Genetic Counseling :

DNA Profiling, Prenatal Diagnostic and Risk

Assesment

Ahmad Hamim Sadewa, MD, PhD

Department of Biochemistry Faculty of Medicine

Gadjah Mada University
DNA Fingerprint
DNA Profiling
DNA profiling : identification of individual by use
their respective DNA profile.
DNA profile : a set or sets of DNA repeat
sequences that reflects genetics makeup of an
individual
Repetitive sequence : nucleotide repeats in
particular regions that are highly variable
(variation range 1->30) (variable number of
tandem repeats/VNTR or short tandem repeat
/STR)
DNA profiling of 12 individuals
1. Restriction Fragment Length Polymorphism
(RFLP)

RFLP is a technique for analyzing the variable lengths

of DNA fragments that result from digesting a DNA
sample with a special kind of enzyme.
RFLP was one of the first applications of DNA analysis
to forensic investigation. With the development of
newer, more efficient DNA-analysis techniques, RFLP is
not used as much as it once was because it requires
relatively large amounts of DNA.
In addition, samples degraded by environmental factors,
such as dirt or mold, do not work well with RFLP.
2. PCR Analysis
Polymerase chain reaction (PCR) is used to make
millions of exact copies of DNA from a biological sample.
DNA amplification with PCR allows DNA analysis on
biological samples as small as a few skin cells. With
combination with RFLP, DNA samples would have to be
about the size of a quarter.
3. STR Analysis
Short tandem repeat (STR) technology is used to
evaluate specific regions (loci) within nuclear DNA.
Variability in STR regions can be used to distinguish one
DNA profile from another.
The Federal Bureau of Investigation (FBI) uses a
standard set of 13 specific STR regions for Combined
DNA Index System (CODIS). CODIS is a software
program that operates local, state, and national
databases of DNA profiles from convicted offenders,
unsolved crime scene evidence, and missing persons.
The odds that two individuals will have the same 13-loci
DNA profile is about one in a billion.
4. Mitochondrial DNA Analysis

Mitochondrial DNA analysis (mtDNA) can be used to

examine the DNA from samples that cannot be analyzed
by RFLP or STR. Nuclear DNA must be extracted from
samples for use in RFLP, PCR, and STR; however,
mtDNA analysis uses DNA extracted from another
cellular organelle called a mitochondrion.
While older biological samples that lack nucleated
cellular material, such as hair, bones, and teeth, cannot
be analyzed with STR and RFLP, they can be analyzed
with mtDNA. In the investigation of cases that have gone
unsolved for many years, mtDNA is extremely valuable.
5. Y-Chromosome Analysis
The Y chromosome is passed directly from father to son,
so analysis of genetic markers on the Y chromosome is
especially useful for tracing relationships among males
or for analyzing biological evidence involving multiple
male contributors.
 Application of DNA Profiling

 1. Paternity and Maternity

VNTR patterns can be used to establish paternity and
maternity. The patterns are specific that a parental VNTR
pattern can be reconstructed even if only the children's
VNTR patterns are known (the more children produced,
the more reliable the reconstruction). Has been used to
solve standard father-identification cases as well as more
complicated cases of confirming legal nationality and, in
instances of adoption, biological parenthood.

 2. Criminal Identification and Forensics

DNA isolated from blood, hair, skin cells, or other genetic
evidence left at the scene of a crime can be compared,
through VNTR patterns, with the DNA of a criminal
suspect to determine guilt or innocence.
 3. Personal Identification
The notion of using DNA fingerprints as a sort of genetic
bar code to identify individuals has been discussed. The
technology required to isolate, keep on file, and then
analyze millions of very specified VNTR patterns is both
expensive and impractical. Social security numbers,
picture ID, and other more feasible methods are much
more likely to remain the prevalent ways to establish
personal identification.
Contoh paternal testing
 Technical Difficulties

Errors in the hybridization and probing process must also

be figured into the probability.
An innocent person should not be sent to jail, a guilty
person allowed to walk free, or a biological mother
denied her legal right to custody of her children, simply
because an experiment in-accurately.
When the analysis of the DNA sample involves
amplification of the sample (creating a much larger
sample of genetically identical DNA from what little
material is available), because if the wrong DNA is
amplified (i.e. a skin cell from the lab technician) the
consequences can be profoundly detrimental.
Standards for determining DNA fingerprinting matches,
and for laboratory security and accuracy which would
minimize error, were neither stringent nor universally
codified.
 Prenatal Diagnostic

