Abstract
Purpose:
To evaluate whether deep prenatal phenotyping of fetal brain abnormalities (FBAs) increases diagnostic yield of trio-exome sequencing (ES) compared with standard phenotyping.
Methods:
Retrospective exploratory analysis of a multicenter prenatal ES study. Participants were eligible if an FBA was diagnosed and subsequently found to have a normal microarray. Deep phenotyping was defined as phenotype based on targeted ultrasound plus prenatal/post-natal magnetic resonance imaging, autopsy, and/or known phenotypes of other affected family members. Standard phenotyping was based on targeted ultrasound alone. FBAs were categorized by major brain findings on prenatal ultrasound. Cases with positive ES results were compared with those that have negative results by available phenotyping, as well as diagnosed FBAs.
Results:
A total of 76 trios with FBAs were identified, of which 25 (33%) cases had positive ES results and 51 (67%) had negative results. Individual modalities of deep phenotyping were not associated with diagnostic ES results. The most common FBAs identified were posterior fossa anomalies and midline defects. Neural tube defects were significantly associated with receipt of a negative ES result (0% vs 22%, P = .01).
Conclusion:
Deep phenotyping was not associated with increased diagnostic yield of ES for FBA in this small cohort. Neural tube defects were associated with negative ES results.
Keywords: Congenital anomalies, Deep phenotyping, Exome sequencing, Fetal brain anomalies, Prenatal diagnosis
Introduction
Brain malformations rank among the most common fetal abnormalities, with approximately 1 to 2 per 1000 live births affected with a central nervous system (CNS) abnormality.1 Fetal brain abnormalities (FBAs) are responsible for 40% to 50% of pediatric developmental delays.2 Ultrasound is the first-line screening tool to identify fetal structural anomalies, and prenatal diagnostic testing to determine genetic causes of congenital abnormalities relies predominantly on chromosomal microarray.3
In the setting of prenatally diagnosed congenital anomalies, in which standard genetic testing including microarray is negative, newer technologies such as exome sequencing (ES) have demonstrated improved diagnostic yield ranging from 15% to 32%.4–7 Accurate interpretation of identified variants on ES is heavily reliant upon available phenotypic information, which is often limited to prenatal ultrasound findings alone.8 Deep prenatal phenotyping includes ultrasound assessment of congenital anomalies plus additional information obtained from prenatal or postnatal magnetic resonance imaging (MRI), autopsy, and phenotypic information from prior affected pregnancies. Emerging data show that deep phenotyping, particularly autopsy, improves diagnostic yield of ES in pregnancies complicated by major fetal anomalies.9–13 For inconclusive or suspicious ultrasound findings in the brain, fetal MRI can provide more phenotypic detail and improve the diagnosis of complex CNS malformations.14–17
Advanced imaging techniques, however, have yet to be intersected with prenatal sequencing efforts. To date, there are no guidelines on specific prenatal phenotypic information for prenatal diagnosticians to obtain in the context of prenatal sequencing. Our objective was to evaluate whether deep prenatal phenotyping of FBAs would increase diagnostic yield of parent-fetus trio-exome sequencing compared with standard prenatal ultrasound phenotyping alone.
Materials and Methods
This was a retrospective exploratory analysis of a cohort study of ES for pregnancies complicated by major structural fetal anomalies. Maternal-paternal-fetal trios were identified from prenatal diagnosis clinics at the University of North Carolina at Chapel Hill and various referring clinics in the United States between January 2015 and March 2021. Approval from the Institutional Review Board at the University of North Carolina at Chapel Hill (IRB 13–4084) was obtained before patient consent and enrollment. Inclusion criteria for this analysis included the following: singleton pregnancy, FBA detected by prenatal ultrasound, non-diagnostic chromosomal microarray, no known infectious or teratogenic etiology of identified anomaly, and fetal and parental DNA available.
Prospectively, trios meeting inclusion criteria were approached for participation after decision regarding continuation of the pregnancy was made. In cases of non-continuing pregnancies, the study was not mentioned until after the couple made the decision to terminate the pregnancy. Additionally, trios were identified retrospectively by querying the UNC Perinatal Database for women with a prior pregnancy complicated by fetal or neonatal death or pregnancy termination due to a congenital anomaly that was not explained by standard prenatal genetic testing. Women who met the inclusion criteria, previously indicated a desire to be contacted if additional fetal testing became available, and had fetal cells archived and available for DNA extraction were approached for enrollment. Additional participants were either self-referred or referred by a clinician aware of study recruitment.
After enrollment, parental blood was collected and stored fetal samples were retrieved for ES analysis. Additional phenotypic information, including prenatal ultrasounds, prenatal or postnatal MRI, and autopsy reports from the affected pregnancy and previously affected pregnancies (when available) were obtained from the referring treatment providers. Phenotyping beyond targeted anatomical ultrasound was not systematically performed nor required for participation in the study and as such was performed at the discretion of the treating provider. Reports and images (when provided) from available phenotyping were reviewed by the research team, but not reinterpreted or assessed for accuracy.
