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CC BY-NC-ND 4.0 · Journal of Fetal Medicine
DOI: 10.1055/s-0045-1810115
Original Article

Targeted Whole-Exome Sequencing for Carrier Screening of Couples Having Previously Affected Pregnancies with Suspected Autosomal Recessive Diseases

Suvarna Magar 
1   Department of Pediatrics, MGM Medical College, Aurangabad, Maharashtra, India
,
Ghansham Magar
2   Department of Obstetrics and Gynaecology, Indian Institute of Medical Science and Research, Jalna, Aurangabad, Maharashtra, India
,
Dheeraj Kotecha
2   Department of Obstetrics and Gynaecology, Indian Institute of Medical Science and Research, Jalna, Aurangabad, Maharashtra, India
,
Ashwini Deshmukh
3   Department of Obstetrics and Gynaecology, Umang Maternity and IVF Center, Shivaji Nagar, Aurangabad, Maharashtra, India
,
Laxmi Rachkonda
4   Department of Obstetrics and Gynaecology, MGM Medical College, Aurangabad, Maharashtra, India
,
Mehak Malhotra
1   Department of Pediatrics, MGM Medical College, Aurangabad, Maharashtra, India
,
Aarti Agrawal
5   Department of Medical Genetics and Medical Biotechnology, MGM Medical College, Aurangabad, Maharashtra, India
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Abstract

Introduction

There are many severe or life threatening autosomal recessive conditions leading to death in the prenatal, early postnatal, or early childhood period without a precise diagnosis being made. Often, genetic counselling and postmortem analysis are not available, and phenotypic descriptions are insufficient. Targeted whole exome sequencing of the couple is the best test when the proband is not available for sampling. It is estimated that couples with previous babies being affected by a particular phenotype are more likely to harbour a heterozygous variant for the given phenotype. Here, we report our experiences with targeted whole exome sequencing for the carrier screening of autosomal recessive lethal disorders in couples with one or more affected foetuses or children.

Methods

We selected 11 consanguineous and four nonconsanguineous consecutive couples at risk for severe autosomal recessive disorders seen at the genetic clinic of the Mahatma Gandhi Mission Medical College and Umang Maternity and IVF Centre. The couples were counseled for carrier screening with targeted next generation sequencing. The inclusion criteria included the loss of at least one child before the age of 2 years due to a severe/lethal condition and/or one or more miscarriages with pathological findings in the fetus. The couple's genetic testing results were reviewed to assess their risk of passing on inherited conditions to their children.

Results

A total of 15 couples were identified with a bad obstetric history in the last 2 years, from June 2022 to May 2024. Out of the 15 couples, 11 had a consanguineous union. Exome was negative in one couple, and only one parent harboured a pathogenic variant in another couple. Of the 15 couples investigated, 4 couples (cases 3, 11, 13, and 15) had lost one child. The remaining 11 couples had lost more than one pregnancy. A likely causative variant for the symptoms of the deceased children was identified in 5 out of the 15 couples investigated (33.33%). Out of the 12 couples, 5 couples were carriers of likely pathogenic variants, and the remaining 7 couples were carriers of variants of uncertain significance.

Conclusions

Our data showed that whole exome sequencing in couples based on phenotype in previous pregnancies or children was a high yield strategy for identifying lethal autosomal recessive disorders.


 

Keywords

whole-exome sequencing - carrier screening - bad obstetric history - autosomal recessive disorders

Introduction

Autosomal recessive diseases are single gene diseases that manifest when pathogenic variants are present on both alleles of a gene. Family history is usually negative, as parents are heterozygous carriers of these diseases and, therefore, they are clinically normal. There are many severe or life threatening autosomal recessive conditions leading to death in prenatal, early postnatal, or early childhood periods without a precise diagnosis being made. Often, genetic counseling and postmortem analysis are not available, and phenotypic descriptions are insufficient. DNA banking for later diagnosis of the affected member is not available, and all these factors pose challenges for future diagnosis and future recurrence risk assessment for the couple. The prerequisite for future prenatal diagnosis or preimplantation diagnosis is having proven the disease in the proband by showing biallelic causative variants in genetic testing of the proband or at least showing that parents are carriers for such diseases. With the availability of proband or parental testing, we can proceed with prenatal diagnosis or preimplantation genetic diagnosis in subsequent pregnancies. Whole exome sequencing (WES) is currently the best test for diagnosing single gene diseases.[1] Targeted WES of the couple is the best test when the proband is not available for sampling. An article by Zhang et al has shown that expanded carrier screening was helpful in otherwise phenotypically normal prepregnancy and early pregnancy couples who intend to have children.[2] It is estimated that couples with previous babies being affected with a particular phenotype are more likely to harbor a heterozygous variant for the given phenotype.

Here we report our experiences with targeted WES for carrier screening of autosomal recessive lethal disorders in couples with one or more affected fetuses or children.


