*3.3.2.2.2. QF-PCR*

QF-PCR method allows the detection of major prenatal numerical chromosome disorders within 24–48 hours. The method identifies polymorphic chromosomal specific repeat sequences (short tandem repeats—STRs) that are amplified by the PCR using fluorescent

to adjacent target sequences on genomic DNA, can be joined together by the DNA ligase. This permits the amplification of all target sites, using a single primer pair that is complementary to the two free ends which are common to all probes. The PCR products are run on a capillary electrophoresis system and MLPA allows relative quantification of up to 50 different target sequences in one reaction. MLPA is a fast method (final results in 2–3 days) and it is less labor intensive and cheaper compared to karyotyping and FISH. The main domain of application of MLPA in prenatal diagnosis is the detection of most common aneuploidies (of chromosomes 21, 18, 13, X and Y) but this technique has some inconvenients associated with the impossibility of detection of triploidy, mosaicisms and maternal contamination. Other applications concern the cases with multiple congenital anomalies and intrauterine growth retardation detected by ultra-sonography. In these situations, using subtelomere probes or specific probes for some specific syndromes (velocardiofacial, Williams, Wolf-Hirschhorn, Prader-Willi, etc.), MLPA could confirm the presence of some subtle unbalanced structural chromosomal anomalies. MLPA can also be used to determine the origin of marker chromo-

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Array CGH is a method that can detect simultaneously sub-microscopic copy number changes across the whole genome, thus overcoming the limitations of karyotyping and locusspecific techniques. Array CGH has become an important tool for clinical diagnostics and gene-identification studies and is having a great impact on the understanding of pathologies, the counseling of families and patient management. Different types of array CGH platforms at an increasingly higher resolution have been developed, differing mainly in the type of the interrogating probes and in their coverage of the genome. The microarray consists of thousands of unlabeled different probes (particular to a specific DNA sequence) fixed on a glass slide or a silicon chip, arranged in orderly rows in the form of a network with a specific density ("DNA chip"). The two samples of DNA (genomic DNA extracted from patient and reference sample) are fragmented and labeled with different fluorochromes (Cy3—green for patient and Cy5—red for test sample), mixed in equal amounts, denatured and co-hybridized on the microarray. The chips are scanned with a microarray scanner and the images obtained are analyzed using a programme that determines the intensities of emissions of both red and green fluorochrome for each spot on the network and calculates their report. This ratio is in proportion to the number of copies of the patient's genome DNA and test sample. If the intensities of the two fluorochromes are equal to a spot (ratio Cy3/Cy5 = 1, or log2 = 0), this region of the patient's DNA is interpreted as normal. If there is a deletion, the test sample hybridizes preferentially to DNA control, and the ratio Cy3/Cy5 will be smaller than 1 (ex. 1:2, log2 = −1). On the other hand, if there is a duplication, the patient DNA will hybridize preferentially, and

The application of array CGH eliminates the majority of fetal chromosomal analysis inconveniences: the long period of waiting for the final result, the possible failures of culture, the poor quality of chromosomal preparations and the reduced number of chromosomal bands. The method uses genomic DNA from fetal cells and can be applied on cells in interphase or division. The sensitivity of method is higher than the standard karyotype, and thus array-CGH allows the detection of all unbalanced chromosomal abnormalities (excepting polyploidy), even the smallest

somes, frequently discovered in chromosomal prenatal diagnosis [113, 114].

the ratio Cy3/Cy5 will be greater than 1 (ex. 3:2, log2 = 0.58.) [115].

*3.3.2.2.4. Array-CGH*

**Figure 7.** Aneuploidies detected using FISH method. (a) Trisomy 21; (b) Trisomy 13; (c) Trisomy 18; (d) Monosomy X; (e) Trisomy XXY; (f) Trisomy XYY (collection of prenatal diagnosis laboratory of "Cuza Vodă" obstetrics and gynecology hospital, Iaşi, Romania).

primers. The amplification is observed and quantified using a genetic analyzer and appropriate software. In a normal case, two peaks of fluorescence activity were obtained that reflect a normal heterozygous fetus (1:1 diallelic normal ration). Trisomic samples demonstrate either three peaks with a ratio of 1:1:1 (trisomic triallelic) or two peaks with a ratio of 2:1 (trisomic diallelic) for each informative probe. For each chromosome four or more polymorphic STR markers are analyzed, and thus only few fetal samples will remain uninformative. By comparison with FISH, QF-PCR can detect mosaicisms that have a rate of 20–30% and also identify the maternal cell contamination. The main advantage of QF-PCR is that it is considerably more cost-effective particularly when larger sample numbers are processed [111]. The main limitation of assay is the impossibility to detect triploidy [112].

