GENETIC SCREENING &

PRENATAL DIAGNOSIS

Dr.Padmesh.V,Dept of Pediatrics, Dr.SMCSI Medical College, Karakonam.

Dr.Padmesh. V

Definition: ‘Prenatal diagnosis is defined as the

detection of abnormalities in the fetus, before birth’

Congenital abnormalities account for 20-25% of perinatal deaths.

Many of these genetic and other disorders can be diagnosed early in pregnancy.

Prenatal diagnosis: 1. Non invasive

Techniques 2. Invasive Techniques

Dr.Padmesh. V

Some Disorders for which

PRENATAL DIAGNOSIS is available: 1. Congenital malformations 2. Chromosomal disorders 3. Non genetic Fetal disorders *Fetal infections, Immune hydrops, Fetal effects of maternal

drugs like ITP,DM

4. Single gene disorders

-Multiple malformation synd *Holt oram, Meckel Gruber, Craniosynostosis, Orofacial

digital synd

-Hematological disorders

*Thalassemias, Hemoglobinopathies, Hemophilia

-Metabolic Disorders

*Tay sach, MCL, Wilson, MPS, CAH, OTC deficiency, Smith-Lemli-Opitz synd

-Neuromuscular disorders *Huntington chorea, Myotonic dystrophy, DMD, Fragile X

Dr.Padmesh. V

4. Single gene disorders: contd…

-Renal Disoders *AD/AR polycystic kidney disease

-Connective tissue dis / Skeletal dysplasia

* Osteogenesis imperfecta, Ehlers Danlos, Achondroplasia, Marfan.

-Skin disorders *Epidermolysis bullosa, Ichthyosis, Ectodermal dysplasia

Dr.Padmesh. V

INDICATIONS OF PRENATAL SCREENING : 1.Advanced parental age

-Maternal age ≥35 yr -Paternal age ≥50 yr

2.Previous child with or family history of

-Congenital abnormality -Dysmorphology

-Mental retardation -Isolated birth defect

-Metabolic disorder -Chromosome abnormality

-Single-gene disorder

3.Consanguinity

4.Teratogen exposure (occupational, abuse)

Dr.Padmesh. V

PRENATAL SCREENING is done in:

5.Repeated pregnancy loss or infertility

6.Pregnancy screening abnormality

-Maternal serum α-fetoprotein

-Maternal triple screen or variant of this test

-Fetal ultrasonography -Fetal karyotype

7.Heterozygote screening based on ethnic risk

-Sickle cell anemia -Thalassemias

-Tay-Sachs, Canavan, Gaucher diseases

 

Dr.Padmesh. V

Prenatal diagnosis: TECHNIQUES: 1. Non invasive

Techniques 2. Invasive Techniques

Dr.Padmesh. V

Prenatal diagnosis: TECHNIQUES: 1. Non invasive Techniques A. Fetal visualization :

B. Screening for neural tube defects (NTDs) :

C. Screening for fetal Down syndrome:

D. Separation of fetal cells from the mother's blood:

2. Invasive Techniquesa.Fetal visualization

b.Fetal tissue sampling

c. Preimplantation biopsy of blastocysts obtained by

in vitro fertilization

d. Cytogenetics e. Molecular genetics

Dr.Padmesh. V

1. NON INVASIVE TECHNIQUES: A. Fetal visualization :

-Ultrasound -Fetal echocardiography -Magnetic resonance imaging (MRI) -Radiography

B. Screening for neural tube defects (NTDs) :

-Measuring maternal serum alpha-fetoprotein (MSAFP)

C. Screening for fetal Down syndrome: -Measuring MSAFP -Measuring maternal unconjugated estriol -Measuring maternal serum beta-human chorionic

gonadotropin (HCG)

D. Separation of fetal cells from the mother's blood:

Dr.Padmesh. V

2. INVASIVE TECHNIQUES:

a.Fetal visualization -Embryoscopy -Fetoscopy

b.Fetal tissue sampling -Amniocentesis -Chorionic villus sampling (CVS) -Percutaneous umbilical blood sampling (PUBS) -Percutaneous skin biopsy -Other organ biopsies, including muscle and liver biopsy

Dr.Padmesh. V

2. INVASIVE TECHNIQUES:

c. Preimplantation biopsy of blastocysts

obtained by in vitro fertilization

d. Cytogenetic investigations -Detection of chromosomal aberrations -Fluorescent in situ hybridization

e. Molecular genetic techniques -Linkage analysis using microsatellite markers -Restriction fragment length polymorphisms

(RFLPs) -Single nucleotide polymorphisms (SNPs)

Dr.Padmesh. V

Noninvasive

Techniques

1. Fetal Visualization2. Screening for NTD3. Screening for Downs4. Separation of fetal cells from the

mother's blood

Dr.Padmesh. V

1. Fetal visualization

-Ultrasound -Fetal echocardiography -Magnetic resonance imaging (MRI)

-Radiography

Dr.Padmesh. V1. Fetal Visualization:

a) USG: Noninvasive procedure for imaging fetal anatomy.

Harmless to both the fetus and the mother.

