CGH array principle

Its principle is to co-hybridize the same amount of DNA from a patient and control, each marked by a different fluorochrome, to a blade on which probes are attached. After a hybridization step, the signals generated by the two fluorochromes are digitized and a ratio of their respective intensity (reflecting the ratio of the patient’s DNA quantity to that of the control) is established at each locus. A graphical representation of the anomaly is obtained through software.

With this new technology, it was possible to detect genomic imbalances that are not visible on the karyotype, i.e., smaller than 5-10 Mb (which corresponds to the size of a chromosome band) (Figure 1).

Subsequently, other types of chips, known as SNP chips or "SNP array" (single nucleotide polymorphism), were developed. These are chips that use short and very specific oligonucleotides that can distinguish sequences that differ from only one nucleotide. On average, one SNP is met every 100 to 1,000 nucleotides. Their number is about 5.10⁶ in the human genome. SNP chips offer the advantage of detecting, in addition to genomic imbalances, situations of single-parent disomy (two chromosomes from the same parent). In France, the term DNA chip chromosome analysis (ACPA) was chosen by the French network "AChro-Puce" to refer to both the techniques of CGH (comparative genomic hybridization) array or comparative genomic hybridization chip as well as the techniques of SNP array (http://www.renapa.univ-montp1.fr/).

Figure 1. Example of the deletion of the short arm of a X Chromosome 7
Size: 159 Mb* (159.10⁶ base pairs)
Chromosome band: 5-10 Mb (5-10.10⁶ base pairs) 
Chromosome band: limit of detection of an anomaly on the karyotype
ACPA allows to detect a gain or loss of genetic material comprising one or more genes whose size is less than a chromosome band (i.e. not visible on the karyotype).

ACPA Medical Applications

The development of the ACPA technique (or chromosome analysis on a DNA chip) has therefore led to a paradigm shift in medical cytogenetics. The first applications of ACPA concerned the cytogenetics of solid tumors due to the complexity of abnormalities detected on the karyotype. Constitutional pathology has been the second field of medical application of this technique with the exploration of patients with isolated or syndromic intellectual deficits. Various studies have shown that ACPA can detect approximately 12% of cryptic genomic imbalances (i.e., not visible on karyotype) in these patients⁽³⁾. For this reason, this technique has become the first-line examination for genomic analysis of patients with intellectual deficiency and/or birth defects. However, the clinical impact of an Anomaly identified by ACPA may be difficult to determine, constituting a major impediment to the application of this technique in prenatal. The technical means pre-empted our understanding of the medical implications of the data generated.

CNVs (copy number variants): classification

The genomic imbalances identified by ACPA are called CNVs (copy number variants) and correspond to quantitative variations of the genome compared to a reference genome*.

It is important to note that a CNV is only a loss or gain of chromosome material without being able to predict its pathogenicity. In fact, a CNV can be mild, i.e. without phenotypic consequences for the individual carrying it. Currently, it is estimated that approximately 16% of the genome would correspond to non-deleterious variations⁽⁴⁾. However, it has been shown that some of these so-called benign CNVs have a role in the immunity or occurrence of common pathology such as systemic lupus^(5,6).

In addition to these benign CNVs, there are pathogenic CNVs because they cause neuro-developmental disorders. Many syndromes, such as the 17q21.31 (Koolen-de-Vries syndrome), could be described as a result of the generalization of postnatal ACPA⁽⁷⁾. It should be noted that the majority of pathogenic CNVs are not recurrent and are scattered across the chromosomes. Thus, a penomic and non-targeted approach is needed to detect them.

In addition to these clear situations, there are CNVs that pose genetic counseling problems. On the one hand, these are CNVs for predisposition to the occurrence of neuro-developmental pathologies. For example, 16p11.2 deletion or 1q21.1 duplication associated with autistic spectrum disorders^(8,9). Case-control studies have shown that these CNVs are susceptibility factors to these conditions because they are more frequently detected in patients than in controls. However, at present, this dichotomy between healthy individuals and controls is not considered to reflect reality, as there is in fact a very broad phenotypic spectrum in individuals with this type of CNV. In fact, a minima cognitive impairment was detected retrospectively in carrier parents when initially considered healthy^(10,11). To date, the genetic, epigenetic, and/or environmental factors leading to the expression of disease are not known. Genetic counseling is therefore very difficult in antenatal because it is not possible to predict the phenotype of a fetus carrying this type of CNV.

