The Importance of Prenatal Whole-Exome Sequencing Testing in the Romanian Population

Abstract

One major cause of prenatal mortality and morbidity is congenital abnormalities. Knowing the prevalence and etiology of congenital malformations is essential for analyzing trends and improving neonatal care. Objective: the team aimed to evaluate the utility of whole-exome sequencing (WES) in Romanian prenatal care, highlighting its diagnostic efficacy in comparison to molecular karyotyping, particularly in cases with negative genetic results prior to WES, unfavorable pregnancy outcomes, and consanguinity. Methods: Initially, we identified pregnancies with abnormal ultrasounds unrelated to known syndromes. Subsequently, we performed SNP (single nucleotide polymorphism)-array testing, yielding negative results. We then applied prenatal WES, utilizing Massive Parallel Sequencing on the NovaSeq 6000 platform (average coverage > 100× read length: 2 × 100 bp) with library preparation using the Twist Human Core Exome kit RefSeq & Mitochondrial panel (Twist Bioscience). The bioinformatic analysis involved direct comparison to the human reference sequence (hg38). Results: We achieved a 50% diagnostic rate. After receiving results, two couples chose pregnancy termination, five had uneventful births, and one pregnancy ended in stillbirth. Additionally, we identified three incidental findings that enhanced patient and at-risk member management. This article details ten prenatal cases tested with WES, highlighting its superior diagnostic performance compared to the SNP array. WES detected the genetic diagnostic in 50% of cases that the SNP array did not. We emphasize the advantages of WES in prenatal diagnostics while acknowledging the need for further investigations to comprehensively evaluate its diagnostic utility in the Romanian population.

1. Introduction

The prevalence of congenital anomalies in the general population is approximately 3–5% [1]. Since the first report of its use in obstetrics, ultrasound has become an important tool for the detection of fetal structural defects, identifying them in 2–3% of pregnancies [2,3].

More than 75–80% of fetal anomalies appear in the first 3 months of gestation. Therefore, a good visualization of the fetus in this stadium allows early detection of structural anomalies [4,5,6]. The detection rates of the major structural anomalies for ultrasound examination in the first and second trimesters are from 12% to 44% and from 20% to 87%, respectively [7,8,9,10,11].

In developed countries, the number of referrals for first-trimester ultrasound (11 weeks to 13 + 6 weeks) has increased, while second-trimester ultrasound is considered the gold standard for detecting structural abnormalities. These patients are referred to chorionic villus biopsy or amniocentesis with karyotype and/or fetal molecular karyotype testing. The result of the classic karyotype identifies a diagnosis in 7–10% of pregnancies and the comparative genomic analysis adds approximately 6% to the etiology of fetal anomalies [4]. However, most cases remain without a genetic diagnosis.

Whole-exome sequencing (WES) is currently clinically available and focuses only on exons or protein-coding regions of the genome. Exons represent 1.5–2% of the total genomic DNA, comprising approximately 22,000 genes. Prenatal WES has the ability to increase diagnostic rates in cases where fetal anomalies are present and enhance our understanding of pathogenic variants. WES also has the potential to expand known disease phenotypes to the prenatal period, which, in turn, can lead to finding new pathognomonic signs that can be observed via ultrasound. The known challenges of prenatal WES include the following:

(1)

Sifting through a giant amount of data in a short timespan;

(2)

Finding pathogenic variants in genetic syndromes or singular genetic pathologies with a reduced disease penetrance and variable expression;

(3)

Providing pre-test and post-test proper counseling, particularly in regard to the stress associated with discovering variants of unknown significance (VUS).

The Aim of the Study

The objective of the present study is to assess whole-exome sequencing’s (WES) usefulness in Romanian prenatal care, emphasizing its diagnostic effectiveness when compared to molecular karyotyping, particularly for patients with adverse pregnancy outcomes, and negative genetic results in gold-standard testing such as molecular karyotyping or consanguinity. The findings could also help the Romanian national authorities to consider offering WES testing for selected cases, just like in other countries that currently offer this for their patients, such as England’s National Health Service (NHS). Currently, this test is not reimbursed in Romania, making it inaccessible in many cases.

