• Group I, Case 3: NDUFS1 gene-associated mitochondrial complex I deficiency: A 32-year-old lady presented for prenatal diagnosis. The family had lost dizygotic twin boys. One of them presented during the neonatal period with progressive encephalopathy and seizures after acute gastroenteritis. He also had high blood pressure, metabolic acidosis, and marginal elevation of lactate, and high plasma and cerebrospinal fluid lactate. The second twin also developed progressive lethargy and encephalopathy. MRI of the brain of both twins revealed similar results. It showed bilateral areas of diffusion restriction involving the white matter of centrum semiovale, periventricular white matter, cortical spinal tract, posterior limbs of internal capsules, middle cerebral peduncles, cervicomedullary junction, and posterior aspects of the cervical spinal cord. Areas of cavities with small foci of hemorrhage in bilateral semiovale and periventricular white matter were also seen. WES analysis of the twins revealed one heterozygous missense likely pathogenic variant in the NDUFS1 gene namely c.1825A>G [p.Thr609Ala] in exon 16. Multiplex ligation-dependent probe amplification (MLPA) of the same gene was performed next, and this showed a duplication of exon 15- 17 on one allele. The missense variant was inherited from the father, and the duplication was inherited from the mother. The present pregnancy was evaluated for these two variants, and the fetus was found to be a carrier of the c.1825A>G variant.

• Group I, Case 9: POMT1 gene-associated Walker-Warburg syndrome: Previous 2 pregnancies of this couple had been terminated. The first affected pregnancy was terminated for antenatally detected fetal encephalocele. In the second affected pregnancy, the mother was referred to us at 13 weeks of gestation and the ultrasound had revealed unilateral cleft lip and palate. A large cyst was identified in the posterior fossa measuring 8.4 mm in diameter, suspected as a precursor of Dandy-Walker malformation. Ventriculomegaly in the brain was observed with significant dilation of ventricular cavities. The chromosomal microarray on CVS was normal. After termination of the second affected pregnancy, fetal autopsy showed unilateral cleft lip and palate, small encephalocele and posterior fossa anomaly (Figure 1a). CES was done which showed a homozygous pathogenic frameshift variant c.1081C>T [p.Gln361Ter] in the POMT1 gene. The results confirmed the genetic diagnosis of congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies type A1. It includes both the more severe Walker-Warburg syndrome (WWS) phenotype, and the slightly less severe muscle-eye-brain disease (MEB).

The option of prenatal diagnosis for future pregnancies was discussed with the couple. However, for the subsequent pregnancy, the couple underwent in vitro fertilization (IVF) with donor gametes.

• Group I, Case 17: LCA5 gene-associated Leber congenital amaurosis: A primigravida consulted at 8-9 weeks of pregnancy as both she and her husband were diagnosed to have childhood-onset retinitis pigmentosa. NGS-based multigene panel testing for retinitis pigmentosa revealed the same homozygous pathogenic variant c.1151del [p.Pro384GlnfsTer18] in the LCA5 gene in both of them, which confirmed the diagnosis of Leber congenital amaurosis in both. There was no history of consanguinity in their parents and also the couple was non-consanguineous. Prenatal testing was not offered in this case due to a 100% risk of biallelic mutations in the ongoing pregnancy.

• Group 1, Case 18: SOX10 gene-associated Waardenburg syndrome type 2E: A 31-year-old second gravida lady (G2P1+0) came to us in early pregnancy. The couple was non-consanguineous, and their five-year-old daughter had motor delay, deafness, heterochromia of iris, and skin hypopigmentation. Deafness was noted at ten months of age, and she had undergone a cochlear implant at 1.5 years of age. She also had history of constipation since 6 months of age. Trio WES was done in the proband and the couple. It identified a heterozygous single base-pair deletion in exon 4 of the SOX10 gene, resulting in a frameshift and premature truncation of the protein. This mutation was not present in the parents. The risk of recurrence was small, but we offered amniocentesis at 16 weeks gestation. Antenatal ultrasound showed no markers of aneuploidy. However, QF-PCR which was done to exclude aneuploidy, revealed trisomy 21. The couple opted for termination of the pregnancy and further processing of the prenatal sample was not done.

• Group II, Case 20: JAGN1 gene-associated congenital neutropenia: A consanguineously married couple were referred in early pregnancy at 6-8 weeks gestation with history of death of two previous male children due to recurrent infections, including respiratory infections and acute gastroenteritis, within 30 days of life. Investigations in the affected children had revealed severe neutropenia, leukopenia with reduced CD3, CD4, CD19, and CD56 cell counts and reduced IgM and IgA. The bone marrow biopsy of one child was suggestive of childhood myelodysplastic syndrome or refractory cytopenia; the child was clinically suspected to be affected with immunodeficiency or bone marrow myelodysplasia. The couple’s NGS testing showed that both were heterozygous carriers for the likely pathogenic variant c.17G>A [p.(Gly6Asp)], in the JAGN1 gene. CVS was performed at 12 weeks of pregnancy and the fetus was found to be heterozygous for this variant.

