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INTRODUCTION to MEDICAL GENETICS

GENETIC DIAGNOSIS, CLINICAL GENETICS and
GENETIC COUNSELLING

GENETIC DIAGNOSIS
A. Two Types
1. Direct Diagnosis
- examine the mutation
2. Indirect Diagnosis
- linked markers used to infer whether the individual has inherited the chromosome segment
containing the disease-causing mutation
B. Direct Diagnosis
1. PCR and Allele-Specific Oligonucleotide (ASO) Probes
a. ASO Probes
- short nucleotide sequences that bind specifically to a single allele of the gene

2. DNA Chips
- involves embedding thousands of different oligonucleotides (represent various mutations and
normal sequences) on a silicone chip
- patient DNA from specific regions amplified by PCR  tagged with a fluorescent label 
exposed to the oligonucleotides on the chip
- ready computerization and miniaturization (hundreds of thousands of oligonucleotides can be
embedded on a single 2-cm2 chip)
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3. Restriction Fragment Length Polymorphism (RFLP) Analysis of PCR Products (RFLP-PCR)
- in some cases, a mutation that creates a disease-producing allele also destroys (or creates in some
instances) a restriction enzyme site

4. RFLP Diagnosis of Myotonic Dystrophy

- RFLP analysis in few cases in which polymorphisms are too large to conveniently amplify with
PCR
- expanded sequence is within the gene region (CTG in the 3′ untranslated region)
- shows anticipation

5. Direct DNA Sequencing

- sequencing of the entire gene (or at least the exons and the intron-exon boundaries)
- time-consuming
- expensive
- sometimes necessary if no specific set of mutations is responsible for most cases of a disease
(ex: familial breast cancer caused by any of several hundred mutations of the BRCA1 or
BRCA2 genes)
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C. Indirect Diagnosis
- if the mutation causing a disease in a family is not known
- infer whether a parent has transmitted the mutation to offspring
- uses genetic markers (restriction fragment length polymorphisms (RFLPs), short tandem repeat
polymorphisms (STRPs), single nucleotide polymorphisms (SNPs)) closely linked (showing <1%
recombination) to disease locus
1. Indirect Genetic Diagnosis Using STRPs

2. Indirect Genetic Testing Using RFLPs

- if RFLP is used as marker for disease-causing gene  data analyzed by Southern blotting and
probe for the gene region

3. RFLP Analysis for X-Linked Disease

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D. Direct vs. Indirect Genetic Diagnosis
- direct genetic diagnosis is used whenever possible (major limitation is the disease-producing mutation/s
must be known )
- if a mutation is not documented  not detected by direct mutation testing  indirect genetic testing

E. Applications of Genetic Diagnosis

a. Carrier diagnosis in recessive diseases
b. Presymptomatic diagnosis for late-onset diseases
c. Asymptomatic diagnosis for diseases with reduced penetrance
d. Prenatal diagnosis
e. Preimplantation testing
1. Prenatal Genetic Diagnosis
- one of the most common applications of genetic diagnosis
- may assist parents  informed decision regarding pregnancy termination
- may prepare parents emotionally and medically for the birth of affected child
a. Amniocentesis
b. Chorionic Villus Sampling
c. Preimplantation Diagnosis
- embryos from in vitro fertilization  remove single cell (from eight-cell stage which
does not harm the embryo)
- DNA  PCR amplification  genetic diagnosis
- pregnancy termination need not be considered (only embryos without mutation are
implanted)
- possibility of diagnostic error as a result of PCR amplification from a single cell

CLINICAL GENETICS and GENETIC COUNSELLING

A. Genetic Disorders
- often no effective therapy  pose considerable health and economic problems because:
- risk of a serious developmental abnormality
- approximately 1 in 30 pregnancies
- approximately 15% of paediatric in-patients
- have multifactorial disorder with a predominantly genetic element
B. Aims of Genetic Counselling
1. Obtain a Full History
- pregnancy history
- drug and alcohol ingestion during pregnancy
- maternal illnesses (ex: diabetes)
2. Establish an Accurate Diagnosis
- examination of the child may help in diagnosing
- genetically abnormal child with characteristic features (ex: trisomy 21)
- a genetically normal fetus damaged in utero
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Child with Down Syndrome

- note - flat broad face
- oblique palpebral fissures
- protruding tongue
- usually have - some degree of mental retardation
- many have cardiac defects

