Presented by Mahmoud Mohammadi

1. Prenatal Cytogenetics
December, 2016
Presented by Mahmoud Mohammadi
PhD student in Molecular Genetics
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2. 12/25/16 2

3. Definition
Prenatal diagnosis is defined as the detection
of abnormalities in the fetus, before birth.
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4. Why Prenatal screening/diagnosis?
 Provide a range of informed choice to the couples at risk of
having a child with abnormality.
 Provide reassurance & remove anxiety, especially among
high risk groups.
 Allow couples at high risk to know that the presence or
absence of the disorder can be confirmed by testing.
 Allow the couples the option of appropriate management
( psychological, pregnancy/delivery, postnatal)
 To enable prenatal treatment of the fetus.
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5. Maternal Risk Factors
 Maternal age > 35 years
 Family history of neural tube defects
 Previous baby born with neural tube defect
 Previous child with chromosomal anomaly
 One or both parents – carriers of sex linked or autosomal
traits
 One parent is known to carry a balanced translocation
 History of recurrent miscarriage
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6. Methods of prenatal diagnosis
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7. Imaging
Ultrasound–
Real time USG can detect
•fetal growth abnormalities-
by biometric measurement of biparietal diameter, femoral
length, or head or abdominal circumference.
•Fetal anomalies eg. Hydrocephalus, NTDs, duodenal atresia,
diaphragmatic hernia, renal agenesis, limb anomalies, omphalocele,
gastroschisis, hydrops.
•Also help in performing BPP, cordocentesis and other invasive
procedures
Doppler USG- for velocimetry and detection of increased vascular
resistence due to fetal hypoxia.
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8. Maternal serum test
(A) α- feto protein:
Incresed Decreased
•Twins Trisomies
•NTDs Aneuploidy
•Intestinal atresia
•Fetal demise
(B) Triple test: can detect 70% of Down syndrome
•Unconjugated estriol ↓
•α- feto protein ↓
•Β-HCG ↑
• (C) Quad test: can detect 80% of Down syndrome.
•Unconjugated estriol ↓
•α- feto protein ↓
•β-HCG ↑
•Inhibin A ↑
• [Note: if only 1st
trimester quad screen is used, α-feto
protein is recommended as a 2nd
trimester follow up]
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9. Maternal cervix
 Fetal fibronectin: indicates risk of preterm birth.
 Bacterial culture: identifies risk of fetal infection (group B
streptococcus, Neisseria gonorrhoeae).
 Fluid: determination of PROM.
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10. Down syndrome screening:
(A) 1st
trimester:
•Fetal nuchal translucency (NT) thickness alone
≤70%
•NT with β-HCG & PAPP-A 87%
(PAPP-A = Pregnancy Associated Palsma Protein- A)
(B) 2nd
trimester:
•Triple test 70%
•Quad test 80%
(C) Integrated screen:
1st
trimester screen + 2nd
trimester screen detect
95%
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11. Risk of DS and Chromosomal Abnormalities at
Term
Maternal Age
at Delivery
(yr)
Risk of DS Risk of Any
Chromosomal
Abnormality
20 1/1650 1/530
25 1/1250 1/480
30 1/950 1/390
35 1/385 1/180
40 1/100 1/65
45 1/30 1/19

12. Neural tube defects
 NTD (Neural tube defects) can affect 1 in 500 infants
 – Commonest forms of NTD known as anencephaly or spina
bifida
 – Neural tube beneath the backbone fails to develop definitive
diagnosis relies on amniocentesis
 – high levels of AFP (Alphafetoprotein) seen in NTD
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13. BIOCHEMICAL MARKERS PRENATAL DIAGNOSIS
1- Alpha-fetoprotein (AFP)
a protein synthesized by the fetus is detectable in
maternal serum from week 6 of pregnancy,with a
peak in week 34 of gestation (4 mg / ml), its value
decreasing in 8-12 months after birth.
Measurement of alpha-fetoprotein can be done
from amniotic fluid or from maternal blood.
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14. 2-Human Chorionic Gonadotropin
 The hormone human chorionic gonadotropin (better known
as HCG) is produced during pregnancy. It is made by cells
that form the placenta, which nourishes the egg after it has
been fertilized and becomes attached to the uterine wall.