 

Methods

Patients and Phenotypic Characteristics

We selected 11 consanguineous and 4 nonconsanguineous consecutive couples at risk for severe autosomal recessive disorders seen at the genetic clinic of the Mahatma Gandhi Mission (MGM) Medical College and Umang Maternity and IVF Center. All the couples sought genetic counseling and testing between June 2022 and May 2024. Couples were counseled for the carrier screening with targeted next generation sequencing. The inclusion criteria included the death of at least one child before the age of 2 years due to a severe/lethal condition and/or one or more miscarriages due to fetal malformations or pregnancy termination done due to pathological findings in the fetus (e.g., complex congenital heart diseases, anencephaly, skeletal malformation, brain malformation, etc.). The need for testing the couples was the absence of the DNA of the proband.


 

Whole-Exome Sequencing

Genomic DNA from peripheral blood leucocytes was extracted as per standard extraction methods. The DNA was enriched for the complete coding regions and splice site junctions of genes using a custom bait capture system. Paired end sequencing was performed with 2 × 100/2 × 150 chemistry using the Illumina Novaseq 6000 platform (Illumina, San Diego, United States). Reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSC hg19.


 

Variant Interpretation

Variant interpretation was performed using an inhouse algorithm or ANNOVAR software. Clinically relevant mutations in both coding and noncoding regions were annotated using published variants in the literature and a set of disease databases like ClinVar, OMIM, HGMD, LOVD, and DECIPHER. Specific genetic variants that might be linked to the proband's condition in previous pregnancies or fetal affection were selected for further analysis. Pathogenicity prediction of the candidate variants was performed using computational tools such as Mutation Taster, REVEL, SIFT, CADD, and searched in ClinVar if already reported. The guidelines and standards of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP)[3] were used as references to describe candidate variants.


 

 

Results

As described in [Tables 1], [2], [3], [4], a total of 15 couples were identified with a bad obstetric history in the last 2 years from June 2022 to May 2024. Out of the 15 couples evaluated, 11 were consanguineous and 4 were nonconsanguineous couples. Of the 15 couples investigated, 4 couples (couples 3, 11, 13, and 15) had lost one pregnancy. The remaining 11 families had lost more than one pregnancy. There were 21 deaths after birth in 15 couples. Five cases had children born with suspected inborn errors of metabolism (cases 5, 8, 9, 11, and 15), and one child had suspected genetic renal disease (case 1). One case was with neuromuscular disease and another one with epileptic encephalopathy in the child (case 12 and case 10). Two cases had children or pregnancies suspected of having congenital malformation syndrome (cases 2, 3, and 14), and two cases had suspected hemophagocytic lymphohistiocytosis (HLH) in children (cases 6 and 13). One couple had pregnancy losses without a clear phenotype and carrier screening revealed a variant of uncertain significance (VUS) for Joubert syndrome and distal arthrogryposis (case 7). Case 4 had a negative exome study. In addition, five of the above mentioned couples (families 3, 4, 6, 8, and 12) had one or more miscarriages due to affected fetuses besides losing an affected child. [Table 1] shows a phenotypic description of the affected children and/or fetuses.

Table 1

Description of families and variants identified by couple whole-exome sequencing with phenotype details of the pregnancy outcomes

Family ID

Consanguinity

Phenotype in the deceased child and DD

Gene

Variant details in couple

Disease (OMIM): increased risk for autosomal recessive disorder

Case 1

N

First pregnancy: spontaneous abortion (SA)

Second pregnancy: increased nuchal thickness, echogenic intracardiac focus, double marker 1:79

Third pregnancy: death at 19 days of life, renal failure, preaxial polydactyly, multicystic kidney disease, cryptorchidism, facial dysmorphism

NPHP3 gene

NM_153240.4

Mother: neg

Father:

c.2694–2_2694–1delAG

Heterozygous (recessive) pathogenic causing

Nephronophthisis-related ciliopathies, Meckel syndrome 7 (MKS7) and renal-hepatic-

pancreatic dysplasia 1 (RHPD1).

Case 2

N

First pregnancy anomaly: tricuspid dysplasia, pulmonary atresia, persistent left superior vena cava, unbalanced atrioventricular septal defect, pericardial effusion, heart block

Second pregnancy anomaly: complex congenital heart disease, bradycardia, gross ascites, fetus hydrops hypoplastic right atrium and ventricle, VSD, narrow pulmonary artery, dilated aorta, arrhythmia, pericardial effusion

DNAI2

NM_023036.4

Father:

c.546C > A

p. Tyr182 Ter

Heterozygous, pathogenic variant

Mother: c.546C > A

p. Tyr182 Ter

Heterozygous pathogenic variant

Primary ciliary dyskinesia, 9, with or without situs inversus

Case 3

Y

First pregnancy anomaly: MTP at 19 weeks; corpus callosum agenesis and perimembranous ventricular septal defect