2. Materials and Methods

After selecting pregnancies with abnormal ultrasound findings, that were not related to a well-known genetic syndrome, the testing algorithm was: first SNP karyotyping and if the result came back negative, the team performed WES, using Massive Parallel Sequencing of the whole coding region of the human genome on Next Generation Sequencing (NGS) Platform, NovaSeq 6000 (average coverage > 100×, read length: 2 × 100 bp). The Twist Human Core Exome kit RefSeq & Mitochondrial panel (Twist Bioscience, San Francisco, CA, USA) was used for the library preparation. Bioinformatic analysis was performed by direct comparison of the genome of the test sample with the human reference sequence (hg38).

Analysis was performed by direct comparison of the genome of the test sample with the human reference sequence (hg38).

Bioinformatic Workflow Regarding Raw Data Analysis (Pipeline 1)

• Alignment: BWA-MEM;
• Mark duplicates: Picard;
• Variant calling: GATK (VCF1), DeepV (VCF2);
• Merge VCF1;
• VCF2 VCF annotations.

In a recent publication by AlDubayan et al., it has been shown that the combination of the two algorithms, GATK and Deep Variant, yields the most accurate results compared to each algorithm separately, with sensitivity approaching 99.9% [12].

The second part of the Bioinformatic Pipeline consists of the following steps.

• Complete list of variants of the patient tested (in comparison to the reference sequence).
• List of variants after the 1st filtering process (MAF > 1%, common variants in dbSNP database, benign/likely benign variants based on ClinVar annotations).
• The 3rd step is the list of variants resulting after the 2nd filtering process based on the annotation of variants as pathogenic/likely pathogenic and VUS—variant of unknown significance.

Reported variants:

• Variants classified as pathogenic and likely pathogenic with documented association with the clinical phenotype of the proband.
• Variants of uncertain significance (VUS) with indications to be associated with the clinical phenotype of the proband and they are predicted as pathogenic by the majority of the in silico tools applied.

Variants not reported:

• Variants classified as benign and likely benign based on the literature search and in silico analysis.
• The process of the variants classification follows the American College of Medical Genetics and Genomics (ACMG) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [13].

Classical karyotype was not performed previous to the SNP array.

Limitations of prenatal WES: WES does not detect small copy number variations, it does not detect aneuploidy, polyploidy, translocations, trinucleotide repeats, or low levels of mosaicism. There are also regions in the genome with low coverage by WES, in particular CpG-rich regions that will not be properly sequenced. The execution time for prenatal WES represents an important limitation [4,5,6].

3. Results

We hereby describe 10 cases of prenatal WES testing for pregnant mothers from the Romanian population between 2022 and 2023 from a private laboratory in Bucharest, Romania.

1. As observed in

Table 1. The cases of prenatal WES testing.

