3.1. Karyotyping

so constitute a serious public health and epidemiological problem [1]. A significant proportion of CAs is detected before birth by routine ultrasound examination and other screening techniques [2]. Prenatal identification of CAs can have significant emotional and psychological consequences for parents and affected babies [2]. Therefore, it is imperative to make the right diagnosis as soon and as accurate as possible. Genetic testing in prenatal period is an area with very sensitive and ethically challenging situations [3]. Determining a genetic diagnosis prenatally permits parents to make informative reproductive decisions and to be counseled about possible fetal outcomes [4]. It adds important information for current pregnancy in the terms of full phenotype beyond ultrasonographically detected abnormalities on the one hand and postnatal prognosis on the other [3]. Consequently, it is important to use appropriate genetic

In recent years new diagnostic algorithms for prenatal genetic testing are being developed with the advent of new genomic technologies, like molecular karyotyping (comparative genomic hybridization) and next-generation sequencing (NGS). These technologies offer testing options that exceed conventional karyotyping of the fetus and provide better diagnostic yield. Despite the evidence of important additional diagnostic yield of new technologies in the etiology of CAs, they have not been systematically implemented in clinical prenatal diagnostic

A CA is defined as any structural anomaly present at birth. Major CAs are anomalies that have medical, surgical, or cosmetic significance and occur in about 2–3% of liveborn and 20% of stillborn infants [1]. Thus, CAs are more prevalent that many chronic childhood diseases, such as autism, pediatric cancers, and type 1 diabetes, are an important cause of neonatal mortality, children morbidity, and long-term disability [1, 5]. Therefore, they represent an important

CAs can be isolated or present in a characteristic pattern affecting one or more organ systems. The overall prevalence of most major CAs does not vary much across ethnic groups [6, 7]. However, the risk for different types of anomalies is variable and may be related to genetic susceptibilities and also to cultural and social differences that can influence exposures (e.g., neural tube defects due to a dietary deficiency of folic acid) [6, 7]. The prevalence of most major birth defects over time has remained constant, but some have shown a significant increase

The etiology of CAs is complex. CAs can be the result of genetic factors, environmental factors, or a combination of both [8, 9], although the underlying etiology often remains unknown. It is estimated that genetic factors represent an important cause of CAs and may result from different genetic mechanisms: the most common are aneuploidies, deletions, and duplications of DNA segments (collectively known as CNV), and single gene disorders [8, 9]. Some disorders have an

testing approach to obtain a specific diagnosis.

398 Congenital Anomalies - From the Embryo to the Neonate

algorithms in several national healthcare systems.

2. The epidemiology and etiology of CA

public health and epidemiological problem.

such as gastroschisis [6, 7].

To identify possible genetic causes underlying ultrasonographically detected CAs or positive result of the screening test (nuchal translucency, combined screening test, triple/quadruple hormone test), a full chromosome analysis has been widely used and regarded as the gold standard from the late 1990s to about 2010. A diagnostic yield of classical karyotype is more than 18% in fetuses presenting with isolated or multiple CAs [10].

3.2. Targeted molecular genetic testing

approach rarely warrants the diagnosis.

3.3. Molecular karyotyping

translocations, and point mutations.

natal genetic diagnosis in high-risk pregnancies.

tic testing in fetuses with ultrasound anomalies [25].

structures [23].

As mentioned above, about 10% of CAs is thought to be associated with monogenic disease [4]. Ultrasound examination can detect many fetal structural abnormalities, from the early anatomic survey in the first trimester to morphology and biometry in the second trimester and monitoring of the fetal growth in the third trimester. In addition to ultrasound, fetal magnetic resonance is now widely used to improve imaging of the central nervous system

Genomic Testing for Prenatal Clinical Evaluation of Congenital Anomalies

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The conventional genetic approach using targeted molecular methods, like Sanger or PCR, is useful (enables) in the diagnostics of cases with ultrasonographically well-defined phenotypes associated with specific diagnostic hypothesis in genetically homogenous CA (e.g., TAR, achondroplasia) and is the method of choice for prenatal testing in cases of familial monogenic condition with known pathogenic variant [24]. In cases with poorly defined phenotype and genetically heterogeneous CAs (many genes responsible for the same phenotype), this

