Opportunities and Limitations of Modern High Throughput Sequencing in Invasive Prenatal Diagnostics

Abstract

Prenatal diagnostics are used to identify the causes of fetal anomalies detected on ultrasound. If ultrasound findings appear to indicate a genetic disorder, sequencing methods offer the opportunity to safely diagnose numerous genetic disorders prenatally with the help of diagnostic puncture and aspiration. Depending on the type of ultrasound abnormality, massive parallel sequencing (MPS) (the terms “high throughput sequencing” and “next generation sequencing” [NGS] are often used synonymously) can identify up to 50% of the causes of fetal malformations (skeletal abnormalities). Confirmation of a genetic disorder makes it possible to inform and advise pregnant women or parents who are looking for advice about the expected development of their unborn child and provides a science-based assessment of the risk of recurrence. This review article describes the benefits and special features of prenatal diagnostic tests using next generation sequencing and looks ahead at the developments in molecular genetic diagnostic procedures which may be used for the prenatal confirmation of genetic disorders in the future.

 

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Keywords

embryo - embryonic period - fetal period - genetics - inheritance - malformation

Introduction

Fetal anomalies are detected on ultrasound in around 3% of all pregnancies [1]. These abnormalities can range from slight anomalies (e.g., slightly increased nuchal translucency or soft markers such as white spot on the fetal heart) to lethal multisystem disorders. The etiology of these findings varies greatly and includes both exogenous and genetic factors. Congenital malformations are detected in 2–6% of all neonates. Prenatal genetic diagnostic testing is used to investigate fetal ultrasound anomalies [2]. Diagnostic puncture plays an important role in prenatal genetic testing and remains the only established and sufficiently evaluated option to diagnose genetic disorders in pregnancy-specific cells [3]. In addition to chorionic villus sampling and amniocentesis, QF-PCR is often carried out prior to classic chromosomal analysis as this allows the most common aneuploidies to be identified within a few hours [4]. In recent years, many human genetics laboratories have also begun to carry out microarray analysis, which has a 5–8.5% higher detection rate of causative submicroscopic chromosomal imbalances compared to conventional cytogenetics due to the method’s higher diagnostic resolution [4]. However, this service is not covered by standard health care plans in Germany (Individuelle Gesundheitsleistung [IgeL]) and the patients has to pay for this type of testing herself. Massive parallel sequencing has become an established, very effective, diagnostic method to diagnose genetic disorders. Although the use of massive parallel sequencing in pediatric diagnostics and in adult patients is usually unproblematic, nevertheless some aspects of this method need to be considered in the context of prenatal diagnostic testing. This overview describes the role of massive parallel sequencing in genetic diagnostic testing and the specifics and limitations of this method in prenatal diagnostic testing. Recent developments in genetic diagnostic testing are additionally presented.

 

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Basic Principles of Massive Parallel Sequencing

For decades, Sanger sequencing was used to identify the base sequences of a gene and determine disease-causing variants. To do this, prior to sequencing, gene sections are first amplified using polymerase chain reaction (PCR). Fluorescence-labeling of bases allows base sequences of the respective amplified DNA sections to be determined using a sequencer.

Although Sanger sequencing continues to be used for targeted gene analyses, the method has some limitations. Laboratory protocols have to be established, adapted and validated for every DNA fragment needing to be sequenced. Depending on the size of the gene, sometimes numerous individual sequences have to be carried out. For quantitative and qualitative reasons, this only makes sense for laboratories if they can carry out numerous diagnostic tests for a specific gene. But this is generally not the case for rare genetic disorders. The important limitation of this method is that only single gene analyses are possible. If several genes could be implicated in a genetic disorder, detailed diagnostic tests of all relevant genes are not possible. For example, around 100 different genes may be implicated in the genetic eye disease retinitis pigmentosa [5]. The diagnostic benefit is even more problematic in cases where single gene analysis cannot be carried out due to the patient’s unspecific clinical manifestations. This is the main reason why single gene analysis is not particularly relevant for prenatal diagnostic testing as ultrasound findings are rarely specific enough for single gene analysis to offer promising results. Even when skeletal anomalies are visible, ultrasound findings are often not specific enough for a clear suspected diagnosis [6]. In most cases, single gene analysis in the prenatal context is and was limited to proving/excluding known pathogenic familial variants.