Parents received pretest counseling about ES and the possible results that may be provided, and consent was obtained separately from each parent. Because of the complexity of the genetic information obtained from ES, consent and return of results were performed by a certified genetic counselor who was not involved in the patient’s clinical care to avoid bias and undue pressure to participate. Additional information regarding genetic counseling points, reported results, DNA extraction, creating duplicate samples, and backup cultures are described in our prior work (Vora et al).18
Although trio DNA samples and prenatal phenotypic information were obtained from multiple sites, all sequencing efforts were performed in a research sequencing lab at our institution. We created ES libraries and exome capture from maternal, paternal, and fetal DNA samples and transferred them to the UNC High Throughput Sequency Facility for sequencing using Illumina Hi-Seq 2500 or Hi-Seq 4000 instruments. Raw-read data were processed, mapped, and aligned, and variants were identified using a standard bioinformatics pipeline developed for the NCGENES project in collaboration with colleagues in the Department of Genetics and the Renaissance Computing Institute. Further information on bioinformatics analysis, quality checks, and workflow, including variant classification, are provided in the supplemental material of our prior study (Vora et al).18
ES results were reviewed by a multidisciplinary committee, including clinical and laboratory geneticists, prenatal geneticist, genetic counselors, and a pediatric geneticist not involved in the patient’s clinical care. Results were classified into 8 categories (Figure 1), and trios could receive more than 1 result. Positive results included those with a pathogenic variant, likely pathogenic variant, and/or variant of uncertain significance that explained the fetal phenotype. All other results were considered negative. Review of all potentially reportable findings was completed by the committee within 2 weeks of variant identification by the primary analyst. Positive results were confirmed in a Clinical Laboratory Improvement Amendments certified molecular genetics clinical laboratory using Sanger sequencing before being reported to parents. All trios with negative sequencing results were periodically reanalyzed to determine variant classification changes or to determine if newly identified genes were responsible for the fetal phenotypes.
Figure 1. Classification scheme of case-level results.
Case level results are classified using guidelines adapted from Clinical Sequencing Exploratory Research (CSER) Consortium. Table adapted from Vora et al.18 AR, autosomal recessive; CNV, copy number variant; CSER, Clinical Sequencing Exploratory Research; Het, heterozygous; VUS, variant of uncertain significance.
The primary analysis was to compare cases of FBA that have positive ES results with those that have negative ES results by available phenotyping. For this purpose, standard phenotyping was defined as phenotype based on ultrasound alone, and deep phenotyping was defined as phenotype based on ultrasound plus additional information from prenatal or postnatal MRI, autopsy, or known phenotypes from prior affected pregnancies. The secondary analysis aimed to evaluate diagnostic yield based on category of FBA findings on prenatal ultrasound, to assess if any group of FBA was associated with receipt of a positive ES result. FBA categories included posterior fossa anomalies, disorders of cerebrospinal fluid, midline defects, cortical disorders, and neural tube defects (NTDs). χ2 was performed for both primary and secondary analyses. In addition, pairwise comparisons were performed using Tukey’s method; P < .05 was considered statistically significant. All analyses were performed using STATA15.
Results
A total of 166 trios were sequenced, of which 76 had a diagnosed FBA on prenatal ultrasound. Of the 76 trios with FBA, 25 (33%) cases had a positive ES result and 51 (67%) had a negative result. The phenotype and sequencing results for positive and negative cases are summarized in Tables 1 and 2, respectively. There was no difference in the number of cases with multisystem organ abnormalities (defined as the presence of 2 or more unrelated major structural anomalies by EUROCAT definition19) between groups (88% vs 78%, P = .30). Table 3 demonstrates the maternal characteristics of the cohort included in this analysis. The majority of the cohort was married, employed with a college education and household income >$75,000/year. A total of 3 cases with positive ES had known consanguinity with multiple regions of homozygosity, whereas 1 case with positive ES and 1 case with negative ES had only 1 region of homozygosity, indicating a more distant relationship. A total of 37 trios (49%) had at least 1 form of deep phenotyping available for review. Sixteen cases (21%) had at least 1 prior affected pregnancy for which phenotypic information was available for review.
