S204- It is All in the Genes: New and Emerging Screening Tests and Genetic Markers

Transcription

1 S204- It is All in the Genes: New and Emerging Screening Tests and Genetic Markers Parul Jayakar, MD, MS Director, Neurogenetics/ Metabolic Program Director, Miami Genetics Laboratories Division of Genetics and Metabolism Miami Children s Hospital Miami, Florida

2 Disclosure of Relevant Relationship Dr. Jayakar (or spouse/partner) has not had (in the past 12 months) any conflicts of interest to resolve or relevant financial relationship with the manufacturers of products or services that will be discussed in this CME activity or in his presentation. Dr. Jayakar will support this presentation and clinical recommendations with the best available evidence from medical literature. Dr. Jayakar does not intend to discuss an unapproved/investigative use of a commercial product/device in this presentation.

3 New, Emerging tests & Markers Historical perspectives & Established tests Evolving Technologies: Micro Array Sequencing Whole Exome Whole Genome Clinical Applications Diagnostic protocols Prenatal diagnoses Genetic counseling Pharmacogenomics What Next- the future

4 Historical Perspectives Established Tests : Walther Flemming discovered chromosomes or DNA 1902: Inherited diseases were first linked to chromosomes 1950: Structure of the DNA molecule :Southern blot analysis onwards : PCR & FISH

5 Developmental Delay (DD/ID) ACMG Practice Guidelines 2005; AAP 2006 Karyotype (yield ~3%) Targeted FISH (~2.6%) Fragile X or other targeted gene testing Metabolic testing Chromosome microarray? Autism Spectrum Disorders (ASD) Multiple Congenital Anomalies (MCA) ACMG Practice Guidelines 2008 Karyotype (yield ~5%) Fragile X (~5%) Chromosome microarray (~10%) Targeted gene testing (PTEN, MECP2)

6 Microarray SNP array (single nucleotide polymorphism) is a type of DNA microarray which is used to detect polymorphisin DNA, is the most frequent type of variation in the genome. There are around 50 million SNPs that have been identified in the human genome The most important applications of SNP arrays are for determining disease susceptibility and for measuring the efficacy of drug therapies designed specifically for individuals. SNPs can also be used to study genetic abnormalities in cancer. Mainly used in gene discovery

7 Microarray Workflow

8 Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies 10% increase in diagnostic yield Cost effective over FISH plus karyotype Competitive with karyotype for low level mosaicism Detects more submicroscopic imbalances than balanced rearrangements that will be missed The American Journal of Human Genetics 86, , May 14, 2010

9 Microarray- Genetic Counseling Issues Variants of uncertain significance Parents may misunderstand VUS as diagnosis Proceed with further testing/diagnosis How to counsel? Parental studies to define VUS Parents not available Parents have same change how to counsel parents? Detection of loss of heterozygosity SNP can detect long stretches of homozygosity Report consanguinity or incest to family/authorities? Lack of balanced translocation /inversion detection Cost- Insurance coverage, 7% population has DD/ID Comfort level of clinicians Ethical and legal issues Access to database for results interpretation Proper GC services and education available?

10 Microarray- Advantages Providing more sensitive test to our patients Diagnostic uncertainty can be traumatic Define management and treatment May shorten diagnostic journey One test views all of genome rather than multiple tests Cost effective for some Defining broader spectrum of known disorders by detecting known del/dup syndromes with abnormal or mild phenotype Increased use of microarray and database development will lead to better understanding of benign and pathogenic variants in the human genome Must eventually find appropriate methods for data storage, sharing, interpretation and ethical/legal issues if we are moving in this direction

11 Case 1 10 year old with Autism Macrocephaly No Dysmorphic features Intellectual disability GI- Gastrointestinal polyps Basic genetic work up- chromosomes, fragile X normal

12 CMA RESULTS PTEN

13 PTEN-Phosphatase and Tensin Homolog PTEN hamartoma tumor syndrome (PHTS) includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS), and Proteus-like syndrome. Genetically Related (Allelic) Disorders Adult-onset Lhermitte-Duclos disease (LDD)-dysplastic gangliocytoma of the cerebellum, a hamartomatous overgrowth known to be a feature of CS Autism/pervasive developmental disorder and macrocephaly. Germline PTEN pathogenic variants were identified in individuals history or family history consistent with CS/BRRS

14 Case 2 3 year old male with Chronic respiratory infections, pneumonias, choking episodes and GERD. Obesity but not hyperphagia. Patient has difficulty breathing at night with multiple obstructive episodes. Parents are 1 st cousins. Examination: Narrow nasal bridge, pointed nose. hypoplastic teeth, thin & pointy lips, polydactaly of right thumb, Conductive hearing loss, vision loss.