TRAPPC12

NM_016030.5

Father:

C.198 G > T; p.met6611e

Heterozygous VUS

Mother: C.198 G > T p.met6611e

Heterozygous VUS

Progressive encephalopathy with brain atrophy and spasticity

Case 4

N

First pregnancy: SA

Second pregnancy: oligohydramnios, severe IUGR, anhydramnios, seizures, succumbed to encephalopathy in neonatal age

Third pregnancy: MTP due to anhydramnios and IUGR

Couple exome study negative

Negative

Case 5

Y

First pregnancy: male—seizure disorder death at 2 months

Second pregnancy: males (twins)—both expired on day 1 on day

Third pregnancy: 4-year-old male—alive

Fourth pregnancy: neonatal death on day 5–6 of life due to seizures

ALDH7A1

NM_001182.5

Father and mother both carrier of variant c.1556G > A

p.Arg519Lys heterozygous, likely pathogenic variant

Pyridoxine-dependent epilepsy.

Abbreviations: DD, differential diagnosis; IUGR, intrauterine growth retardation; MTP, medical termination of pregnancy; SA, spontaneous abortion; VSD, ventricular septal defect; VUS, variant of uncertain significance.


The couple in case 1 had a fetal phenotype related to renal malformations and only one pathogenic variant was detected in the NPHP3 gene in the father, causing nephronophthisis-related ciliopathy ([Table 1]). A couple, in case 4, had two spontaneous miscarriages and one neonatal death due to neonatal encephalopathy, but the couple's exome study was negative ([Table 1]). So, the couple in cases 1 and 4 could not be offered any diagnostic possibilities in the affected pregnancy or proband.

The couple in case 2 had two fetuses with complex congenital heart diseases and the couple was detected as having a heterozygous pathogenic variant c.546C > A in the DNAI2 gene, causing primary ciliary dyskinesia (PCD) 9, with or without situs inversus ([Table 1]). The first pregnancy anomaly scan detected tricuspid dysplasia, pulmonary atresia, persistent left superior vena cava, unbalanced atrioventricular septal defect, pericardial effusion, and heart block, and the second pregnancy fetal anomaly scan detected complex congenital heart disease, bradycardia. gross ascites, fetal hydrops, hypoplastic right atrium and ventricle, ventricular septal defect (VSD), narrow pulmonary artery, dilated aorta, arrhythmia, and pericardial effusion. There was no heterotaxy associated with these complex heart diseases in the two fetuses.

The couple in case 3 with fetal brain malformation were carriers of heterozygous VUS c.198 G > T in the TRAPPC12 gene, probably matching progressive encephalopathy with brain atrophy and spasticity ([Table 1]). This variant is described as VUS in the ClinVar database.[4]

The couple in case 5 had four babies expired and the couple's exome revealed both parents were a heterozygous carrier for the pathogenic variant c.1556G > A in the ALDH7A1-gene-causing pyridoxine-dependent epilepsy ([Table 1]). The first child was male with a global developmental delay (GDD) with drug-refractory seizures and expired at 8 months of life. The second was a twin pregnancy, both male babies, expired on the second day of life, and the cause was not known. The third pregnancy was a male child alive and well, and the fourth pregnancy was a male, with a history of seizures on day 1 of life and death on day 7 of life. The first child was evaluated, and a magnetic resonance imaging (MRI) brain showed a few tiny cysts seen at the foramen of Monro as gemistocytic cysts; EEG of the child showed frequent sharp and slow waves over the left frontocentral, bilateral occipital region with focal features with incomplete subcortical spread. All the babies had typical symptoms of early onset encephalopathy mimicking hypoxic ischemic encephalopathy, which is seen in pyridoxine-dependent epilepsy.

The couples in cases 6 and 13 were both detected to be carriers of likely pathogenic and pathogenic variants causing HLH type 3 in the UNC13D gene and type 2 in the PRF1 gene, respectively, matching the phenotype in affected children ([Tables 2] and [4]). The couple in case 6 had a first child normal and the second child expired at 5 months and the third child expired at 1 month of age. The second and third children were evaluated for recurrent febrile illness, with hepatosplenomegaly and requiring packed cell transfusions. Bone marrow biopsy in the second child showed hemophagocytosis and genetic testing was advised, but was not done by the parents. The couple came with a third child with febrile illness from birth, with hepatosplenomegaly. Diagnosis of HLH was suspected, but the child expired at 1 month of age. The couple in case 13 had a neonate with full term pregnancy who was diagnosed with hepatomegaly, jaundice, liver failure, and pancytopenia. Initially, a diagnosis of hemochromatosis, tyrosinemia, or galactosemia was suspected. This baby expired on day 8 of life due to liver cell failure.

