Underlying Genetic Etiologies of Congenital Diaphragmatic Hernia

Abstract

Congenital diaphragmatic hernia (CDH) is often detectable prenatally. Advances in genetic testing have made it possible to obtain a molecular diagnosis in many fetuses with CDH. Here, we review the aneuploidies, copy number variants (CNVs), and single genes that have been clearly associated with CDH. We suggest that array-based CNV analysis, with or without a chromosome analysis, is the optimal test for identifying chromosomal abnormalities and CNVs in fetuses with CDH. To identify causative sequence variants, whole exome sequencing (WES) is the most comprehensive strategy currently available. Whole genome sequencing (WGS) with CNV analysis has the potential to become the most efficient and effective means of identifying an underlying diagnosis but is not yet routinely available for prenatal diagnosis. We describe how to overcome and address the diagnostic and clinical uncertainty that may remain after genetic testing, and review how a molecular diagnosis may impact recurrence risk estimations, mortality rates, and the availability and outcomes of fetal therapy. We conclude that after the prenatal detection of CDH, patients should be counseled about the possible genetic causes of the CDH, and the genetic testing modalities available to them, in accordance with generally accepted guidelines for pretest counseling in the prenatal setting.

Introduction

Congenital diaphragmatic hernia (CDH) is present in approximately 1 in 4,000 newborns and accounts for 8% of all major congenital anomalies (1, 2). Additional congenital anomalies are identified in 40-60% of CDH cases (3–5). Among all CDH cases, congenital heart defects are present in 25-40%, urogenital anomalies in 18%, musculoskeletal anomalies in 16% and central nervous system anomalies in 10% (3). Although CDH is defined as an abnormal opening in the diaphragm that allows the abdominal viscera to protrude into the thorax, this definition is sometimes expanded to include diaphragmatic eventrations (DE) in which the diaphragm continuity is not disrupted, but the diaphragm is abnormally elevated due to a congenital defect in the diaphragmatic musculature (6). In this review, this expanded definition of CDH will be used since the developmental processes and underlying molecular causes of CDH and DE often overlap, their associated medical problems are similar, and individuals and families are called upon to make the same decisions regarding genetic testing when either of these defects are identified (7–9).

When CDH is identified prenatally, parents must make a variety of decisions including whether or not to pursue genetic testing (10). CDH is a genetically heterogeneous disorder, in which each known genetic cause is responsible for only a fraction of cases (11, 12). The penetrance of CDH varies widely among CDH-related genetic syndromes, and it is likely that other genetic, environmental, epigenetic, and stochastic factors play a role determining if an individual affected by a specific genetic disorder will ultimately develop CDH (13, 14). In this review, we provide information on the chromosomal abnormalities and genes that have been clearly associated with the development of CDH in humans. We review the prenatal tests most commonly used in the setting of prenatally diagnosed CDH and provide information that can help medical providers choose the most appropriate genetic tests. Since providers are charged with informing individuals and families about genetic testing, we also review the limitations, benefits, and risks associated with these tests.

Aneuploidies, Copy Number Variants, and Single Genes Associated with CDH

A variety of aneuploidies, copy number variants (CNVs), and single genes have been clearly associated with an increased risk of developing CDH as summarized in Tables 1, 2, and 3 respectively (12, 13, 15–17). The CDH-related genes listed in Table 3 were curated through a literature review with emphasis being placed on identifying specific genes that cause CDH rather than identifying genetic diagnoses associated with CDH. For example, there are twelve types of Coffin-Siris syndrome, each of which is defined molecularly by their causative gene, but only variants in ARID1B, DPF2, SMARCA4, SMARCB1 and SMARCE1 have been reported in association with CDH (18–23). Other excellent reviews on the genetic causes of CDH and their associated phenotypes include those by Yu et al. and Longoni et al. (13, 16).

Table 1.