15 CASE 2 CMA FINDINGS

16 CASE 2 CMA FINDINGS - CONTINUED

17 CASE 2 CMA FINDINGS - CONTINUED

18 Case 2- Bardet Biedl Syndrome (BBS) Clinical Picture: Rod-cone dystrophy, Truncal obesity, Postaxial polydactyly, Cognitive impairment, Male hypogonadotrophic hypogonadism, Renal abnormalities Testing-Eighteen genes are associated with BBS Positive- BBS5+

19 NIPT- Non-Invasive Prenatal Testing Fetal Cell-free DNA in Maternal Blood Fetal DNA likely arises from cytotrophoblastic cells of placenta which is released into bloodstream as small DNA fragments ( bp) 10-15% of the cell-free DNA circulating in maternal blood is from the fetu Analyzes cell-free fetal DNA circulating in maternal blood-reliable after 7+ weeks gestation Undetectable within hours postpartum Uses mass-parallel sequencing to amplify fetal DNA in order to perform genetic testing Increases in fetal DNA for chromosomes 13, 18 and 21 and X and Y aneuploidies

20 CRITERIA FOR NIPT TESTING NIPT will be offered to any high risk patient who would normally be offered invasive testing NIPT technologies have been validated in singleton pregnancies at high risk for Trisomy 21 due to: Advanced maternal age Abnormal serum screen Personal or family history of aneuploidy Abnormal ultrasound Turners Syndrome Cystic hygroma

21 NIPT- Limitations False Positives have been reported Downs Syndrome :0.2% Trisomy 18: 1% Trisomy 13 and Sex chromosomes aneuploidies: 8-15% Sampling error and percentage of Free fetal DNA Does not screen for Open Neural Tube Defects A positive test will need to be followed with an amniocentesis or CVS Time constraints for reproductive decision making day turnaround & need for conformational testing Insurance coverage is variable

22 Exome Sequencing What is an Exon? Portion of the human genome that contains functionally important sequences of DNA - referred to as exons which make essential proteins. Why are exons important? Approximately 180,000 exons arranged in about 22,000 genes in the human genome which represents about 3% of the genome. Most of the errors are located in the exons. Sequencing of the exome analyzes a patient's DNA to discover the genetic cause of diseases. What is the principle? Sequence nucleotide by nucleotide, the human exome of an individual. The Patients sequence is then compared to standards and references in the normal population and any variations in an individual's DNA sequence can be identified and used diagnose mendelian and common diseases. Indications for testing undiagnosed genetic syndrome, neurological conditions including seizure disorders, movement disorders, severe developmental delay, and intellectual disability.

23 Exome Sequencing What is captured and what is not CAPTURED: Carrier status for common autosomal recessive conditions, such as cystic fibrosis and Tay-Sachs disease Variants in genes involved in drug metabolism. Expanded report will contain information about genes unrelated to the phenotype: deleterious mutations and unclassified variants in genes unrelated to the disease phenotype; and deleterious mutations in genes with no currently known association with disease in humans. NOT CAPTURED Triplet repeat expansions, large deletions and duplications Detection of small indels may not be as accurate as detection of base substitutions. Mitochondrial genome Genes that have closely related pseudogenes are not uniquely captured by this method Introns

24 CASE 3 16 yr old female- haitian/hispanic with normal development till about 7-8 yrs followed by: Pseudobulbar episodes of excessive crying started 5-7 yrs Abnormal MRI- bilateral cerebral hemisphere white matter disease, basal ganglia involvement and multiple symmetric lesions in the brainstem, putamen bilaterally, caudate nuclei. Spastic diplegia, abnormal gait with intoeing stable, no improvement with baclofen noted at 7-8 yrs Mod/severe (left>right) femoral anteversion with compensitory external tibial torsion, Neuromuscular kyphosis/scoliosis Loss of bladder and bowel control Encephalopathy- Neuroregression with Learning Disability stable in ESE, struggles in school Extensive infectious/genetic work up in past 7 yrs non revealing.