Child with Down Syndrome

- broad hand with a single transverse (simian) crease

3. Family Tree Drawing

- essential
a. Questions to be Asked
- abortions
- stillbirths
- deaths
- marriages
- consanguinity
- medical history of family members
b. Diagnoses
- may need verification from other hospital reports
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4. Future Pregnancy Risk Estimation of Being Affected or Carrying a Disorder
- based on the pattern of inheritance
a. Mendelian Disorders
- carry a high risk
b. Chromosomal Abnormalities
- carry a low risk
c. Empirical Risks
- may be obtained from population or family studies
5. Information Giving on Prognosis and Management with Adequate Time Given
- all information discussed openly, freely and repeated as necessary
6. Continued Support and Follow-Up
- explanation of the implications for other siblings and family members
7. Genetic Screening
- includes
a. Prenatal Diagnosis
- if requested
b. Carrier Detection
c. Data Storage
- in genetic registers
C. Genetic Counselling is Non-Directive
- the couple making their own decisions on the basis of an accurate presentation of the facts and risks in a
way they can understand
D. “Exome” Sequencing
- in the near future?
- development of cheap high-throughput sequencing  couples could be tested for all genes prior to
starting a family to assess if they are carriers of recessive mutations in the same disease-associated
gene  information could be used in prenatal diagnosis

GENETIC ANTICIPATION
A. Successive Generations of Individuals with Genetic Disorders
- ex: dystrophia myotonica
Huntington’s chorea
- present earlier and with progressively worse symptoms  anticipation
B. Anticipation
- due to unstable mutations occurring within the disease gene
1. Trinucleotide Repeats
- ex: CTG (dystrophia myotonica)
CAG (Huntington’s chorea)
- expand within the disease gene with each generation
- somatic expansion with cellular replication is also observed
- can occur within the translated or untranslated (and presumably regulatory) regions of the
target genes
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PRENATAL DIAGNOSIS
- analyze DNA of family members of the effected individual  establish diagnostic protocol for a
genetic disease
- identify markers (RFLPs) that are tightly linked to the disease trait  RFLP analysis 
prenatal diagnosis
- allows for an informed reproductive choice if the fetus is affected
1. Risks of Down’s Syndrome
- increase disproportionately and rapidly for children born to mothers older than 35 years
2. Infants Born to Mothers with History or Family History of Other Conditions due to Chromosomal
Abnormalities
- may be at increased risk
A. Personal Choice
- there should be a detailed discussion with all mothers as to the possible consequences of each screening
test before they are offered it
- should have an understanding of the
- failure rates
- detection rates
- false positive rates
- false negative rates of each test
 can properly exercise choice
B. Methods Available
1. Fetal Visualization
a. Ultrasound
b. Fetoscopy
- useful only if the genetic abnormality results in gross anatomic defects (neural tube defects)
2. Amniotic Fluid Chemical Composition Analysis
a. -Fetoprotein
- high levels is associated with neural tube defects
3. Fetal Cell Analysis
a. From amniotic fluid
b. From biopsy of the chorionic villi
- can be used for karyotyping (assesses the morphology of metaphase chromosomes)
4. New Staining and Cell Sorting Techniques
- rapid identification of
- trisomies
- translocations that produce chromosomes of abnormal lengths
5. Fetal DNA Molecular Analysis
- promises to provide most detailed genetic picture  informed reproductive decisions
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C. Sources of DNA
1. WBCs
2. Amniotic Fluid
3. Chorionic Villi
D. Investigations Depend on Gestation
1. 7-11 Weeks
a. Vaginal Ultrasound
- confirm
- viability
- fetal number
- gestation by crown-rump measurement
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2. 11-13 Weeks and 6 Days
a. Combined Test
- more accurate than the triple test alone at 16 weeks
i. Ultrasound
- for nuchal translucency measurement (normal fold < 6 mm) to attempt to
detect major chromosomal abnormalities (ex: trisomies, Turner’s
syndrome)
- useful if the genetic abnormality results in gross anatomic defects (ex: neural
tube defects)

First Trimester Nuchal Translucency

- anechoic space located behind the fetal neck
- visible and measurable in almost all fetuses from
10 to 14 weeks

Normal nuchal translucency (NT)