Aneuploidies associated with CDH

Chromosomal Anomaly	Syndrome (MIM#)†	Frequency of CDH in individuals with this syndrome	Commonly affected organ systems and specific anomalies potentially identifiable prenatally	Commonly reported neuro-developmental phenotypes	References
Trisomy 13	Patau syndrome	+	FGR, CNS, eye, orofacial clefting, cardiovascular, omphalocele, renal, limb, genitourinary, skeletal, neural tube defects, single umbilical artery	DD, ID, Sz	(17, 79)
Trisomy 18	Edward syndrome	+	FGR, polyhydramnios, CNS, orofacial clefting, cardiovascular, omphalocele, renal, limb, single umbilical artery	DD, ID, Sz	(79, 80)
Trisomy 21	Down syndrome (MIM# 190685)	+	Increased nuchal thickness, cardiovascular, gastrointestinal	DD, ID	(79, 80)
Trisomy 22	Complete or mosaic trisomy 22	+	FGR, body asymmetry, cardiovascular, genitourinary	DD, ID	(17, 81)
45,X	Turner syndrome	+	Cystic hygroma, fetal hydrops, cardiovascular, renal	LD	(17, 79)
47,XXX	Trisomy X, triple X syndrome	+	Renal	DD, LD, ADHD	(16, 43)

^†Online Mendelian Inheritance in Man (https://omim.org/) number if available.

ADHD = Attention deficit hyperactivity disorder, ASD = Autism spectrum disorder, DD =Developmental delay, FGR = fetal growth restriction, ID = intellectual disability, LD = learning disabilities, Psy = Psychiatric disorders, Sz = seizures

⁺CDH is a relatively rare feature of this syndrome with an incidence estimated at <10% or, for less extensively characterized syndromes, CDH is not generally considered to be a component of the syndrome (13)

Table 2.

Copy number variants associated with CDH

Chromosomal Anomaly	Syndrome (MIM#)†	Frequency of CDH in individuals with this syndrome	Commonly affected organ systems and specific anomalies potentially identifiable prenatally	Commonly reported neuro-developmental phenotypes	References
Deletion 1q41q42	1q41q42 microdeletion syndrome (MIM# 612530)	++	CNS, orofacial clefting, cardiovascular	DD, ID, Sz	(33)
Deletion 4p16.3	Wolf-Hirschhorn syndrome (MIM# 194190)	++	FGR, CNS, orofacial clefting, cardiovascular, splenic, genitourinary	DD, ID, Sz	(82)
Deletion 8p23.1	8p23.1 microdeletion syndrome	++	FGR, cardiovascular	DD, ID	(34)
Tetrasomy 12p	Pallister-Killian syndrome (MIM# 601803)	++	Fetal macrosomia, polyhydramnios, orofacial clefting, cardiovascular, renal, genitourinary, skeletal, limb	DD, ID, Sz	(30, 31)
Deletion 15q24	15q24 microdeletion syndrome (MIM# 613406)	++	FGR, CNS, genitourinary	DD, ID	(83)
Deletion 15q25.2	15q25.2 microdeletion syndrome (MIM# 614294)	++	Cardiovascular, orofacial clefting, limb	DD, ADHD, ID, ASD	(57)
Deletion 15q26	15q26-qter deletion syndrome (MIM# 612626)	++	FGR, cardiovascular, genitourinary, skeletal	DD, ID	(17, 35)
Deletion 16p11.2	Chromosome 16p11.2 deletion syndrome, 593kb (MIM# 611913)	+	Cardiovascular, renal, genitourinary	DD, ID, ASD, Sz	(8, 42)
Deletion 17q12	17q12 deletion syndrome (MIM# 614527)	++	Genitourinary	DD, ID, ASD, Psy	(84)
Deletion 22q11	DiGeorge syndrome (MIM# 188400)	+	Orofacial clefting, cardiovascular	DD, ID, ADHD, Psy, Sz	(85)
Supernumerary der(22),t(11;22)	Emanuel syndrome (MIM# 609029)	+	CNS, orofacial clefting, cardiovascular, renal, genitourinary	DD, ID, Sz	(17)

^†Online Mendelian Inheritance in Man (https://omim.org/) number if available

⁺CDH is a relatively rare feature of this syndrome with an incidence estimated at <10% or, for less extensively characterized syndromes, CDH is not generally considered to be a component of the syndrome

⁺⁺CHD with an estimated incidence of 10-40%, or for less extensively characterized syndromes, multiple case reports of CDH are present in the published literature (13).

