1. Genetic testing and Gene Therapy
Chapter 13
Genetic testing and Gene Therapy

2. Genetic Testing
analysis of chromossomes, DNA, RNA, proteins, or other analytes
prenatal diagnosis, heterozygote carrier detection, and presymptomatic diagnosis

3. Population Screening for Genetic Disorders
Types of Genetic Screening and Prenatal Diagnosis
Population Screening for Genetic Disorders
Newborn Screening: from blood
Phenylketonuria
Galactosemia
Hypothyroidism
Hemoglobinopathies
Others: maple syrup urine disease, homocystinuria, tyrosinemia, and several other diseases
*Universal newborn hearing screening (>60% of congenital hearing loss is due to genetic factors)
Heterozygote Screening
Tay-Sachs disease, Ashkenazi Jewish population
Sickle cell disease, African-American population
Thalassemias in at-risk ethnic groups
Cystic fibrosis in some populations (persons of European descent, Ashkenazi Jews)

4. Prenatal Diagnosis of Genetic Disorders
Types of Genetic Screening and Prenatal Diagnosis
Prenatal Diagnosis of Genetic Disorders
Diagnostic Testing (Invasive Prenatal Diagnosis)
Amniocentesis
Chorionic villus sampling
Percutaneous umbilical blood sampling (PUBS)
Preimplantation genetic diagnosis
Population Screening
Maternal age >35 years
Family history of condition diagnosable by prenatal techniques
Quadruple screen: maternal serum α-fetoprotein, estriol, human chorionic gonadotropin, inhibin-A
First trimester screening: ultrasonography, PAPP-A, and free β subunit of human chorionic gonadotropin
Fetal Visualization Techniques
Ultrasonography
Radiography
Magnetic resonance imaging

5. Family Screening for Genetic Disorders
Types of Genetic Screening and Prenatal Diagnosis
Family Screening for Genetic Disorders
Family history of chromosomal rearrangement (e.g., translocation)
Screening female relatives in an X-linked pedigree (e.g., Duchenne muscular dystrophy, fragile X syndrome)
Heterozygote screening within at-risk families (e.g., cystic fibrosis)
Presymptomatic screening (e.g., Huntington disease, breast cancer, colon cancer)

7. The distribution of creatine kinase in DMD
Genetic screening
The distribution of creatine kinase in DMD
67% sensitivity
95% specificity
The distribution of creatine kinase (CK) in normal women and in women who are heterozygous carriers for a mutation in the Duchenne muscular dystrophy gene. Note the overlap in distribution between the two groups: About two thirds of carriers have CK levels that exceed the 95th percentile in normal women. If the 95th percentile is used as a cutoff to identify carriers, then the sensitivity of the test is 67% (i.e., two thirds of carriers will be detected), and the specificity is 95% (i.e., 95% of normal women will be correctly identified).

8. Genetic screening

9. Newborn screening

10. Heterozygote screening

11. Public Policy Guidelines for Heterozygote Screening
Recommended guidelines
Screening should be voluntary, and confidentiality must be ensured.
Screening requires informed consent.
Providers of screening services have an obligation to ensure that adequate education and counseling are included in the program.
Quality control of all aspects of the laboratory testing, including systematic proficiency testing, is required and should be implemented as soon as possible.
There should be equal access to testing.

12. Pedigree for autosomal dominant breast cancer
Presymptomatic Diagnosis
Pedigree for autosomal dominant breast cancer
In this pedigree for autosomal dominant breast cancer, the analysis of a closely linked marker on chromosome 17 shows that the mutation is on the same chromosome as marker allele 1 in the affected mother in generation II. This indicates that the daughter in generation III has inherited the mutation-bearing chromosome from her mother and is highly likely to develop a breast tumor.

13. Psychosocial Implications of Genetic Screening
A positive screening test does not necessarily indicate disease presence.
Problems
cancellation of health insurance, employer discrimination
invasion of right to choose and privacy
no effective treatment (eg. Huntington disease) → stress
Some benefits
diagnosis of BRCA1 and BRCA2 carriers → prophylactic mesotectomy and oophorectomy
negative results → avoiding unpleasant procedures
privacy and confidentiality/accurate communication of risk information

14. RFLP mutations causing diseases eg. sickle cell disease
Molecular Tools for Screening and Diagnosis
RFLP
mutations causing diseases
→ altering recognition sites of restriction enzymes → RFLP
eg. sickle cell disease
→ an MST II site of β globin gene
family information not needed, no errors due to recombinations
disadvantages: only 5% of disease-causing mutations → affecting known restriction sites