While the conventional genetic diagnostic approach is time-consuming, labor-intensive, and with limited diagnostic yield, the new genomic approaches and technologies, like molecular karyotyping and next-generation sequencing, offer new possibilities to establish specific pre-

Growing knowledge and important technical evolution in the last two decades enabled us, in the clinical context, to detect and interpret smaller and smaller genomic imbalances. The classical karyotyping has been replaced first by comparative genomic hybridization and soon thereafter with array-based CGH (aCGH). It is becoming widely applied in the prenatal setting, where it is recommended by many professional societies for routine prenatal diagnos-

The comparative genomic hybridization using microarrays (aCGH) is based on competitive hybridization of short segments of whole genome DNA to preprepared probes (short sequences of DNA), spotted on a glass slide in a precise grid (microarray). The DNA of a patient and reference sample DNA are both digested with restriction enzymes to generate short fragments and after that labeled with two different fluorescent dyes. Both patient and reference DNAs are combined and hybridized to the same microarray, thereby competing for the same probes. A specialized scanner measures signal intensities and dedicated software links signals to specific genomic regions (Figure 1). When there is a deletion in the patient, we see it as a predominance of reference DNA in that genomic region. As a result, a relative log ratio of patient's signal compared to reference signal gives a curve with negative values. Despite enabling the detection of progressively smaller genomic imbalances, one needs to be aware of the limitations—the technique will not detect low-level mosaicism, triploidy, balanced

As chromosome analysis is subjective and experience-dependent [13], its insufficiencies have been complemented by fluorescent in situ hybridization (FISH) analysis, where a DNAspecific fluorescent probe is hybridized to the complementary sequence in a cell preparation. In contrast to conventional chromosome analysis, FISH can be used to study cultured or direct cell preparations (metaphase/interphase FISH). FISH allows for the detection of repetitive regions like satellites in the acrocentric chromosome or variable length of pericentromeric heterochromatin not covered by genomic methods [13]. Currently, FISH analysis is a valuable tool for the identification of the origin of the marker chromosome composed of heterochromatin [14] and complex chromosome rearrangements and mechanism of the chromosome rearrangement [15, 16]. Locus-specific fluorescent probes detect subtelomere and interstitial submicroscopic chromosomal rearrangements associated with clinically recognizable phenotypes with diagnostic yield of 3–6% for chromosome abnormalities [17]. However, screening with FISH for tandem duplications seems to be of limited value.

FISH is a more targeted approach, because it requires prior knowledge of chromosome region of interest but has limited utility as a first-tier investigation [18].

Chromosome analysis may also be considered as a quantitative method, which can accurately detect the proportion of the mosaicism. As many as 0.16% of cases with low-level yet clinically significant chromosome mosaicism would be undetected by array CGH method [18]. In addition, in mosaic cases with two abnormal cell lines resulting in a no-net gain or loss (i.e., 45,X/47, XXX) array, CGH would return to normal result [18].

Different types of mosaicism can be found in prenatal diagnostics, like confined placental mosaicism (CPM), true fetal mosaicism, and clonal expansion because of in vitro cultivation [20]. CPM is found in about 1–2% of chorionic villi samples, and certain chromosome trisomies are typically found, like trisomy of chromosome 2, 7, or 16 [19].

Over the last decades, chromosome analysis has been the cornerstone in prenatal genetic diagnosis. In fetuses with multiple CA, there is a chance of more than 18% to detect a chromosomal abnormality [11], while a chance for a chromosomal aberration in cases with an isolated CA is not well determined [20, 21].

There are some pros to why fetal karyotyping remains in the everyday genetic practice. The chromosome analysis assesses the number (aneuploidies) and the structure of chromosomes (chromosome rearrangements) in a single assay (i.e., free trisomy 21 versus unbalanced Robertsonian translocation involving chromosome 21 or balanced reciprocal translocations) [18].

However, because of its low resolution, the need for cell cultivation, which is time-consuming and artifact prone, and the inability to detect complex abnormalities, the chromosome analysis is placed behind other high-throughput genomic investigations [19].

Currently, karyotyping remains the investigation of choice for low-risk pregnancies with normal fetal morphology, like advanced maternal age with increased risk for trisomy 21 [22].