 

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Massive Parallel Sequencing (MPS). A Game Changer in Genetic Diagnostic Testing

In contrast to Sanger sequencing, MPS consists of parallel high-throughput sequencing of short DNA fragments (including fragments of a complete human genome, if necessary). With this sequencing method, many overlapping fragments are generated for the target region. Bioinformatics is used to assign sequence information to a specific genomic target/reference region. The sequencing coverage describes the average number of sequence bases per base in the target regions. Deviations from the reference region are identified and filtered using detailed algorithms. Filtering is necessary as every individual will have innumerable deviations (> 3 million in a genome analysis) from the reference sequence but only a few of these variants will be important for the disorder. Bioinformatic processing filters out all the variants which are not pathogenic because of their genomic position, their frequency in healthy controls, and other criteria (for example, variants in genes not associated with the disorder). Results are also compared with information from databases on pathogenic variants to directly identify potentially relevant variants [7]. Classifying the variants is therefore the greatest challenge in massive parallel sequencing, and diagnostic evaluations are currently only carried out by highly qualified experts.

In principle, two different technical approaches are used for diagnostic MPS [8]:

• Gene panel sequencing: Groups of genes which are diagnostically relevant for a condition, e.g., epilepsies, are grouped for testing. Median number per group is 150–200 genes [9].

• Exome sequencing: All DNA sequences of the human genome which carry genetic information für protein-coding genes are enriched; this corresponds to about 1% of the genome (around 20000 genes) [10].

• Genome sequencing: The whole genome is sequenced, including non-protein-coding sections which could have clinically relevant pathogenic variants; testing focuses on the detection of structural chromosomal disorders.

• NIPT: For this MPS-based non-invasive prenatal test (NIPT), cell-free fragments obtained from maternal plasma are sequenced untargeted. Based on the obtained sequence of sequenced fragments, the fragment can be assigned to a specific chromosome using bioinformatics. If a trisomy is present, significantly more fragments of the relevant chromosome (e.g., chromosome 21 in trisomy 21) will be detected [11].

[Table 1] presents an overview of the different genetic testing methods.

Table 1 Comparison of prenatal genetic diagnostic methods.

Method	Advantage	Disadvantage
* These methods are currently not routinely used in prenatal molecular genetic diagnostics.
NIPT	Non-invasive	Scope of investigation is limited (usually only detects trisomies 13, 18 and 21 and numerical aberrations of chromosomes X and Y) Abnormal findings have to be confirmed using invasive diagnostic testing.
Chromosome analysis	Established, cost-effective method to identify numerical and structural chromosomal disorders	No detection of sequence variants or smaller chromosomal aberrations
Single gene sequencing	Targeted identification of familial variants (segregation studies)	Scope of investigation is very limited
Panel diagnostics	Clearly defined scope of investigation based on clinical phenotype; therefore, lower risk of unclear variants and incidental findings	No detection of causative variants outside the scope of the investigation The set of relevant genes is only unambiguously defined for a limited number of fetal phenotypes.
(Trio) exome diagnostics	Covers all potentially relevant genes	Higher risk of incidental findings Unclear variants identified more often
Microarrays	Established method to detect pathogenic gains/losses (e.g., microdeletion and duplication syndromes)	Method is limited to detecting gains and losses.
Optical genome mapping (OGM)*	Detects structural chromosomal disorders (e.g., inversions, translocations)	Requires very good DNA quality Very expensive No detection of sequence variants
Genome sequencing*	Detects structural chromosomal disorders and sequence variants	High sequencing costs Handling of variants in non-coding genome regions Detection of variants in non-coding genome sections

 

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Panel Diagnostics

In panel diagnostics, only a specific set of defined genes from the genome of the person being investigated are enriched and only these are sequenced. The primary advantages of this method are the low sequencing performance required and the focused approach with this method, as only genes directly associated with the clinical problem are analyzed. There is no expectation of any additional or incidental findings.