• Group II, Case 21: ELOVL4 gene-associated developmental delay and ichthyosis: This consanguineous couple was referred prior to their second pregnancy, for genetic counseling and evaluation, as they had lost their first daughter at 14 months of age. The child had global developmental delay and seizures. She also had ichthyosis noticed since birth. As the proband was not available for testing, we did carrier screening by NGS. A heterozygous missense variant c.257T>A [p.(Met86Lys)] in the ELOVL4 gene was identified in both the husband and wife. Though this variant was classified as a VOUS, the clinical history of the affected deceased child was consistent with the features of ELOVL4-associated ichthyosis-spastic quadriplegia-mental retardation syndrome. Therefore, this variant was considered to be the disease-causing variant and we offered prenatal testing by CVS at 12 weeks after explaining the limitations of prenatal testing based on VOUS. The fetus was found to be homozygous for the ELOVL4 gene variant.

5 Discussion

In this case series, we discuss the findings of NGS-based testing in families referred for prenatal diagnosis in view of history of a suspected genetic disorder in the family. In 18 cases out of the total 25, the proband was available for testing (Group I). Out of these 18, work up was done in the preconception period in fifteen families and in early pregnancy in 2 cases; only the case with biotinidase deficiency came in the second trimester. Couple carrier testing by NGS was performed in 5 cases where the proband was no longer alive and where the proband’s evaluation had not been completed (Group II). Two cases underwent direct fetal sampling as they presented to us in the second trimester.

Evaluation of the proband during the preconception period is the best way of approaching genetic counseling and planning for prenatal diagnosis. Referral during early pregnancy also allows the option of prenatal testing. Late referral is associated with limitations in providing prenatal diagnosis due to time constraints. Sometimes prenatal sampling may be done simultaneously with testing of the proband or the couple. The limitations in such a situation need to be documented as in spite of collecting the fetal sample through invasive methods, accurate prenatal diagnosis may not be possible.

Prenatal genetic testing should be offered when pathogenic or likely pathogenic variant(s) consistent with the clinical history is/ are detected. VOUS present counseling challenges to the clinician and dilemmas to the family for decision-making.

In group III, trio testing (in prenatal sample and the couple simultaneously) was done. Trio analysis has higher diagnostic yields compared with non-trio analysis. It allows for the identification of de novo variants, determination of phase for biallelic variants, and confirmation of carrier status in both parents when a homozygous variant is detected. (Monaghan et al., 2020).

Pretest counseling is ideally provided by a professional trained in genetics. In this study, the biggest challenge we faced was of clinical correlation of the NGS-based analysis. Most of the cases referred to us were in the preconception period or early pregnancy, so the prenatal diagnosis was done at 12 weeks and analysis was completed within the 20 weeks limits of termination. In six cases where we performed amniocentesis, four showed normal results and so termination of pregnancy was not needed. So, early referral is the key factor in the management but women should not be denied prenatal diagnosis if they present later in pregnancy as some of them would abort without testing and this may be a normal baby! This is because risk of recurrence is 25% in autosomal recessive disorders. With the use of prenatal exome sequencing, the turn-around time has to be rapid to maintain all aspects of reproductive choice.

One-fifth of our referrals were related to ultrasound-detected anomalies. These included two cases of polycystic kidneys detected as large bright echogenic kidneys on the ultrasound. As the index case was not available, we utilized NGS-based carrier analysis. Exome sequencing can identify genetic etiologies in cases with fetal anomalies, particularly recurrent cases or when there is a history of consanguinity or when clinical features are suggestive of a monogenic syndrome e.g., Meckel-Gruber syndrome.

Two recent large cohort studies have studied the diagnostic yield of exome sequencing in fetuses with one or more ultrasound anomalies and normal karyotype and microarray. The PAGE study included 610 fetuses with ultrasound anomalies, from the United Kingdom. They reported a pathogenic variant in 8.5%, and VUS in 3.9% of cases that was considered possibly pathogenic (Lord et al., 2019). Petrovski et al. reported on 234 fetuses with ultrasound anomalies and normal results on karyotype and CMA. They reported a pathogenic variant in 10% of such fetuses. Detection rate was 6% in fetuses with a single anomaly, whereas 19% of fetuses with more than one anomaly had a pathogenic genetic variant (Petrovski et al., 2019). These studies suggest that prenatal exome sequencing may provide clinically relevant information for the management of fetal anomalies identified in pregnancy and this will guide genetic counseling and prenatal diagnosis in future pregnancies.

6 Conclusion

The application of NGS-based testing has the potential to facilitate prenatal testing, and this can be employed in various scenarios as illustrated by our experience. The importance of diagnosis of the proband with a possible monogenic disorder preconceptionally needs to be stressed. Equally important is genetic testing of stillbirths or neonatal deaths, or at least storing a piece of umbilical cord or blood sample in an EDTA tube for further testing. The powerful genomic technique of NGS needs to be appropriately used for the benefit of families with poor reproductive outcomes.

Acknowledgments: Parag Agarwal and Anisha Raju, THB, Gurugram for assisting in the final formatting of the manuscript.

Funding: No funding received

Conflict of interests: None

References