Prenatal Cytogenetics

December, 2016Presented by Mahmoud Mohammadi

PhD student in Molecular Genetics

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در حدیث نبوی آمده است:

وَ التَنکُِحوا الِقرابَۀ= القریبۀ= فَانَّ وَلَدَ یُخلَقُ ضاویاً.

با اقوام نزدیک ازدواج نکنید چرا که منجر به ناتوانی فرزند می گردد.

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Definition

Prenatal diagnosis is defined as the detection

of abnormalities in the fetus, before birth.

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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.05/02/23 4

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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Methods of prenatal diagnosis

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ImagingUltrasound– 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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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]05/02/23 8

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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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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Risk of DS and Chromosomal Abnormalities at Term

Maternal Age at Delivery

(yr)

Risk of DS Risk of Any Chromosomal Abnormality

20 1/1650 1/53025 1/1250 1/48030 1/950 1/39035 1/385 1/18040 1/100 1/6545 1/30 1/19

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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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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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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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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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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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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Biochemical markers in the second trimester

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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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Mid Trimester Amniocentesis20-23g needle

Ultrasound guided

Usually 20cc amniotic fluid

Results – 2 to 3 weeks

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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) determination05/02/23 21

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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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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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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Chorionic villus sampling Diagnosed by italian biologist Giuseppe simoni, 1983 percutaneous transabdominal with 19-20g needle transvaginal transcervical

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15-30mg each aspiration

20mg ideal for cytogenetic testing

30-40mg for cytogenetic and other direct molecular and biochemical tests

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Transvaginal or transabdominal chorionic villous biopsy, whichprovides 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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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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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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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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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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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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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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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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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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Method of Cordocentesis:Under ultrasound guidance needle is insertedin the umbilical vein within the umbilicalcord at its placental end or fetal end. Uponentering the umbilical cord, the stylet isremoved 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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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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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 fibroblastsFluid for biochemical study

Fetal white blood cells(others-infection and biochemical study)

KARYOTYPE RESULT Direct preparation: 24-48 hrsCulture: 10-14 days

Culture:3-4weeks 24-48hrs

FETAL LOSS 0.5-1% 0.5% 1-2%ACCURACY Accurate Highly accurate Highly accurateTERMINATION OF PREGNANCY

1st trimester-safe 2nd trimester-risky 2nd trimester-risky

MATERNAL EFFECTS Very little More traumatic More traumatic

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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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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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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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Cytogenetic Investigations Chromosome Analysis (Karyotype Analysis)

Fluorescence in situ Hybridization (FISH)

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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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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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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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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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Direct DNA Analysis

Linkage Analysis(indirect DNA analysis)

DNA Sequencing

Molecular genetics

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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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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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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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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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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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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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