Management of Genetic Disorders

Management of Genetic Disorders     Screening Clinical Tests Genetic Consultation Legal and Ethical Considerations Screening Definition  the application of specific tests (cytogenetic, biochemical or molecular) to an informed population to detect variation which may lead to the identification of carriers or other individuals at risk Criteria  Screening criteria –  characteristics of the disorder     frequent severe prior to clinical onset quan/qual alterations reliable valid inexpensive adaptable easy to collect, analyze, ship sample informed consent benefit screening laws  characteristics of the test      characteristics of the system     Frequecies of Some Inborn Errors of Metabolism for which Newborn Screening Is Available Cystic Fibrosis Congenital hypothyroidism Cystinuria Alpha-1-antitrypsin deficiency Phenylketonuria Histidinemia Galactosemia Biotinidase deficiency Adenosine deaminase deficiency Maple Syrup Urine Disease Homocystinuria 1 in 2500 liveborn 1 in 6000 1 in 7000 1 in 8000 1 in 12000 1 in 17000 1 in 57000 1 in 60000 1 in 100000 1 in 200000 1 in 200000 Note: Frequencies may vary among ethnic groups Variation by Ethnic Group for Cystic Fibrosis F508 Mutation Ethnic group/Location African American Askenazai Jews Spanish; Italians French Canadians Northern European Caucasians (French, German, English, Swiss) Frequency F508 30 37 50 70 76 Cystic Fibrosis Facts        1 in 2500 Caucasians (liveborn) 1/20 to 1/25 carriers (No. European Caucasians) 4 of 5 without a family history CFTR gene/protein Most common mutation is F508 > 350 other mutations identified DNA “brush” test Clinical Tests Peripheral Blood (PB)  Procurement  venipuncture  5-10cc in vacutainer tube (red, green, purple, yellow)  heel stick (newborn): 5-10 drops Source of cells C B Enzyme/protei n assays; increased substrate M WBC Karyotype Direct gene analysis with probes Isolate mRNA then make cDNA RBC Hb; enzyme assays Enzyme/protei n assays; increased substrate; AFP/HCG/UnE3 Serum/plasma Fetal cells Karyotype Enzyme/protei n assays; increased substrate Direct gene analysis with probes PB:  Screening Pregnancies for NTD & CA Maternal serum (MS) screening:  a-fetoprotein (AFP) human chorionic gonadotropin (HCG)   unconjugated estriol (UnE3)  a-fetoprotein (AFP):     globulin protein produced by the fetal liver, yolk sac, GIT and kidneys enters amniotic fluid (AF) via excreted urine crosses placenta and enters maternal serum (MS) increases to max in MS between 12-18 weeks  Human chorionic gonadotropin (HCG):    produced by the placenta crosses into maternal circulation concentration increases and peaks between 8-11 weeks, then decreases in MS  Unconjugated estriol (UnE3):     produced by the fetal adrenal cortex travels to fetal blood, then to fetal liver (hydroxylated) crosses placenta and enters MS measure in MS as the unconjugated steroid  MSAFP+/Triple Test/AFP Xtra    use of all three markers (AFP, HCG, UnE3) determine risks for NTDs determine risks for specific CA: Trisomies18 and 21 Marker AFP levels ONTD (open Neural Tube Defects) OVWD (open Ventral Wall Defects) Amniotic band syndrome Fetal demise Renal agenesis Obstructive uropathy Ectopia vesicae Hydrocephalus Gestational diabetes Genital herpes Toxemia Placental abnormality TWINS levels Trisomy 21 Trisomy 18 45,X 47,XXY False pregnancy Molar pregnancy “missed abortion” HCG UnE3 Trisomy 21 Trisomy 21  Summary of screening for CA:  AFP alone detects 15-20% DS pregnancies  AFP + age + race + gest diabetes + wt detects 35-49% DS pregnancies  AFP + HCG + UnE3 + age detects 65-70% DS pregnancies  Other indicators can be added such as free Beta, inhibin and PAPP-a that can increase detection to 80+% When How Pro & Con Con Borderline cases difficult to interpret 85% reliability for NTDs 65-70% reliability for trisomies Must screen 4% of ALL females to achieve 70% detection rate Must have accurate gest age It’s only a screen Ave 15.5-18.5 weeks (14-21 max) Collect MS in red top clot tube Do ultrasound to confirm gestational age Use radioimmunoassay or monoclonal Ab test  Pro No need to correct for weight, gest diabetes, race if use all markers Prepares family for birth of affected child Provides an option to the