Prenatal diagnosis or prenatal screening is test

for diseases or conditions in a fetus or embryo
before it is born, to
1. enable timely medical or surgical treatment of a
condition before or after birth,
2. give the parents the chance to abort a fetus with
the diagnosed condition,
3. give parents the chance to "prepare"
psychologically, socially, financially, and
medically for a baby with a health problem or
disability, or for the likelihood of a stillbirth.
 Methods

1. Non Invasive : USG, blood chemistry, cytogenetics

2. Invasive : villi choriales or amniotic fluid
 Prenatal Diagnostic

Spinal muscular atrophy (SMA)

autosomal recessive neuromuscular disease
incidence 1 : 10,000; carrier frequency 1:50
muscle atrophy due to destruction of alpha motor
neuron in cornu anterior medulla spinalis
3 types based on onset and motoric function
Type I : < 6 month, unable to sit and walk
Type II : 6 – 18 month, able to sit
Type III : > 18 month, able to sit and walk
Responsible gene : survival motor neuron 1 (SMN1)
mapped on chromosome 5q
SMN2 is a modifying factor
SMN1 produced full length, SMN2 produced delta7 protein
 Problem : SMA family
expect healthy offspring

Prenatal diagnosis is essential

Methods to detect deletion in the prenatal periods :

SMN1 Deletion Test and Ag1CA Analysis
 Subject

six SMA families (father, mother, SMA patient)

attending hospital after informed consents were
obtained
first sibling is SMA patient 2 – 5 -year old
healthy pregnance mothers 8 – 12 week when
amniotic fluid or chorionic villi were withdrawn
 Methods
AF, CV, leukocyte

DNA extraction

SMN1 Deletion Test

(PCR – RFLP) Ag1CA Analysis

Termination
Continue : delivery, follow up until 18 month
SMN1 Deletion Test (PCR – RFLP)
PCR amplification of SMN Exons 7 and 8
Primers exon 7 : R111 and X7-Dra
Primers exon 8 : 541C960 and 541C1120
Restriction endonuclease Dra1 (exon 7) and Dde1 (exon 8)

Ag1CA Analysis
PCR amplification with fluorescence labeled primer
 Results and Discussion
Family Sample Deletion Test Ag1CA

I CV non del confirm

II AF non del confirm
III CV non del confirm
IV CV del confirm
V AF non del confirm
VI AF del confirm

del : termination
non-del follow up : normal delivery, development,
no symptoms of neuromuscular diseases
Non-deleted case

Nishimura Family SMN1 Exons 7, 8 and NAIP Exon 5 Deletion Test

Mk Father Mother Patient AF

SMN1 Exon 7

SMN2 Exon 7

SMN1 Exon 8
SMN2 Exon 8
SMN2 Exon 8
Non-deleted case
Deleted case
Miyake Family SMN1 Exons 7, 8 and NAIP Exon 5 Deletion Test

Mk Father Mother Patient AF

SMN1 Exon 7

SMN2 Exon 7

SMN1 Exon 8
SMN2 Exon 8
SMN2 Exon 8
Deleted case

Father

Mother

Patient

AF
Problems on prenatal diagnosis

Contamination with maternal cells

Low DNA concentration
Similar Ag1CA pattern of mother, father and sibs
Risks : spontaneous abortion, bleeding, infection
 Conclusion

Genetic Testing is a good method for parentage

testing and prenatal diagnostic

Many problems arise in this method should be

considered carefully.
Qualitative Trait
Absence or presence of disease
Concordant : two individual in a family have the
same disease
Discordant : only one of a pair in a family has the
disease
Quantitative Trait
Measurable physiological quantities : blood
pressure, cholesterol level, BMI
Such variation due to genetic and non-genetic
factors.
Measuring Familial Aggregation
1. Relative Risk

comparing the frequency in relative with that of

general population
Relative risk is high in some disease :
schozophrenia (12), autisme (150), DM Type 1
(35)
2. Case control study

case is compared with control with respect

of family history, geographical location,
parity, previous illnes etc.
confounding factor : choice of controls
Twin Study

Disease Concordance in Twin Study

powerful for determining whether genotype

alone is sufficient to produce a disease in
monozygotic or dizigotic twin
High concordance rate : epilepsy, DM type
1, osteoarthritis, psoriasis, cleft lip/palate