 Levels can first be detected by a blood test about 11 days
after conception and about 12 - 14 days after conception
by a urine test.
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15. 3-Unconjugated estriol
 Estriol (E3) is one of the three major naturally occurring estrogens, the
others being estradiol (E2) and estrone (E1).
 In non-pregnant females, the major estrogen is estradiol produced by the
ovaries.
 During pregnancy, estriol is secreted by the placenta and fetus and
becomes the dominant estrogen form.
 The primary form of estriol measured during pregnancy is unconjugated
estriol (also referred to as “free” estriol or uE3).
 Maternal serum uE3 levels have been used as a functional marker of the
fetal-placental unit and in the evaluation of pregnancy complications.
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16. 4-Inhibin - A
 Inhibins are glycoprotein hormones of which there are two
molecular forms, inhibin A and inhibin B.
 Classically, inhibin is known to have a negative feedback effect
on pituitary follicle-stimulating hormone secretion.
 Inhibin A is the predominant molecular form of inhibin in maternal
circulation from 4 weeks of gestation.
 The precise biological function of inhibin A in pregnancy is could
be a better marker of placental function than human chorionic
gonadotropin because of its shorter half-life.
 The possible clinical applications for the measurement of inhibin
A in early pregnancy could be in predicting miscarriage, Down's
syndrome, preeclampsia, and fetal growth restriction in the first
and/or second trimester before the onset of the clinical
symptoms.
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17. 5-Pregnancy-associated plasma protein-A
 It largest of the pregnancy associated proteins produced by
both the embryo and the placenta during pregnancy
 Detection of this protein is also suggested as a first and second
trimester diagnostic test for aneuploidies, including Trisomies
21 or Down’s Syndrome
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18. Biochemical markers in the second
trimester
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19. Amniocentesis
 First introduced by Serr and Fuchs and Riis in the 1950s for
fetal sex determination
 Only at the late 70th
a static ultrasound was used to locate
the placenta and amniotic fluid pocket
 Only In 1983, Jeanty reported a technique of amniocentesis
’’under ultrasound vision’’
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20. Mid Trimester Amniocentesis
20-23g needle
Ultrasound guided
Usually 20cc amniotic fluid
Results – 2 to 3 weeks
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21. USG guided percutaneous
withdrawal of amniotic
fluid for diagnostic purpose.
Timing: between 14- 16wks.
Indications:
•Karyotype (advanced maternal age)
•Fetal maturity
(L:S ratio, phosphatidylcholine or
phosphatidylglycerol)
•Biochemical enzyme/amino acid/hormone
analysis.
•Molecular genetic DNA diagnosis.
•α- fetoprotein(for NTDs) and
17-ketosteroid (for adrenogenital
syndrome) determination
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22. Method of amniocentesis:
Performed transabdominally (TA). During the procedure, a
needle is passed through the abdomen and into the
amniotic sac under continuous ultrasound guidance. The
needle stilette is removed once the needle is in the correct
position. A small sample of amniotic fluid (10–20ml) is then
removed using a syringe attached to the needle.
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23. 12/25/16 23

24. Complications
 Pregnancy loss 0.3-1.0%.
 Increase risk:
 Needle larger than 18g
 Multiple needle insertion
 Discoloration of the fluid
 High AFP, multiple late abortions, previous vaginal bleeding
 Placental perforation – recent studies didn’t find correlation
 Leakage of amniotic fluid (better prognosis than spontaneous
leakage)
 Amnionitis
 Vaginal bleeding
 Needle puncture of the fetus
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25. Risk of invasive procedure
 Early amniocentesis:
 High pregnancy loss
 High fetal malformations
 High rate of multiple needle insertions (4.7%)
 High rate laboratory failures (1.8%)
 Late amniocentesis:
 “Low” pregnancy loss (0.3-1%)
 Low rate of multiple needle insertions (1.7%)
 Low rate laboratory failures (0.2%)
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26. Chorionic villus sampling
 Diagnosed by italian biologist Giuseppe simoni, 1983
 percutaneous transabdominal with 19-20g needle
 transvaginal
 transcervical
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27.  15-30mg each aspiration
 20mg ideal for cytogenetic testing
 30-40mg for cytogenetic and other direct molecular and
biochemical tests
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28.  Transvaginal or transabdominal
chorionic villous biopsy, which
provides fetal cells.
