2014 January-March; 8(1/2): 1–10. ISSN: 1971-3282 ISSN: 1971-3290

Introducing the next generation sequencing in genomic amnio and villuos sampling. The so called “Next Generation Prenatal Diagnosis” (NGPD)

Claudio Giorlandino,1 Alvaro Mesoraca,2 Domenico Bizzoco,2 Claudio Dello Russo,2 Antonella Cima,2 Gianluca Di Giacomo,2 Pietro Cignini,1 Francesco Padula,1 Nella Dugo,1 Laura D’Emidio,1 Cristiana Brizzi,1 Raffaella Raffio,1 Vincenzo Milite,1 Lucia Mangiafico,1 Claudio Coco,1 Ornella Carcioppolo,1 Roberto Vigna,1 Marialuisa Mastrandrea,1 and Luisa Mobili1

1Artemisia Fetal Maternal Medical Centre, Department of Prenatal Diagnosis, Rome, Italy
2Artemisia Fetal Maternal Medical Centre, Department of Genetics, Rome, Italy


In the last 30 years, invasive prenatal diagnosis has predominantly involved research into chromosomal anomalies, in particular Down’s Syndrome (1).

In the last 10 years, parents have been requesting ever more information during pregnancy (2,3) and there has been an increase in the number of cases with ultrasound markers concerning possible fetal complications of unknown origin. This has led to the introduction of prenatal diagnosis and increasingly detailed techniques such as CGH Array (46).

These techniques have become standard diagnostic practice in cases where the ultrasound scan provides a conflicting result. However, in reality, such procedures are thought to cover only 10% of the fetal anomalies linked to genetic malformations discovered at birth (7).

Prenatal diagnosis is becoming more and more detailed due to the continual legal action taken by parents regarding diagnostic ultrasound which fails to identify fetal anomalies and regarding unwanted births in general (810).

In fact, the continuous evolution of human genetics has led to the development of extremely detailed methodologies, which are able to evaluate not only the errors in chromosomes, both “big errors” (karyotype) and “small errors” (microdeletions, microduplications), but also gene mutations.

To date, approximately 19,000 coding genes contained in the human exome have been identified. The recent introduction of NGS (Next Generation Sequencing) has made it possible, in theory, to explore the entire exome and reveal every form of mutation (1115).

Therefore, it is possible, today, to open up a completely new diagnostic scenario which would have been considered impossible only a few years ago. However, if this development is not controlled, it could lead to a so-called genetic “deviation”, i.e. a genetics that could have unforeseen repercussions on the life and dignity of the individual.

In fact, the risks concerning possible social, emotional and financial consequences in the family and individual is very high. The potential negative impact of prenatal genetic testing must respect the “right not to know”. The exaggeration in ever more detailed testing concerning the genetic structure of the embryo creates tension within a family. In the future, this could create genetic discrimination regarding employment or health insurance costs (16,17).

Despite the fact there is theoretically no technical limit to these methodologies, it is important to establish ethical and moral guidelines, at least regarding how these new methodologies are used in prenatal diagnosis.

Technical limits of prenatal diagnostic methodologies

Prenatal diagnosis, unlike screening, is not simply limited to selecting populations that risk living birth to Down’s Syndrome children. In fact, depending on the method used, it can explore all the chromosomal and genetic pathologies that can be diagnosed following birth (1825).

In fact, the following methods can be used in prenatal diagnosis on a routine basis or in high risk populations:

  • - traditional cytogenetics, introduced in the 1950s, makes it possible to identify chromosome anomalies, which can be numerical (such as trisomy, monosomy), or structural (translocations, deletions and inversions) (26).
  • - QF-PCR (Quantitative Fluorescent Polymerase Chain Reaction). This technique which was initially introduced in the USA in 1993, produces a precise and quick diagnosis concerning the most common fetal aneuploidies responsible for the most frequent neonatal pathologies (Down’s Syndrome, Patau, Edwards, Turner, Klinefelter) (27).
  • - Gene sequencing; the first generation of genomic sequencing was developed by Sanger in 1975 (chain-termination method) and by Maxam and Gilbert in 1977 (chemical sequencing methods). Sanger’s method was found to be technically less complicated and has evolved considerably over the years. The time and costs needed to sequence the DNA represent a limit of this technique (2830).
  • - Array-Comparative Genomic Hybridization (CGH) was introduced in 1992 and is based on the comparative genomic hybridization of the patient and a reference genome which is considered normal. In this way, it is possible to identify microdeletions and microduplications (46, 31).
  • - NGS (Next-generation sequencing), which was introduced in 2005, involves the sequencing of DNA molecules amplified clonally or of single molecules of DNA which are spatially separated in flow cells. This strategy represents a radical change compared to Sanger’s sequencing method, which is based on the electrophoretic separation of fragments of varying lengths obtained through single sequencing events and which, therefore, has the advantage of reducing time and costs, but above all with this technique it is possible to obtain a considerable quantity of information with one single sequencing cycle (1115).

Ethical limits concerning prenatal diagnostic methodologies

One of the consequences regarding the wide range of genetic tests available today is that it is necessary to establish a series of moral, ethical and ideological principles in order to define limits concerning the utilization of these techniques.

The principles that should be taken into consideration are as follows:

  • - Freedom for the couple to procreate responsibly and to know, in accordance with the rules and regulations established in the country of origin, the state of health of their child.
  • - The right to life of the foetus in cases where the presence of an altered genetic structure is not serious enough to classify them as wrongful life.