In addition, the second category of CNV that raises genetic counseling problems corresponds to SUVs (variation of certain significance, or variant of uncertain meaning). These are "private" and often inherited CNVs whose clinical consequences for the developing fetus are not established. This creates great anxiety when the information is passed on to the parents and can lead to an unjustified request for a medical interruption of pregnancy.

Finally, exceptionally, a predisposing CNV at the onset of a late revelation pathology can also be identified⁽¹²⁾. The application of this new technology in prenatal care should therefore lead to a consideration of the ethical problems it raises.

Due to the difficulty of interpreting some CNVs (SUVs) or the possible detection of a CNV that has a medical impact that is not related to the initial indication ("Incidental discovery CNVs"), international recommendations call for a pre-test consultation to be offered to the patient so that accurate and clear information can be provided by trained health personnel (doctor, doctor, midwife, genetic counselor...)^(13,14). The gathering of specific consent for this analysis is essential. Finally, when a CNV is notified on the report, it must be explained to the patient during a post-test consultation.

Benefits and limitations of prenatal ACPA

In recent years, the feasibility and benefits of applying ACPA in prenatal have been widely demonstrated⁽¹⁵⁾. The main advantages of this prenatal technique are:

• the possibility of detecting cryptic pathogenic CNVs;
• faster detection than the non-cryptic CNV karyotype, as a result can be achieved in less than a week by the fact that the technique is carried out on uncultivated cells (from the collection of amniotic fluid, choral villi or fetal blood);
• the possibility of analyzing cells that are not capable of dividing. An ACPA can be performed from fetal tissues (skin, lung, thymus..) after a fetal death in utero for example;
• a fine characterization of the genomic imbalance identified (size of the CNV and content of genes). The molecular characterization of a chromosome reshuffle allows a more accurate phenotype - genotype correlation to be established and thus provides more adequate genetic advice.

While CAPA is a high-performance technology, it is important to note that it has limitations. Since the principle of the technique is to compare the amount of DNA of a patient with a witness, balanced reshuffles (without loss or gain of chromosome material) are not highlighted. Moreover, cell-by-cell analysis is not possible, unlike karyotype or FISH (fluorescence in situ hybridization) and, as a result, low mosaics (< 10-20%) are not detected. Finally, triploidia can only be detected by SNP chips and not by CGH array.

Another limitation to be considered is that the ACPA provides quantitative information without specifying the type of anomaly identified (e.g., translocation or insertion derivative). The identification of a CNV should therefore be supplemented by a FISH study. In addition, a FISH study in parents is required to determine the character of the anomaly de novo or inherited, which in some situations will assist in the classification of the CNV (pathogen or non-pathogen).

Indications of prenatal ACPA 

A prenatal chromosome diagnosis is proposed where there is a significant increase in the risk of unbalanced chromosomal abnormality in the fetus that could lead to sufficiently serious and/or lethal developmental disorders to propose a medical interruption of pregnancy. Various studies have shown that the rate of cryptic CNV pathogens detected by ACPA is about 9% in polymalformed fetuses. In the case of isolated malformation, this rate is lower at around 6%⁽¹⁶⁾. For example, the realization of a penomics ACPA in fetuses with morphological abnormality(s) is a diagnostic strategy that has been adopted in many countries, notably in France (ACLF Recommendations http://www.eaclf.org/docs/ ACPA/GBP_ACPA-v10.pdf). According to Wit et al. metaanalysis, cardiac, central nervous system, and musculoskeletal system malformations are the most important sources of pathogenic chromosome abnormalities⁽¹⁶⁾.