The placenta contains tissue
that is genetically identical to fetus.
 Timing: In first trimester,
shouldn’t be performed before 10wk,
commonly performed between
11 and 13 wks.
 Indications: for karyotype, enzyme assay, molecular DNA genetic
analysis.
 The CVS procedure collects larger samples and provides faster
results than amniocentesis.
 Different from amniocentesis in that it does not allow for testing for
neural tube defects.
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29. Associated risk/ Complications:
1.miscarriage/ fetal loss (1% - 2%).
2. Oromandibular limb hypoplasia.
3.Isolated limb reduction defect-
•Increased risk is associated with decreased gestational age at the time
of CVS, highest susceptibility when CVS if performed before 9 wks.
•Mechanism: thromboembolization or fetal hypoperfusion through
hypovolemia or vasoconstriction (based on assumption that caused by
some form of vascular disruption). The limbs and mandible are
susceptible to such disruption before 10 weeks’ gestation.
•Overall risk for transverse limb deficiency from CVS is 0.03%–0.10%
4.Rh- isoimmunization.
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30. Risk of invasive procedure - CVS
 Transabdominal CVS as safe as second trimester
amniocentesis
 Trans abdominal and transcervical CVS are equally safe
and efficacious, provided that centers have expertise with
both approaches
 In approximately 3–5% of cases, clinical circumstances
will support one approach over the other
 Limb reduction – not after 9 weeks
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31. Mosaicism
 True chromosomal mosaicism is when two or more
abnormal cells lines are detected in two or more culture
flasks from the same individual.
 Pseudomosaicism is a term used to describe two
abnormal cell lines that are found in only one culture
flask (not reported to the patient)
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32. Mosaicism (trisomic cells) in CVS
 Four possible conditions:
 Mosaicism only in the placenta not affecting the fetus or placental
function.
 Mosaicism only in the placenta not affecting the fetus but alter
placental function (IUGR)
 Trisomy cells are both in the placenta and in the fetus
 Trisomy cells in the placenta and uniparental disomy in the fetus
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33. Factors considered when trying to
predict the outcome of mosaicism
 method of ascertainment
 CVS shows that the placenta is affected
 Amniotic fluid suggests that at least one fetal tissue may
be affected
 Fetal blood sampling confirms the diagnosis of
chromosomal mosaicism
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34. Uniparental Disomy
 Arises when an individual inherits two copies of a
chromosome pair from one parent and no copy from the
other parent
 Maternal UPD – two copies from the mother
 Paternal UPD – two copies from the father
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35. Cordocentesis
 Described in 1983 by Fernand Daffos.
 Procedure to obtain fetal blood.
 Cordocentesis, or PUBS
(Percutaneous Umbilical Blood Sampling),
is the sampling of blood from the umbilical cord.
 Objective: (a) prenatal diagnosis and (b) fetal therapy.
 Timing: can be performed as early as 16 wks of gestation but
commonly performed between 18-22 wks of gestation for
prenatal diagnosis.
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36.  Indication of cordocentesis:
(a) Prenatal diagnosis:
• Detection of anemia, hemoglobinopathies, thrombocytopenia,
acidosis, hypoxia, polycythemia
• Immunoglobuline M antibody response to infection
• Rapid karyotype and molecular DNA genetic diagnosis.
(b) Fetal therapy:
• Transfusion or administration of drugs.
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37. Method of Cordocentesis:
Under ultrasound guidance needle is inserted
in the umbilical vein within the umbilical
cord at its placental end or fetal end. Upon
entering the umbilical cord, the stylet is
removed and fetal blood is withdrawn into a syringe attached to the
hub of the needle.
The needle is withdrawn, then the puncture site is monitored for
bleeding, and the fetal heart rate is assessed. After this procedure,
the fetal heart rate and uterine contraction are monitored for 1-2
hours.
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38. Complications:
• Pregnancy loss, overall fetal loss risk of 1- 2%.
• Transient fetal bradycardia, manifestations of a vasovagal
response caused by local vasospasm, more with umbilical
artery puncture.
• Bleeding from the puncture site, cord hematoma.
