Seminar prenatal genetic screening

59
PRENATAL GENETIC SCREENING By Dr Sreelasya Kakarla Dept of OBG, SSMC, Tumkur

Transcript of Seminar prenatal genetic screening

Page 1: Seminar prenatal genetic screening

PRENATAL GENETIC

SCREENING

By Dr Sreelasya KakarlaDept of OBG,

SSMC, Tumkur

Page 2: Seminar prenatal genetic screening

Congenital defects may be anatomical or functional.

Causes of these defects may be preconceptional or because of post conceptional exposure

Chromosomal abnormalities : 0.2% Single gene defects : 0.4% Multifactorial : 0.7% Unknown : 0.6% Anomalies due to exposure to teratogen

: 0.1%

Page 3: Seminar prenatal genetic screening

Prenatal diagnosis is the science of identifying structural and functional abnormalities in the developing fetus.

Page 4: Seminar prenatal genetic screening

Diagnostic evaluation typically involves 3 major categories :

1. Fetuses at a high risk for a genetic or congenital disorder.

2. Fetuses at high risk for common congenital abnormalities.

3. Fetuses discovered ultrasonographically to have structural or functional abnormalities.

High risk describes ‘a risk greater than the chance of fetal death associated with the diagnostic procedure considered’.

Page 5: Seminar prenatal genetic screening

INDICATIONS FOR PRENATAL DIAGNOSIS Increased risk for fetal chromosomal

abnormalities based on advanced maternal age, previous pregnancy affected by fetal chromosomal abnormality

Family history of a known genetic condition or if the couple is a known carrier of gene mutation or balanced translocation.

Either of the couple affected by congenital disorder : eg ; congenital cardiac defects

Previous history of a pregnancy affected by congenital anomalies

Page 6: Seminar prenatal genetic screening

Family history of congenital abnormalities

Maternal medical condition : diabetes, autoimmune diseases, hypertension, hypothyroidism etc.,

Abnormal ultrasound findings-soft markers.

Medications : anticonvulsants, oral anticoagulants, chemotherapeutic agents.

Congenital infections : rubella, cytomegalovirus, chickenpox, syphilis.

Page 7: Seminar prenatal genetic screening

METHODS OF PRENATAL DIAGNOSISNON INVASIVE Ultrasound Fetal MRI Free fetal DNA

INVASIVE Amniocentesis Chorionic villus sampling Fetal blood sampling Fetal biopsy Fetal surgery

Page 8: Seminar prenatal genetic screening

CONFIRMATORY TESTS FOR PRENATAL DIAGNOSISChromosome abnormalities

Karyotype by conventional chromosome analysis Fluoroscent in situ hybridization (FISH) Multiplex ligation dependent probe amplification

(MLPA) Comparative genomic hybridization (CGH)

Single gene analysis

Sanger sequencing Next generation sequencing(NGS) Others : amplification refractory mutations

system(ARMs)

Page 9: Seminar prenatal genetic screening

SCREENING FOR COMMON CONGENITAL ABNORMALITIESNEURAL TUBE DEFECTS : Screening for NTDs is recommended if

the following RISK FACTORS are present Family history of neural tube defects Exposure to certain environmental

agents Diabetes Hyperthermia Drugs : anticonvulsants

isotretinoin Antifolate receptor antibodies

Page 10: Seminar prenatal genetic screening

ALPHA FETO PROTEIN Glycoprotein Synthesized early in gestation by fetal

yolk sac ; later by fetal gastro intestinal tract and liver

Concentration increases steadily in both fetal serum and amniotic fluid until 13 weeks, after which these levels rapidly decrease.

Passes into maternal circulation by diffusion across the placental membranes and may also be by placental circulation.

Page 11: Seminar prenatal genetic screening

FIST TRIMESTER SCREENING NUCHAL TRANSLUCENCY

Anechoic stripe visible just internal to the skin stripe at the level of back of the fetal neck.

Consequent to the subcutaneous accumulation of fluid in the fetal neck in the 1st trimester.

Incidence of chromosomal abnormalities and structural anomalies is related to the thickness rather than the appearance.

The translucency usually resolves in the 2nd trimester but may persist as a cystic hygroma or nuchal oedema.

Page 12: Seminar prenatal genetic screening
Page 13: Seminar prenatal genetic screening

Chromosomal abnormalities are found in 20-30% of fetuses with increased nuchal translucency.