Table 2

Description of families and variants identified by couple whole-exome sequencing with phenotype details of the pregnancy outcomes

Family ID

Consanguinity

Phenotype in the deceased child and DD

Gene

Variant details in couple

Disease (OMIM): increased risk for autosomal recessive disorder

Case 6

Y

First pregnancy: female, 6 years, alive and well

Second pregnancy: male, died at 5 months of age due to febrile illness

Third pregnancy: male, died at 1 month of age due to

hemophagocytic lymphohistiocytosis

Forth pregnancy: MTP

UNC13D

NM_199242.3

CFTR

NM_000492.4

1.Both mother and father are heterozygous carrier of

c.615G > A

p.Trp205Ter, likely pathogenic variant

2. Both parents heterozygous carrier of

C.223C > T, p. Arg 75Ter pathogenic variant

Familial hemophagocytic lymphohistiocytosis, 3,

cystic fibrosis

Case 7

Y

First pregnancy: SA, 2 months

Second pregnancy: IUD at 8 months

Third pregnancy: SA at 9 weeks

POC, CMA-N

CEP290

NM_025114.4

PIEZO2

NM_001378183.1

Both mother and father are carrier of

1) c.4676A > G P.Tyr 1559 Cyst VUS, heterozygous variant

2) Both mother and father are carrier of

c.4542G > T P.G1n1514 His

Heterozygous VUS

Joubert syndrome BBS-14,

distal arthrogryposis with impaired proprioception and touch

Case 8

Y

First pregnancy: male, expired at 18 months, GDD with seizures with LRTI

Second pregnancy: male, GDD, and death at 8 months due to a febrile illness

GRHPR

NM_012203.2

MAN2B1

NM_000528.4

GCDH

NM_000159.4

GBA2

NM_020944.3

Both mother and father are carrier of heterozygous pathogenic variant C.494G> A

p.Gly165Asp

Both mother and father are carrier of variant

c.2579C > T

p.Ala860val, VUS

Both mother and father are carrier of variant

c.471C > G

p.Ser157Arg VUS

Both mother and father are heterozygous carrier of variant

c.1408 del

p.Arg470AspfsTer102

Likely pathogenic

Primary hyperoxaluria type II,

α mannosidosis 17 type I and II,

glutaric aciduria type I

spastic paraplegia 46, autosomal

recessive

Abbreviations: CMA, chromosomal microarray; DD, differential diagnosis; IUD, intrauterine death; GDD, global developmental delay; MTP, medical termination of pregnancy; POC, product of conception; SA, spontaneous abortion; VUS, variant of uncertain significance.


Table 3

Description of families and variants identified by couple whole-exome sequencing with phenotype details of the pregnancy outcomes

Family ID.

Consanguinity

Phenotype in the deceased child and DD

Gene

Variant details in couple

Disease (OMIM): increased risk for autosomal recessive disorder

Case 9

Y

G1: male, died at 11 months, GDD (global developmental delay) with seizures

G2: female, IUD at 8 months

G3: 2 months SA

G4: female, failure to thrive, GDD died at 8 months.

ETHE1

NM_014297.5

OTOA

NM_144672.4

G6PD

NM_001360016.2

Both mother and father are heterozygous carrier of VUS

c.461A > T

p.Asp154Val

c.2359G > T

P-Gly787 Ter likely pathogenic

Mother heterozygous carrier of pathogenic variant c.949G > A

p.Gly317lys.

Ethylmalonic encephalopathy

AR deafness

G6PD deficiency

Case 10

N

First: male, death at 18 months. GDD and seizure disorder

Second: GDD with neuroregression with seizure disorder and myopic astigmatism

ITPA

NM_033453.4

Both parents are carrier of heterozygous VUS c.533G > A

p.Arg178His

Developmental and epileptic encephalopathy 35 (AR)

Case 11

Y

First: female, hepatosplenomegaly with cirrhosis with pancytopenia, expired at 3 years

FAH

NM_000137.4

SDR9C7

NM_148897.3

DUOX2

NM_001363711.2

Both parents are carrier of heterozygous VUS

c.941T > C

p.11e314Thr

Both parents are carrier of heterozygous pathogenic variant

c.112G > A

p.Gly38Arg

Both parents are carrier of heterozygous likely pathogenic variant

c.618dup p.Pro207AlafsTer

Tyrosinemia type 1

ichthyosis, congenital, autosomal recessive 13

thyroid dyshormonogenesis 6

Case 12

Y

First: congenital bilateral vocal cord paralysis. Respiratory distress and death at 2 months

Second: congenital bilateral vocal cord paralysis. Death at 1 month of life.

IGHMBP2

NM_002180.2

CFTR

NM_000492.3

Both parents are carrier of heterozygous likely pathogenic variant

c.1817G > A

p.Arg606His

Both parents are carrier of heterozygous likely pathogenic variant

c.473G > A

p.Ser158Asn

Charcot–Marie–Tooth disease, type 2 (CMT2S)

cystic fibrosis (CF).