• Fetomaternal hemorrhage
• Premature labor
• Infection
• Rh iso- immunization
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39. CVS AMNIOCENETESIS CORDOCENTESIS
TIME Transcervical 10-
13wks,
Transadominal 10
weeks to term
After 15
weeks(early 12-14
weeks)
18-20 weeks
MATERIALS FOR
STUDY
Trophoblast cells Fetal fibroblasts
Fluid for biochemical
study
Fetal white blood
cells(others-infection and
biochemical study)
KARYOTYPE RESULT Direct preparation:
24-48 hrs
Culture: 10-14 days
Culture:3-4weeks 24-48hrs
FETAL LOSS 0.5-1% 0.5% 1-2%
ACCURACY Accurate Highly accurate Highly accurate
TERMINATION OF
PREGNANCY
1st
trimester-safe 2nd
trimester-risky 2nd
trimester-risky
MATERNAL EFFECTS Very little More traumatic More traumatic
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40. Fetal Tissue Sampling
 Preimplantation Biopsy or Preimplantation Genetic
Diagnosis:
 The most frequent candidates are parents with family histories of
serious monogenic disorders & translocations, who are therefore
at increased risk for transmitting these conditions to future
generations.
 Polar body & blastomere testing are the two primary
methods.
 In Polar Body Testing, positive test results in two polar bodies
ensure that the egg itself is unaffected – therefore, the mutation
has segregated to the polar body, not to the developing ovum.
Once an egg is found to be unaffected, it is fertilized via
traditional in vitro fertilization (IVF) & implanted into the uterus.
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41. Blastomere PGD first requires traditional in vitro fertilization,
after which cells are grown to the 8-cell stage. One or two cells
are harvested & analysed, & an unaffected blastocyst is
implanted into the uterus.
An advantage to preconception testing over traditional
postconception prenatal diagnosis is that it allows parents to
avoid the possibility of receiving abnormal prenatal diagnosis
results, & thus the difficult decisions associated with pregnancy
management and/or maintenance.
PGD can be laborious, time-consuming & expensive.
Complicating factors include a high rate of polyspermia, & a
small amount of DNA in polar bodies (making it difficult to
amplify) which can produce less definitive test results.
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42. Fetal Tissue Sampling
 Other organ biopsies, including muscle & liver
biopsy:
 Fetal liver biopsy is best performed between 17-20
weeks to diagnose inborn errors of metabolism, such
as glucose-6-phosphatase deficiency, glycogen
storage disease type IA & nonketotic hyperglycemia.
 Fetal muscle biopsy is carried out under ultrasound
guidance at about 18 weeks' gestation to analyze the
muscle fibers histochemically for prenatal diagnosis of
Becker-Duchenne muscular dystrophy.
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43. Cytogenetic Investigations
 Chromosome Analysis (Karyotype Analysis)
 Fluorescence in situ Hybridization (FISH)
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44. Cytogenetic Investigations
 Chromosome Analysis:
 The most common method of detecting aneuploidy is karyotype
analysis, wherein metaphase cells are examined microscopically & the
number of chromosomes counted.
 Typically 10–15 cells are analysed to rule aneuploidy.
 Karyotype Analysis : Each chromosome pair has a unique banding
pattern that can be seen with various stains like Giemsa.
 Counting the number of staining chromosomes allows for detection of
aneuploidies.
 Analysing for the absence, presence, rearrangement, etc. of these bands
allows for detection of larger deletions, duplications and structural
aberrations
 Although G banding is typically used first to analyse prenatal specimens,
various other banding techniques (including quinacrine (Q), reverse (R),
centromeric heterochromatin (C) & high-resolution banding) may be
used to analyse different portions of particular chromosomes.
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45. Cytogenetic Investigations
 Fluorescence in situ Hybridization (FISH):
 FISH is mainly used to detect the presence or absence of
microdeletions, microduplications & aneuploidy without the full
effort associated with DNA sequencing or complete karyotype
analysis
 This three-step technique allows specific DNA sequences or
chromosomes to be visualized microscopically.
1. A specific, single-stranded DNA probe is hybridized to its
complementary, target DNA sequence, while the cell is in
prophase, metaphase or interphase;
2. fluorescent antibodies are then hybridized to the probe DNA
sequence;
3. finally, the fluorescent signals are examined under the
microscope.
