Important points of diagnostic tests during pregnancy

Important points of diagnostic tests during pregnancy

1- Full Karyotype

2- FISH

3- QF-PCR

4- MLPA

5- Array CGH


1- Full karyotype

It is a technique that can detect the ability of almost all chromosomes, both in terms of number (pyloid) and large structural disorders (in the normal type, structural changes above 5 to 10 MB and in the High resolution type above 3 MB).

During this process, chromosomes are differentiated based on length, location of the centromere, bonding pattern (chromosomes are seen as light or dark colored stripes during the staining process), differentiation of sex chromosomes (alzomes) from autosomal (non-sexual) chromosomes. And other physical properties are differentiated and put together.

There are a number of disorders such as Down syndrome (trisomy 21), Edward syndrome (trisomy 18), Pato syndrome (trisomy 13), Klein-Felter syndrome (47, XXY), Turner syndrome (45, XO) and trisomy 9 (as the fourth most common trisomy). It leads to the birth of a living baby and causes speech problems in a person (recognizable).

Structural equilibrium and non-equilibrium chromosomal abnormalities that range in size from 5 to 10 megawatts in routine methods and about 3 megawatts in High Resolution Karyotyping.

These disorders include:

- Cry du chat syndrome (part of the short arm of chromosome 5 is removed, which leads to problems in the structure of the larynx or larynx).

- Monosomy 1p36 or 1p36 Deletion syndrome (part of the short arm of chromosome 1 is removed and it is also called monosomy 1p36. This syndrome leads to the development of resistant epilepsy, which is associated with mental retardation, growth disorder, hypotension, seizures). , Speech impediment, vision and hearing problems)

Angelman syndrome (removal of a large piece of the long arm of a chromosome 15 of maternal origin that results in shrinking of the head, specific physical structure of the face, severe mental disability, motor disability, seizures, motor and balance problems) and

- Prader-Willi syndrome (removal of a large piece of the long arm of a chromosome 15 of paternal origin that leads to muscle hypotension in the affected infant, reduction of lactation, reduction of the growth process).

Chromosomal disorders in the form of mosaicism and translocation can also be detected in this way.

This is the only legal way to issue an abortion permit.

Disadvantages:

Diagnosis time is long (between 12 and 18 working days and an average of 14 working days), which is due to the long history of response due to the need for amniocentesis cells.

- Unable to diagnose a parental dysplasia (Uniparental disomy) (when the child inherits both chromosomes from one parent and is the only detectable method in these methylation disorders).

- Disorders related to single genes are not detectable.

- Much depends on the skill and experience of the test worker (More labor-intensive).



FISH or (Fluorescence in situ hybridization)

It is a molecular cytogenetic technique that uses a series of fluorescent probes that attach to a portion of the nucleocyte acid sequences that complement the probe.

With the help of special probes, 5 chromosomes 21, 18, 13, X and Y, which cause 60 to 80% of chromosomal disorders at birth, can be identified.

Advantages:

- The preparation time of the answer is short and reaches 1 to 2 days.

Using Lucus-specific probes (a specific part of a gene on a single chromosome), it can detect microdeletion in contiguous gene genes.

- No need for cell culture.

- Different types of biological samples can be used in this method.

- Able to diagnose structural disorders between 190 kbps and 1 MB.

- There is the ability to reject or confirm positive NIPT answers, with high speed and accuracy.

Disadvantages:

- Unable to detect unknown chromosomal abnormalities,

- Unable to diagnose structural disorders smaller than 190 kbps,

- Unable to diagnose a parental dysplasia (Uniparental disomy) (when the child inherits both chromosomes from the same parent)

- Unable to detect inversions,

- Relatively expensive,

- We will not be able to receive legal abortion by answering this method alone.

- Not able to evaluate all chromosomes,

- Not easily available throughout the country.

QF-PCR

Based on the rapid diagnosis of common numerical chromosomal abnormalities (aneuploidy in chromosomes 13, 18, 21, X, and Y), this method makes it possible with high accuracy and speed (about 48 hours) in non-mosaic specimens.

QF-PCR (‌ Quantitative Flurescent-Polymerase Chain Reacation) is based on the quantitative fluorescence reaction.

The kit can be used on samples of DNA purified from blood, amniotic fluid, placental chorionic villi (CVS) and free DNA inside amniotic fluid.