Case No.	Phenotype	Gestational Age	Reason for WES Testing Referral	Fetal WES	Pregnancy Outcome	Incidental/Secondary Findings
1	Fetal sinus bradycardia, supraventricular extrasystoles	22 weeks	Negative SNP array Known genes linked to fetal phenotype	Negative	Stillbirth	PALB2:c.93dupA(p.Leu32thrfsTer11)—inherited from the mother
2	Aberrant right subclavicular artery, rocker bottom feet, fetal hydrops, fetal ascites	20 weeks	Negative SNP array	LMX1B:c.718G > A (p.Val240Ile) Focal segmental glomerulosclerosis 10 Nail–patella syndrome	Born, affirmatively without symptoms at the age of 1 y.o.	-
3	Right aortic arch, severe hydronephrosis, caudal regression syndrome, mesoaxial hand polydactyly	18 weeks	Negative SNP array Clinical suspicion of VACTERL syndrome	HOXD13:c.820C > T (p.Arg274Ter) Brachydactyly-syndactyly syndrome Brachydactyly, type D Brachydactyly, type E Syndactyly, type V Synpolydactyly 1	Couple decided on termination of pregnancy	-
4	Mega Cisterna Magna, ventriculomegaly	22 weeks	Negative SNP array	ADNP:c.1612G > A (p.Glu538Lys) Helsmoortel–Van der Aa syndrome De novo	Ongoing pregnancy	-
5	Clubfeet, microretrognathia, arachnodactyly, agenesis of the corpus callosum, intrauterine growth restriction	24 weeks	Negative SNP array	TGFBR1:c.734A > C (p.Glu245Ala) Loeys Dietz syndrome	Born, affirmatively without symptoms at the age of 1 y.o.	HBB: c.-151C > T Beta-thalassemia— inherited from the mother
6	Corpus callosum agenesis Oligohydramnios	23 weeks	Consanguinity Negative SNP array	Negative	Born, affirmatively without symptoms at the age of 2 y.o.	BRCA2: c.793 + 1G > A Susceptibility to breast–ovarian cancer, pancreatic cancer, prostate cancer—inherited from mother
7	Intracardiac echogenic focus, pyelectasis. Long QT syndrome of the father, family history of sudden cardiac death, arrhythmias due to affected father (without genetic diagnosis)	19 weeks	Negative SNP array	KCNQ1:c.605-28A > G Long QT syndrome 1 Susceptibility to Short QT syndrome 2 Atrial fibrillation, familial, 3 Inherited from the father	Born, affirmatively without symptoms at the age of 1 y.o.	-
8	Right aortic arch	17 weeks	Negative SNP array	Negative	Born, affirmatively without symptoms at the age of 1 y.o.	-
9	Sexual ambiguity on ultrasound morphology. Intrauterine growth restriction, hypospadias, polyhydramnios.	20 weeks	SNP array: arr [GRCh38]12p13.33p11.22(148769_30138756)x2 hmz, 12q21.31q24.22(84757938_117685540)x2 hmz	Negative	Couple decided on termination of pregnancy	-
10	Borderline bilateral ventriculomegaly, suspicion of hydrocephaly with Sylvian stenosis	19 weeks	Negative SNP array	Negative LAMB1:c.3499C > T (p.Arg1167Ter) Lissencephaly 5 (AR) PTPN23:c.2248C > A (p.Pro750Thr) Neurodevelopmental disorder and structural brain anomalies with or without seizures and spasticity (AR)	Ongoing pregnancy	-

, in the first case, we present a 26-year-old pregnant woman for whom the second-trimester ultrasound revealed fetal sinus bradycardia and supraventricular extrasystoles. There is a considerable number of genes associated with channelopathies, which increases the chance of diagnosis using whole-exome sequencing. The purpose of the test was to detect the presence of point mutations (nucleotide substitutions and deletion/insertion of some base pairs) in 62 genes associated with cardiac arrhythmias. The result was negative for pathogenic or likely pathogenic variants in channelopathy genes but with incidental findings of a pathogenic variant in the PALB2 gene (inherited from the mother).

Familial history was negative for cancers. Following the genetic counseling, the couple decided to keep the pregnancy, and also an adequate monitoring plan was made for the mother and relatives at risk.

The c.93dupA variant inserts one nucleotide in exon 2 of the PALB2 gene, creating a frameshift and premature translation stop signal, resulting in an absent or non-functional protein. Loss-of-function variants in the PALB2 gene are known to be pathogenic. This variant is not found in the gnomAD database. This variant has been detected in multiple individuals with a personal and/or family history of breast cancer.

Women with a pathogenic or likely pathogenic variant in the PALB2 gene have a risk of breast cancer that is estimated to be 9.47 times higher than the average. Particularly, women carriers of PALB2 pathogenic variants have a 14% risk of developing breast cancer by age 50 and a 35% risk by age 70. They also have an increased risk for ovarian and pancreatic cancer with an estimated lifetime risk of 3–5% and 5–10%, respectively.

2. In the second case, ultrasound anomalies included aberrant right subclavicular artery, rocker bottom feet, fetal hydrops, and fetal ascites. Familial history for genetic disorders was negative. Fetal WES revealed a variant of unknown significance (VUS) mutation in the LMX1B gene. The LMX1B (LIM homeobox transcription factor 1-beta) gene is located on chromosome 9q33.3, which encodes a transcription factor that belongs to the LIM-homeodomain family of proteins.