25 Whole Exome Results Juvenile Alexander: JUVENILE ALEXANDER DISEASE The juvenile form usually presents between Ages four and ten years, occasionally in the mid-teens. Bulbar/pseudobulbar signs, Ataxia, Gradual loss of intellectual function, Seizures, Megalencephaly, Survival ranges from the early teens to the 20s-30s.

26 Next Generation Sequencing Panels These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such have revolutionized the study of genomics and molecular biology. Examples: Hearing Loss Ataxia Cardiomyopathy Intellectual disability Autism Storage disorders

27 Case 4 7 yr old Caucasian female Severe autism Non Verbal Aggression Self injurious behavirors Intellectual disability Extensive genetic work up- non contributory to clinical symptomatology Testing- Next Generation Sequencing Panel

28 Smith-Magenis Syndrome Developmental delay, Cognitive impairment, and behavioral abnormalities. Hypotonia, hyporeflexia, Intellectual disability. Sleep disturbance, Stereotypies, and maladaptive and self-injurious behaviors, Inattention, distractibility, hyperactivity, impulsivity RAI1 -Deletion 17p11.2 ~95% RAI1- Sequence variants - 5%-10%

29 Pharmacogenomics Personalized Medicine Diagnosis predicting risk of disease Determining whether a treatment is working Producing safer drugs by predicting potential for adverse effects earlier Targeting groups of people most likely to benefit from a drug, while keeping its use from those who may be harmed by it Producing better medical products Ready access to information Decreasing health care costs The Goal of Personalized Medicine The Right Dose of The Right Drug for The Right Indication for The Right Patient at The Right Time.

30 Codeine and Cytochrome P450 CYP2D6 1. Codeine is a commonly used opioid Codeine is a prodrug It must be metabolized into morphine for activity 2. Cytochrome P450 allele CYP2D6 is the metabolizing enzyme in the liver 3. 7% of Caucasians are missing one copy of the Cytochrome P450 -CYP2D6 gene-codeine does not work effectively in these individuals 4. CYP2A6, CYP2B6, CYP2C9,CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are responsible for metabolizing most clinically important drugs

31 Warfarin: Significant Problems for Humans! Ranks #1 in total mentions of deaths for drugs causing adverse events (from death certificates) Ranks among the top drugs associated hospital emergency room visits for bleeding Overall frequency of major bleeding range from 2% to 16% (versus 0.1% for most drugs) Minor bleeding event rates in randomized control trials of new anticoagulants has been as high as 29% per year.

32 Warfarin Levels Depend on Two Enzymes CYP2C9 & VKORC1

33 Estimated Warfarin Dose (mg/day) Based on Genotypes

34 Warfarin Genetic Analysis Permits More rapid determination of stable therapeutic dose. Better prediction of dose than clinical methods alone. Applicable to the 70-75% of patients not in controlled anticoagulation centers. Reduces between 4,500 and 22,000 serious bleeding events annually. Genetic testing now required by FDA

35 Personalized Cancer Care

36 Whole Genome sequencing Determines the complete DNA sequence of an organism's genome at a single time. Sequencing all of an organism's chromosomal DNA as well as DNA contained in the mitochondria Full genome sequencing will provide information on all six billion bases in the human genome, >3,000 x data Common Applications: Discover common and rare mutations Assemble novel sequence de novo Compare DNA variations between multiple strains or individuals Associate DNA variants with a phenotype, such as a disease Discover biomarkers and therapeutic targets

37 AND AFTER THAT?

38 Questions to ponder 1. Variants of unknown significance/who does the analysis 2. Cost : Startup and price for each test 3. Reimbursements 4. Cost effectiveness in clinical testing 5. Ethical considerations

39 QUESTIONS? The woods are lovely, dark, and deep, But I have promises to keep, And miles to go before I sleep, And miles to go before I sleep. - Robert Frost