50% of these are trisomy 21, Rest are contributed by trisomy 13, 18,

turners syndrome. Majority of the cases, NT < 4.5 mm

Page 14: Seminar prenatal genetic screening

Aetiology of increased nuchal translucency

Multifactorial cardiac failure Superior mediastinal compression

causing venouscongestion, Altered composition of extracellular

matrix, Abnormal or delayed development of

lymphatic system Consequent to decreased fetal

movements, Fetal anemia

Page 15: Seminar prenatal genetic screening

CRITERIA FOR ASSESSMENT OF NT

Fetus to be in true sagital section Ideal image includes : nasal skin,

echogenic tip of nose, nasal bone, palate in rectangular shape, the translucent diencephalon in the centre and the nuchal translucency posteriorly in the same image.

It should definitely not include any part of the zygoma between the nose and the palate.

CRL should range between 45 and 84 mm.

It is important to exclude the presence of umbilical cord near the fetal neck.

Page 16: Seminar prenatal genetic screening

FETAL NASAL BONE Nasal bone is absent or hypoplastic in around 69% of

fetuses with trisomy 21 in 11-13 weeks. Technically ,the section for assessment and

measurement is same as for nuchal translucency. Transducer should be parallel to the direction of the

nose.

3 lines are evident :

skin represented by the top line , Echogenic nasal bone just below this which is thicker

than overlying skin and A line in front of the nose which represents tip of the

nose

Page 17: Seminar prenatal genetic screening
Page 18: Seminar prenatal genetic screening
Page 19: Seminar prenatal genetic screening

MATERNAL SERUM AFP SCREENING Done between 14-22 weeks Measured in ng/dl Reported as multiples of the median

(MoM) Weight, race, diabetic status,

gestational age, number of fetuses influence the level.

2.0-2.5 MoM : upper limit of normal. 2.5-3.5 MoM : indiscriminate zone >3.5 MoM : increased fetal risk . Sensitivity : 90% PPV: 2-6%

Page 20: Seminar prenatal genetic screening

CONDITIONS ASSOCIATED WITH ABNORMAL MS AFP

ELEVATED LEVELS : Neural tube defects. Pilonidal cysts Esophageal or intestinal obstruction Liver necrosis Cystic hygroma Sacrococcygeal teratoma Abdominal wall defects : omphalocele,

gastroschisis Multifetal gestation Undetermined gestation.

Page 21: Seminar prenatal genetic screening

LOW LEVELS :

Chromosomal trisomies Gestational trophoblastic disorders Fetal death Overestimated gestational age.

Page 22: Seminar prenatal genetic screening

A combination of the MSAFP test + Ultrasonography detects almost all cases of anencephaly and most cases of spina bifida.

Also, a NTD can be distinguished from other fetal defects, such as abdominal wall defects, by the use of an acetylcholinesterase test carried out on amniotic fluid.

If the level of acetylcholinesterase rises along with AFAFP, it is suspected as a condition of a NTD.

However, the MSAFP levels also increase with gestational age, gestational diabetes, twins, pregnancies complicated by bleeding, and in association with intrauterine growth retardation.

Page 23: Seminar prenatal genetic screening

SCREENING FOR DOWNS SYNDROME

Depending on the gestational age at pregnancy booking,testing options that can be offered include

Combined screening (11-13 weeks) : NT + serum PAPPA & free B-Hcg

Quadruple screening (15-18 weeks) : serum AFP,uE3, free B-Hcg & inhibin A

Integrated screen: NT + serum PAPPA+Quadruple screening

Page 24: Seminar prenatal genetic screening

Stepwise screening: Combined screening+ Quadruple marker test in all patients with down syndrome risk (DSR) <1 in 30 on the Combined screen.

Contingent screen: Combined screening+ Quadruple marker test only if the DSR is 1 in 30 to 1500

Page 25: Seminar prenatal genetic screening

SOFT MARKERS Femur length < 0.91MoM Echogenic intracardiac focus Increased nuchal skinfold thickness ≥

6mm Humerus length < 0.89 MoM Moderate or marked echogenic bowel major malformations Pyelectasis ≥3mm ventriculomegaly

Page 26: Seminar prenatal genetic screening

INVASIVE TECHNIQUES MOST COMMONLY USED Amniocentesis Chorionic villus sampling (CVS) Cordocentesis

Page 27: Seminar prenatal genetic screening

AMNIOCENTESIS Usually performed between 16-20 weeks of

gestation. Procedure performed using ultrasound

guidance and sterile technique. Typically performed by two operators. The main operator performs the invasive

procedure while the assistant performs the ultrasound examination and guides the needle insertion.