 

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Exome Testing

In exome diagnostics, the sections coding for proteins in all ca. 20000 genes in the genome are sequenced in parallel using MPS [12]. Bioinformatic algorithms are then used to filter out the genes from the full dataset which are connected to the respective clinical problem. Human Phenotype Ontology terms (HPO terms) have been found to be useful in postnatal diagnostic testing. HPO terms are used to describe a clinical phenotype and associate the phenotype with genes known to be clinically connected to the respective phenotype. This individually tailored gene selection allows diagnostic testing to be adapted to the patient’s specific clinical picture according to the current state of knowledge. In many cases, the quality of the sequencing data now also provides reliable evidence of copy number variants (CNVs), i.e., of small losses and gains of genetic material comparable to microarray analysis. The high flexibility of exome diagnostics means that in most laboratories, panel diagnostics have been replaced by exome diagnostics. The challenge for exome testing, however, is the huge number of genetic variants which need examining (ca. 15000–40000) to assess whether they are connected to the patient’s disorder. Internationally valid criteria have been defined for this to allow consistent classification and clinical evaluation of genetic variants [13] [14] [15]. But even though classifying variants is carried out with the help of bioinformatic methods, the expertise of specialist research assistants and medical staff who jointly assess the genetic and phenotypic information in the clinical context remains indispensable. Variants of uncertain significance (VUS) are particularly challenging. To begin with, it is not possible to definitively determine whether these variants are the cause of the disorder or not [14]. For patients seeking advice and for the investigating physicians, this leads to uncertainty as there are no unambiguous answers to their clinical questions. Prior to carrying out exome testing, it is important to inform patients about the possibility that findings may be uncertain and explain what this means. As the sequence information of almost all known genes is available in exome sequencing, it is also possible to find pathogenic variants in genes not directly connected to the clinical picture of the patient being investigated [16]. Because of this known issue, patients must be fully informed in accordance with the German Genetic Diagnostics Act before exome testing is carried out [17]. Prenatal massive parallel sequencing is included in the current Doctors’ Fee Schedule in Germany (EBM).

Trio exome diagnostics are a special form of exome diagnostics. This type of testing does not just analyze the exome data of the affected patient/fetus but also the exome data of the affected parents and compares the findings. Bioinformatic algorithms are used to rapidly identify de novo mutations and parental inheritance patterns in autosomal-recessive diseases. Obtaining parental sequence information improves the classification of detected variants, as the number of variants of uncertain significance is reduced. Studies have shown that trio exome diagnostics has been able to solve significantly more cases than single exome analysis [18].

 

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Uses and Challenges of Prenatal Exome Diagnostics

Numerous international studies have shown the diagnostic benefit of using (trio) exome diagnostics to confirm genetic disorders [19] [20]. Reverse phenotyping is increasingly becoming the preferred method of analysis. A classic diagnostic workup consists of starting with a precise clinical characterization of the patient. The clinical characteristics are then used as the basis for a suspected diagnosis and other potential differential diagnoses, which are then confirmed or disproved based on the findings of the genetic investigation. But the large number of rare genetic diseases, many of which show wide clinical variability, means that this diagnostic approach is often unsuccessful. Reverse phenotyping uses the opposite approach. The genetic data obtained from exome sequencing is used to identify variants which could potentially lead to a specific disorder. If the genetic data give rise to the suspicion of one or more diagnoses, the clinical symptoms are specifically reviewed and the associated disorder can be diagnosed. In prenatal diagnostics, reverse phenotyping may be used to clarify prenatal anomalies, although it is important to be aware that there is only limited information available on the prenatal phenotype of many rare genetic disorders. Targeted ultrasound examinations can often determine whether certain phenotypical features can be detected which would confirm the presence of a specific genetic disorder.

MPS is now a recognized and accepted part of prenatal diagnostics. In a statement issued by the German Society for Human Genetics in 2023, the Society explicitly highlighted the use of microarray and panel or exome diagnostics after excluding the most common trisomies with NIPT or QF-PCR, even without carrying out karyotyping [2]. Across the world, prenatal MPS is now part of the standard diagnostic workup in many countries and is included in guidelines and recommendations [21] [22] [23] [24].

Because of the speed of diagnosis and greater precision, MPS is also the method of choice for prenatal diagnostic testing. But it is important to be aware of specific aspects of the prenatal setting:

1. Ultrasound examinations often only provide limited phenotypic information.

2. Many clinical features are only detectable in the later weeks of pregnancy.

3. Many identifiable structural anomalies in early pregnancy are unspecific and permit only limited conclusions to be drawn about defined clinical pictures.