 Microdeletions/microduplications detectable by fluorescence in
situ hybridization (FISH)
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46. FISH interphase view
• Probes to
Chromosome 21 are
RED and for 13 are
GREEN
•PRSENCE OF THREE
RED SIGNALE S
INDICATE TRISOMY 21
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47. DISORDER CHROMOSOMAL BAND FINDING
Angelman syndrome 15q12(maternal) Microdeletion
Duchenne Muscular Dystrphy Xp21 Microdeletion
Prader-Willi Syndrome 15q12(paternal) Microdeletion
Retinoblastoma 13q14 Microdeletion
α- thalassemia 16p13 Microdeltion
WAGR syndrome 11q13 Microdeletion
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48.  Direct DNA Analysis
 Linkage Analysis(indirect DNA analysis)
 DNA Sequencing
Molecular genetics
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49.  Direct DNA analysis:
• Direct mutation analysis involves analysing a target
segment of DNA for the presence of a specific
mutation. Like FISH, it requires knowledge of the
correct sequence for the specific gene or DNA
segment before analysis. Once known, the sample
sequence may be compared to the known, ‘model’,
genomic sequence in a variety of methods, as
described below :
 Mutation analysis with restriction enzymes.
 Sequencing of restriction enzyme products.
 Allele-Specific Oligonucleotide (ASO) analysis.
Molecular genetics
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50.  Direct DNA analysis:
 Mutation analysis with restriction enzymes:
If the putative mutation is known to alter the recognition for
a splice site, direct analysis by restriction enzyme assay is
possible. The presence of a mutation can be detected by
digesting control and sample DNA with the same restriction
enzymes (known to cut the DNA at a specific splice site) and
then analysing resultant DNA fragments (called Restriction
Fragment Length Polymorphisms, or RFLPs ) for differences
by Southern blotting. Those segments containing mutation(s)
at or near a splice site are identifiable because they were not
cut by a restriction enzyme, and are therefore longer,
appearing higher on the Southern blot gel. This technique is
used in genetic testing for sickle cell anaemia.
Molecular genetics
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51.  Direct DNA analysis:
 Sequencing of restriction enzyme products:
 Disorders secondary to deletion of DNA ( e. g α thalassemia, DMD, CF &
growth harmone deficiency) can be detected by the altered size of DNA
segments produced following a Polymerase Chain Reaction(PCR)
 DNA sequences that have been cut with restriction enzymes can also be
sequenced by a specialized amplification technique. Copies of a particular
piece of DNA(cut by restriction enzymes) are placed into four vials &
amplified by polymerase chain reaction (PCR).
 Fragments from the vials are then allowed to migrate, in parallel, down a
Southern blot gel.
 The shortest fragments travel furthest, the longer segments remain closer
to the top. From top to bottom, the banding pattern produced represents
fragments that decrease in size by one nucleotide base. The DNA sequence
can therefore be read from the shortest, single-base strand at the bottom
of the gel, up to the entire sequence length at the top
Molecular genetics
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52.  Direct DNA analysis:
 Allele-Specific Oligonucleotide (ASO) analysis:
• Direct detection of a DNA mutation can also be
accomplished by allele specific oligonucleotide analysis.
• If the PCR –amplified DNA is not altered in size by deletion
or insertion, recognition of mutated DNA sequence can
occur by hybridization with the known mutant allele.
• ASO analysis allows direct DNA diagnosis of Tay-Sachs
Disease, alpha & beta thalassemia, Cystic fibrosis &
phenylketonuria
Molecular genetics
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53.  Linkage Analysis(indirect DNA analysis):
 Linkage analysis is a means of indirectly detecting a
patient’s mutation status, when several family members are
known to be affected with the same genetic disorder, &
when an exact mutation is not known.
 DNA from affected & unaffected family members is analysed
for polymorphisms such as microsatellite repeats, restriction
fragment length polymorphisms (RFLPs) and variable
number tandem repeats (VNTRs)
Molecular genetics
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54.  DNA Sequencing :
• DNA sequencing for many disorders has revealed that a
multitude of different mutations within a gene can result
in same clinical disease.
• For Example,Cystic Fibrosis can result from more than
1,000 different mutations.
• Therefore, for any specific disease, prenatal diagnosis by
DNA testing may require both Direct & Indirect methods.
Molecular genetics
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