The limits governing the application of these techniques must, therefore, vary depending on the populations examined.

High risk populations (family, maternal age, the presence of genetic markers) can be advised to test one or more specific problems or advised to use all the methodologies currently available, above all if the objective of these interventions is to guarantee the two above-mentioned principles. In particular, regarding the quality of life of the new-born child, some investigations, such as the search for the mutations responsible for congenital deafness or for cystic fibrosis, can make it possible to set up interventions that can improve the outcome of the new-born child (3234).

In low risk populations, there is an increasing number of couples that, for various reasons, request precise details regarding the health of the foetus. These range from serious situations such as anxiety or social and financial difficulty to less ethical and hedonistic considerations. Whatever the underlying reason, these people want to know exactly the state of health of the foetus.

Even though this will never be possible, it is however evident that the use of various technologies, such as the ones listed above, in low-risk prenatal diagnosis can identify much more than the 10% of genetic anomalies that are currently revealed with traditional methods.

Is it now possible, therefore, to offer this population a complete diagnostic test?

In Italy, in 1978 a law came into force which establishes that mothers have the right to obtain all the information that medical science is able to provide regarding the health of their child, so that pregnancy can progress responsibly (35).

The High Court of Cassation, recently stated that, in an important and significant sentence, medics are to be considered entirely responsible should they fail to inform the mother that there are tests which can provide certain diagnoses regarding anomalies that could arise (36).

Medics must, therefore, provide information regarding the availability of sophisticated diagnostic techniques, although they are not obliged to propose or impose their utilization.

What will these ethical limits be?

We are of the opinion that prenatal diagnostic techniques should remain within certain limits and in particular, there should be:

  • - no investigation into genetic errors which do not provide a clear clinical picture
  • - no investigation into SNPs (single Nucleotide Polymorphisms) which simply indicate a predisposition towards the onset of degenerative diseases or tumors
  • - no investigation into pathologies that are, however, compatible with a normal or acceptable quality of life, such as diabetes, hypertension and metabolic diseases
  • - no investigation into diseases that start in later life, such as Alzheimer.

The clinical use of NGS

Taking into consideration our knowledge regarding genetic diseases, their frequency and the clinical correlation between the alteration of the DNA and resulting pathologies which follow the above-mentioned technical and ethical criteria, a system could be proposed whereby, instead of investigating the 19,000 genes currently known on the exome, investigations could be limited to only 300 of these genes, whose mutations codify for approximately one hundred well-known and well-defined pathologies (Tab. 1).

Table 1Table 1
Syndromic disorder caused by mutation of genes.

Together with these, it is possible to utilize traditional genomic technologies, such as CGH array, in association with NGS (Tab. 2).

Table 2Table 2
Syndromic disorder caused by microdelection/microduplication of genetic locus.

Traditional cytogenetic analyses can also be added to these genetic techniques. In fact, these methods can be used to diagnose approximately 350 pathologies, which, being the most frequent, represent more than 80% of the 6,760 pathologies currently known today (37).

On the basis of what we know today, this type of system would be able to cover almost all the pathologies that occur in less than 1 case for every 50,000. Therefore, it becomes very unlikely that the gynaecologist or sonographer can make a mistake in the diagnosis or discover, at birth, the presence of an unexpected pathology.

In fact, the introduction of such a technique in the future could guarantee that couples receive precise information and also that medics could be “protected” from “incidents” where professional responsibility would be involved.

Such important prenatal investigations, however, cannot disregard an accurate and complete genetic consultation, which not only provides parents information regarding diagnostic certainties but also the uncertainties and doubts which can arise from such ample molecular investigations (despite the fact the selection of genes and mutations to be analysed could be limited).

Final considerations

There is considerable innovation and relevant confusion regarding the world of prenatal genetic testing at the moment.

While on the one hand, the recent introduction of non-invasive tests through the research of foetal DNA on maternal blood is reducing the field of investigation to the screening of only a few aneuploids which offer no guarantees, on the other, there is a low but progressive growth of studies carried out directly on the foetal DNA through invasive techniques.

Therefore, we are heading in two seemingly opposing directions towards unreliable tests which provide limited information on the one side and towards precise tests for an excessive quantity of information on the other.

Which direction should we take? Which category of patients should be directed one way and which should be directed in the other?

While for high-risk populations such as those where NIPT or an ultrasound scan reveals a possible anomaly, there seems to be general consensus towards the use of invasive genomic testing, considerable doubt remains regarding low-risk populations.

In this larger latter category, of particular importance are the expectations of the couple, the correct information provided by medics and, above all, the legal medical implications.

In Italy, the Civil Supreme Court has twice convicted medics for having proposed screening tests instead of diagnostic testing (36).

This has generated great interest in obtaining compensation for any diagnosis of a genetic disease considered to be responsible for a “wrongful life” which “potentially” could have been discovered using the scientific methods currently available.

Therefore, the Legislator, in Italy, has practically told the personal gynaecologist not to accept responsibility regarding recommendations to their colleague concerning screening tests for Down’s Syndrome. A consequence of this is that they are effectively “obliging” them to propose genetic diagnostic tests. In other words, to guarantee themselves from a legal point of view, they inform the expectant mother of the existing differences in various strategies and they request very precise consensus from the parent.

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