Anatomical lesions, including some genitourinary, gastrointestinal, skeletal, and central nervous system abnormalities and congenital cardiopathies, can be visualized by ultrasound between 16-20 weeks' gestation.

Ultrasound also is used to guide invasive sampling, such as amniocentesis, CVS, cordocentesis, and various fetal biopsies.

Dr.Padmesh. V Fetal USG

Dr.Padmesh. V

1. Fetal Visualization: contd..

b)Fetal ECHO: -Fetal echocardiography can be performed at 15 weeks' gestation

and beyond.

-When this technique is used with duplex or color flow Doppler, it can identify a number of major structural cardiac defects and rhythm.

-Fetal echocardiography is recommended in cases where cardiac defects are suspected.

c) MRI: Because of fetal movements, its application has been limited.

d)RADIOGRAPHY: -Fetal skeleton can be visualized by radiography from 10 weeks'

gestation onward.

-Used for the diagnosis of inherited skeletal dysplasias, particularly osteochondrodysplasia, in the second and third trimesters.

Dr.Padmesh. V1. Fetal Visualization: FETAL ECHO

Dr.Padmesh. V

2. Screening for neural tube defects:

Screening for NTDs is recommended if the following are present:

Ultrasound findings indicate NTDs. A child with NTDs is already in the family. A family history of NTDs exists, especially a mother with NTDs. The mother has type 1 diabetes mellitus during pregnancy. Maternal exposure to drugs, such as valproic acid, is associated

with NTDs. Elevated level of MSAFP is present.

Dr.Padmesh. V

2. Screening for neural tube defects:

The developing fetus has 2 major blood proteins, albumin and alpha-fetoprotein (AFP), while adults have only albumin in their blood.

AFP is produced by the yolk sac and later by the liver; it enters the amniotic fluid and then the maternal serum via fetal urine. Therefore MSAFP level can be used to determine the AFP levels from the fetus.

In the condition of an open NTD (eg, anencephaly, spina bifida) and abdominal wall defects in the fetus, AFP diffuses rapidly from exposed fetal tissues into amniotic fluid, and the MSAFP level rises.

The MSAFP test has the greatest sensitivity between 16-18 weeks' gestation, but it also can be performed between 15-22 weeks' gestation.

Dr.Padmesh. V

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.

Dr.Padmesh. V3. Screening for Downs syndrome:

1st Trimester Screening TestsMaternal Serum Markers-Preg. asso. Placental Protein A (PAPP-A)

-Free ß hCG

Fetal Marker- Nuchal fold> 4mm

at 8-12 wks GA

2nd Trimester Screening Tests Maternal Serum Markers

AFP

E3 Triple Test

hCG

Inhibin A Quadruple Test

65%

57-73%85%

69% 76%

94%

Dr.Padmesh. V

4. Separation of fetal cells from the mother's blood

Fetal blood cells enter maternal circulation through the placental villi.

These cells can be collected safely from approximately 12-18

weeks' gestation onward.

Fetal blood cells can then be analyzed for the diagnosis of genetic disorders using FISH, PCR etc.

Fetal cells separated from a mother's blood have been successfully used in the diagnosis of cystic fibrosis, sickle cell anemia, and thalassemia in a fetus.

Dr.Padmesh. V

Invasive Techniques

Dr.Padmesh. V

1. Fetal visualization: Embryoscopy: Embryoscopy is performed in the first trimester.

In this technique, a rigid endoscope is inserted via the cervix in the space between the amnion and the chorion, under sterile conditions and ultrasound guidance, to visualize the embryo for the diagnosis of structural malformations.

Dr.Padmesh. V

1. Fetal visualization: Fetoscopy Fetoscopy is performed during the second trimester

(after 16 weeks’ gestation).

In this technique, a fine-caliber endoscope is inserted into the amniotic cavity through a small maternal abdominal incision, under sterile conditions and ultrasound guidance, for the visualization of the embryo to detect the presence of subtle structural abnormalities.

It also is used for fetal blood and tissue sampling.

Fetoscopy is associated with a 3-5% risk of miscarriage;

Dr.Padmesh. V

1. Fetal visualization: Fetoscopy

Dr.Padmesh. V

2. Fetal tissue sampling Amniocentesis Amniocentesis is an invasive, well-established, safe, reliable,

and accurate procedure performed between 14-20 weeks of pregnancy.

It is performed under ultrasound guidance.

A 22-gauge needle is passed through the mother's lower abdomen into the amniotic cavity inside the uterus, and 10-20 mL of amniotic fluid that contains cells from amnion, fetal skin, fetal lungs, and urinary tract epithelium are collected.

Dr.Padmesh. V

2. Fetal tissue sampling Amniocentesis 1.The Cells are grown in culture for chromosomal,

biochemical, and

molecular biologic analyses.

2. The Supernatant amniotic fluid is used for the measurement of

substances such as AFAFP, hormones, and enzymes.

3. In the third trimester of pregnancy, the amniotic fluid can be

analyzed for determination of fetal lung maturity.

The results of cytogenetic and biochemical studies on amniotic cell cultures are more than 90% accurate.