Pathogenic variants in the LMX1B gene are causing focal segmental glomerulosclerosis 10 (MIM# 256020) and Nail–patella syndrome (MIM# 161200). Focal segmental glomerulosclerosis-10 (FSGS10) is an autosomal dominant kidney disease characterized by isolated glomerulopathy without extrarenal manifestations. This renal disease is highly variable in severity and pathology, even within the same family. Nail–patella syndrome is also an autosomal dominant disease characterized by dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, iliac horns, glaucoma, and focal segmental glomerulosclerosis. Renal involvement is the major determinant of the prognosis for Nail–patella syndrome. Patients often present with varying degrees of proteinuria or hematuria, and can occasionally progress to chronic renal failure.

When following up on this case, the baby was born with no complications and presented no clinical symptoms, until the present, at around 1 year of age.

3. The third case presented multiple abnormal findings on the second-trimester ultrasound exam including the right aortic arch, severe hydronephrosis, caudal regression syndrome, and mesoaxial hand polydactyly. The fetal WES result shows a heterozygous pathogenic variant in HOXD13 gene c.820C >T (p.Arg274Ter). The c.820C >T (p.R274*) alteration, located in exon 2 of the HOXD13 gene, consists of a C to T substitution at nucleotide position 820. This changes the amino acid from an arginine (R) to a stop codon at amino acid position 274. Based on data from the gnomAD database, the HOXD13 c.820C > T alteration was observed in <0.01% of the total alleles studied. This couple decided on the termination of pregnancy.

4. In the fourth case, the WES referral was an abnormality of the cerebral structure—mega cisterna magna and ventriculomegaly.

Whole-exome analysis for the detection of point mutation variants (nucleotide substitutions and deletion/insertion of some base pairs) associated with the clinical phenotype of the fetus (focus on 189 brain malformation gene panel) was performed and the result revealed a missense heterozygous variant in the ADNP gene with uncertain clinical significance (VUS).

The ADNP gene is located on chromosome 20q13.13, which encodes a homeodomain-containing zinc finger protein with transcription factor activity that is essential for brain formation. Pathogenic variants in the ADNP gene cause autosomal dominant Helsmoortel–Van der Aa syndrome (MIM# 615873), a neurodevelopmental disorder characterized by impaired intellectual development, motor delay, autism spectrum disorder, facial dysmorphisms, hypotonia, congenital heart disease, visual difficulties, and gastrointestinal issues. Morphological brain particularities include wide ventricles, cerebral atrophy, underdevelopment of the corpus callosum, delayed myelination, white matter lesions, and cortical dysplasia. The c.1612G > A (p.Glu538Lys) variant replaces glutamic acid with lysine at codon 538. The glutamic acid residue is strongly conserved and there is a moderate physicochemical difference between glutamic acid and lysine. The observed variant is absent in the gnomAD database.

In cases such as this, when the observed variant is a VUS variant, yet there are clinical characteristics to be observed in the patient as well as facial features, the variant should be very much taken into consideration and the case be thoroughly analyzed, as to corroborate other studies or other cases reported by various researchers worldwide. The multidisciplinary team who managed this case advised for continuous follow-up of the pregnancy and the medical geneticists explained to the family the implications of the current finding.

5. In the fifth case, the ultrasound revealed the agenesis of the corpus callosum, limb malformation, and intrauterine growth restriction. The prenatal WES testing identified the following variant: c.734A > C (p.Glu245Ala), in the TGFBR1 gene in a heterozygous status. This is a likely pathogenic (class 4—PM5, PP3, PM1, PM2) variant, de novo, which means the identified variant is disease-causing.