family Fibroblasts  Procurement  skin biopsy  1-3 mm “punch” Source of cells C B Enzyme/protei n assays; increased substrate M skin Karyotype Direct gene analysis with probes Bone Marrow (BM) Procurement  1-5 cc BM in a red top vacutainer tube  iliac crest or sternum  Source of cells C B M Bone marrow Karyotype: direct or 24 hour culture for leukemia to determine type, treatment, assess remission Assess surface antigens to determine if transplantatio n was successful Ultrasound (Sonogram) Procurement  use of high frequency sound waves that bounce off tissues and produce echoes which are converted to pictures (sonogram) and viewed on a screen  fluid = dark; tissue = white  Detect Proper implantation Location of placenta Confirm pregnancy Multiple pregnancies IUGR AF volume (poly or oligo) Heart rate Growth rate (serial US) Cord position Cord blood flow Fetal anomalies: NTDs, cardiac defects Renal tract anomalies Limb reduction defects Omphalocoele Gastroschisis Assess Correct gestational age via BPD*, chest diameter, femur length Need for postnatal treatment With PND Used in conjunction with CVS*, amnio, PUBS**, etc *BPD = biparietal diameter *Chorionic villous sampling **Percutaneous umbilical blood sampling Amniocentesis (Amnio)  Reason for Referral  AMA  previously affected child  translocation carrier  family history (AR, XR) Amniocentesis (Amnio)  Procurement  removal of AF transabdominally with US* at 14-16 weeks gestation determined via last menstrual period (LMP)  *Ultrasound (US):      locates the placenta (anterior or fundal in 70%) assess amount of AF date pregnancy via BPD, femur length, chest size locate fetus assess anatomy (brain, spinal cord, stomach, kidneys, HR) Amniocentesis (Amnio)  Procurement (cont)  removal of 10-20 cc fluid which contains amniocytes and fluid  use 20-21 gauge spinal needle, strict asepsis w/ or w/o local anesthetic  AF is produced by the fetus  amniocytes are fetal in origin: skin, amnion, eyes, GU tract, GIT, respiratory system Source Amniotic fluid C B AFP for NTD (no correlation with DS) AchE for NTD Enzyme assays Increased substrate M Amniocytes (fetal cells) Karyotype Enzyme assays Increased substrate Direct gene analysis using probes Amniocentesis (Amnio)  Risks  1 in 200 overall  Fetus:      placental or cord hemorrhage puncture fetal scratches infection loss of AF (oligohydramnios) due to leakage may lead to Potter’s Syndrome, a pressure deformation  Mom:        1 in 300 miscarriage risk uterine contractions abdominal tenderness and swelling infection fluid loss (oligohydramnios) vaginal spotting/bleeding decision leading to 2nd trimester termination Amniocentesis (Amnio)  Problems/complications         anterior placenta fibroids twins Rh incompatibility maternal cell contamination culture failure TAT decision to terminate Early Amnio     Prior to 14 weeks, some as early as 12 Data over 7-8 years indicates a ½% miscarriage risk Some docs use smaller gauge (25) needle and collect 14-15 cc “Conference on PND and Therapy” (1998)     N=892 11-12 weeks Sab = 4% leakage = 2.5% clubfoot = 1.5% concluded that CVS was safer than amnio at 11-12 weeks Chorionic Villous Sampling (CVS)  Reason for Referral  AMA  previously affected child  translocation carrier  family history (AR, XR) Chorionic Villous Sampling (CVS)  Procurement       biopsy of chorion frondosum to obtain villi US guidance 8-11 weeks gestation ~25 mg wet tissue via catheter transvaginally or transabdominally villi are assumed to reflect fetal composition Source CVS cells C Karyotype (24 hour direct culture or 1 week) B Enzyme assays Increased substrate NOTE: Tissue not suitable for AFP M Direct gene analysis using probes Chorionic Villous Sampling (CVS)  Risks  1-3% overall for experienced doc  Fetus:     infection (<1/200) limb defects due to disruption of fetal development; severe arm/leg anomalies (5/289 pregnancies – UK data) oromandibular hypogenesis amniotic puncture leading to amnionitis  Mom:  ½-1½% miscarriage risk  cramping/bleeding  infection (<1/200) Chorionic Villous Sampling (CVS)  Problems/complications      cannot do AFP for NTD; must do MSAFP at 15+ weeks do villi really reflect fetal composition? mosaicism Rh limb defects  Greatest advantage  TIME