Abbreviations: DD, differential diagnosis; G6PD, glucose 6 phosphate dehydrogenase; GDD, global developmental delay; IUD, intrauterine death; SA, spontaneous abortion; VUS, variant of uncertain significance.


Table 4

Description of families and variants identified by couple whole-exome sequencing with phenotype details of the pregnancy outcomes

Family ID

Consanguinity

Phenotype in the deceased child and DD

Gene

Variant details in couple

Disease (OMIM): increased risk for autosomal recessive disorder

Case 13

Y

First pregnancy: female neonatal cholestasis, hepatosplenomegaly, hyperbilirubinemia and liver failure and expired on day 8 of life.

PRF1

NM_005041.6

Both parents are carrier of heterozygous pathogenic variant

c.386G > C,

p.Trp129Ser,

Familial hemophagocytic

lymphohistiocytosis-2

Case 14

Y

First: female child. Born preterm, NVD, polyhydramnios noted, birth weight 1.5 kg, NICU stay for 40 days. Presented with extension deformities in wrist and ankle, cleft lip and palate, micrognathia, GDD, hypertonia; CHD—PDA, ASD, VSD, severe pulmonary hypertension;

ventriculomegaly. Passed away at 4 years of age.

Second: female child. Born FTNVD, polyhydramnios noted, CIAB, birth weight 2.5 kg. Presented with cleft palate, possible CHD, cleft palate, similar looks to first child, cortical thumb, possible neural tube defect. Passed away in sleep at 2 months of age.

Third: male fetus. IUD at 8 months of gestation. Polyhydramnios noted. Preterm, weight 1.5 kg noted.

Fourth: male. USG at 19 weeks of gestation revealed prominence of bilateral lateral ventricles, possible pulmonary stenosis, PDA.

Postnatally detected pulmonary atresia, ASD, and PDA; dysmorphism—dolichocephaly, long forehead, megalocornea, long philtrum,

possible micropenis. MRI was suggestive of hypoplastic corpus callosum. Passed away at 1.5 months of age.

KLHL7

NM_001031710.3

PIEZO1

NM_001142864.4

Both parents are carrier of heterozygous VUS

c.1361A > G

p.Tyr454Cys

Both parents are carrier of heterozygous likely pathogenic variant

c.5389C > T

p.Arg1797Cys

Perching syndrome

Lymphatic malformation 6

Case 15

Y

First pregnancy: male child, hyperammonemia, respiratory failure, convulsions, refractory neonatal seizures, neonatal encephalopathy, hypoglycemia, cerebral edema, small fossa ovalis atrial septal defect, mild tricuspid regurgitation, mild pulmonary hypertension, moderate biventricular dysfunction, and dilated left ventricle. Expired at 10 days of life

ASS1

NM_054012.3

Both parents are carrier of heterozygous VUS

c.361A > G (p.Lys121Glu)

Citrullinemia type 1

Abbreviations: ASD, atrial septal defect; CHD, congenital heart disease; FTNVD, full-term normal vaginal delivery; GDD, global developmental delay; IUD, intrauterine death; NVD, normal vaginal delivery; PDA, patent ductus arteriosus; VSD, ventricular septal defect; VUS, variant of uncertain significance.


The couple in case 7 had three pregnancy losses without any malformations noted, and the couple were carriers of VUS in two genes, CEP290 and PIEZO2 ([Table 2]). Phenotype matching could not be ascertained.

The couple in case 8 had two children affected with GDD and the couple were carriers of variants in four different genes ([Table 2]). Out of the four, two genes were associated with neurological phenotype matching with the disease in children (MAN2B1 and GCDH gene) and the remaining two were causing progressive renal disease and neuroregression (GRHPR and GBA2 gene). The couple in case 8 was a heterozygous carrier of c.2579C > T in the MAN2B1 gene. This variant was a VUS and caused disease, specifically α-mannosidosis types I and II. Both parents were also heterozygous carriers of variant c.471C > G in the GCDH gene, which had conflicting classifications of pathogenicity ([Table 2]). We did not have clinical details of the previously affected children.

The couple in case 9 had two children who died of GDD and encephalopathy. The first child was a male with GDD and seizure disorder who died at 11 months of age. The second pregnancy was a female fetus with intrauterine death at 8 months of gestation. The third pregnancy was a spontaneous abortion at 2 months of gestation. The fourth pregnancy was a female child, full term normal delivery with a birth weight of 3.5 kg, failure to thrive, and GDD. The baby expired at the age of 6 months due to encephalopathy. Her investigations showed lactate: 4 micromoles/L (elevated); HCO3: 14.3 meq/L (metabolic acidosis). MRI brain showed T2-weighted hyperintense signal intensity in periaqueductal gray matter, showing restricted diffusion on diffusion weighted imaging and corresponding low apparent diffusion coefficient values and the T2 hyperintense signal intensity in bilateral putamen. Dilated subarachnoid spaces in bilateral cerebral hemispheres are suggestive of metabolic disorders like Leigh syndrome. The couple's exome analysis revealed variants in three genes, amongst which a heterozygous VUS in the ETHE1 gene matched with the symptoms in children. Both parents were heterozygous for the variant c.461A > T, which was reported as a VUS in ClinVar. Another heterozygous pathogenic variant in a couple was in the OTOA and G6PD genes ([Table 3]). For the ETHE1 gene variant with a matching phenotype, extensive post test counseling was done for the uncertain status of the variant and the need for further evidence to attribute it to the phenotype in the previous child.