15. Hybridization of allele-specific oligonucleotides (ASO)
Molecular Tools for Screening and Diagnosis
Hybridization of allele-specific oligonucleotides (ASO)
determination of homozygote and heterozygote of mutated genes
advantages : alternation of restriction sites not necessary
disadvantages : need cloning and sequencing the disease gene, different probes for different mutations
eg. useful for sickle cell disease and α1-antitrypsin deficiency, but not for CF

16. Allele-specific oligonucleotide (ASO)
Molecular Tools for Screening and Diagnosis
Allele-specific oligonucleotide (ASO)
A, A 21-bp allele-specific oligonucleotide (ASO) probe (yellow) is constructed to undergo complementary base pairing only with the normal β-globin sequence, and another ASO probe (green) is constructed to undergo complementary base pairing only with a β-globin sequence that contains a missense mutation that produces a substitution of valine for glutamic acid at position 6 of the β-globin polypeptide (seeChapter 3), causing sickle cell disease in homozygotes. B, In this family, the parents are both heterozygous carriers of the missense mutation, so their DNA hybridizes to both ASO probes. The first female offspring has a homozygous normal genotype, the second offspring is heterozygous, and third offspring is an affected homozygote. A variety of methods, including microarrays (see Chapter 3), can be used to detect these ASO hybridization patterns.

17. Tandem mass spectrometry
Molecular Tools for Screening and Diagnosis
Tandem mass spectrometry
PCR-amplified DNA molecules (CFTR and APOE)
tandem mass spectrometry (accurate and rapid)
1st spectrometer : separation of ionized molecules, and fragmentation
2nd spectrometer : assessing the charge and mass of fragments

18. Molecular Tools for Screening and Diagnosis

19. Amniocentesis 15-17 weeks after last menstrual period (LMP)
Prenatal Diagnosis
Amniocentesis
15-17 weeks after last menstrual period (LMP)
culturing amniocytes for 7 days → cytogenetic studies (for days), FISH (1-2 days), AFP analysis (spina bifida, anencephaly, and aneuploidy)
0.5% higher risk of fetus loss at weeks post-LMP
to confirm real mosaicism from pseudomosaicism
analysis of all cells from a single colony
fetal blood sampling
A schematic illustration of an amniocentesis, in which 20 to 30 mL of amniotic fluid is withdrawn transabdominally (with ultrasound guidance), usually at 15 to 17 weeks' gestation.

20. Indications for Prenatal Diagnosis by Amniocentesis
Maternal age >35 years
Previous child with chromosome abnormality
History of structural chromosome abnormality in one parent
Family history of genetic defect that is diagnosable by biochemical or DNA analysis
Increased risk of neural tube defect due to positive family history

21. Transcervical chorionic villus sampling (CVS)
Prenatal Diagnosis
Transcervical chorionic villus sampling (CVS)
at weeks post-LMP
culturing trophoblasts → cytogenetic studies and diagnosis of metabolic defects
some misleadings (1%-2%) by confined placental mosaicism
need amniocentesis to confirm mosaicism
no AFP measurement
1-1.5% higher risk of fetus loss
A schematic illustration of a transcervical chorionic villus sampling (CVS) procedure. With ultrasound guidance, a catheter is inserted, and several milligrams of villus tissue are aspirated.

22. Percutaneous Umbilical Blood Sampling (PUBS)
Prenatal Diagnosis
Percutaneous Umbilical Blood Sampling (PUBS)
medical-dictionary.thefreedictionary.com
at 16th week post-LMP, rapid diagnosis in 2-3 days
puncture of the umbilical cord with ultrasound guidance
applications
diagnosis of structural anomalies
diagnosis of hemotologic or immunologic disorders
rapid detection of true misaicism

23. Prenatal Diagnosis

24. Selected Disorders Diagnosed by Ultrasound in the Second Trimester
Prenatal Diagnosis
Selected Disorders Diagnosed by Ultrasound in the Second Trimester
SYMPTOM COMPLEX
Hydrops
Oligohydramnios
Polyhydramnios
Intrauterine growth retardation
CENTRAL NERVOUS SYSTEM
Anencephaly
Encephalocele
Holoprosencephaly
Hydrocephalus
CHEST
Congenital heart disease
Diaphragmatic hernia
ABDOMEN, PELVIS
Gastrointestinal atresias
Gastroschisis
Omphalocele
Renal agenesis
Cystic kidneys
Hydronephrosis
SKELETAL SYSTEM
Limb reduction defects
Many chondrodystrophies, including thanatophoric dysplasia and osteogenesis imperfecta
CRANIOFACIAL
Cleft lip

25. Ultrasonography of meningomyelocele
Prenatal Diagnosis
Ultrasonography of meningomyelocele
A, Photograph of an ultrasound result, revealing a fetus with a normal spinal column. B, Ultrasound result for a fetus with a meningomyelocele(척추수막류), visible as fluid-filled sacs (arrow) located toward the base of the spinal column.

