is-a-chromosome GENETIC TESTING – Background GENETIC TESTING – Background WHAT ARE CHROMOSOMES and HOW ARE THEY INHERITED? HOW DO WE GET MUTATIONS? INHERITED (Germline) MUTATIONS - Most cells have two copies of each chromosome => Inherited mutations are present at birth and are in all of the cells of the body - One is inherited from your father and the other from Recessive mutations – Need two copies of the your mother mutation (one from mom and one from dad) - Each child has a unique for disease combination of parents’ DNA Dominant mutations – Only need one copy of the mutation (from mom or dad) for disease https://scratchcradle.wordpress.com/2012/07/29/gms5-chicken-chromosomes/ 3 4/24/2017 How does medical genetic testing ordered by your doctor for bone GENETIC TESTING marrow failure differ from commercially available DNA testing? Examines your DNA to look for mutations in the genetic code “Next Generation Sequencing” Ted.com Panel of genes related to hereditary marrow failure Usually testing is only needed once => Samples sent for testing can be: Saliva, Buccal Swab, Blood, Bone Marrow, Skin biopsy, or Hair follicle Ancestry DNA testing Commercially Available DNA testing – Genetic Traits Similar: 23andMe.com => DNA sequencing techniques Asparagus Odor Detection Hair Curliness Different: Back Hair (available for men only) Light or Dark Hair The genes tested Bald Spot (available for men only) Male Hair Loss (available for men only) Bitter Taste Perception Newborn Hair Amount Nuclear or mitochondrial DNA Cheek Dimples Photic Sneeze Reflex genes that vary based on: Cleft Chin Red Hair . patterns of human migration Earlobe Type Skin Pigmentation . ethnic populations Earwax Type Sweet Taste Preference Eye Color Toe Length Ratio Examples: Finger Length Ratio Unibrow National Geographic Genographic Project Freckles Widow's Peak Ancestry.com 23andMe https://www.geni.com/blog/dna-testing-for-genealogy-getting-started-part-two-376163.html 4 4/24/2017 Commercially available DNA testing: Health Genetic Mutations Causing Hereditary Bone Marrow Failure Disorders 23andMe - 35 Health related Genes currently • tested including: Limited amount of information Inheritance FANCC (3 variants related to Ashkenazi Jewish decent) related to small number of gene Gene Pattern Cystic Fibrosis mutations well studied by Fanconi anemia over 19 genes encoding FANC proteins AR, XLR Sickle Cell Anemia scientists Telomeropathies, Dyskeratosis cong. TERT, TERC, DKC and others AD, AR, XLR Alpha-1 antitrypsin deficiency (lung and liver disease) Diamond Blackfan anemia RPS19, RPS24, RPL11 AD Schwachman Diamond SBDS AR Late-onset Alzheimer’s disease • Nearly all of the genes related Celiac disease (a digestive disorder, can’t eat gluten) Familial MDS/AML GATA2, RUNX1, DDX41 AD to BMF and MDS are not Early-onset primary dystonia (a movement disorder) Familial AA SRP72 AD Factor XI deficiency (a blood-clotting disorder) included (with the exception of FANCC) Familial Thrombocytopenia/AA/MDS ANKRD26, ETV6 AD Gaucher disease type 1 (an organ and tissue disorder) Familial AML, ET, CMML, PMF ATG2B, SKIP duplications AD Glucose-6-phosphate dehydrogenase deficiency • Not sufficient for diagnosing Hereditary hemochromatosis (an iron overload disorder) hereditary marrow failure Hereditary thrombophilia (a blood clot disorder) disorders Parkinson’s disease Bone marrow failure syndromes: How can genetic testing help to distinguish and guide therapy? Medical Genetic Testing for Bone Marrow Failure AA/PNH PNH LGL Purpose: Autoimmune SDS Disease: AA => To diagnose hereditary bone marrow failure MS, IBD, uveitis, HYPOCELLULAR DM type 1, etc. TELOMERE MDS disorders (DKC) GATA2 MDS AML FA Acquired AA SDS => Sequence genes known to cause BMF, looking for mutations AA, aplastic anaemia; AID, autoimmune disease; AML, acute myelogenous leukemia; DKC, dyskeratosis congenita; IBD, inflammatory bowel disease; LGL, large granular lymphocyte leukemia; GATA2, Gata2 deficiency; FA, Fanconi anemia; SDS, Shwachman–Diamond syndrome; MDS, myelodysplastic syndrome; MS, multiple sclerosis; DM, diabetes mellitus; PNH, paroxysmal nocturnal hemoglobinuria Modified from Young NS, et al. Blood 2006;108:2509–19 5 4/24/2017 GENETIC TESTING – Reasons Your Doctor May Order Genetic Testing Why is it important to identify an inherited BMF gene mutation? If other family members have: • Proper diagnosis Bone marrow failure MDS • Guides therapy Leukemia Physical malformations: small height, abnormal fingers or nail growth, etc. • Implications for family members Stiffening (fibrosis) of the lung and liver Early hair graying • Implications for future pregnancies => Evaluate for an inherited genetic mutation • Donor selection and screening for bone marrow transplantation Most common onset of disease is in children and young adults Genetic Testing for Inherited Mutations Genetic Testing - Informed Consent Examples of Genes Commonly Tested in Sequencing Panels for Bone Marrow Failure RPL11 SBDS VPS45 BRCA2 FANCA G6PC3 RPL3A SBF2 WAS Patient or guardian must fully understand: CSFR3 FANCB GATA2 RPL5 SLX4 WRAP53 CTC1 FANCC* GFI1 RPS10 SRP72 DKC1 FANCD2 HAX1 - Testing procedure RPS19 TERC - Benefits and limitations of the test ELANE FANCE MPL FANCF NHP22 RPS24 TERT - Possible consequences of the test ERCC4 FANCG NOP10 RPS26 TINF2 - Implications for family members and future pregnancies FANCI PALB2 RPS7 USB1 - Voluntary agreement to have the test done FANCL RAD51C RTEL1 XRCC2 FANCM RBM8A RUNX1 * The only gene included in 23andMe testing 6 4/24/2017 Example of Genetic Testing Results What does “Variant of unknown significance” mean? Patient 1 Result Patient 1 Result, cont. DNA Protein “Variant of Unknown Significance” VUS: Zygosity: Not sure if the DNA change is a normal Heterozygous - one copy of gene is mutated and the other copy is normal variation, or if it is a mutation. Homozygous – both copies of the gene are mutated Inheritance (pattern for the specific gene): Recessive - need both copies of the gene mutated to have increased risk of disease Dominant – only need one copy of the gene mutated for increased risk of disease Question: Do all family members with an inherited mutation develop