The TGFBR1 gene is located on chromosome 9q22.33 and encodes a transmembrane serine/threonine kinase receptor for transforming growth factor beta. Monoallelic pathogenic variants in this gene have been associated with Loeys–Dietz syndrome 1 (Loeys–Dietz syndrome 1, MIM #609192) and are inherited in an autosomal dominant manner. Loeys–Dietz syndrome is characterized by hypertelorism, bifid uvula, and/or cleft palate, and arterial tortuosity is characterized by widespread vascular aneurysm and dissection. Clinical features include microretrognathia, hypertelorism, exotropia, blue sclerae, craniosynostosis, malar hypoplasia, arachnodactyly, camptodactyly, talipes equinovarus, translucent skin, joint laxity, pectus deformity, and dolichostenomelia. Loeys–Dietz syndrome patients have a high risk of aortic dissection or rupture at an early age and at aortic diameters that ordinarily are not predictive of these events.

The c.734A > C (p.Glu245Ala) variant replaces glutamic acid with alanine at codon 245 and it is located in the kinase domain. The glutamic acid residue is highly conserved, and it shows large differences in physicochemical properties compared with alanine. According to the ACMG criteria (PM1, PM2, PM5, PP3), the c.734A > C (p.Glu245Ala) variant detected in the TGFBR1 gene is classified as likely pathogenic.

This case also yielded a pathogenic variant in the HBB gene, inherited from the mother: c.-151C > T, which is responsible for the minor trait, Beta-thalassemia (AR).

The follow-up of this case shows that this baby was born, affirmatively without symptoms at the age of 1 year. The Doppler echocardiography showed a slightly dilated abdominal aorta, with no current hemodynamic importance.

6. In the sixth case, the couple was referred to genetic counseling due to being consanguineous and because the fetus showed corpus callosum agenesis and oligohydramnios at the fetal morphology. They are second-degree cousins. At the time of the consultation, the gestational age of the pregnancy was 18 weeks.

The analysis through WES yielded a negative result, but with an incidental pathogenic variant in the BRCA2 gene, inherited from the mother. The variant is classified as pathogenic (class 5—PVS1, PP5, PM2): c.793 + 1G > A, allowing the multidisciplinary team to also perform cascade testing and find that this variant was inherited from the mother. The team then tested all first-degree relatives of the mother.

Genetic counseling was offered to the mother and all the relatives at risk.

7. This case was referred to our clinic by the attending obstetrician at 19 weeks of gestation for intracardiac echogenic focus, pyelectasis, long QT syndrome of the father, family history of sudden cardiac death, and arrhythmias (without a genetic diagnosis).

The variant discovered was a VUS variant: KCNQ1:c.605-28A > G. Pathogenic monoallelic variants are responsible for long QT syndrome 1, susceptibility to short QT syndrome 2, atrial fibrillation, and familial 3. After this result, we recommended Sanger sequencing for both parents to test the known mutation found in the fetus. The mutation was also present in the father and corresponded to his clinical phenotype, long QT syndrome 1, so we concluded that this variant was inherited from the father. In this manner, prenatal WES was also conducted for known mutation testing of the father, who was diagnosed with long QT syndrome.

8. This case was referred for a right aortic arch by our obstetrician colleague at 17 weeks of gestation. After we performed the SNP array and yielded a negative result, the team decided to undergo WES testing.

The prenatal WES yielded a negative result, the couple decided to continue with the pregnancy, and the baby was born, affirmatively without symptoms at the age of 1.

9. For the ninth case, the fetus had sexual ambiguity on ultrasound morphology, intrauterine growth restriction, hypospadias, and polyhydramnios. Because the pregnancy was already 20 weeks old, we decided to perform the SNP array and WES at the same time.

The SNP array result was arr[GRCh38]12p13.33p11.22(148769_30138756)x2 hmz,12q21.31q24.22(84757938_117685540)x2 hmz.

The prenatal WES was negative.

In the meantime, the couple decided to terminate the pregnancy, based on the ultrasound findings, and the multidisciplinary team decided to perform constitutional karyotyping from the product of conception, keeping in mind the result of the SNP array.

The constitutional karyotype of the fetus was 47,XY,+12[7]/46,XY [23] (ISCN 2020), revealing a mosaic trisomy 12, in 23% of cells, which could explain the fetal phenotype.