It is noteworthy that the test can be performed on tissue samples, blood of adults and newborns.

It consists of 14 Short Tandem Repeat (STR) markers, which are multiplied in a multiplexer PCR reaction.

Finally, the results are determined using capillary electrophoresis.

As the number of markers for a chromosome increases, so does the detection power. These markers cover important trisomy areas of chromosomes (critical regions).

Advantages:

- The main advantage of these tests is the ability to respond in a short time (1 to 3 working days).

- Also has high sensitivity and specificity (due to the use of several STR markers)

- It is possible to detect maternal cell contamination in this way. (First, the embryo should be differentiated from the embryo by the mother cell microscope and, if necessary, the two samples should be differentiated using agarose gel).

- The price is right

- No need for less labor-intensive personnel to perform the test,

- Not being affected by gestational age is also one of the important benefits of these techniques.

Disadvantages:

- The limitation of QF-PCR, MLPA and FISH tests is the inability to detect low degrees of mosaicism.

- It is also incapable of detecting disorders in chromosomes other than chromosomes 13, 18, 21, X or Y.

- The cost of the test depends on the number of samples.

- Fragment analysis of these markers is done by capillary electrophoresis. So to view and interpret the results of this kit, you need a Genetic Analyzer or similar devices. Access to this device is not possible everywhere.

- The possibility of legal abortion alone is not possible by this method,



MLPA or (Multiplex Ligation-dependent Probe Amplification)

This method of multiplication of the multi-attachment probe (MLPA) is a sensitive and powerful method for examining changes in the number of copies (Copy Number Variation) at the genome level.

This method is widely used in medical diagnosis and research to identify pathogenic gene deletions and proliferations that are caused by genetic diseases.

In this method, using a set of probes, CNV‌ can be examined in more than 40 different sequences of genomes in one reaction.

First, two separate probes are hybridized to specific areas of the genome (target location) and adjacent to each other, so that they are only as far apart as a phosphodiester band.

Then in the ligation stage or the addition of these two probes are connected by phosphodiester bonding. It is then propagated by a pair of universal primers.

The electrophoresis product is then separated by capillary electrophoresis, and by comparing it with the normal sample, the gene dose can be obtained at the junction of each pair of probes.

Depending on the presence or absence of a proliferative component, a small number of gene copies can be obtained.

Compared to the CGH method, which detects changes in the number of copies (CNV) in all genomes, the MLPA method examines these changes in small genomic locations.

Advantages:

- Investigating cases of deletion and chromosomal duplication

- Response speed to less than 24 hours

It is capable of detecting any CNV‌ in the range of 50 to 75 nucleotides, and can distinguish between even one nucleotide.

- Almost affordable.

- At the same time is able to perform 60 simultaneous reactions.

Disadvantages:

- A small number of samples can be done in each thigh.

- Very sensitive to contaminants.

- It requires a high amount of DNA to perform the test, so the DNA content in a cell is not enough to perform the test (the best way to extract DNA in this method is by salting out using the K‌ proteinase).

- Unable to identify unknown point mutations.

- Unable to detect new benign polymorphisms near probe connections.

- Unable to identify disorders related to all chromosomes.

- Legal abortion alone is not possible with this test.

- Unable to identify the types of mosaics.

Array CGH

This method is a high-resolution technique used to detect unbalanced deletions or additions throughout the genome.

This test, also called the molecular karyotype, has a higher resolution than the karyotype.

Many diseases are caused by differences in the number of copies of the chromosome (Copy Number Variation-CNV).

How to perform a CGH Array test

To perform the Array CGH test, the DNA sample of the person in question is compared with the DNA control sample.

For this purpose, these DNA samples are stained with different fluorescent dyes (in the picture, two colors, red and green, for the test sample and control sample, respectively).

Slides are also connected to DNA oligonucleotide probes or BAC clones.

The control sample and the test specimen sample are poured on these slides and the time and conditions required for the binding and hybridization of DNA fragments are provided.

In areas of DNA where there is no difference between the control sample and the sample being tested, both red and green are banded equally and are yellow.

However, in areas where there is a deletion in the test sample, the color of the control sample (green) is more common, and in areas where there is a duplication, the color of the sample being sampled (red) is more common.