Table 1.
Phenotype and sequencing results from cases with a positive ES result
| Prenatal FBAa | Multisystemic Anomalies | ES Resultb | Deep Phenotype | Category of FBA |
|---|---|---|---|---|
| Smooth brain (HP:0001339) | Arthrogryposis; micrognathia; bilateral pleural effusion; skin edema; VSD; polyhydramnios |
MUSK (Possible) NM_005592.3c.1724T>C [p.Ile575Thr]/ c.2408A>G [p.Tyr803Cys] |
Autopsy MRI | Cortical disorder |
| Intracranial cyst in posterior fossa, no flow on ultrasound (HP:0007291) Suspected absent cerebellum (HP:0007360) |
Dolichocephaly; echogenic kidneys; pericardial effusion; oligohydramnios |
TMEM67 (Definite) NM_153704.5 c.579_580delAG [p.Gly195fs]/ c.622A>T [p.Arg208Ter] |
Posterior fossa | |
| Hydrocephalus (HP:0000238) Hypoplastic cerebellum (HP:0001321) “Obliterated” cisterna magna (N/Ac) Arnold Chiari malformation (HP:0002308) |
Cystic hygroma; abdominal ascites and skin edema; omphalocele; echogenic bowel; broad abducted thumbs |
WDR81 (Probable) NM_001163809 c.218delT [p.Gly74fs]/ c.2836_2839delTTTG [p.Phe946fs] |
CSF disorder Posterior fossa | |
| Mild bilateral ventriculomegaly (HP:0002119) Enlarged cisterna magna (HP:0011427) |
Hydrops; urinary tract malformation, urinary tract dilation; malrotated gut |
ITPR1 (Probable) NM_001168272 c.7591G>A [p.Val2531Met] de novo |
CSF disorder Posterior fossa | |
| Arachnoid cyst (HP:0100702) Abnormal “intracranial anatomy” (N/A) Two affected pregnancies. |
Micrognathia; low set ears; proptotic orbits; renal pyelectasis; all long bones short in arms and legs, clenched hands, clubbed right foot; suspected overriding aorta. Prior pregnancy with large VSD, Tetralogy of Fallot; low set ears, micrognathia; ambiguous genitalia; shortened long bones in upper and lower extremities, bilateral clenched hands and clubbed, rocker-bottom feet; scoliosis and kyphosis |
ALG3 (Possible) NM_005787 c.487C>T [p.Arg163Cys]/ c.1154G>C [p.Arg385Thr] |
Autopsy Prior affected pregnancy | Midline defect |
| Hypoplastic cerebellum (HP:0001321) Absent corpus callosum (HP:0001274) |
All long bones with at least a 5-week growth lag at 20 weeks, by 27 weeks, entire fetus was at the 0%ile; short ribs; AVSD; abnormal flattened profile with short nasal bone; bilateral postaxial polydactyly; ambiguous genitalia |
DHCR7 (Probable) NM_001360 c.964-1G>C splice site/ c.439G>A [p.Gly147Ser] |
Midline defect Posterior fossa | |
| Enlarged cisterna magna (HP:0011427) Ventriculomegaly (HP:0002119) Three affected pregnancies. |
Enlarged bladder with distorted abdomen; extremely short long bones and small chest; bilateral polydactyly on hands; neck fixed in a flexed position. Prior pregnancies have also shown thick nuchal translucency/cystic hygroma. |
TRAF3IP1 (Possible) NM_015650.3 c.169G>A [p.Glu57Lys]/ c.988-1G>C splice site |
Prior affected pregnancy | CSF disorder Posterior fossa |
| Brachycephaly (HP:0000248) Bilateral choroid plexus cysts (HP:0002190) Abnormal cavum septum pellucidum (N/A) |
Adrenal hypoplasia (microscopic remnants on autopsy); multiple renal arteries; 2-vessel cord; sloping forehead; micrognathia; low set ears; bilateral clenched hands; pectus excavatum; ambiguous genitalia |
TRAIP (Possible) NM_005879 c.140 C>T [p.Pro47Leu]/ c.553 C>T [p.Arg185*] |
Autopsy | Midline defect |
| Dandy Walker malformation (HP:0001305) Four affected pregnancies (2 prior with encephalocele). |
Bilateral polydactyly of hands, bilateral overlapping toes; bilateral enlarged echogenic kidneys with cortical rays and microcysts. |
CEP290 (Definite) NM_025114 c.384_387 delTAGA [p.Asp128Glufs]/ c.1936 C>T [p.Gln646Ter] |
Autopsy Prior affected pregnancy | Posterior fossa |
| Bilateral ventriculomegaly (HP:0002119) | Bilateral cleft lip and palate; hypertelorism; absent stomach bubble; diaphragmatic hernia; possible right sided aortic arch |
CHD7 (Definite) NM_017780.3 c.282delT [p.Asn96fs] de novo |
Autopsy MRI | CSF disorder |
| Absent corpus callosum (HP:0001274) | Growth restriction <3rd percentile; small right eye or coloboma; ambiguous genitalia; ventricular septal defect |
DPH1 (Probable) NM_001383.3 c.374T>C [p.Leu125Pro] homozygous |
MRI | Midline defect |
| Severe ventriculomegaly, normal head circumference (HP:0002119) Thin cortical mantle (N/A) Vermian hypoplasia (HP:0001320) Cerebellum <1st percentile with small cystic regions (HP:0001321) Possible rhombencephalosynapsis (HP:0031913) |
Arthrogryposis - contractures of elbows, hips, knees, feet; elbows show bilateral pterygium; cleft palate with micrognathia |
DYNC1H1 (Probable) NM_001376.4 c.7999A>G [p.Asn2667Asp] de novo |