The couple in case 10 had two children who expired due to GDD with seizure disorder and neuroregression. MRI brain of the third child was suggestive of symmetrical altered signal intensities involving the periventricular and subcortical white matter of bilateral fronto-parieto-temporo-occipital regions and internal capsules. Eye examination of the daughter was suggestive of myopic astigmatism. Both parents were carriers of a VUS in the ITPA gene c.533G > A, causing developmental and epileptic encephalopathy 35 ([Table 3]). The couple in case 11 were carriers of a VUS c.941 T > C in the FAH gene, causing tyrosinemia 1, matching the symptoms of chronic liver disease with jaundice, with deranged prothrombin time and bleeding tendencies ([Table 3]). The succinyl acetone in urine was negative, and the tyrosine levels were normal. Serum α-fetoprotein was high, and the child also had features of rickets and a history of one fracture in the humerus at the age of 18 months. This child expired at 3 years of age due to liver cell failure. Given the VUS and no biochemical evidence of tyrosinemia, a trial of the tyrosinemia diet was advised, but it was not followed up by the parents because of financial constraints. The couple was also carriers of likely pathogenic variants in the other two genes (SDR9C7 and DUOX2 genes). The couple in case 12 had two children who expired after vocal cord paralysis within 1 to 2 months of life, and both parents were carriers of a likely pathogenic variant c.1817G > A in the IGHMBP2 gene, causing Charcot–Marie–Tooth disease, type 2 (CMT2; [Table 3]). The couple were also carriers of the CFTR gene. Both neonates of the couple suffered severe respiratory distress and stridor at birth and were diagnosed with bilateral vocal cord palsy at the age of 1 month and 15 days of life.

The couple in case 14 had four pregnancy losses with multiple congenital malformations and GDD, and was a carrier of VUS c.1361A > G in the KLHL7 gene, causing Perching syndrome ([Table 4]). The first pregnancy had polyhydramnios with a male child having a birth weight of 1.5 kg, and a neonatal intensive care unit stay for 40 days. This baby presented with extension deformities in wrists and ankles, cleft lip and palate, micrognathia, GDD, hypertonia, congenital heart defect (CHD) (patent ductus arteriosus [PDA], atrial septal defect, and ventricular septal defect), severe pulmonary hypertension, and ventriculomegaly. This child passed away at 4 years of age. The second pregnancy was a female child born with a full term normal delivery. Polyhydramnios was again noted in this pregnancy. This baby had a birth weight of 2.5 kg and presented with a cleft palate, possible CHD, and similar looks to the first child, cortical thumb, and passed away in sleep at 2 months of age. The third child was a male fetus, and an intrauterine death at 8 months of gestation and polyhydramnios were noted. The fourth pregnancy was a male with antenatally detected anomalies like pulmonary stenosis, PDA, and pulmonary atresia. This fourth baby was born preterm, weighing 1.5 kg. Dysmorphism was noted in this baby, like dolichocephaly, long forehead, megalocornea, long philtrum, and micropenis. MRI of the brain performed on this baby was suggestive of hypoplastic corpus callosum. The baby expired at one and a half months of age. The clinical features in the previous child and pregnancies matched with the diagnosis of Perching syndrome as described in [Table 4]. This couple were carriers of another disease in the PIEZO1 gene. Couples in case 15 were heterozygous for a VUS in the ASS1 gene, and typical manifestations in the previous baby with encephalopathy, causing citrullinemia type 1 ([Table 4]). The child had hyperammonemia with ammonia levels of more than 800 micromoles/L. Tandem mass spectrometry and urine gas chromatography-mass spectrometry were advised, but the child expired at 10 days of life, and it was not sent.

A total of 6 pathogenic, 8 likely pathogenic, and 10 VUSs were detected in the study based on the ACMG/AMP classification. Couples in cases 6, 8, 9, 11, and 14 were consanguineous couples and were carriers of pathogenic variants in more than one disease ([Tables 2] [4]).


 

Discussion

In this study, we describe the WES carrier testing results of couples with a previous bad obstetric history when no proband genetic diagnosis was available.