Pre procedure ultrasound examination is performed to identify the placental location and fetal position in an attempt to avoid both during the needle insertion.

Page 28: Seminar prenatal genetic screening

The desired area of the maternal abdomen is cleaned, sterilized and draped with sterile drapes.

Ultrasound probe covered by sterile sleeve an continuous ultrasound guidance is provided during the procedure.

Ultrasound probe held vertically and the desired target is centered on the screen.

Needle guide is attached to the probe laterally , which provides a needle track ,at a 45◦ angle to the horizontal plane.

Page 29: Seminar prenatal genetic screening

Alternative : Free hand needle insertion can be

done , the needle is inserted 3 cm lateral to the probe, in the same plane and at 45◦ angle.

The guide increases the ease of needle insertion & reduces the risks of failed attempts and complications.

5 inch length 22 gauge spinal needle is used.

Rarely 7 inch length needle is used in obese patients.

Page 30: Seminar prenatal genetic screening

Amniotic sac is entered and fluid is aspirated using sterile syringes.

The first 1-2ml of the amniotic fluid may be contaminated by maternal cells and can be discarded.

Fluid subsequently aspirated can be sent for fetal chromosomal analysis after tissue culture or direct fluorescent insitu hybridization techniques.

Amount required for chromosomal analysis : 15-20 ml.

Page 31: Seminar prenatal genetic screening
Page 32: Seminar prenatal genetic screening

Pregnancy loss rate : 1 in 200

Complications :

Infection Inadvertent trauma to the fetus or placenta Leakage of amniotic fluid Miscarriage. Feto maternal hemorrhage, Isoimmunization may occur in Rh negative

women and it should be covered by prophylactic antiD in non sensitized women.

Page 33: Seminar prenatal genetic screening

EARLY AMNIOCENTESIS 12-14 WEEKS Done in order to obtain the results

earlier in gestation Increase in risk of talipes equinovarus. For patients desiring earlier diagnosis ,

transabdominal CVS should be preferred over early amniocentesis.

Page 34: Seminar prenatal genetic screening

CHORIONIC VILLUS SAMPLINGEarly prenatal diagnosisFirst successful CVS was reported in 1983.

Page 35: Seminar prenatal genetic screening

TRANSABDOMINAL CVS

TECHNIQUE Performed between 10-12 weeks Later weeks preferred as the thicker

placenta increases the success and the ease of the procedure.

A semi full or a full bladder is essential. Ultrasound guided sterile technique can

be performed using the needle guide or the free hand.

After sterilizing the skin over the maternal abdomen, local anaesthesia is administered.

Page 36: Seminar prenatal genetic screening

A 19 or 20 gauge needle is used Insertion done at 45◦angle and the path

of the tip is being continuously visualized on the ultrasound moniter.

Once the tip of the needle has reached the target, the stillete is removed & a 10 ml luer-lock syringe is attached to the needle hub for an air tight seal.

The tissue is aspirated by applying the negative pressure in the syringe.

Page 37: Seminar prenatal genetic screening

Due to more solid nature of the CVS sample, tip of the needle has to be moved back and forth 5-10 times while applying continuous negative pressure with the syringe to get adequate amount of tissue.

Page 38: Seminar prenatal genetic screening

TRANSABDOMINAL CVS

Page 39: Seminar prenatal genetic screening

TRANSCERVICAL CVS Ultrasound guidance provided abdominally Patient placed in lithotomy position, Vulva cleaned and sterilized. Speculum inserted into the vaginal canal and

the cervix is exposed 1.5mm diameter plastic catheter which has

been threaded over a solid malleable aluminum stylet is introduced into the cervical canal under the ultrasound guidance.

Once the target tissue reached, aluminum obturator is withdrawn carefully, avoiding the tear or puncture of the plastic catheter.

Page 40: Seminar prenatal genetic screening

A syringe is attached to the hub of the catheter and suction applied.