10. The last case was referred for genetic counseling and testing based on borderline bilateral ventriculomegaly, and suspicion of hydrocephaly with Sylvian stenosis at fetal morphology. The SNP array was negative, so we continued with WES, which also yielded a negative result.

Even if the result for WES was negative in this case, we found two variants in accordance with the fetal phenotype, but without clinical impact on the diagnosis because the inheritance is autosomal recessive. The variants reported were LAMB1:c.3499C > T (p.Arg1167Ter) and PTPN23:c.2248C >A (p.Pro750Thr). Biallelic variants in the LAMB1 gene are known to cause lissencephaly 5, and biallelic variants in PTPN23 cause neurodevelopmental disorder and structural brain anomalies with or without seizures and spasticity.

The couple was offered genetic counseling and the whole multidisciplinary team had a very important role in managing this family.

We conclude that WES yielded a positive result for 50% of the cases where the SNP array was negative.

Two couples decided to terminate their pregnancy after receiving the results and five gave birth without incidents and are still being monitored, but regrettably, one pregnancy resulted in a stillbirth.

The results reveal a superior yield for prenatal whole-exome sequencing in comparison to a conventional molecular karyotype providing results in 50% of families that would not have an accurate diagnosis otherwise.

4. Discussion

The reasons why SNP arrays may not detect certain mutations that are detected by WES comparisons and detailed discussion are as follows.

4.1. Scope of Analysis

SNP arrays are designed to detect single nucleotide variations (SNPs) and copy number variations (CNVs) at specific known positions in the genome. They are limited to the specific SNPs included in the array and do not cover the entire genome. WES, on the other hand, sequences and analyzes a substantial portion of an individual’s genome, focusing on the protein-coding regions known as exons. WES has the potential to detect a wider range of mutations, including point mutations, insertions, deletions, and structural variations.

4.2. Mutation Type

SNP arrays are primarily designed for detecting single nucleotide polymorphisms (SNPs), which are single-base-pair variations. They are less effective at detecting other types of mutations, such as small insertions and deletions (indels) or complex structural variations. WES is capable of detecting various types of mutations, including SNPs, indels, and larger structural variants. It provides a more comprehensive view of genetic variations within the exome.

4.3. Resolution

SNP arrays typically have a lower resolution compared to sequencing techniques like WES. They may miss rare or novel mutations that are not represented on the array. WES has a higher resolution because it sequences specific regions of the genome. This allows for the detection of a broader range of genetic variations, including rare and novel mutations.

4.4. Limitations of Array Design

The design of SNP arrays is based on a fixed set of known SNPs. Mutations that are not represented by the selected SNPs on the array will not be detected. WES is not limited by a predefined set of markers, and it can identify mutations in regions that are not covered by SNP arrays.

4.5. Data Analysis

Data analysis for SNP arrays is focused on genotyping specific SNPs and identifying CNVs. It may not include the same level of variant calling and annotation as WES analysis, which can identify a broader range of mutations. WES data analysis is more comprehensive and includes variant calling and annotation, which can identify various types of mutations in the exonic regions.

4.5.1. Limitations of Prenatal WES

WES does not detect small copy number variations, aneuploidy, polyploidy, translocations, trinucleotide repeats, or low levels of mosaicism. There are also regions in the genome with low coverage by WES, in particular, CpG-rich regions that will not be properly sequenced. The execution time for prenatal WES represents an important limitation of the testing method [4,6,14,15].

Prenatal genomic diagnosis, genetic counseling, and decisions based on informed consent require the intervention of a multidisciplinary team that includes an expert in maternal–fetal medicine with competence in prenatal ultrasound, a geneticist, a neonatologist, and a psychologist [16,17,18,19].

Prenatal diagnosis of a lethal genetic disease allows parents to make decisions related to the evolution of the pregnancy, including terminating the pregnancy under local legal conditions or preparing for the birth to take place in a neonatal intensive care center. In addition, it is possible to establish adequate genetic counseling, to estimate the risk of recurrence and the available reproductive options [20,21,22,23].