MRI | CSF disorder Posterior fossa |
| Echogenic borders of the cerebral ventricles with calcifications (HP:0007229) Hyperechoic parenchyma surrounding ventricles (N/A) Additional calcifications in the choroid plexus, corpus callosum, thalamus (HP:0002514) |
Bitemporal bowing; micrognathia; hypoplastic/absent nasal bone; cleft palate; exophthalmos; possible VSD; head circumference and long bones measuring small; narrow thorax with short ribs |
FAM20C (Definite) NM_020223.3 c.456dupC [p.Gly153fs] de novo |
Midline defect | |
| Holoprosencephaly (HP:0001360) | Midline abnormal proboscis, anophthalmia; omphalocele; heterotaxy with a complex heart defect; echogenic bowel; echogenic kidneys; ambiguous genitalia |
TBC1D32 (Probable) NM_152730.5 c.3325_3326delAG [p.Ser1109fs] homozygous |
Autopsy | Midline defect |
| Absent cavum septum pellucidum (HP:4000138) Cortical dysplasia (HP:0002539) |
Cardiac defect; hydronephrosis; hypoplastic nasal bone; hydrops; polyhydramnios |
PTPN11 (Definite) NM_002834.4 c.1381G>A [p.Ala461Thr] de novo |
MRI | Midline defect Cortical disorder |
| Hydrocephalus with 3rd and 4th ventricle dilation (HP:0000238, HP:0002198, HP:0007082) | Nuchal translucency at 12 weeks 4.5 mm; flat profile; VSD vs Tetralogy of Fallot; single umbilical artery |
ARID1A (Definite) NM_006015.4 c.2527_2528insC [p.Gly843fs] de novo |
CSF disorder | |
| Small cerebellum <1% (HP:0001321) Microcephaly (HP:0000252) |
Brachycephaly; low set ears; abnormal profile with micrognathia; sloped forehead; possible heart defect |
EFTUD2 (Definite) NM_004247.3 c.2698_2701delGTCT [p.Val900fs] de novo |
Posterior fossa | |
| Cerebellar hypoplasia, onset in third trimester (HP:0001321) Three affected pregnancies. | Third trimester onset of low fetal movement, arthrogryposis, and polyhydramnios |
TK2 (Probable) NM_004614.4 c.469_470insTGGG [p.Asp157fs] homozygous |
MRI Prior affected pregnancy | Posterior fossa |
| Cerebellar hypoplasia <1%ile at 18 weeks (HP:0001321) Hypoplastic corpus callosum (HP:0002079) Microcephaly <2nd percentile at 18 weeks (HP:0000252) |
Echogenic bowel |
EFTUD2 (Probable) NM_004247.3 c.1187T>C [p.Leu396Pro] de novo |
Midline defect Posterior fossa | |
| Absent cavum septum pellucidum (HP:4000138) Absent corpus callosum (HP:0001274) Splayed cerebellum (N/A) Incomplete vermis (HP:0002951) 4th ventricle dilation (HP:0002198) |
Right cleft lip; small omphalocele; cardiac defect including double outlet right ventricle vs Tetralogy of Fallot; suspected bilateral colobomas |
CHD7 (Probable) NM_017780.3 c.5074G>T [p.Gly1692*] de novo |
MRI | Midline defect Posterior fossa |
| Dandy Walker malformation (HP:0001305) Bilateral choroid plexus cysts (HP:0002190) |
Nasal bone hypoplasia |
TUBB (Probable) NM_178014.4 c.185G>A [p.Arg62His] de novo |
Posterior fossa | |
| Severe hydrocephalus vs holoprosencephaly on 15 week ultrasound (HP:0000238 vs HP:0001360) Hypoplastic cerebellar vermis (HP:0006817) |
Arthrogryposis of upper and lower extremities |
TUBB2B (Probable) NM_178012.5 c.1172G>T [p.Arg391Leu] de novo |
CSF disorders Posterior fossa | |
| Holoprosencephaly (HP:0001360) Three affected pregnancies. |
None |
ZIC2 (Probable) NM_007129.5 c.217dupC [p.Gln73fs] (Maternal) |
Prior affected pregnancy | Midline defect |
| Dandy Walker (HP:0001305) Splayed cerebellum (N/A) Absent vermis (HP:0006817) 3rd and 4th ventricle dilation (HP:0002198, HP:0007082) Hydrocephalus (HP:0000238) Absent cavum septum pellucidum (HP:4000138) |
None |
POMT1(Probable) NM_001077365.1 c.1031G>A [p.Cys344Tyr]/ c.1436delG [p.Gly479fs] |
CSF disorder Posterior fossa | |
| Hydrocephalus (HP:0000238) Third ventricle dilation (HP:00007082) Small cerebellum (HP:0001321) Lemon sign (HP:0032269) Dangling choroids (N/A) Absent cavum septum pellucidum (HP:4000138) Prior affected pregnancy with occipital encephalocele. |
None Prior affected pregnancy with cleft lip |
B3GALNT2 (Possible) NM_152490.5 c.652-1G>C / c.23T>G [p.Leu8Arg] |
Prior affected pregnancy | CSF disorder Midline defect Posterior fossa |
AVSD, atrioventricular septal defect; CSF, cerebrospinal fluid; ES, exome sequencing; FBA, fetal brain abnormality; MRI, magnetic resonance imaging; VSD, ventricular septal defect.
Prenatal FBA written as description from available phenotyping as well as associated HPO term, when available.
Case level results are classified using guidelines adapted from Clinical Sequencing Exploratory Research (CSER) Consortium. “Definite” results indicate pathogenic variants only. “Probable” results indicate likely pathogenic variants. “Possible” indicates that at least 1 variant is a variant of uncertain significance according to ACMG criteria at the time of analysis.
N/A denotes HPO term not available for this fetal phenotype. HPO terms do not include many prenatal specific phenotypes at this time.
Table 2.