ADHD = attention deficit hyperactivity disorder, ASD = autism spectrum disorder, DD = developmental delay, FGR = fetal growth restriction, ID = intellectual disability, Psy = psychiatric disorders, Sz = seizures

Table 3.

CDH-associated genes and their related genetic syndromes

Gene†	Inheritance pattern(s)	Disorder (MIM#)‡	Frequency of CDH in individuals with this syndrome	Commonly affected organ systems and specific anomalies potentially identifiable prenatally	Commonly reported neuro-developmental phenotypes	References
ABL1	AD	Congenital heart defects and skeletal malformations syndrome (MIM# 617602)	++	FGR, cardiovascular, skeletal	DD	(86)
ALG12	AR	Congenital disorder of glycosylation, type 1g (MIM# 607143)	++	Cardiovascular, genitourinary, skeletal	DD, ID, Sz	(20)
ARID1B	AD	Coffin-Siris syndrome 1 (MIM# 135900)	+	CNS, cardiovascular	DD, ID, Sz	(19)
B3GAT3	AR	Multiple joint dislocations, short stature, craniofacial dysmorphism, with or without congenital heart defects (MIM# 245600)	+	Cardiovascular, skeletal	DD	(87, 88)
BRCA2	AR	Fanconi anemia (MIM# 605724)	+	FGR, cardiovascular	Not common	(20)
CHD7	AD	CHARGE syndrome (MIM# 214800)	+	Eye, orofacial clefting, cardiovascular, gastrointestinal, renal, genitourinary	DD, ID, Sz	(89)
CHRNG	AR	Escobar syndrome (MIM# 265000); Multiple pterygium syndrome, lethal type (MIM# 253290)	+	Orofacial clefting, genitourinary, pterygia	Not common in Escobar syndrome	(71, 90)
COL3A1	AD; AR	Ehlers-Danlos syndrome, vascular type (MIM# 130050); Polymicrogyria with or without vascular-type EDS (MIM# 618343)	+	CNS, cardiovascular	Not common in Ehlers-Danlos syndrome, vascular type; A subset of those with polymicrogyria have DD, ID, Sz	(91, 92)
COX7B	XL	Linear skin defects with multiple congenital anomalies 2 (MIM# 300887)	++	Cardiovascular, renal	DD, ID	(93)
DLL3	AR	Spondylocostal dysostosis 1, autosomal recessive (MIM# 277300)	+	Skeletal	Not common	(94)
DPF2	AD	Coffin-Siris syndrome 7 (MIM# 618027)	+	Cardiovascular, skeletal	DD, ID	(23)
EFEMP2	AR	Cutis laxa, autosomal recessive, type IB (MIM# 614437)	+	Skeletal	Not common	(95)
EFNB1	XL	Craniofrontonasal dysplasia (MIM# 304110)	+	Orofacial clefting, skeletal	Not common	(96)
EP300	AD	Rubinstein-Taybi syndrome 2 (MIM# 613684)	+	Cardiovascular, renal	DD, ID, ASD	(20)
FBN1	AD	Marfan syndrome (MIM# 154700)	+	None	Not common	(97, 98)
FGFR2	AD	Apert syndrome (MIM# 101200)	+	Cranial, orofacial clefting, cardiovascular, gastrointestinal, limb	DD, ID	(99, 100)
FGFRL1	AR	FGFRL1-related disorder (MIM# 605830)	?	None	?	(101)
FOXP1	AD	Mental retardation with language impairment and with or without autistic features (MIM# 613670)	+	CNS, cardiovascular, renal, genitourinary	DD, ID, ASD	(20, 36)
FOXP4	AD	FOXP4-related disorder (MIM# 608924)	++	None	DD, ID	(102, 103)
FRAS1	AR	Fraser syndrome 1 (MIM# 219000)	+	Orofacial clefting, genitourinary, renal, limb	DD, ID	(104)
FREM1	AR	Bifid nose with or without anorectal and renal anomalies (MIM# 608980); Manitoba oculotrichoanal syndrome (MIM# 248450)	+	Eye, renal	Not common	(105)
FREM2	AR	Fraser syndrome 2 (MIM# 617666)	+	Orofacial clefting, genitourinary, renal, limb	Not common	(104)
GATA4	AD	GATA4-related disorders (MIM# 600576)	+	Cardiovascular	Not common	(106)
GATA6	AD	GATA6-related disorders (MIM# 601656)	+	Cardiovascular, gastrointestinal	DD in some patients	(107)
GPC3	XL	Simpson-Gobali-Behmel, type 1 (MIM# 312870)	+	LGA, orofacial clefting, cardiovascular, pulmonary, splenic, renal, skeletal	DD, ID	(108, 109)
HCCS	XL	Linear skin defects with multiple congenital anomalies 1 (MIM# 309801)	++	CNS, cardiovascular, genitourinary	DD, ID, Sz	(110, 111)
