Cytogenetic and molecular m ethods for the detection of chromosomal aberration Dr. Maryam Tahmasebi Birgani PhD in Molecular Genetics Tarbiat Modares University of Tehran University of Florence

How can we decide ? A number of different methods can now be used to detect chromosomal abnormalities: Cytogenetic methods Molecular methods (DNA-Based methods)

Molecular methods Polymerase chain reaction (PCR) Similar to DNA replication What does the cell need for DNA replication?

Polymerase chain reaction (PCR) PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.

Electrophoresis Agarose preparation

Loading Dye Size marker

Sanger sequencing

Amplification Refractory Mutation System (ARMS) Amplification refractory mutation system (ARMS) is a PCR-based method developed in the late 1980s also for the analysis of known point mutations. ARMS is based on the fact that DNA amplification is inefficient if there is a mismatch between the template DNA and the 3′ terminal nucleotide of a PCR primer. A primer with a 3′ terminal nucleotide that is complementary to the wild-type allele will not have efficient extension when a mutation is present and vice versa. Therefore, one can differentiate between two alleles by simple PCR amplification.

Restriction Fragment Length Polymorphism(RFLP)

Allele-Specific Oligonucleotide (ASO) test

Cytogenetic methods Karyotype

… Fluorescence in situ hybridization This can be used for rapid detection of aneuploidy on interphase chromosomes in prenatal testing Fetal sex Detect unbalanced translocation

Probe

Prenatal diagnosis Prenatal diagnosis includes all aspects of embryonic and fetal diagnosis. Prenatal diagnosis for genetic conditions is presently indicated in: about 8% of all pregnancies for these couples at increased risk of serious genetic disease Sex determination Disease that are not sever In practice, 93% of prenatal tests provide reassurance for the couple concerned, and selective termination of pregnancy is undertaken in only 7%. At - risk pregnancies may be identified prior to or during a pregnancy

Amniocentesis Amniocentesis is the withdrawal of amniotic fluid. This is usually performed at 16 – 18 weeks of gestation when there is about 180 ml of liquor and the ratio of viable to non – viable cells is maximal. Under aseptic conditions, and after prior placental localization with ultrasound, a needle is introduced under ultrasound guidance into the amniotic cavity via the maternal abdomen. A volume of 10 – 20 ml of liquor is withdrawn and this can be used for several different tests

The chance of maternal cell contamination is greatly reduced if a stilette is used in the needle and if the first few drops of amniotic fluid withdrawn are discarded.

Fetal sexing: This is required for female carriers of serious X - linked disorders. Now, using X and Y chromosome - specific polymorphic DNA markers permits fetal sexing. This can include an analysis by PCR of intron 3 of the amelogenin ( AMEL ) gene in the X/Y homologous region. In the latter, the presence of a 6 b deletion in the allele on the X but not the Y chromosome permits determination of the sex of the fetus.

fetal karyotyping: the amniotic fluid cells are grown in culture and a result is available in 2 – 3 weeks. About 1.5% of samples fail to grow: if the fluid was heavily blood stained was less than 5 ml in volume if transport to the laboratory was delayed if a local anaesthetic was used. In experienced laboratories, contamination of the fetal amniotic cells with maternal cells is unlikely but should be suspected if the cell cultures take longer than usual to grow and if fibroblasts rather than epithelioid cells predominate. Chromosomal mosaicism can pose difficult diagnostic problems. Mosaicism: True false 46,XX/46,XY ‘ mosaicism ’ is an exception to this as it almost invariably represents maternal cell contamination.

QF-PCR Fetal Karyotyping is very labour - intensive, and attempts have been made to circumvent this and to shorten the time to diagnosis

electrophoretograms of four chromosome specific multiplex quantitative fluorescent polymerase chain reactions (QF-PCR). From top to bottom: multiplex PCR for chromosome 21 detecting Down's syndrome (trisomy 21), multiplex PCR for chromosome 13 detecting Patau syndrome (trisomy 13), multiplex PCR for chromosome 18 detecting Edward's syndrome (trisomy 18) and multiplex PCR for chromosome X detecting Turner's syndrome (45,X).

Amniocentesis carries a small additional risk of miscarriage for the pregnancy. In one randomised controlled trial of over 4000 women, this risk was estimated to be 1.0%. In addition, for any pregnancy at 16 weeks of gestation, there is a 2.5% chance of spontaneous miscarriage.

Chorionic villus sampling Sampling of chorionic villi from the fetus is performed from 10 weeks of gestation onwards and is now available in most major obstetric centres. The biopsy is usually taken under ultrasound guidance via a transabdominal approach. Each biopsy yields about 5 – 30 mg of tissue, which can be used for fetal sexing, fetal karyotyping, biochemical studies and DNA analysis. A direct fetal chromosomal analysis is possible within 24 h, but, in view of the problem of mosaicism in CVS samples, this should always be followed by chromosomal analysis on cultured cells from the sample 2 – 3 weeks later (a sample of 5 mg should be adequate for chromosomal analysis). DNA analysis or biochemical tests can be completed in 1 – 2 weeks, usually without the need to culture the cells

Cordocentesis, fetal skin biopsy and fetal liver biopsy Under ultrasound guidance, a fine needle can be passed transabdominally into the fetal umbilical cord where it enters the placenta in order to take a fetal blood sample or to perform an in utero transfusion. The procedure is possible from 18 weeks of gestation onwards and the procedure - related fetal loss rate is about 1%. Some serious skin disorders (e.g. epidermolysis bullosa) can be diagnosed in a fetal skin biopsy taken via a fetoscope, and in occasional metabolic disorders a fetal liver biopsy is necessary for diagnosis.

Ultrasonography Visualization of the fetus by ultrasound carries no proven hazard for either mother or fetus. Over 280 different congenital malformations may be diagnosed by an experienced ultrasonographer. Anencephaly may be detected on ultrasound as early as 10 – 12 weeks gestation but for most abnormalities 16 – 18 weeks is the optimal time. Serial scans may be required, especially to detect abnormal growth of, for example, the head or limbs. Although fetal genitalia can be visualized from 16 weeks of gestation, ultrasonography would not be adequate for fetal sexing for a serious genetic condition.

Free fetal DNA (cfDNA) In recent years, it has become clear that, in addition to fetal cells, cell - free fetal DNA can be found in the maternal circulation. The source is most likely to be apoptosis (programmed cell death) of cells in the placenta, and fetal DNA has been found in maternal serum and plasma as early as 7 weeks of gestation.

Preimplantation genetic diagnosis PGD single cell can be removed at day 3 post - fertilization from a 6 – 10 - cell embryo following in vitro fertilization. Using either PCR or FISH, the fetal sex is determined

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