Phenotype and sequencing results from cases with a negative ES result
| Prenatal FBAa | Multisystemic Anomalies | Deep Phenotype | Category of FBA |
|---|---|---|---|
| Cerebellar hypoplasia (HP:0001321) Intracranial cysts (HP:0010576) Abnormal calvarium (HP:0002683) Non-visualization of cavum septum pellucidum (HP:4000138) |
Growth restriction, echogenic kidneys (polycystic), echogenic bowel, hypotelorism | Midline defect Posterior fossa |
|
| Schizencephaly (HP:0010636) | Arthrogryposis | Prior affected pregnancy | Cortical disorder |
| Dandy Walker malformation (HP:0001305) Absent corpus callosum (HP:0001274) Cerebellar and vermian hypoplasia (HP:0001321, HP:0001320) |
Small to absent left orbit | MRI | Midline defect Posterior fossa |
| Vermis abnormality of the cerebellum (HP:0002334) Dandy Walker variant (HP:0001305) |
Truncus arteriosis with VSD | Posterior fossa | |
| Hypoplastic cerebellum (HP:0001321) | Cleft lip/palate; unilateral absent radius | Posterior fossa | |
| Bilateral ventriculomegaly (HP:0002119) Agenesis of corpus callosum (HP:0001274) |
Hypertelorism; omphalocele; likely VSD | CSF disorder Midline defect |
|
| Severe hydrocephalus vs hydranencephaly (HP:0000238 vs HP:0002324) | Amniotic bands arising from umbilical cord, acute angulation of lumbar spine without an ONTD, bilateral facial clefts (lips, palate, nose), fused eyes, left ear small and low set, absent left arm, giant omphalocele with liver, stomach and bowel, bilateral clubbed feet with rocker-bottom appearance, syndacytyly of toes on left foot | CSF disorder | |
| Bilateral ventriculomegaly (HP:0002119) Cerebellar hypoplasia (HP:0001321) Abnormal cavum septum pellucidum (HP:4000138) |
Bilateral hydronephrosis; arthrogryposis with clubbed hands and feet; polyhydramnios | CSF disorder Midline defect Posterior fossa |
|
| Anencephaly (HP:0002323) Previous pregnancy with anencephaly. |
Previous pregnancy with hand webbing | Autopsy Prior affected pregnancy |
Neural tube defect |
| Anencephaly (HP:0002323) | Diaphragmatic hernia, lower lip cleft | Neural tube defect | |
| Enlarged cavum septum pellucidum (N/Ab) Abnormal posterior fossa (HP:0000932) |
Diaphragmatic hernia; shortened long bones; Y shaped gluteal cleft; abnormal facial appearance (hypotelorism, flattened nasal bridge) | Autopsy | Midline defect Posterior fossa |
| Holoprosencephaly (HP:0001360) | Absent nose, absent or hypoplastic orbits (possible single orbit) | Midline defect | |
| Abnormal posterior fossa (HP:0000932) Absent corpus callosum (HP:0001274) Large cystic structure (N/A) |
Pseudocleft of upper lip with prenasal edema; AVSD; borderline platyspondyly; upper extremities: bilateral polydactyly and syndactyly, short radius and ulna; lower extremities: short femurs, short tibias, absent fibulae, polydactyly, clubbed feet. | Midline defect Posterior fossa |
|
| Hypoplastic cerebellum (HP:0001321) Absent cavum septum pellucidum (HP:4000138) Ventriculomegaly (HP:0002119) Two affected pregnancies. |
IUGR, micrognathia, heart defect, arthrogryposis. | Prior affected pregnancy | CSF disorder Midline defect Posterior fossa |
| Ventriculomegaly (HP:0002119) Absent cavum septum pellucidum (HP:4000138) Absent corpus callosum (HP:0001274) |
IUGR, renal agenesis, cleft lip/palate, esophageal pouch, heart defect, arthrogryposis. Two affected pregnancies. | Prior affected pregnancy | CSF disorder Midline defect |
| Severe hydrocephalus with no normal brain anatomy seen (HP:0000238) | Macrosomia: Dating was based on an early first trimester ultrasound. At 20 wk 3 d - measured 23 wk 6. Head, abdomen, femurs, all large (+4 weeks) At 25 wk - measured 31 wk 1 d (+6 weeks) At 31 wk 1 d - measured 37 wk 2 d (+6 weeks) Frontal bone (head) scalloping; Heart defect - hypoplastic left heart. Right sided heart; Bilateral hydronephrosis/hydroureter; Bilateral clubbed feet. |
MRI | CSF disorder |
| Open neural tube defect (HP:0034237) | Scoliosis/kyphosis; Coarctation of the aorta; Absent left arm with small left hand (no humerus, forearm, no bones in small hand), Rocker bottom feet | Neural tube defect | |
| Hydrocephalus (HP:0000238) | First trimester cystic hygroma; Bilateral radial ray malformation with single forearm bone on the right. Absent stomach (possible esophageal atresia); Abnormal cardiac axis, “flat” AV valve, VSD, possible double outlet right ventricle; Right renal agenesis/cystic kidney; Intrabdominal cyst with echogenic rim and associated peritoneal calcifications concerning for early bowel perforation. |
CSF disorder | |
| Small cerebellum (HP:0001321) Smooth brain (HP:0001339) Calcifications (HP:0002514) Arachnoid cyst (HP:0100702) |
IUGR, frontal bossing, Hydroureter, hydronephorosis, long bones short, clubbed feet, left hand fixed, hyperextended, clinodactyly | Posterior fossa Cortical disorder |
|
| Encephalocele (HP:0002084) | Heart defect, omphalocele, cleft lip/palate, right humerus, radius, ulna are missing with 3 digits arising from the scapula, polyhydramnios | Neural tube defect | |
| Microcephaly (HP:0000252) Lissencephaly (HP:0001339) One prior affected pregnancy. |
IUGR, heart defect, pancytopenia, sloping forehead, inverted nipples, Pancreatic atrophy | Autopsy MRI and autopsy on first affected (lived 16 months) Prior affected pregnancy |
Cortical disorder |
| Severe hydrocephalus (HP:0000238) | Cleft lip/palate, horseshoe kidney, polyhydramnios, sacral dimple, VSD | MRI | CSF disorder |
| Enlarged cisterna magna (HP:0011427) Absent corpus callosum (HP:0001274) |
VSD, thickened myocardium, IUGR, ambiguous genitalia. IUFD. | Midline defect Posterior fossa |
|
| Alobar holoprosencephaly (HP:0006988) | Midface hypoplasia, cyclopia. Heart defect - single ventricle. Dilated bowel, enlarged bladder. Cystic mass in umbilical cord. Ambiguious genitalia |