HDAC8	XL	Cornelia de Lange syndrome 5 (MIM# 300882)	+	Orofacial clefting, cardiovascular, limb	DD, ID, Sz	(112)
KDM3B	AD	Diets-Jongmans syndrome (MIM# 618846)	+	CNS	DD, ID	(113)
KMT2D	AD	Kabuki syndrome 1 (MIM# 147920)	+	FGR, orofacial clefting, cardiovascular, gastrointestinal, renal, genitourinary	DD, ID, Sz	(20, 114)
LRP2	AR	Donnai-Barrow syndrome (MIM# 222448)	+++	CNS, cardiovascular, gastrointestinal	DD, ID	(115)
LTBP4	AR	Cutis laxa, autosomal recessive, type IC (MIM# 613177)	+++	None	Not common	(116, 117)
MN1	AD	CEBALID syndrome (MIM# 618774)	+	CNS, skeletal	DD, ID, Sz, ASD	(118)
MYOD1	AR	Myopathy, congenital, with diaphragmatic defects, respiratory insufficiency, and dysmorphic facies (MIM# 618975)	+++	Polyhydramnios, cystic hygroma, orofacial clefting, renal, skeletal	DD	(119, 120)
MYRF	AD	Cardiac-urogenital syndrome (MIM# 618280)	+++	Cardiovascular, pulmonary, genitourinary	DD	(36, 40)
NIPBL	AD	Cornelia de Lange syndrome 1 (MIM# 122470)	+	Cardiovascular, orofacial clefting, limb	DD, ID, Sz	(121, 122)
NR2F2	AD	Congenital heart defects, multiple types, 4 (MIM# 615779); 46,XX sex reversal 5 (MIM# 618901)	++	Cardiovascular, genitourinary	DD	(42, 123)
PBX1	AD	Congenital anomalies of kidney and urinary tract syndrome with or without hearing loss, abnormal ears, or developmental delay (MIM# 617641)	++	Oligohydramnios, Cardiovascular, genitourinary, renal	DD, ID	(124)
PIGN	AR	Multiple congenital anomalies-hypotonia-seizures syndrome 1 (MIM# 614080)	+++	CNS, orofacial clefting, renal, genitourinary	DD, ID, Sz	(125, 126)
POGZ	AD	White-Sutton syndrome (MIM# 616364)	+	Genitourinary	DD, ID, Sz, ASD	(127)
PORCN	XL	Focal dermal hypoplasia (MIM# 305600)	+	CNS, eye, orofacial clefting, renal, genitourinary, limb	DD, ID in 15-20%	(128–130)
RAD21	AD	Cornelia de Lange syndrome 4 (MIM# 614701)	+	Cardiovascular, orofacial clefting, limb	DD, ID, Sz	(131)
RARB	AD, AR	Microphthalmia, syndromic 12 (MIM# 615524)	+++	Eye, cardiovascular, genitourinary	DD, ID	(132)
RLIM	XL	Tonne-Kalscheuer syndrome (MIM# 300978)	+++	Cardiovascular, genitourinary	DD, ID	(133)
SIN3A	AD	Witteveen-Kolk syndrome (MIM# 613406)	+	FGR, CNS, genitourinary	DD, ID, Sz, ADHD, ASD	(36)
SLC2A10	AR	Arterial tortuosity syndrome (MIM# 208050)	++	Cardiovascular	ID has been described	(134)
SLIT3	AR	SLIT3-related disorder (MIM# 603745)	?	None	?	(135)
SMARCA4	AD	Coffin-Siris syndrome 4 (MIM# 614609)	+	CNS, cardiovascular	DD, ID, Sz	(20)
SMARCB1	AD	Coffin-Siris syndrome 3 (MIM# 614608)	+	CNS, cardiovascular	DD, ID, Sz	(21)
SMARCE1	AD	Coffin-Siris syndrome 5 (MIM# 616938)	+	CNS, cardiovascular	DD, ID, Sz	(22)
SMC1A	XL	Cornelia de Lange type 2 (MIM# 300590); Developmental and epileptic encephalopathy 85, with or without midline brain defects (MIM # 301033)	+	Cardiovascular, orofacial clefting, upper-limb reduction defects	DD, ID, Sz	(136)
SPECC1L	AD	Opitz GBBB syndrome type II (MIM# 145410); Teebi hypertelorism syndrome (MIM# 145420)	+	CNS, orofacial clefting, cardiovascular, gastrointestinal, genitourinary, renal, skeletal	DD, ID, Sz	(137)
STAG2	XL	Mullegama-Klein-Martinez syndrome (MIM# 301022); Holoprosencephaly 13, X-linked (MIM# 301043)	+	CNS, orofacial clefting, cardiovascular	DD, ID	(138, 139)
STRA6	AR	Microphthalmia, syndromic 9 (MIM# 601186)	+++	Eye, cardiovascular, splenic, renal, genitourinary	DD, ID	(140, 141)
WT1	AD	Denys-Drash syndrome (MIM# 194080); Meacham syndrome (MIM# 608978); Frasier syndrome (MIM# 136680)	+	Genitourinary	Not common	(142–144)
ZFPM2	AD	Diaphragmatic hernia type 3 (MIM# 610187); Tetralogy of Fallot (MIM# 187500)	++	Cardiovascular	Not common	(7, 8)

