Principles Of Genetic Screening, Diagnosis, And Gene Therapy

Question 1

Prenatal screening

Answer 1

. Assists at risk families in having healthy children
. Assists families at risk for having child w/ genetic disease in gathering medical and surgical tools needed
. Assists mother in potentially problematic pregnancies

Question 2

Biochemistry procedures in prenatal screening

Answer 2

. Measure of conc. Of fetalproduced analytes in maternal fluids (AFP, HCG, UE or all 3 together at 15 weeks)
. Measure con. Of fetal produced analytes in amniotic fluid from transabdominal amniocentesis (15-17 weeks)
. Karyotyping or fluorescence in situ hybridization (FISH) of fetal chromosomes in amniocytes to detect aneuploidies and major chromosomal deletion/duplication
. DNA analyses on fetal DNA via amniocentesis (15-17 weeks) or chorionic villus sampling (10-11 weeks) to detect mutation in genes

Question 3

Maternal serum alpha feto protein (MSAFP)

Answer 3

. In NTD, the con. Of AFP is higher than normal in maternal serum and amniotic fluid
. MSAFP test done at 16 weeks for open NTD
. Noninvasive, no risk, sensitivity is lower than amniotic AFP diagnosis
. Also does screening that inc. ability to detect Down’s

Question 4

Triple screen

Answer 4

. Inc. sensitivity for detecting down’s

. Measures 3 blood markers

Question 5

Quad test

Answer 5

. Also measures inhibin A
. Slightly better than triple test at estimating probability that baby has down’s
. Second trimester serum screen
. Inhibin A is inc. in down’s, No value for NTD screening

Question 6

First trimester markers

Answer 6

. Free beta-hCG: helpful in both 1st and 2nd trimester
. Total beta-hCG
. Pregnancy-assoc. plasma protein A (PAPP-A): glycoprotein produced by trophoblast reduced in Down syndrome and trisomy 18 used as quantitative screen in 1st trimester

Question 7

Nuchal translucency

Answer 7

. Scan using ultrasound to help identify higher risks for Down’s and trisomy 16 in fetus

Question 8

Cell free fetal DNA testing (cffDNA)

Answer 8

. Small amount of DNA released from placenta and into pregnant woman’s blood stream
. Cell free DNA in blood is screened for Down’s, trisomy 13 and 18, and problems w/ number of sex chromosomes
. Test done starting at 10 weeks
. Positive test followed by diagnostic test amniocentesis and chorionic villi sampling

Question 9

Amniocentesis

Answer 9

. Performed at 15-17 week
. Rate of fetal loss: 1/200
. Provides cells for growth to use chromosomal analysis, detecting enzyme deficiencies, extraction of DNA and subsequent DNA analysis
. FISH analysis w/o growing cells
. Amniotic fluid for AFP and other enzyme assays

Question 10

Indications for amniocentesis

Answer 10

. Advanced maternal age
. Abnormal maternal serum triple marker screening test
. Family history of chromosomal abnormality
. Neural tube defect/abnormal maternal serum alpha feto protein
. Mendelian disease detectable by biochemical or DNA analysis

Question 11

Chorionic villi sampling

Answer 11

. Performed at 10-11 weeks
. Aspirate fetal trophoblastic tissue by trans cervical or transabdominal approach
. Risk of fetal loss: 1-1.5%
. CVS may inc. risk of limb reduction defects

Question 12

Tandem mass spectrometry

Answer 12

. Screening for multiple disorders by measuring elevations of compounds from dried blood sampling obtained after birth by heel prick
. All states screen for 29 conditions
. New conditions added if condition can be detected 24-48 hrs after birth and can’t be detected by doctor exam, test is specific and sensitive, early detection and intervention has effective treatment

Question 13

Diagnostic testing

Answer 13

. Used to confirm/rule out known suspected genetic disorder in symptomatic individual

Question 14

Predictive testing

Answer 14

. Asymptomatic individuals w/ family history of genetic disorder
. Presymptomatic and predisposition always testing

Question 15

Carrier testing

Answer 15

. Identifies individuals who have gene mutation for disorder inherited in autosomal recessive or x-linked manner
. Carriers usually do not have symptoms related to mutation
. Offered to individuals who have family members w/ genetic condition, family members of identified carrier, and individuals in ethnic or racial groups w/ higher rate for certain condition

Question 16

Cytogenetic testing

Answer 16

. Identifies large structural changes
. Helpful in Syndrome phenotypes and symptoms assoc. w/ abnormalities on scale of chromosomes rather than single exons or genes
. Chromosomal analysis, FISH, spectral karyotyping
. Comparative genomic hybridization (CGS) allows detection of amplifications and deletions of smaller regions fo DNA along lengths of all chromosomes

Question 17

Mutation detection

Answer 17

. Targeted and limited to predefined number of sequence changes selected in advance
. Selection based on assoc. w/ clinical phenotype
. Polymerase chain reaction assay and allele and specific oligonucleotide hybridization (ASO)
. Allele specific short DNA sequences immobilized in separate well and arranged in chip Grid pattern in combo w/ fluorescent labeled test DNA
. Drawback: in diseases w/ several known mutations, currently available assays only test for partial panel

Question 18

In vitro fertilization and diagnosis

Answer 18

. Begins w/ removal of single cell from 8-16 cell blastomere generated in invitro fertilization
. w/ PCR, DNA is amplified then used for single gene condition diagnosis
. FISH analysis done to diagnose aneuploidy

Question 19

Polar body analysis

Answer 19

. Examine polar body shed at same time as ovum
. Early detection w/ avoiding pregnancy termination
. Provides info on mother’s genetics
. Most useful when aneuploidies, x-linked recessive conditioned or carrier risks related to mom’s genes are suspected

Question 20

Gene therapy

Answer 20

. Expression of an exogenously introduced gene in target host tissue
. Agent used to facilitate delivery of therapeutic gene to its target (vector)
. Vectors can be viral or non viral, by non-viral have not been as successful

Question 21

Advantages and disadvantages of using retrovirus in gene therapy

Answer 21

. A: stable expression from integration into host DNA, prolonged expression
D: potential for insertional mutagenesis, requires dividing cells

Question 22

Advantages and disadvantages of using lentivirus in gene therapy

Answer 22

A: broad range of target cells, doesn’t require cell division
D: potential for insertional mutagenesis, no clinical trials yet

Question 23

Advantages and disadvantages of using adenovirus in gene therapy

Answer 23

A: broad range of target cells, does not require division
D: transient expression, immune response to viral proteins, insert size limit fo 7.5 kb

Question 24

Advantages and disadvantages of using adeno-assoc. virus in gene therapy

Answer 24

A: infects proliferating and non-proliferating cells, stable gene expression
D: insert size limit of 4.5 kB

Question 25

Clinical trials of gene therapy in people have shown some success in treating what diseases?

Answer 25

. Severe combined immune deficiency
. Hemophilia
. Blindness caused by retinitis pigmentosa
. Leukemia (CAR-T therapies)

Question 26

CRISPR technology

Answer 26

. Appears to overcome issues assoc. w/ viral gene therapy
. Challenge is get to desirable gene to right place inside the body
. Other approaches include gene silencing and epigenetic modifications