#### *3.3.2.2.3. MLPA*

MLPA is a PCR-based method used for quantification of the copy numbers of specific sequences of DNA. This method uses a two-part probe of unique length that, when hybridized to adjacent target sequences on genomic DNA, can be joined together by the DNA ligase. This permits the amplification of all target sites, using a single primer pair that is complementary to the two free ends which are common to all probes. The PCR products are run on a capillary electrophoresis system and MLPA allows relative quantification of up to 50 different target sequences in one reaction. MLPA is a fast method (final results in 2–3 days) and it is less labor intensive and cheaper compared to karyotyping and FISH. The main domain of application of MLPA in prenatal diagnosis is the detection of most common aneuploidies (of chromosomes 21, 18, 13, X and Y) but this technique has some inconvenients associated with the impossibility of detection of triploidy, mosaicisms and maternal contamination. Other applications concern the cases with multiple congenital anomalies and intrauterine growth retardation detected by ultra-sonography. In these situations, using subtelomere probes or specific probes for some specific syndromes (velocardiofacial, Williams, Wolf-Hirschhorn, Prader-Willi, etc.), MLPA could confirm the presence of some subtle unbalanced structural chromosomal anomalies. MLPA can also be used to determine the origin of marker chromosomes, frequently discovered in chromosomal prenatal diagnosis [113, 114].

#### *3.3.2.2.4. Array-CGH*

primers. The amplification is observed and quantified using a genetic analyzer and appropriate software. In a normal case, two peaks of fluorescence activity were obtained that reflect a normal heterozygous fetus (1:1 diallelic normal ration). Trisomic samples demonstrate either three peaks with a ratio of 1:1:1 (trisomic triallelic) or two peaks with a ratio of 2:1 (trisomic diallelic) for each informative probe. For each chromosome four or more polymorphic STR markers are analyzed, and thus only few fetal samples will remain uninformative. By comparison with FISH, QF-PCR can detect mosaicisms that have a rate of 20–30% and also identify the maternal cell contamination. The main advantage of QF-PCR is that it is considerably more cost-effective particularly when larger sample numbers are processed [111]. The main

**Figure 7.** Aneuploidies detected using FISH method. (a) Trisomy 21; (b) Trisomy 13; (c) Trisomy 18; (d) Monosomy X; (e) Trisomy XXY; (f) Trisomy XYY (collection of prenatal diagnosis laboratory of "Cuza Vodă" obstetrics and gynecology

MLPA is a PCR-based method used for quantification of the copy numbers of specific sequences of DNA. This method uses a two-part probe of unique length that, when hybridized

limitation of assay is the impossibility to detect triploidy [112].

*3.3.2.2.3. MLPA*

hospital, Iaşi, Romania).

384 Congenital Anomalies - From the Embryo to the Neonate

Array CGH is a method that can detect simultaneously sub-microscopic copy number changes across the whole genome, thus overcoming the limitations of karyotyping and locusspecific techniques. Array CGH has become an important tool for clinical diagnostics and gene-identification studies and is having a great impact on the understanding of pathologies, the counseling of families and patient management. Different types of array CGH platforms at an increasingly higher resolution have been developed, differing mainly in the type of the interrogating probes and in their coverage of the genome. The microarray consists of thousands of unlabeled different probes (particular to a specific DNA sequence) fixed on a glass slide or a silicon chip, arranged in orderly rows in the form of a network with a specific density ("DNA chip"). The two samples of DNA (genomic DNA extracted from patient and reference sample) are fragmented and labeled with different fluorochromes (Cy3—green for patient and Cy5—red for test sample), mixed in equal amounts, denatured and co-hybridized on the microarray. The chips are scanned with a microarray scanner and the images obtained are analyzed using a programme that determines the intensities of emissions of both red and green fluorochrome for each spot on the network and calculates their report. This ratio is in proportion to the number of copies of the patient's genome DNA and test sample. If the intensities of the two fluorochromes are equal to a spot (ratio Cy3/Cy5 = 1, or log2 = 0), this region of the patient's DNA is interpreted as normal. If there is a deletion, the test sample hybridizes preferentially to DNA control, and the ratio Cy3/Cy5 will be smaller than 1 (ex. 1:2, log2 = −1). On the other hand, if there is a duplication, the patient DNA will hybridize preferentially, and the ratio Cy3/Cy5 will be greater than 1 (ex. 3:2, log2 = 0.58.) [115].