4.5.2. Genetic Counseling in Prenatal WES

There is currently no consensus related to the indications for the use of WES prenatally in the absence of fetal anomalies, so pre-test genetic consultation and counseling are essential.

Genetic counseling begins before the actual test for all cases, without exception, and is carried out by a medical genetic specialist. Aspects related to the benefits and limitations of the test, the parents’ expectations, and the necessity and usefulness of obtaining a definite diagnosis instead of one that is uncertain will be discussed. In addition, the pre-test discussion must include information about the possible results (interpretation of variants)—the identification of variants that most likely explain the fetal phenotype, and the identification of variants with uncertain clinical significance in genes that could be involved in the fetal phenotype or a negative result, without identifying any variant possibly linked to the phenotype. Variants with uncertain clinical significance can be reinterpreted later, as new information emerges online and becomes available in the literature [23,24,25,26,27,28,29,30].

4.5.3. Ethical Aspects

The statement by de Jong and de Wert [31] starts a debate about how prenatal testing should be state-insured so that couples have the option to terminate a particular pregnancy where the future child will be born with severe disorders that might lead to the child’s death or have the child disabled for life. State health services should remain impartial concerning couples’ pre- and post-test choices regarding whether the couple would like to keep or terminate the pregnancy, whilst offering the best support to uphold their wish. For example, if the couple would like to keep a pregnancy where the newborn would have either congenital affections or various other pathologies, the state services could book a consultation prenatally for a particular type of surgeon or organize a multidisciplinary team to better treat and tend to the needs of the patient. On the other side, should the couple choose to terminate the pregnancy, the state services could offer a variety of ob-gyn specialists who could provide them with the best care before and after terminating the pregnancy [10,11].

A crucial step in genetic testing is the requirement of informed consent from the tested patients or the prenatally screened couple’s pregnancy. The informed consent form should comprise pre- and post-test options, the lack of legal constraint, a specialized pathology management plan, assuring optimal perinatal care, or, in other cases, as far as palliative care, the limitations of the genetic testing, and the limitations of the current worldwide knowledge.

Diagnostic yields for this technique vary from 8 to 80%, depending on cohort characteristics such as size, family history, ethnicity, and patient selection.

Another aspect we want to bring attention to is the similar diagnostic yield our study discovered in comparison to other published studies in the medical literature. For example, Mellis et al. discover a diagnostic yield of 31% but acknowledge that the percentage is higher—42% for pre-selected cases with a high likelihood of monogenic disorders [14]. Our cohort was selected and made up only of fetuses that had a high risk of having a Mendelian disorder.

Another recent work by Miceikaite et al. has demonstrated the overall diagnostic yield of WES or whole-genome sequencing (WGS) of 40%. The authors selected 40 pregnancies with fetal anomalies or increased nuchal translucency [16].

A multicentric study from 2019 on 45 families shows an overall diagnostic yield of 28.9%, while, among families with structural ultrasound (US) anomalies, the diagnostic yield is 31.7% and even higher—55.6% for fetuses with structural anomalies and relevant family history [17].

Further testing is needed to obtain a clearer picture of the prenatal diagnostic yield in the Romanian population. Genetic screening programs should be developed and implemented by the Department of Health, together with healthcare professionals, and a coordinating team should be assigned to evaluate and supervise these programs to maintain and ensure their good use [15,31,32,33,34,35,36,37].

5. Conclusions

The diagnostic yield of WES testing among our patients was 50%, similar to other studies recently published in the literature.

In this article, we present 10 cases that were tested through prenatal WES, showing the great diagnostic yield in comparison to the SNP array in our selected cases. As a closing remark, we wish to highlight the benefits of WES testing in prenatal cases. Not only have we discovered the etiology of the fetal structural abnormalities in US, but also we discovered some incidental and secondary findings that diagnosed the parents or made the medical multidisciplinary team aware of some high risks for the parents and their families. The benefits for the family members and the expecting parents justify the evaluation of the need for national health insurance in our state to provide this testing free of charge for patients with a medical genetics recommendation.