ACPA in fetuses with heart disease

Heart disease is one of the most common birth defects at birth. Their prevalence is estimated to be between 4 and 8 per 1,000 live births. In antenatal, the incidence of chromosome abnormalities in fetuses with heart disease is high, about 25%⁽¹⁷⁾. These are mainly cases of trisomy 21, trisomy 18, trisomy 13, monosomy X and microdeletion 22q11.21. A recent meta-analysis of Jansen et al. including 1131 fetuses with cardiac disease revealed that ACPA detected approximately 7% of abnormalities (after excluding aneuploidy and microdeletion cases 22q11.21)⁽¹⁸⁾. This rate is higher in syndromic forms (9.3%) than in isolated forms (3.4%). In addition, chromosome abnormality is more frequently found in the case of syndromic interventricular communication and left ventricular ejection pathway abnormalities. It should be noted that pathogenic CNVs have also been identified in fetuses with large vessel transposing or heterotaxis. Currently, it is therefore recommended to perform ACPA regardless of the type of heart disease^(18-20).

ACPA in fetuses with a central nervous system defect  

Central nervous system abnormalities are often severe and are a frequent cause of medical termination of pregnancy⁽²¹⁾. They concern 0.14-0.16% of live births and 2-6% of stillborn children. The rate of pathogenic CNVs in fetuses with a central nervous system defect is approximately 7-11%, according to studies^(22,23). This is one of the most common congenital malformations associated with a pathogenic CNV. These genomic imbalances are most common in fetuses with Dandy-Walker malformation, cerebellar hypoplasia, or holoprosencephaly.

ACPA in fetuses with nucal hyperclarity

It was shown from the early 1990s that nucal hyperclarity is associated with a risk of chromosomal abnormalities such as trisomies 21, 18 and 13, triploidia, and gonosomal aneuploidia. It should be noted that trisomy 21 represents approximately 50% of the chromosomal abnormalities observed in the first quarter of nucal hyperclarity^(24,25). More recently, several studies have shown the contribution of ACPA in this indication^(16,23,26). In a recent meta-analysis of Grande et al. including 1,696 fetuses from 17 studies, the pathogenic CNV rate is 4% for isolated nucal hyperclarity and increases to 7% if another sign of ultrasound call is associated⁽²⁷⁾.

ACPA in fetuses with intrauterine growth retardation

It is known that most chromosomal abnormalities are associated with intrauterine growth retardation (IUCN). These include trisomy 18, trisomy 13, 4p16.3 (Wolf-Hirschhorn Syndrome), 5p15.2 (Cat Cree Syndrome) and 15q26 (including IGF1R gene)⁽²⁸⁾. With respect to the contribution of ACPA in this indication, the Shaffer et al. study showed that a pathogenic CNV was found in 2.7% of cases⁽²³⁾. This rate increases to about 10% if congenital malformation is associated with the NCIU. The recent meta-analysis of Borrell et al. revealed similar rates with about 4% pathogenic CNVs in isolated NCIU and about 10% if associated with congenital malformation⁽²⁹⁾. It is therefore entirely lawful to propose an ACPA in the case of CIDR. below the 3th percentile without identified etiology, even if no other fetal morphological abnormality is associated.

What type of ACPA should be used in prenatal?

The main obstacle to the application of ACPA in prenatal is the presence of SUVs and CNVs predisposing to pathologies that are difficult to predict phenotype (especially in the absence of ultrasound call signs, or in the case of minor ultrasound call signs). As a result, a reflection was made on the type of DNA chip to be used in antenatal: the objective was to detect the maximum number of pathogenic CNVs while limiting the detection of CNVs that raise genetic counseling problems. However, it has been shown that the resolution level of the chip used is correlated with the probability of detecting SUVs^(30,31). As a result, some centers have chosen to adopt a different chip and/or with a different resolution than the one used in postnatal. In France, an enriched genomic chip on regions associated with known microdeletional or microduplicational syndromes and genes involved in severe diseases is often used⁽³²⁾. In addition, the detection threshold for a CNV was set at 1.5 Mb by most teams in France. In the future, the development of public databases (DECIPHER http://decipher.sanger.ac.uk/; Database of Genomic Variants http://projects.tcag.ca/variation/) or private will help clarify the clinical impact of some of these CNVs.

Conclusion

The advent of DNA chips is a major advance in the field of cytogenetics. Indeed, this tool offers the possibility of performing a penomic examination at a very high level of resolution. However, the detection of genomic imbalance sometimes raises many questions about its clinical consequences, which is a major limitation on the application of ACPA in prenatal diagnosis. However, ACPA is currently the preferred technique for fetal genome analysis when an invasive gesture for chromosome study is indicated.

*A reference genome is used for the CGH array but not for SNP chips.