Midline defect | |
| Aprosencephaly (HP:0007268) Microcephaly (HP:0000252) |
Hypoplastic adrenal glands, hypotelorism, hypoplastic palpebral fissures, absent orbits, proboscis, small mouth, anal atresia, heart defect, hypoplastic right lung with single lobe | Autopsy | Midline defect |
| Hydrocephalus (HP:0000238) Thin cortex (N/A) Dangling choriods (N/A) Hypoplastic cerebellum (HP:0001321) |
Right sided aortic arch, VSD, thoracic kyphosis, clubbed feet | CSF disorder Posterior fossa |
|
| Exencephaly (HP:0030769) Large open neural tube defect, similar to rachichisis (HP:0034237) |
Kyphosis - could not see diaphragm, lungs or stomach. Micrognathia | Neural tube defect | |
| Large right posterior encephalocele (HP:0002084) | Large facial midline cleft, clenched hands, 2 VC. Patient reports one hand had no digits, the other had nubs only without bones. Syndactyly of toes. Cloudy corneas. | Neural tube defect | |
| Bilateral ventriculomegaly (HP:0002119) Small cerebellum (HP:0001321) |
Clubbed feet, contracted arms, Abnormal facial profile, NOS. Consanguinity | CSF disorder Posterior fossa |
|
| Encephalocele (HP:0002084) | Cleft lip and palate, anophthalmia, absent external nasal structures with wide spaced nares, anotia, contractures of arms and hands, oligodactyly and syndactyly of hands and feet. | Autopsy | Neural tube defect |
| Lobar holoprosencephaly (HP:0006870) | Thick NT, short arms and legs - fixed position, absent tibia and fibia bilaterally, absent left kidney, transposition of the great arteries, cleft lip palate | Midline defect | |
| Unilateral severe ventriculomegaly vs porencephalic cyst (HP:0002119 vs HP:0002132) | Unilateral clubbed foot, possible heart defect (suboptiomal images) | CSF disorder | |
| Cloverleaf skull (HP:0002676) Dandy Walker malformation (HP:0001305) | IUGR <1%ile, hypoplastic kidneys with oligohydramnios, left sided diaphragmatic hernia, kyphosis. Suboptimal heart due to CDH. | Autopsy | Posterior fossa |
| Absent vs severely hypoplastic cerebellum (HP:0007360 vs HP:0001321) | SUA, anhydramnios, cystic hygroma, echogenic bowel, growth restriction, IUFD 20 weeks. | Posterior fossa | |
| Agenesis corpus callosum (HP:0001274) Arachnoid cyst (HP:0100702) Vermian agenesis (HP:0006817) |
Micropthalmia of the right eye, abnl positioning of left fingers, short femurs and humeri, multiple hemivertebrae | Midline defect Posterior fossa |
|
| Dandy Walker malformation (HP:0001305) Hydrocephalus (HP:0000238) Dysplastic cerebellum and vermis (HP:0006893) Dysplastic midbrain including brainstem (N/A) |
IUGR, clenched hands, echogenic kidneys | MRI | CSF disorder Posterior fossa |
| Dandy Walker malformation (HP:0001305) Agenesis corpus callosum (HP:0001274) Ventriculomegaly (HP:0002119) Midline cyst (N/A) Vermian hypoplasia (HP:0001320) Brainstem hypoplasia (HP:0007362) Excessive cortical malformation, possible tectal thickening (N/A) |
Bilateral anophthalmia, midface hypoplasia, multiple areas of scoliosis/kyphosis. | MRI | CSF disorder Posterior fossa Cortical disorder |
| Bilateral ventriculomegaly (HP:0002119) Hypoplastic cerebellum (HP:0001321) Absent corpus callosum (HP:0001274) Dandy Walker variant (HP:0001305) |
Right clubbed foot. Micrognathia. Echogenic bowel. | CSF disorder Midline defect Posterior fossa |
|
| Alobar holoprosencephaly (HP:0006988) Microcephaly <1%ile (HP:0000252) Prior pregnancy with holoprosencephaly and microcephaly. |
Cleft lip/palate, absent stomach, second pregnancy also with absent nose, left micropthalmia | Prior affected pregnancy | Midline defect |
| Bilateral ventriculomegaly (HP:0002119) Hypoplastic cerebellum (HP:0001321) |
Heart defect - double outlet right ventricle and VSD | CSF disorder Posterior fossa |
|
| Open neural tube defect (HP:0034237) | Cleft lip/palate, very low set ears (noted at chin), micrognathia, tetrology of fallot with pulmonary stenosis, VSD, polyhydramnios | Neural tube defect | |
| Midline intrahemispheric cyst (HP:0010576) Absent cavum septum pellucidum (HP:4000138) Absent corpus callosum (HP:0001274) |
None | MRI | Midline defect |
| Acrania/anencephaly (HP0030716, HP:0002323) Prior pregnancy with acrania/anencephaly. Family history of males with anencephaly. |
None | Prior affected pregnancy | Neural tube defect |
| Vein of galen malformation (HP:0030713) | None | MRI | |
| Absent corpus callosum (HP:0001274) Absent cavum septum pellucidum (HP:4000138) Teardrop shaped ventricles (HP:0002118) |
None | Midline defect | |
| Holoprosencephaly, semilobar to alobar (HP:0001360) | None | MRI | Midline defect |
| Unilateral ventriculomegaly (HP:0002119) Hypoplastic corpus callosum (HP:0002079) Possible schizencephaly (HP:0010636) Polymicrogryria (HP:0002126) |
None | MRI | CSF disorder Midline defect Cortical disorder |
| Absent corpus callosum (HP:0001274) Bilateral ventriculomegaly (HP:0002119) Absent cavum septum pellucidum (HP:4000138) |
None | CSF disorder Midline defect |
|
| Anencephaly HP:0002323 Prior pregnancy with anencephaly. | None | Prior affected pregnancy | Neural tube defect |
| Anencephaly HP:0002323 Prior pregnancy with encephalocele. |
None | Prior affected pregnancy | Neural tube defect |
| Polymicrogyria (HP:0002126) Bilateral ventriculomegaly (HP:0002119) |
None | Autopsy MRI |
CSF disorder Cortical disorder |
AV, atrioventricular; AVSD, atrioventricular septal defect; CDH, congenital diaphragmatic hernia; CSF, cerebrospinal fluid; d, day; FBA, fetal brain abnormality; IUFD, intrauterine fetal demise; IUGR, intrauterine growth restriction; MRI, magnetic resonance imaging; NOS, not otherwise specified; ONTD, open neural tube defect; SUA, single umbilical artery; VSD, ventricular septal defect; wk, week.