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Slightly enlarged NT

Very abnormal NT

ii. Maternal Serum Tests

- all serum marker measurements are corrected for gestational ages (multiple of
the mean, MOM) value for the appropriate week of gestation
iia. Pregnancy-Associated Plasma Protein-A (PAPP-A)
- from the syncytial trophoblast
iib. β-Human Chorionic Gonadotrophin
- for trisomy 21
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b. Further Investigations
- if abnormalities are detected
i. Chorionic Villus Sampling (CVS)
- at 11-13 weeks age of gestation
- under ultrasound control
- sample the placental site
- removal of small sample of chorionic villus material (transcervical or
transabdominal approach)
- villi are of fetal origin  large sample of actively dividing fetal cells for
diagnosis
- provide diagnosis earlier in pregnancy
- placental mosaicism (multiple cell types in the villi)  small possibility of
diagnostic
- risk of fetal demise due to chorionic villus sampling approximately 1/100

ii. Amniocentesis
- amniotic fluid sample (10-20 mL) collected at 15 weeks age of gestation
- fetal cells present in amniotic fluid  obtain fetal cells  cytogenetic testing
 diagnose single-gene disorders, chromosome abnormalities,
biochemical disorders
- risk of fetal demise due to amniocentesis approximately 1/200
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iia. Karyotyping
- assesses morphology of metaphase chromosomes
3. 14-20 Weeks (Serum Triple or Quadruple Test)
- if the pregnancy is too advanced for the earlier tests or if the combined test was not offered
a. Triple Test
- for chromosomal abnormalities
- testing maternal serum for
i. α-Fetoprotein
- low
- high for neural tube defects
ii. Unconjugated Estradiol
- low
iii. Human Chorionic Gonadotrophin
- high for
- Down’s syndrome
- neural tube defects
b. Quadruple Test
i. α-Fetoprotein
- low
- high for neural tube defects
ii. Unconjugated Estradiol
- low
iii. Human Chorionic Gonadotrophin
- high for
- Down’s syndrome
- neural tube defects
iv. Inhibin A
- high in Down’s syndrome
4. 14-22 Weeks
a. Ultrasound
i. Structural Abnormalities
ia. Neural Tube Defects
- the gestation period for detection depends on severity
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Case of Anencephaly
- no brain or bone seen superior to the orbits
diagnosed at 11 weeks of
gestation

ib. Congenital Heart Defects

- best done at 18-22 weeks age of gestation
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The typical ventricular septal defect and

aortic override are seen on a long axis view of the left
ventricle in a fetus with tetralogy of Fallot
(AO - aorta)

ii. Detection Rates

- 14 to 61% for hypoplastic ventricle
- 97 to 100% for anencephaly
5. Others
a. Salvage of Fetal Cells
- from - the maternal blood
- cervical secretions
b. Retrieve Maternal Plasma Cell Free Fetal DNA
c. Fetal Circulating Nucleic Acids
- detect - myotonic dystrophy
- Huntington’s chorea
E. Sickle Cell Disease Diagnosis Using Recombinant DNA Technology
a. Genetic Hemoglobin Disorders
- most common genetic diseases in humans
1. Early Efforts to Diagnose Sickle Cell Anemia
- prenatal diagnosis of hemoglobinopathies
- involves determination of the amount and kinds of hemoglobin synthesized in red
cells obtained from fetal blood
- ex: presence of hemoglobin S in hemolysates indicates sickle cell anemia
- invasive procedures to obtain fetal blood have high mortality rate (5%)
- diagnosis cannot be carried out until late in the 2nd trimester of pregnancy when HbS
begins to be produced
2. RFLP Analysis
a. Sickle Cell Anemia
- example of genetic disease caused by a point mutation
- sequence altered by the mutation abolishes the recognition site of the restriction
endonuclease MSTII that recognize the sequence CCTNAGG (N is a
nucleotide)
- A to T mutation within a codon of the s-globin gene
- eliminates a cleavage site for the enzyme
- DNA digested with restrictive endonuclease MSTII yields 1.15 kb fragment
- 1.35 kb fragment
- generated from the S gene as a result of the loss of MSTII cleavage site
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b. Diagnostic Techniques for Analyzing Fetal DNA

- from - amniotic cells
- chorionic villous sampling
- safe
- early detection of sickle cell anemia as well as other genetic diseases
F. Indirect, Prenatal Diagnosis of Phenylketonuria Using RFLP
1. Phenylalanine Hydroxylase (PAH) Gene
- deficient in phenylketonuria (PKU)
- located on chromosome 12
- spans about 90 kb of genomic DNA
- contains 13 exons separated by introns
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2. Restriction Fragment Length Polymorphism (RFLP) Analysis

- abnormal phenylalanine hydroxylase (PAH) gene
- can be shown using DNA polymorphism as markers to distinguish between normal
and mutant genes
- DNA from WBCs  cleaved with appropriate restriction enzyme  electrophoresis
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- presence of polymorphic site  creates fragment “b” in the autoradiogram

- absence of polymorphic site  yields only fragment “a”
3. Value of DNA-Based Screening
- determine
- if an unborn fetus is affected
- carriers of PKU gene
- PKU - autosomal recessive trait
- important to identify heterozygotes for future family planning