A single aspiration will typically provide adequate amount of sample for chromosomal analysis.

Page 41: Seminar prenatal genetic screening

In both the transabdominal and the transcervical CVS, extreme caution is taken to avoid injury to amnion and chorion.

Injury will increase risk of amniotic fluid leakage and repeated pregnancy loss.

It is important to avoid losing the negative pressure which can occur, if the negative pressure applied is too high and the piston of the syringe comes off.

Page 42: Seminar prenatal genetic screening

TRANSCERVICAL CVS

Page 43: Seminar prenatal genetic screening

CORDOCENTESIS Cordocentesis or percutaneous umbilical

blood sampling (PUBS). It was initially described for fetal

transfusion of red blood cells in the setting of anemia from alloimmunization

Fetal blood sampling is also performed for fetal karyotype determination, particularly in cases of mosaicism identified following amniocentesis or CVS.

Fetal blood karyotyping can be accomplished within 24 to 48 hours.

Page 44: Seminar prenatal genetic screening
Page 45: Seminar prenatal genetic screening

 Under direct sonographic guidance using a 22- or 23-gauge spinal needle into the umbilical vein, and blood is slowly withdrawn into a heparinized syringe.

Adequate visualization of the needle is essential.

Fetal blood sampling is often performed near the placental cord insertion site, where it may be easier to enter the cord if the placenta is anterior .

Alternatively, a free loop of cord may be punctured.

Page 46: Seminar prenatal genetic screening

Arterial puncture is avoided, because it may result in vasospasm and fetal bradycardia. After the needle is removed, fetal cardiac motion is documented, and the site is observed for bleeding.

fetal loss rate is approximately 1.4 % Other complications – cord vessel bleeding in 20

to 30 % of cases, fetal-maternal bleeding in 40 % of cases in

which the placenta is traversed fetal bradycardia in 5 to 10 % Most complications are transitory, with complete

recovery, but some result in fetal loss.

Page 47: Seminar prenatal genetic screening

RECENT ADVANCES IN PRENATAL DIAGNOSIS Fluorescent in situ hybridization Chromosomal microarray analysis Free fetal DNA

Page 48: Seminar prenatal genetic screening

FLUORESCENCE IN SITU HYBRIDIZATION Involves detection of aneuploidies using

probes derived from specific sub regions of the chromosomes in uncultured amniocytes.

Results can be available within 24-48 hrs.

Page 49: Seminar prenatal genetic screening
Page 50: Seminar prenatal genetic screening

CHROMOSOMAL MICROARRAY ANALYSIS CMA can detect the abnormality when the

genetic abnormality involves more than 300 kilo bases.

limitations It cannot detect balanced translocations Point mutations Low level mosaicism Previously not described gene deletions

/duplications involving <300 kb There is also a possibility of an abnormality

detected on CMA which have no clinical implications.

Page 51: Seminar prenatal genetic screening

FREE FETAL DNA

Page 52: Seminar prenatal genetic screening

PREIMPLANTATION GENETIC TESTING Genetic testing performed on oocytes

or embryos before implantation in vitro fertilization (IVF), may provide valuable information regarding the chromosomal complement and single-gene disorders.

1. polar body analysis 2. blastomere biopsy 3. trophectoderm biopsy

Page 53: Seminar prenatal genetic screening

POLAR BODY ANALYSIS Maternally inherited genetic disorder. The first and second polar bodies are

extruded from the developing oocyte. Sampling does not affect fetal

development Disadvantages : paternal genetic

contribution is not evaluated.

Page 54: Seminar prenatal genetic screening
Page 55: Seminar prenatal genetic screening

BLASTOMERE BIOPSY Done at the 6- to 8-cell stage limitation : mosaicism of the

blastomeres may not reflect the chromosomal complement of the developing embryo.

The technique is associated with a 10%reduction in the pregnancy rate.

Page 56: Seminar prenatal genetic screening

BLASTOMERE BIOPSY

Page 57: Seminar prenatal genetic screening

TROPHECTODERM BIOPSY 5 to 7 cells from a 5- to 6-day old

blastocyst

Advantage no embronyal cells are removed as

trophectoderm cells give rise to the trophoblast.

Disadvantage performed later in development.

Page 58: Seminar prenatal genetic screening
Page 59: Seminar prenatal genetic screening