The application of array CGH eliminates the majority of fetal chromosomal analysis inconveniences: the long period of waiting for the final result, the possible failures of culture, the poor quality of chromosomal preparations and the reduced number of chromosomal bands. The method uses genomic DNA from fetal cells and can be applied on cells in interphase or division. The sensitivity of method is higher than the standard karyotype, and thus array-CGH allows the detection of all unbalanced chromosomal abnormalities (excepting polyploidy), even the smallest such subtelomeric rearrangements. In the prenatal diagnosis, "targeted" arrays are commonly used, containing genomic clones for subtelomeric regions and those that are frequently involved in microdeletion/microduplication syndromes. The major advantage of array-CGH is the very high resolution, this technique allowing the detection of genomic changes of 50–100 kb. The main inconvenient is the possibility of detecting copy number variants (CNVs) with unknown clinical consequences. In this case, with limited possibility of fetal phenotype investigation, the evaluation of functional consequences of genomic changes is very difficult. A CNV discovered in these conditions is most probably pathogen if it is de novo, has a size >1 Mb, contains a deletion rather than a duplication and involves a gene-rich area [116–118].

results are done during the second trimester which increases the anxiety of the couple and

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The chromosomal disorders have an important impact on the health of future infants. For this reason, in the last decade, important efforts were made to improve the prenatal screening and diagnosis. The prenatal screening uses non-invasive methods that allow the detection of pregnancies with risk of chromosomal anomalies. These methods can be done in the first or second trimesters of pregnancy. The first-trimester screening methods are biochemical and sonographic. Biochemical screening for first trimester uses the detection of PAPP-A and β-hCG in the maternal serum. Sonographic examination in the first trimester allows the detection of some markers—nuchal translucency, absence of the nasal bone, tricuspid regurgitation or abnormal blood flow through the ductus venosus—that are associated with high risk for aneuploidy. The prenatal screening during the second trimester of pregnancy can be done by biochemical or sonographic examinations as well. The biochemical screening is based on the detection of aFP, hCG (or β-hCG), μE and inhibin A in maternal serum. The sonographic examinations in the second trimester can identify some structural defects (cardiac, cerebral, renal, etc.) but more frequent are the sonographic markers like nuchal thickening, hyperechoic bowel, shortened limbs, pyelectasis, echogenic intracardiac foci, widened pelvic angle, and so on. The best choice for prenatal screening of aneuploidies is the use of combined biochemical (in the first and second trimesters) and sonographic examination. Such protocol has a detection rate higher than 90% at 5% false positive rate. The prenatal diagnosis requires an invasive procedure (chorionic villus sampling, amniocentesis, cordocentesis) to obtain fetal material. The fetal cells can be used for cytogenetic or molecular analyses. The cytogenetic analyses (fetal karyotype) require a long-time cell culture and have a limited resolution but have the advantage of diagnosis of all chromosomal anomalies. Some molecular analyses (FISH, QF-PCR, MLPA) are targeted methods and can identify only specific anomalies, such as aneuploidies of chromosomes 21, 18, 13 X and Y. Array-CGH (molecular karyotype) eliminates the major inconvenience of karyotype (long-time culture and limited resolution) but is expensive and thus it's use remains prohibitive in countries with limited economical resources. However, the implementation of prenatal screening and diagnosis allows many couples the opportunity to take an

limits its reproductive options [119, 120].

informed decision in relation to their baby's future.

\*Address all correspondence to: vgord@mail.com

\*, Demetra Gabriela Socolov2

1 Medical Genetics Department, "Grigore T. Popa" University of Medicine and Pharmacy,

2 Obstetrics and Gynecology Department, "Grigore T. Popa" University of Medicine and

and Răzvan Vladimir Socolov2

**Author details**

Iaşi, Romania

Eusebiu Vlad Gorduza1

Pharmacy, Iaşi, Romania

**5. Conclusions**

Nowadays, in developed countries, the array-CGH represents the first option in the prenatal genetic investigation of fetuses with multiple congenital anomalies detected by ultra-sonography.