Percutaneous Umbilical Blood Sampling (PUBS)  Procurement      1st used in 1982 for IV intrauterine transfusion for severe hemolytic anemia accompanied by fetoscopy: the viewing of the fetus using an endoscope and fiber optic illumination done at ~16 weeks removal of fetal blood from the umbilical vein (16 mm thick) use of a 20-22 gauge needle to obtain 1-2 ml blood Source Fetal blood C Karyotype (WBC) B Enzyme assays Increased substrate (WBC, RBC, serum) M Direct gene analysis using probes (WBC) Percutaneous Umbilical Blood Sampling (PUBS)  Treatment  blood gases  Rh status  drug administration for fetal infection  BM transplant  hemophilia/clotting deficiencies  hemolytic anemia  Ab testing Percutaneous Umbilical Blood Sampling (PUBS)  Risks  1-10% overall for experienced doc  Fetus:    infection fetal death (1-1½ %) injury  Mom:  ranges from 0.8% to 3.5%  infection  bleeding Percutaneous Umbilical Blood Sampling (PUBS)  Problems/complications   unknown fetal/postnatal effects on development repeated treatments requiring repeated risks Fetal Biopsy  Procurement    accompanied by fetoscopy: the viewing of the fetus using an endoscope and fiber optic illumination transabdominally removal of specific tissue (eg: liver, muscle) Source Fetal tissue C B Enzyme assays (eg: PKU, DMD) M Direct gene analysis using probes Fetal Biopsy  Risks  1-10% overall for experienced doc  Fetus:    infection fetal death (1-1½ %) injury  Mom:  ranges from 0.8% to 3.5%  infection  bleeding Assisted Reproduction (AR) & Preimplantation Genetics (PG)  Procurement (AR)  IVF        ova obtained via laparoscopy and Pergonal transvaginal US needle through top of vagina reaches ovary retrieve 5-7 eggs embryo freezing after fertilization increased miscarriage risk with age Does the technology itself lead to an increase in damage of the genes/chromosomes? Assisted Reproduction (AR) & Preimplantation Genetics (PG)  Procurement (PG)  biopsy of blastomere, morula or polar body  one cell can be PCRed for DNA Source Blastomere C Interphase cytogenetics using FISH for #13, 18, 21, Y B Enzyme assays (eg: LN) M Direct gene analysis using probes Morula cell Polar body PCR to detect gene of interest CF gene (if PB has CF gene, egg must have normal gene) Assisted Reproduction (AR) & Preimplantation Genetics (PG) Risks  once pregnancy is achieved, risks similar to those of other PND except for miscarriage with PG  multiple pregnancies (naturally high risk)  Assisted Reproduction (AR) & Preimplantation Genetics (PG) Problems/complications        sperm contamination use only female techs to avoid Y chromosome contamination time of enzyme expression oocyte enzyme contamination uterine lavage may not be successful viability of frozen embryos vs. frozen eggs Products of Conception (POC)  Procurement   Sab Tab Source Fetal tissue or placenta (EEM) C Karyotype B Enzyme assays (limited) M Direct gene analysis using probes Products of Conception (POC) Risks  Mom:  hemorrhaging  infection  uterine damage  Products of Conception (POC)  Problems/complications      infection use of urea for Tab leads to non-viable cells tissue will not grow non-sterile conditions for Tab/Sab Does EEM really reflect the status of the fetus? Genetic Consultation/Parental Options Genetic Consultation  Definition/purpose:      to relay information on diagnosis, prognosis, risks, implications, options, tests, therapies, services and mode of inheritance for a genetic disorder to maintain confidentiality to be understandable and accurate to relieve anxiety to provide management and follow-up   “It is a kind of social work which is often medical, but not always so.” Sheldon Reed Directive vs. non-directive Reasons for Referral        Advanced maternal age (AMA) Previous family history  single gene defects  chromosomal anomalies  multifactorial disorders  cancer history Repeated miscarriages Ambiguous genitalia Infertility Abnormal prenatal diagnosis Teratogen exposure Ideal Consultation Schedule Session 1: 1. 2. 3. 4. 5. Obtain family and medical history Discuss available options and tests Discuss costs of tests Arrange for collection of samples Schedule next session Session 2: 1. 2. 3. 