^†For syndromes in which more than one gene can be causative, we have only listed genes that have been specifically associated with CDH.

^‡Online Mendelian Inheritance in Man (https://omim.org/)

^?= not known due to the low number of cases reported

⁺CDH is a relatively rare feature of this syndrome with an incidence estimated at <10% or, for less extensively characterized syndromes, CDH is not generally considered to be a component of the syndrome

⁺⁺CHD is a variable feature of this syndrome with an estimated incidence of ~10-40% or, for less extensively characterized syndromes, multiple case reports of CDH are present in the published literature

⁺⁺⁺= CDH is a common finding in this syndrome with an incidence of >40% or, for less extensively characterized syndrome, it is considered a core feature (13)

AD = autosomal dominant, ADHD = attention deficit hyperactivity disorder, AR = autosomal recessive, ASD = autism spectrum disorder, DD =developmental delay, FGR = fetal growth restriction, ID = intellectual disability, Sz = seizures, XL = X-linked

A detailed description of the clinical features associated with each CDH-associated chromosomal abnormality and gene is beyond the scope of this review. However, in Tables 1–3 we provide Online Mendelian Inheritance in Man numbers (MIM#; https://www.omim.org) which can be used to access detailed lists the phenotypes associated with each genetic disorder. We have also indicated how common CDH is among individuals with each genetic disorder (13), and we have listed common anomalies associated with these disorders that can be detected prenatally.

