Seminar prenatal genetic screening
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Transcript of Seminar prenatal genetic screening
PRENATAL GENETIC
SCREENING
By Dr Sreelasya KakarlaDept of OBG,
SSMC, Tumkur
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%
Prenatal diagnosis is the science of identifying structural and functional abnormalities in the developing fetus.
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’.
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
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.
METHODS OF PRENATAL DIAGNOSISNON INVASIVE Ultrasound Fetal MRI Free fetal DNA
INVASIVE Amniocentesis Chorionic villus sampling Fetal blood sampling Fetal biopsy Fetal surgery
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)
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
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.
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.
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
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
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.
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
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%
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.
LOW LEVELS :
Chromosomal trisomies Gestational trophoblastic disorders Fetal death Overestimated gestational age.
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.
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
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
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
INVASIVE TECHNIQUES MOST COMMONLY USED Amniocentesis Chorionic villus sampling (CVS) Cordocentesis
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.
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.
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.
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.
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.
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.
CHORIONIC VILLUS SAMPLINGEarly prenatal diagnosisFirst successful CVS was reported in 1983.
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.
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.
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.
TRANSABDOMINAL CVS
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.
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.
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.
TRANSCERVICAL CVS
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.
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.
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.
RECENT ADVANCES IN PRENATAL DIAGNOSIS Fluorescent in situ hybridization Chromosomal microarray analysis Free fetal DNA
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.
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.
FREE FETAL DNA
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
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.
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.
BLASTOMERE BIOPSY
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.