Risks with amniocentesis are rare but include 0.5-1.0% fetal loss and maternal Rh sensitization.

Dr.Padmesh. V

Amniocentesis

Dr.Padmesh. V 2. Fetal tissue sampling

Chorionic villus sampling CVS is performed very early in gestation between 9-12 weeks,

ideally at 10 weeks' gestation.

A catheter is passed through the cervix or through the abdominal wall into the uterus under ultrasound guidance, and a sample of chorionic villi surrounding the sac is obtained.

The villi are dissected from the decidual tissue, and chromosome analysis is carried out on these cells to determine the karyotype of the fetus.

DNA can be extracted from these cells for molecular analysis.

DNA analysis of CVS specimens is helpful for early diagnosis of hemoglobinopathies. Tissue culture can be initiated on these cells for further studies.

Dr.Padmesh. V 2. Fetal tissue sampling

Chorionic villus sampling The major advantage of CVS over amniocentesis is getting

quick results and its use in early pregnancy.

Abnormalities can be identified at an early stage, and decisions about termination of the pregnancy can be taken early.

Abortion is also much safer at this early stage.

Disadvantages of CVS as compared to amniocentesis are:

-2-3% risk of causing miscarriage, and,

-Rarely limb defects in the fetus.

-Maternal Rh sensitization.

Dr.Padmesh. V

Dr.Padmesh. V 2. Fetal tissue sampling

Percutaneous umbilical blood sampling: (PUBS) / Cordocentesis:

Method for fetal blood sampling

Performed after 16 weeks' gestation.

A needle is inserted into the umbilical cord under ultrasound guidance, and fetal blood is collected from the umbilical vein for chromosome analysis and genetic diagnosis.

An advantage of PUBS is the rapid rate at which lymphocytes grow, allowing prompt genetic diagnosis.

This technique is also useful for evaluating fetal metabolism and hematologic abnormalities.

Dr.Padmesh. V PUBS

Dr.Padmesh. V

COMPARISON OF THREE MAIN PROCEDURES:

CVS Amniocentesis FBS

Gest.age 11-12 wks >15-16 wks >18 wks

Samplesuccess% 96-99% 99.5%

90%Abortion risk 1-2% 0.5%

2%Reporting time 3-15 days 10-15 days

1 weekCulture failure <1% <1%

Nil

Dr.Padmesh. V 2. Fetal tissue sampling

Percutaneous skin biopsy:

Fetal skin biopsies are taken under ultrasonic guidance between 17-20 weeks' gestation

To prenatally diagnose serious skin disorders, such as anhidrotic ectodermal dysplasia, epidermolysis bullosa letalis, epidermolysis bullosa dystrophica, hypohidrotic ectodermal dysplasia, oculocutaneous albinism, and genetic forms of ichthyosis.

Dr.Padmesh. V 2. Fetal tissue sampling

Other organ biopsies, including liver and muscle biopsy:

Fetal liver biopsy is best performed between 17-20 weeks' gestation under ultrasound guidance.

Fetal liver biopsy is needed to diagnose inborn errors of metabolism, such as ornithine transcarbamylase deficiency, glucose-6-phosphatase deficiency , glycogen storage disease type IA, nonketotic hyperglycemia, and carbamoyl-phosphate synthetase deficiency.

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.

Dr.Padmesh. V

Preimplantation biopsy of blastocysts obtained by in vitro fertilization: Newer concept..

Techniques are being developed to test cells obtained from biopsy of blastocysts of pregnancies conceived through in vitro fertilization.

These techniques will be helpful for selective transfer and implantation of those pregnancies into the uterus that are not affected by a specific genetic disorder.

Dr.Padmesh. V

Cytogenetic Investigations

Dr.Padmesh. V

Detection of chromosomal aberrations:

Chromosomal aberrations, such as deletions, duplications, translocations, and inversions diagnosed in affected parents or siblings, can be detected prenatally in a fetus by chromosomal analysis. 

This analysis can be undertaken on fetal cells obtained through such techniques as amniocentesis and CVS.

Dr.Padmesh. VFISH

Dr.Padmesh. VFISH

Dr.Padmesh. V

(Fluorescent in situ hybridization )

FISH is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes.

FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity.

Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes.

FISH

Dr.Padmesh. V

(Fluorescent in situ hybridization )

FISH

Chromosomal DNA

Heat Denaturation

Probe DNA labelled with Flurochrome

..

.

.

Hybridization

Dr.Padmesh. V

(Fluorescent in situ hybridization )

The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (up left) is the one where the wrong rearrangement is present.

FISH

Dr.Padmesh. V Benefits of Prenatal Diagnosis:

Prenatal diagnosis determines the outcome of pregnancy.

It is helpful for couples to decide whether to continue the pregnancy.

It indicates possible complications that can arise at birth process.

Prenatal diagnosis is helpful for the management of remaining weeks of pregnancy.

It prepares the couple for the birth of a child with an abnormality.

Prenatal diagnosis can be helpful for the improvement of the outcome of pregnancy using fetal treatment.

Dr.Padmesh. V

Thank you..