4. Present and discuss test results Explain mode of inheritance (if applicable) Present additional options (if available) Explain risks to future children and/or other family members Treatment/Therapy    Fetal surgery Enzyme replacement therapy Gene replacement therapy Fetal Surgery: Congenital Diaphragmatic Hernia Congenital Diaphragmatic Hernia Major Clinical Features polyhydramnios dyspnea cyanosis pulmonary hypoplasia pneumothorax hypoxia “hole” or hernia failed to close at 8 weeks 75% die before birth organs press on lungs stomach fills w/ fluid acidosis            Congenital Diaphragmatic Hernia Treatment best performed at 22-28 weeks halothane for uterine relaxation anesthesia for mom and fetus open abdomen, open uterus use Gore-Tex patch to repair and enlarge fetal abdomen to accommodate organs must control post-operative uterine contractions       Enzyme Replacement Therapy Enzyme Replacement: Questions to be Asked       How is the enzyme to be given? What is the source of the enzyme? How can sufficient quantities be obtained? How is the enzyme targeted to the needed tissue? What is the half life of the enzyme? Will the enzyme alleviate/reverse the clinical symptoms? Enzyme Replacement: Examples     Tay Sachs Disease Gaucher’s Syndrome Fabry’s Disease Hurler Syndrome Gene Replacement Therapy Gene Replacement: Questions to be Asked        How will gene expression be controlled? What is/are the side effect(s) of the virus used? Will the gene product be produced in sufficient quantities? Must the gene be targeted to the needed tissue? Will the gene product cleave its substrate? Will the gene product alleviate/reverse the clinical symptoms? Can the gene product correct mental retardation? Methods for Gene Replacement Allotransplantation = replacement of cells (eg: bone marrow) of a patient with histocompatible cells from an acceptable donor Methods for Gene Replacement Stem Cells = pluripotent cells that can give rise to any/all specialized cells of the body Stem Cell Transplantation = when transplanted, bone marrow stem cells, for example, can give rise to all types of blood cells Methods for Gene Replacement Autotransplantation = use of the patient’s own cells (eg: bone marrow) to introduce the needed gene via retroviral insertion Methods for Gene Replacement Autotransplantation procedure:       “good” gene added to attenuated retrovirus BM cells removed from patient BM cells infected with modified, attenuated retrovirus BM cells returned to patient “good” gene copies increase with increase in BM cells needed gene product provided to patient by “good gene” Problems/Advantages with Autotransplantation       Control of gene expression? Will substrate be cleaved? Will all clinical problems be solved? Can MR be corrected? Are there side effects of virus? Will enzyme be produced in sufficient quantity? No immune reaction Decrease in many clinical signs\ Decrease in accumulated substrates    Gene Replacement: Examples    Maroteaux-Lamy Syndrome (MPS VI) Hurler Syndrome ADA deficiency Adenosine Deaminase Deficiency Major Clinical Features complete absence of ADA severe combined immunodeficiency (SCID) recurrent and severe infections failure to thrive intestinal malabsorption      Legal & Ethical Considerations Definition of Terms  LEGAL = according to the law  ETHICAL = the study of the general nature of morals and of the specific moral choices to be made by the individual in his relationship with others  MORAL = of or concerned with the judgment of the goodness or badness of human action and character Definition of Terms (cont)  BRAIN DEATH = human death requires cessation “of all functions of the entire brain including the brain stem” (President’s Commission on Ethics in Medicine and Research”)  WRONGFUL LIFE = child sues for being born with a “burden”  WRONGFUL DEATH = right of beneficiaries (relatives of deceased) to recover for the loss they suffer from the death of the affected Genetic/Medical Professionals & Their Responsibilities Primary Care Physician Source of referral; oversees care; receives and conveys test results Provides clinical diagnosis for M.D. Clinical Geneticist genetic disorders Ph.D. Medical Geneticist/Genetic Counselor Provides genetic consultation and risk assessment Clinical Laboratory Provides diagnostic confirmation