Prenatal FBA written as description from available phenotyping as well as associated HPO term, when available.
N/A denotes HPO term not available for this fetal phenotype. HPO terms do not include many prenatal specific phenotypes at this time.
Table 3.
Maternal characteristics
| Maternal Characteristic | N = 76 |
|---|---|
| Age (mean, SD) | 30 (4.4) |
| Employed | 42 (55) |
| Married | 56 (74) |
| College education | 46 (61) |
| Household income >$75,000/year | 42 (55) |
| Prior affected pregnancy | 16 (21) |
Data shown as N (%), unless otherwise noted.
Figure 2 depicts the findings of our primary analysis comparing cases of FBA that have a positive ES result with those that have a negative ES result by available phenotyping. There was a trend toward receipt of a positive ES result in cases that had at least 1 form deep phenotyping available (56% (N = 14) positive ES vs 45% (N = 23) negative ES); however, the results were not statistically significant (P = .37). This trend persisted when evaluating the individual modalities of deep phenotyping. Cases with MRI, autopsy, or information from prior affected pregnancies were associated with receipt of positive ES result, though these findings were not statistically significant. In pairwise comparisons, there was no significant difference between methods of phenotyping in achieving a positive ES result (MRI vs autopsy, P = .06; MRI vs family history, P = .13; autopsy vs family history, P = .06).
Figure 2. Deep phenotyping and ES results.
ES, exome sequencing; MRI, magnetic resonance imaging.
Table 4 shows the findings of our secondary analysis evaluating diagnostic yield based on the category of FBA. Prenatal ultrasound findings were reviewed for each case and FBAs were categorized into 5 groups: posterior fossa anomalies, disorders of cerebrospinal fluid, midline defects, cortical disorders, and NTDs. Of note, many of these cases had more than 1 FBA identified and presence of 1 FBA does not mutually exclude the presence of another. The most common FBAs identified were posterior fossa anomalies and midline defects, though there was no difference in receipt of a positive ES result in either group. The only statistically significant finding was that NTDs were associated with receipt of a negative ES result. Similarly, pairwise comparisons were not statistically significant except for when comparing cortical anomalies with NTDs; compared with cortical anomalies, NTDs were statistically less likely to produce a positive ES result (P = .03).
Table 4.
ES result stratified by FBA category
| FBA Category | Positive ES | Negative ES | P value |
|---|---|---|---|
| Posterior fossa anomalies | 15 (60) | 19 (37) | 0.06 |
| Disorders of cerebrospinal fluid (CSF) | 9 (36) | 18 (35) | 1.0 |
| Midline defects | 11 (44) | 21 (41) | 0.8 |
| Cortical disorders | 2 (8) | 6 (12) | 0.6 |
| Neural tube defects (NTDs) | 0 | 11 (22) | 0.01 |
Data shown as N (%). Values do not add up to 100%, as findings are not mutually exclusive.
Posterior Fossa Anomalies: vermian dysgenesis/hypoplasia without NTD.
Midline Defects: holoprosencephaly, abnormal cavum septum pellucidum/corpus callosum.
Disorders of CSF: ventriculomegaly, hydrocephaly.
Cortical Disorders: lissencephaly, schizencephaly, polymicrogyria.
Neural Tube Defect: spina bifida, anencephaly, encephalocele.
ES, exome sequencing; CSF, cerebrospinal fluid; NTDs, neural tube defects.
Discussion
Principal findings
Within this cohort, individual modalities of deep phenotyping did not show an association with increased diagnostic yield of ES; however, there was a trend toward receipt of a positive ES result in cases of FBAs that had deep phenotyping. Notably, NTDs were more frequently associated with negative sequencing results. It is important to note that all NTDs identified in this cohort were in conjunction with other multiorgan system anomalies or there was history of recurrent pregnancies with NTD, which increased suspicion for a possible genetic etiology. Prior literature suggests that ES significantly improves the yield of prenatal diagnosis in the setting of FBAs, particularly when multisystem anomalies are present.