 Applications of the CGH Array Test

- Identification of microdermabrasion (microsurgery) and microdermabrasion syndromes (microscopy)

- Identification of sub-telomere recombinations or other unbalanced chromosomal recombinations

Investigating the Causes of Evolutionary Delay and Mental Retardation: Chromosomal abnormalities are found in 30-25% of patients with developmental delay and mental retardation.

- Multiple congenital anomalies (heart failure, primary microcephaly, short stature, growth retardation, etc.)

Autism Spectrum Disorders - A significant proportion of people with idiopathic autism have chromosomal aberrations and recurrences.

- Behavioral abnormalities

Symptoms such as epilepsy and seizures - pathogenic CNVs have been reported in 5-10% of people with epilepsy.

Dysmorphisms - Chromosomal abnormalities are the main cause of dysmorphic features.

Prenatal Diagnosis (PND): Array CGH In addition to detecting numerical changes in chromosomes and aneuploidy in cases where there is a history of birth of a child with congenital disorders (which is the addition or complete removal of chromosomes, such as chromosome 21 trisomy) Down syndrome is also effective in detecting small structural imbalances.

Advanced Embryo Selection in IVF This method is used to select the fetus before transferring it to the mother's uterus (PGS).

- In cancer research and diagnostic studies - CNV and SNP changes can be identified by microbial kits.

Areas without heterozygosity (AOH or LOH) and single parental dyes (UPD) are identified by platforms with SNP study probes.

The advantages of the Array CGH method

- This method is able to analyze DNA from almost all tissues, including archived tissues or tissues that cannot be cultured.

All 46 human chromosomes are tested.

- In this experiment, the exact location of deletions and additions in the genome is identified.

- It is more sensitive and accurate than conventional karyotype methods. This technique is able to detect openings and additions of 5 to 10 kg open.

- This test can help identify breakdown points in known chromosomal imbalances.

- Marker chromosomes are identified by this method.

- The lack of time-consuming techniques for cell culture increases the speed of this procedure.

- Array CGH is more comprehensive than techniques such as FISH and QF-PCR. In the FISH technique, only one gene or pre-known part of the genome is examined according to the clinical symptoms. However, the Array CGH test allows a large number of deletions and additions to be made with just one test, regardless of the clinical symptoms.

In people who report normal, normal karyotype results, a microbiological test can detect chromosomal imbalances or microscopy and duplication.

Limitations

- The Array CGH method is not able to detect balanced chromosomal displacements and inversions. In this case, no part of the chromosomes is removed or added, and only the movement of parts of the chromosomes causes the disease.

Low-percentage mosaicism for unbalanced changes and aneuploidy may not be detected in this way. Microbial susceptibility to mosaicism is influenced by the platform, sample type, number of copies, DNA quality, data quality, and size of the unbalanced area.

Chromosomal mechanism of genetic imbalance may not be apparent.

Tetraploid or other pluride may not be detected or difficult to detect.

It does not detect abnormalities caused by trinucleotide repeat expansion disorders.

- Changes in the number of copies are not detected in genomic locations that do not exist on the platform.

- Removals and duplications that are smaller than the detection level based on the coverage and performance of the probes, point mutations, gene expression and methylation anomalies that may be involved in the patient's phenotype, in this way. They are not detected.

No microbial platform can detect all mutations in a particular syndrome. Therefore, it should be noted that not recognizing the change in the number of copies in each locus does not rule out the diagnosis of a disease related to that locus.

- Interpretation of aCGH results is based on information in databases and sometimes by studying changes in parents. Sometimes, based on this evidence, it is not possible to interpret the results definitively, and the result is reported as a variant of uncertain significance (VUS).

- Sometimes changes that are not related to the patient's current phenotype (Incidental findings) are also identified with this method.

Required sample

- Array CGH test is performed on peripheral blood samples from children or adults (5 ml of peripheral blood). Blood samples are taken in laboratory EDTA tubes, which must be stored in a cool place to prevent them from freezing.

For prenatal diagnosis, a sample of amniotic fluid or embryonic placental villi (20 mg of embryonic villi or 20 ml of embryonic amniotic fluid in two separate tubes) is also examined. Embryonic specimens should be sent to the laboratory as soon as possible.

Blastomeres, trophoblasts, or polar bodies can be used to diagnose pre-implantation (PGS). In this regard, previous coordination with the laboratory should be done.