We included couples with a positive family history, with at least one deceased child who was suspected to be suffering from a rare undiagnosed disease or because of multiple affected fetuses with congenital anomalies. A likely causative variant for the symptoms of the deceased children was identified in 5 out of the 15 couples investigated (33.33%). Out of the 12 couples, 5 couples were carriers of pathogenic/likely pathogenic variants, and the remaining 7 couples were carriers of VUS with phenotype matching the deceased child or fetal anomalies. In total, 11/15 couples were consanguineous ([Tables 1] to [4]). Without a genetic diagnosis through WES in the proband, pinpointing causative variants in the parents is challenging. However, based on phenotypic features, many reports in our study have identified VUSs. A detailed discussion of the variants and other studies describing similar phenotypes is discussed below.

The couple in case 2, with complex congenital heart diseases in previous pregnancies, had a pathogenic variant in the DNAI2 gene, causing PCD. According to a study by Kennedy et al, at least 6.3% of patients with PCD have heterotaxy, and most of those have cardiovascular abnormalities. The prevalence of congenital heart disease with heterotaxy is 200 fold higher in PCD than in the general population (1:50 vs. 1:10,000).[5]

The couple in case 3 with fetal brain malformation were carriers of heterozygous VUS c.198 G > T in the TRAPPC12 gene, probably matching progressive encephalopathy with brain atrophy and spasticity. This variant is described as VUS in the ClinVar database.[4] In a study by Milev et al, three patients with TRAPP12 gene variants are described and all of them have either the absence or severe thinning of the corpus callosum.[6] Loss-of-function variants are pathogenic for this gene, and most missense variants are VUS.

The couple in case 5 exome revealed a heterozygous carrier state in the couple for the pathogenic variant c.1556G > A in the ALDH7A1 gene, causing pyridoxine-dependent epilepsy. This variant has already been reported in a homozygous state in another Indian girl who presented with neonatal seizures and GDD.[7]

The couple in case 6 had two children who expired of suspected hemophagocytosis. The parents' exome detected that they both were carriers of pathogenic variants in the UNC13D gene, C.615G > A. This variant is novel, as it is not found in ClinVar or existing literature. The couple in case 13 were heterozygous carriers of the c.386G > C variant in the PRF1 gene. This variant is already listed in ClinVar and has been reported in Indian patients.[8] [9] [10]

The couple in case 7 did not have a direct fetal anomaly, and chromosomal microarray was done at the time of the second spontaneous abortion, as indicated for suspected numerical or structural chromosomal aberration as a cause. WES was advised as the couple was consanguineously married, and there was a late intrauterine death or stillbirth. Assuming that many genetic conditions yet remain to be discovered and the full phenotypic spectrum of many known Mendelian disorders is not fully understood, WES has been advised for the couple. In a study by Stanley et al, exome sequencing had an 8.5% yield for stillbirth.[11]

The exome analysis of the couple in case 9 revealed heterozygous VUS in the ETHE1 gene, which was matching with the symptoms in children. Both parents were heterozygous carriers for the variant c.461A > T, and this variant was reported as a VUS in ClinVar. Six cases of ethylmalonic acidemia were reported recently from India, where the phenotype matched with our patient.[12]

A couple in case 10 had a VUS in ITPA gene c.533G > A, causing developmental and epileptic encephalopathy 35. The review by Scala et al showed phenotype closely matching with the disease. The c.533G > A variant is listed as a VUS in ClinVar, with strong computational evidence suggesting pathogenicity (https://varsome.com/variant/hg19/ITPA, accessed on November 19, 2024).[13]

The couple in case 11 had a proband with features suggestive of tyrosinemia, negative urine succinyl acetone, and normal tyrosine levels. False negatives have been reported with succinyl acetone testing, and since the couple's variant is classified as a VUS with uncertain pathogenicity, the diagnosis remains unresolved for now.[14]

In case 12, the couple's two children died following vocal cord paralysis, and genetic testing revealed both parents were carriers of the likely pathogenic variant c.1817G > A in the IGHMBP2 gene, associated with CMT2. Congenital vocal cord paralysis is reported most frequently in CMT1 and is a known feature of CMT.[15] Another study by Cottenie et al described 15 patients, and Schottmann et al described 3 patients with different phenotypes of CMT, but none had congenital vocal cord paralysis. It was the first time described in our probands.[16] [17] The couple in case 14 had four pregnancy losses with multiple congenital malformations with GDD, and the couple was a carrier of VUS c.1361A > G in the KLHL7 gene, causative of Perching syndrome. Bruel et al described the phenotypic features of six patients whose features matched those described in the previous four pregnancy outcomes in our cases. The variant is not described in ClinVar and is a novel variant.[18] As the couple had babies with recurrent congenital malformations, a chromosomal microarray test performed in the last pregnancy was reported as unremarkable. A couple of karyotypes can definitely be documented in this couple, as the current classification of the variant in the KLHL7 gene is a VUS.