Individuals and families often inquire about the neurodevelopmental phenotypes associated with a specific diagnosis. These can be difficult to predict since many CDH-related genetic disorders are associated with a wide spectrum of neurodevelopmental issues. This spectrum may be even broader in individuals with CDH since neurodevelopmental dysfunction has been recognized as one of the most common comorbidities in CDH survivors (24). However, as a starting point, we have listed commonly described neurodevelopmental phenotypes associated with each genetic disorder in Tables 1–3.

Common Causes of CDH

With the exception of Down syndrome, all CDH-associated disorders are rare with an incidence of less than 1 in 2,000. Trisomy 18, which accounts for approximately 2-5% of CDH cases, and trisomy 13, which accounts for <1% of CDH cases, are among the most commonly identified aneuploidies in fetuses with CDH (25–27). The prenatal sonographic pattern of trisomy 18 is characterized by fetal growth restriction, polyhydramnios, brachycephaly, a narrow frontal cranium, choroid plexus cysts, overlapping digits, congenital heart defects, omphalocele, and single umbilical artery (28). Trisomy 13 fetuses may show various brain anomalies including holoprosencephaly, ventriculomegaly, an enlarged cistern magna, and agenesis of the corpus callosum, microcephaly, midfacial hypoplasia, microphthalmia, hypotelorism, cleft lip and palate, nuchal thickening or cystic hygroma, neural tube defects, omphalocele, kidney and urogenital anomalies, hyperechogenic bowel, cardiac defects, single umbilical artery, radial aplasia, polydactyly and flexion deformities of the fingers (29).

Pallister-Killian syndrome (PKS), caused by full or mosaic tetrasomy of chromosome 12p, accounts for 2-5% of CDH cases (15, 30). Sonographic markers for PKS in the first trimester include increased nuchal translucency and nasal bone hypoplasia, and in the second trimester include polyhydramnios, femur shortening, fetal macrosomia, a thickened nuchal fold, a typical facial profile, ventriculomegaly, congenital heart defects, renal pyelectasis, polydactyly and cleft palate (31).

CNVs commonly seen in fetuses with CDH include deletions of 1q41q42, 8p23.1 and 15q26 (15, 17, 32). Deletions of 1q42q42 account for 1–3% of CDH cases and are associated with variable features that may include midline defects such as structural brain anomalies, cleft palate, and congenital heart defects (33). 8p23.1 deletions account for 3-5% of CDH cases and are characterized prenatally by fetal growth restriction and cardiac anomalies (34). Deletions of 15q26 account for 1–2% of CDH case and are characterized by fetal growth restriction, cardiac anomalies, genitourinary anomalies, hypertelorism, and skeletal anomalies (17, 32, 35).

Among single gene causes of CDH, pathogenic variants affecting KMT2D, MYRF, ZFPM2 are commonly identified (20, 36, 37). Variants in KMT2D cause Kabuki syndrome 1 whose prenatal presentation may include an abnormal maternal serum screening, polyhydramnios, fetal growth restriction, single umbilical artery, orofacial clefting, and cardiovascular, gastrointestinal, renal, and/or genitourinary anomalies (38, 39). Variants in MYRF are associated with cardiac-urogenital syndrome which is characterized by a wide variety of cardiac anomalies, pulmonary hypoplasia which can be independent of CDH, and genitourinary anomalies (36, 40). Pathogenic ZFPM2 variants can cause both isolated CDH and CDH associated with cardiovascular anomalies (7, 37).