26. Maternal serum α-fetoprotein (MSAFP)
Prenatal Diagnosis
Maternal serum α-fetoprotein (MSAFP)
MSAFP and neural tube defects
women carrying a fetus with NTD: times higher level of MSAFP than normal median
1%-2% of pregnant women → amniocentesis → 6% (1 in 15) elevated amniotic fluid AFP: 6% positive predictive value
90% of anencephaly, 80% of spina bifida
Maternal serum α-fetoprotein (MSAFP) levels in mothers carrying normal fetuses and in mothers carrying fetuses with Down syndrome and open spina bifida. MSAFP is somewhat lowered when the fetus has Down syndrome, and it is substantially elevated when the fetus has an open spina bifida. (From Milunsky A: Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, 4th ed. Baltimore: Johns Hopkins University Press, 1998.)

27. Maternal serum α-fetoprotein (MSAFP)
Prenatal Diagnosis
Maternal serum α-fetoprotein (MSAFP)
MSAFP and Down syndrome
40% of sensitivity
women older than 35 : 1/380 risk factor
women younger than time lower than normal median of MSAFP : 3-4 fold higher
women at 25 years old : a risk of 1/1250
women at 25 years old of median : a risk of 171/1
a risk of 1/380: indication for amniocentesis
Maternal serum α-fetoprotein (MSAFP) levels in mothers carrying normal fetuses and in mothers carrying fetuses with Down syndrome and open spina bifida. MSAFP is somewhat lowered when the fetus has Down syndrome, and it is substantially elevated when the fetus has an open spina bifida. (From Milunsky A: Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, 4th ed. Baltimore: Johns Hopkins University Press, 1998.)

28. New Diagnostic Techniques
Prenatal Diagnosis
New Diagnostic Techniques
Preimplantation genetic diagnosis (PGD)
removal of 1 or 2 cells from blastomere (6 or 8 cells) or blastocyst (100 cells)→ FISH for aneuploidy diagnosis → PCR for diagnosis of single gene mutation
Polar body diagnosis
collecting polar bodies → FISH → PCR
no evaluation of paternal mutations
Analysis of fetal DNA in maternal circulation
cell-sorting techniques → nucleated red cells (at 6-8 weeks post-LMP) → FISH → PCR
Evaluation of cell-free fetal DNA : for identification of sex and Rh blood-type

29. Fetal Treatment treatment for rare inborn errors surgical treatment
biotin responsive multiple carboxylase deficiency → biotin treatment to mother from 23 weeks post-LMP → a normal baby
congenital adrenal hyperplasia (CAH)→ dexamethasone treatment to the mother from 100 weeks post-LMP diminished musculinization
surgical treatment
spina bifida : successful
transplantation of hematopoietic stem cells to X-SCID : some success

30. Gene therapy

31. Gene therapy using a retroviral vector
Gene therapy using a retroviral vector. The retrovirus is prevented from replicating by removal of most of its genome, and a normal human gene is inserted into the retrovirus. Incubated with human somatic cells allows the retrovirus to insert copies of the normal human gene into the cell. Once integrated into the cell's DNA, the inserted gene produces a normal gene product.

32. Gene therapy
Adenoviral Vectors

33. Adeno-associated viral vectors
Gene therapy
Adeno-associated viral vectors

34. Gene therapy
Lentiviral vectors

35. Gene therapy using an antisense technique
Gene therapy using an antisense technique. Binding of the abnormal mRNA by the antisense molecule prevents it from being translated into an abnormal protein.

36. Gene therapy using a hammerhead ribozyme
B, Gene therapy using a hammerhead ribozyme, which binds to a mutated mRNA, cleaving and eliminating it.

37. Gene-blocking therapy using RNA interference (RNAi)
Gene-blocking therapy using RNA interference (RNAi). A dicer cleaves double-stranded RNA (dsRNA) into 20-bp single-stranded RNA fragments called short interfering RNAs (siRNAs). These fragments form a template that is recognized and bound by the RNA-induced silencing complex (RISC), which cleaves and destroys the complementary RNA strand. In RNAi, a dsRNA is engineered to produce siRNA strands that are complementary to a mutated mRNA, causing the RISC complex to destroy the mRNA.

38. Gene-blocking therapy using RNA interference (RNAi)