Our study suggests that couple exome sequencing is a valuable diagnostic strategy when a proband DNA-based diagnosis is unavailable. In 9 out of 15 families, more than one child or pregnancy was affected, highlighting the need for greater awareness about couple exome sequencing as a diagnostic option. There is a need for increased awareness among pediatricians and obstetricians to facilitate early suspicion of genetic diseases. Performing the DNA banking of the affected fetuses or children of the couple is the next most important step for a definitive diagnosis of the problem. WES is a valuable tool for optimizing future pregnancy outcomes, as illustrated in this case series. Janssens et al have described the need for adequate pre- and post-test counseling in couples with consanguineous marriages and couples with previous losses to foster informed, autonomous reproductive decision making and provide support for couples found to be at risk.[19] The National Family Health Survey-4 conducted in 2015 to 2016 showed that the overall prevalence of consanguineous marriage was 9.9% in India.[20] In a study by Reuter et al, the yield of WES was 36.8% in consanguineous couples.[21] In the study by Huang et al, WES was a high yield strategy in three groups of couples, which are those with a past history of adverse pregnancy outcomes, previous genetic disease, and couples with consanguineous marriage. The detection rate of causative variants in couples was maximum with consanguineous marriage and followed by couples with adverse pregnancy outcomes.[22] Preconceptional or premarital counseling becomes valuable in regions of high consanguinity in the community. In the study by Sallevelt et al, 100 consanguineously married couples underwent the WES test for detection of the pathogenic and likely pathogenic disease-causing variants for the known autosomal recessive diseases, and 28 couples were harboring the variants (28%).[23] In another study by Zhang et al, 5.34% of the couples at risk (n = 56) were heterozygous for the same autosomal recessive disease (56 out of 1,048 couples).[2] We had 5/15 cases with pathogenic and likely pathogenic variants in heterozygous states in the couples (33.33%).

We had a higher prevalence of consanguineous marriages in our study couples (73%) and a higher yield of the same heterozygous variants in those couples. Seven couples were carriers for more than one disease variant in the WES, which was also helpful for consideration of prenatal testing in future pregnancies. ACMG guidelines recommend prenatal diagnostic testing for families with pathogenic or likely pathogenic variants. In our series, prenatal testing can be performed in cases 2, 5, 6, 12, and 13 (33.33%). When a phenotype closely matches a VUS in a relevant gene, further diagnostic approaches are warranted to reclassify the VUS. Reclassification of a VUS to pathogenic or likely pathogenic status could facilitate prenatal testing in future pregnancies. Functional studies, RNA sequencing, and RT-PCR (reverse transcription-polymerase chain reaction) quantification can help establish the pathogenicity of the variant. Reanalysis of next generation sequencing data can uncover previously missed pathogenic or likely pathogenic variants matching the phenotype. A study by Zastrow et al described several approaches to help reclassify inconclusive or negative exomes into actionable results.[24] In the study by Komlosi et al, the authors have described the results of 13 couples by using a gene panel of a set of 430 genes known to be causative for rare autosomal recessive diseases with poor prognosis. Five couples received the causative diagnosis out of the 430 genes of the panel. An additional two cases were diagnosed based on WES.[25] The small number of cases is a limitation of the study and further such studies with larger sample sizes should identify common autosomal recessive conditions relevant to the Indian population. The strategies to resolve the VUS status of variants were beyond the scope of this article. More publications are needed on multidisciplinary approaches to determine VUS pathogenicity, enabling accurate and ethical prenatal testing for couples. In general, couple exome sequencing is particularly valuable when proband diagnosis is unavailable, especially if it identifies heterozygous pathogenic or likely pathogenic variants matching the proband's condition. Pre- and post-test counseling by a clinical geneticist is essential for couples to grasp complex genetic test results.


 

Conclusion

Our data showed that WES in couples based on phenotype in previous pregnancies or children was a high yield strategy for identifying lethal autosomal recessive disorders.


 

 

Conflict of Interest

None declared.

Acknowledgments

The authors thank the patients' parents who helped them with their children's biochemical testing.

Name of the Department and Institution to which the Work should be Attributed

Institution: Department of Pediatrics, MGM Medical College, N-6, CIDCO, Aurangabad -431002; MGM Institute of Health Sciences (MGM IHS, Navi Mumbai) Maharashtra, India.


Author Contribution

S.M., G.M., and A.A. were involved in the review of literature, data collection, and wrote the first draft. S.M., G.M., D.K., A.D., L.R., and M.M. were involved in management of patients, designing of study, drafting the article, analysis and interpretation of data, and will act as guarantor. S.M., G.M., D.K., A.D., L.R., M.M., and A.A. were involved in the management of patients and data collection. The final manuscript was approved by all the authors.


Ethical Clearance

The above study has been approved by ethical committee of MGM Medical College and University.



Address for correspondence

Suvarna Magar, MBBS, DCH, DNB
Department of Pediatric, MGM Medical College
N-6, CIDCO, Aurangabad 431002, Maharashtra
India   

Publication History

Article published online:
25 July 2025

© 2025. Society of Fetal Medicine. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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