Recommendations for Prenatal Genetic Testing for CDH

Due to advances in molecular diagnostic techniques and our understanding of the genetic basis of CDH, an underlying genetic cause can be identified in a steadily increasing percentage of individuals with both isolated and non-isolated forms of CDH (8, 15, 20). These advances, and a lack of large studies in which optimal genetic testing is performed on all fetuses with CDH, make it difficult to determine the exact percentage of CDH cases in which a molecular diagnosis can be made. However, based on studies of various cohorts tested using chromosome analysis, array-based CNV analysis, and whole exome sequencing (WES), it is reasonable to conclude that a molecular diagnosis can be made in ~5-10% of isolated CDH cases and ~30-50% of non-isolated CDH cases (8, 16, 20, 25–27, 36, 41–53). Often, structural birth defects, neurodevelopmental abnormalities, and other medical problems are ultimately identified in individuals that appear to have isolated CDH prenatally. Hence, genetic testing should be offered whenever CDH is identified prenatally regardless of the presence or absence of additional anomalies.

Array-based CNV analysis—often referred to as chromosomal microarray analysis (CMA)—with its relatively fast turnaround time, is the test of choice for identifying the location and extent of CNVs in isolated and non-isolated cases of CDH (8, 49, 50). The ability to perform array-based CNV analyses on uncultured samples can also result in improved detection rates for mosaicism when compared to techniques that require cells to be grown in culture (54, 55).

Given its relatively low resolution, the need to culture cells, and the time required for manual scoring, chromosome analysis—which involves the generation of karyotype, or picture of the number and visual appearance of the chromosomes in a cell—is not considered a first-line test for most cases of CDH. However, chromosome analysis is considered the test of choice for determining if a fetus has Down syndrome due to trisomy 21 or a Robertsonian translocation affecting chromosome 21, and can be used to detect balanced chromosomal anomalies that cannot be detected by array-based CNV analysis (56). Similarly, fluorescent in situ hybridization (FISH) is not a first-line test, but may be employed if there is a need to rapidly obtain evidence in support of the presence of a chromosomal abnormality that would change medical management, to test for a previously defined familial cytogenetic anomaly, or as an adjunct test when mosaicism is suspected (54).

Since sequence variants can also cause both isolated and non-isolated CDH, the genetic evaluation of a fetus with CDH cannot be considered complete unless it includes a screen for these variants. Gene panel tests for CDH can be ordered clinically but are not preferred in the prenatal setting since they are unlikely to include newly described CDH-related genes and may not include genes associated with low CDH penetrance (20). WES is available on a clinical basis and is the preferred sequencing test since it is designed to screen the protein coding regions of all genes. Since panel tests may not include CNV analyses for CDH-associated genomic regions for which a causative gene(s) has yet to be clearly identified, and CNV analyses that utilize data generated by WES cannot reliably identify all of the CNV associated with CDH, a separate array-based CNV analysis should be ordered with these tests unless previously obtained (8, 42, 57, 58). In contrast, whole genome sequencing (WGS) with CNV analysis, which is currently available clinically for postnatal diagnosis, and is being used prenatally on a research basis, can be ordered as a stand-alone test (59, 60). We therefore recommend WES after array-based CNV analysis—or, in selected cases, concurrent with array-based CNV analysis to reduce the time to diagnosis—or WGS as the preferred methods for prenatal genetic testing in fetuses with CDH.

In some cases, additional testing may be needed to determine if a variant identified in a CDH-related gene is likely to be causative or benign. In cases where the variant is predicted to affect splicing, RNA-sequencing (RNA-seq) may be used to determine the effects of the variant on the mature mRNA (61). In other cases, functional or biochemical studies may be available which can help determine the effect of the variant in question on protein function. An increasing number of CDH-related disorders have also been shown to be associated with a specific epigenetic pattern of DNA methylation that can be identified clinically, and methylation studies are indicated in cases where there is reason to suspect that Beckwith-Wiedemann syndrome is the underlying cause of CDH (62, 63). Currently, these types of adjunct tests are rarely undertaken or completed prenatally, and in some cases, may only be available on a research basis. A variety of services are currently available through which clinicians can connect with physicians and researchers who may have expertise on particular gene including GeneMatcher (https://genematcher.org/) and Matchmaker Exchange (https://www.matchmakerexchange.org/)(64, 65).