4. At present, no or very little information is available about the prenatal phenotypes of many rare diseases over the course of pregnancy.

5. Laboratories carrying out the diagnostic tests require defined procedures to deal with variants of uncertain significance which could be connected to the fetal clinical picture. The physician informing the parent(s)/the specialist in prenatal medicine must be informed of this.

It is also important to be aware that uncertain findings can be a heavy psychological burden and an additional stressor during pregnancy. Prior to carrying out diagnostic testing, it is important to be aware that medical staff who provide information to affected parents bear a special responsibility, as the German Genetic Diagnostics Act mandates that parents need to be informed about the extent but also the limitations of planned tests. The laboratory commissioned to do the tests must also take the special situation of prenatal diagnostic testing into account. This especially applies to processing times and dealing with detected variants. Experience has shown that, in principle, it is better to only report pathogenic and probable pathogenic variants. Reporting a variant of uncertain significance may be justified in individual cases, for example, when the fetal phenotype indicates that the detected variant may be causative or when additional targeted tests to investigate causation need to be arranged. In Germany, some human genetics laboratories have joined together to form a National Alliance of Rare Genetic Diseases (Nationale Allianz Seltener Genetischer Erkrankungen, www.nasge.de) to provide an interdisciplinary online discussion of uncertain genetic variants within 24 hours.

 

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Prenatal Trio Exome Testing in Pregnancies without Ultrasound Anomalies

More and more couples are requesting prenatal genetic testing even if the pregnancy is normal. Given the fact that numerous genetic disorders, especially disorders with cognitive impairments, cannot be detected on ultrasound, the wish for extra security is understandable. The inclusion of non-invasive prenatal testing (NIPT) in standard prenatal care means that a screening method to exclude the most common aneuploidies is already being used in numerous pregnancies without abnormal ultrasound findings. Trio exome testing of pregnancies without ultrasound anomalies is therefore already being offered by some facilities, but this service is not covered by standard health care plans in Germany. For this type of testing, prior counseling of the parents about the extent and limitations of testing is indispensable as is a strict policy about the reporting of findings, with parents only being told about established pathogenic genetic variants in genes associated with severe infantile disorders. Reporting variants of uncertain significance and of late-manifesting disorders is not justifiable [25].

 

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Diagnostic Value of (Trio) Exome Testing

Numerous international studies have confirmed the additional value of exome testing for the detection of the genetic causes of fetal malformations [26] [27] [28]. A number of different meta-analyses have now been published which attest to the benefit of prenatal MPS to clarify different issues. In one meta-analysis of prenatal brain anomalies in 1583 affected fetuses, the detection rate using exome sequencing was 32% higher compared to karyotyping and microarray analysis [29]. Comparable cross-study results have also been achieved for intrauterine growth retardation (IUGR), increased nuchal transparency, and congenital anomalies of the kidney and urinary tract (CAKUT) [30] [31]

A recently published study of 500 pregnancies, most of them from Germany, with different ultrasound anomalies was able to show that it was possible to determine the cause of the anomalies in more than 30% of cases with exome diagnostics [32]. Surprisingly high detection rates have even been reported for cases with higher isolated nuchal transparency measurements (NT > 3 mm), a finding that was confirmed in a recent international study [33]. Although the majority of causative genetic variants were de novo mutations (47.1%), genetic inheritance was confirmed in 41.8% of cases. In addition to an asymptomatic mother passing X-chromosome variants to a male fetus, autosomal-recessive diseases are primarily detectable where both parents are unknowing carriers of a pathogenic variant. Disorders with reduced penetrance have also been detected in some cases. This term is used to describe diseases such as holoprosencephaly where not every carrier will go on to develop the disease, meaning that a pathogenic variant has been passed on from an unaffected parent to the affected fetus. In summary, prenatal exome testing is a rapid, effective, and useful method to identify genetic causes of fetal malformations [33]. The integration of prenatal exome diagnostics into diagnostic procedures is shown in [Fig. 1].

Fig. 1 Diagnostic procedure for massive parallel sequencing carried out in pregnancies with ultrasound anomalies.

 

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New Methods and Approaches

Technological progress means that new diagnostic procedures in human genetics are constantly becoming available which offer patients better and faster results. But with every new technology there is the challenge how to translate research-based applications into standard care.