Results in the context of what is known
In a study of 114 cases of major CNS anomalies, Yaron et al estimated diagnostic yield of greater than 50% with use of ES, positing that ES should be considered the first-tier test in the prenatal diagnosis of major fetal CNS anomalies.10 Likewise, Baptiste et al identified ES as an important tool in improving diagnostic rate in the setting of CNS anomalies, particularly when multisystem anomalies are present.9 Our current analysis of data related to FBAs adds to the overall literature, demonstrating the improvement in diagnostic yield when multiple anomalies are present.18
These studies do not specifically address how phenotypic information affects variant detection and interpretation. As demonstrated with ES analysis in pediatric and adult populations, the addition of diagnostic data through imaging can significantly improve molecular diagnosis and clinical consultation. Phenotype-driven approaches have been shown to significantly enhance variant prioritization and improve clinical counseling when a diagnosis is made.20 For example, neuro-imaging has been shown to aid variant interpretation in the setting of monogenic causes of clinically diagnosed cerebral palsy.21 In regard to the prenatal period, the addition of phenotypic data can aid in precision diagnosis, particularly because prenatal phenotypes and their postnatal correlates are better documented.8 However, this raises several practical considerations for documentation of fetal phenotype. First, the Human Phenotype Ontology, though having recently expanded terms to include prenatal findings, is not uniformly used for ultrasound-detected anomalies because it is in pediatric and adult populations.22 Additionally, given the limited scope of ES in the prenatal period at this time, there are a paucity of data available to correlate prenatal and postnatal phenotypes outside of study populations, limiting the data available for study-based or commercial ES variant interpretation.
Interestingly, our paper suggests that we were statistically less likely to have a positive ES result in the setting of NTDs. This particular finding is within the context of an overall small sample size, and an even smaller numbers of pregnancies with NTDs. The genetic basis of NTDs is being studied, with several papers published between 2019 and 2022 discussing novel genes that may be associated, causative, or impacting folic acid metabolism.23,24 However, as this is a clinical ES study, we may not have identified the novel genes that are yet to be included in clinical databases that would provide a positive sequencing result. In addition, other anomalies, such as posterior fossa anomalies and talipes, may be within the constellation of findings that are associated with NTD. Thus, despite the presence of multiple anomalies, the methods of variant identification in sequencing in clinical studies may limit identification of novel genes that are currently being described in nonclinical contexts.
Strengths and limitations
There are several strengths of this study. First, it included sequencing of maternal-paternal-fetal trios, which helps to determine pathogenicity of identified variants and evaluate inheritance. Additionally, all potentially causative variants were reviewed by a multidisciplinary committee, which improves the accuracy of variant interpretation. Lastly, all positive results were confirmed using Sanger sequencing at a Clinical Laboratory Improvement Amendments certified lab.
There are several limitations that likely contribute to the inability to show statistical significance of our findings. First, this was an exploratory analysis of a small cohort where deep phenotyping was not systematically pursued. Although there was a trend toward increased yield in cases with deep phenotyping, our cohort may be underpowered to identify statistically significant differences. Several factors may have influenced which patients received phenotyping, such as the type of abnormality detected, individual institutional practices, providers involved in care, and pregnancy disposition. This is inherent to the current study design of a secondary analysis of a convenience prospective cohort. Second, postnatal phenotypic information was unavailable for many of the cases in this cohort, because of a high rate of pregnancy termination, as well as missing pregnancy outcome data because of prenatal care/delivery at outside institutions. Third, this was a highly selected cohort where the majority of included fetuses had multisystem abnormalities; thus, our high diagnostic yield should be interpreted with caution in cohorts in which the pretest probability of a genetic etiology is lower (eg, single organ system abnormalities or anomalies associated with a multi-factorial etiology). Finally, most of the maternal participants in this cohort were married with higher socioeconomic status that self-selected into the study. As such, the results of our study may not be applicable to a general obstetric population.
Conclusions
Further study using a larger cohort is needed to better assess the impact of deep phenotyping on diagnostic yield of prenatal ES for fetal anomalies. In addition, study design should include standard phenotyping for commonly encountered brain anomalies, thus eliminating potential individual and institution level variation. Improvement of current imaging techniques, standard documentation of prenatal phenotypes using universal terminology, and systematic or standardized approaches to obtaining deep phenotypic information are needed to better optimize our ability to interpret ES findings and care for families with a pregnancy complicated by congenital birth defects.
Funding
Funding for this research was supported by the following grants:
R21 TR002770 (National Center for Advancing Translational Sciences: PIs: Vora; Davis)
R01 HD105868 (National Institute for Child and Human Development; PI; Vora)
Additional funding sources:
NICHD R01HD105868 (PI: Vora); NICHD K23HD088742 (PI: Vora); NICHD: K12HD00144116 (PI: Boggess); NCATS R21TR002770 (PI: Vora).
Footnotes
Ethics Declaration
This study was approved by the Institutional Review Board at the University of North Carolina at Chapel Hill (IRB 13–4084). Informed consent was obtained from all participants before enrollment. All reported and stored individual data were de-identified.
This study was selected for oral presentation at the 42nd annual meeting of the Society for Maternal-Fetal Medicine, held virtually between January 31 and February 5, 2022.
Conflict of Interest
Neeta Vora receives supplies in kind from Illumina for an NIH grant on whole genome sequencing unrelated to the data in this paper. All other authors declare no conflicts of interest.
Data Availability
The data that support the findings of this study are available from the corresponding author on reasonable request. We define reasonable as a request to review data for purposes of further study or clarification of any data or analyses presented in the paper. The parent fetal exome study from which the data for this study are derived is registered in dbGaP and available to the public.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author on reasonable request. We define reasonable as a request to review data for purposes of further study or clarification of any data or analyses presented in the paper. The parent fetal exome study from which the data for this study are derived is registered in dbGaP and available to the public.