Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

Management of Genetic Disorders by sammyc2007

VIEWS: 706 PAGES: 78

									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



characteristics of the test

  

reliable valid inexpensive adaptable
easy to collect, analyze, ship sample informed consent benefit screening laws



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 WBC

C Karyotype

B
Enzyme/protei n assays; increased substrate

M 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

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
Trisomy 21

levels
Trisomy 21 Trisomy 18 45,X 47,XXY False pregnancy Molar pregnancy “missed abortion”

AFP

HCG UnE3

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 skin

C Karyotype

B
Enzyme/protei n assays; increased substrate

M 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 PCR to detect gene of interest CF gene (if PB has CF gene, egg must have normal gene)

Morula cell
Polar body

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


								
To top