 

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Ethical Aspects of MPS

In addition to the technical aspects and the health care policies and health economic issues which need to be considered when new diagnostic options are introduced, the extensive collection of genetic data raises a number of unanswered questions which society will need to address. One such example is the collection of genetic data outside of diagnostic testing. An oft-cited example of this is the inadvertent detection of a predisposition to develop breast or ovarian cancer based on the identification of a pathogenic BRCA-1 or BRCA-2 variant. The detection could potentially be lifesaving for the affected person as suitable preventive measures (close monitoring and screening programs) are available [34]. But collecting such data also has its risks which need to be considered. How can “genetic discrimination” be prevented, for example in the context of health insurance? The German Genetic Diagnostics Act accords special protected status to knowledge of genetic constellations and emphasizes that patients have the right to decide for themselves whether they want to know their own genetic findings or not [35]. But the problem is that the German Genetic Diagnostics Act was passed at a time (2009) when MPS and the collection of large amounts of genetic data was not yet fully possible. This has given rise to conflicting situations, specifically in the case of individuals incapable of giving consent (for example, the unborn child), for example when a predisposition to breast cancer is detected (as an additional or incidental finding in the context of prenatal single exome analysis) but it is not permitted to report this finding, meaning that this knowledge will be lost.

To address the different ethical, legal, and social challenges of MPS and provide recommendations for action in unclear situations, reference should be made to the guidelines of the German Gene Diagnostics Commission [36] [37].

 

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New Genetic Diagnostic Methods

Recent methods which are beginning to be used in human genetic diagnostics include whole genome sequencing and genome mapping. As described above, around 1–2% of the whole genome is sequenced with exome sequencing. In whole genome sequencing, the entire genome is sequenced. With whole genome sequencing, pathogenic variants can be confirmed in areas not covered by exome diagnostics. Moreover, whole genome sequencing can be used to identify structural chromosomal disorders if it is combined with bioinformatic methods [38]. Long-read sequencing will also play an important role in genome analysis in future. This is an NGS-based sequencing method which provides sequence information about genes which cannot usually be displayed with standard short-read sequencing because of their base sequences. This method can additionally be used to diagnose repeat expansion disorders (e.g., fragile X-syndrome) and imprinting disorders caused by aberrant DNA methylation patterns [39].

The problem with whole genome sequencing is the high costs involved and the difficulty of evaluating variants, as large numbers of variants (> 3 million) are obtained for which a reliable clinical assessment is currently not possible because the reference data is inadequate. But a cost-benefit analysis will have to be carried out before these methods can be included as part of standard clinical care. The German government has therefore launched the “Genome Sequencing Pilot Project” which aims to evaluate the clinical benefits of genome sequencing outside primary care genetics and create the necessary variant databases. Another new method increasingly being used in human genetic diagnostics is optical genome mapping (OGM), an imaging technique for structural variant identification. OGM represents (with certain limitations) an advance on the classic karyotyping used to identify structural chromosomal disorders [40]. The resolution of OGM is 10000 times higher than that of the standard light microscopy used for chromosomal analysis and can identify small structural variants which could not previously be detected. Other developments in genetic diagnostics, such as the use of cell-free fetal DNA (cffDNA) in NIPT, indicate that whole exome screening for monogenic disorders in maternal serum will be possible in future [41].

 

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Summary/Outlook

The detection of prenatal ultrasound anomalies puts a severe psychological strain on affected parents. Waiting for the results of testing is very stressful and adds to worries about their child and fears about the diagnostic puncture. Detection of a genetic disorder in the fetus explains the cause of the identified anomalies but also has far-reaching consequences for the affected couple who may have to make a decision about whether to continue the pregnancy, depending on the genetic findings. In many cases, future pregnancies may also be at risk. However, detection of a de novo mutation is associated with a very low probability of recurrence, and this is a key factor if parents wish to have other children in future.

The detection rate of prenatal exome diagnostics in pregnancies with abnormal genetic findings is high. Identifying the genetic cause of the anomalous ultrasound findings allows affected parents to make an informed independent decision about whether to continue the pregnancy and to obtain a risk assessment for future pregnancies.

 

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Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Received: 26 September 2024

Accepted after revision: 25 March 2025

Article published online:
11 July 2025