The risks associated with genetic testing in the setting of a prenatal diagnosis of CDH are not significantly different than those encountered whenever genetic testing is employed and include risks associated with obtaining a DNA sample as well as the potential for negative financial, emotional and social consequences. Genetic testing may be expensive, and those costs may have to be paid by individual or family requesting them. Learning the underlying diagnosis, failing to identify a molecular cause, being unsure about a diagnosis, or identifying a genetic diagnosis unrelated to CDH, could provoke feeling of anxiety, anger, sadness, depression, and/or guilt in parents and family members (66, 67). In some cases, one or both parents may be identified as a carrier. This could, in turn, engender tension between family members. These potential consequences should be acknowledged when counseling individuals and families regarding genetic testing.

Overcoming and Addressing Diagnostic and Clinical Uncertainty

Although imaging studies can provide clues to a possible etiology, the lack of pathognomonic prenatal features for most CDH-related disorders makes it difficult to arrive at a prenatal diagnosis without molecular testing. At the same time, we note that genetic testing may not be definitive since the significance of some changes may remain unclear including copy number and sequencing variants affecting genes that have not been associated with CDH, or variants in CDH-associated genes whose effects on gene function cannot be clearly determined. In such cases, sonographic and, when available, fetal MRI findings may play a critical role in determining the likely etiology of the CDH by providing evidence in favor or against a specific molecular diagnosis. Determining the inheritance patterns of the variant among family members may also provide evidence for, or against, the pathogenicity of a specific variant.

Even in cases where genetic testing clearly identifies an underlying genetic cause, there often exist significant clinical uncertainty regarding the medical problems that may be encountered. For example, many CDH-related genetic syndromes are associated with neurodevelopmental or other phenotypes that are incompletely penetrant and whose severity varies significantly among affected individuals (13). The presence of CDH itself is associated with an increased risk for a variety of medical problems, including neurodevelopmental phenotypes, making prognostication even more difficult (68, 69). Hence, pre-test counseling should address the potential for both diagnostic and prognostic uncertainty.

The Effects of an Underlying Molecular Cause on Mortality

Some genetic causes of CDH are independently associated with an increased risk of mortality and/or the development of other major malformations. For example, trisomy 18 and trisomy 13 are, by themselves, associated with a high level of mortality prior to one year of age (25–27, 70). Multiple pterygium syndrome, lethal type, caused by variants in CHRNG, by definition, leads to death in the prenatal/perinatal period with individuals manifesting less severe phenotypes being diagnosed with Escobar syndrome (71). Some other aneuploidies (e.g. trisomy 21 and trisomy 22), genomic syndromes (e.g. 8p23.1 and 22q11.2 microdeletions), and single gene disorders (e.g. Kabuki syndrome and CHARGE syndrome) that can cause CDH are also associated with the development of congenital heart defects and other major malformations that could lead to an elevation in mortality rates (72, 73).

Recurrence Risk Determination

The sibling recurrence risk for isolated CDH with a negative family history is often quoted as <2% based on a mathematical model of multifactorial inheritance risk (74–76). It is important to recognize that this recurrence risk does not apply to cases of non-isolated CDH and may not accurately represent the recurrence risk for isolated CDH in some families. For example, isolated CDH caused by pathogenic variants ZFPM2 are inherited in an autosomal dominant fashion with incomplete penetrance (37). In this case, 50% of all pregnancies conceived by an unaffected, carrier parent or sibling will inherit the pathogenic ZFPM2 variant and, consequently, would have a significantly increased risk of developing CDH with or without a coexisting congenital heart defect. Similar patterns of increased risk among the unaffected carrier parents and siblings of a proband can occur in families transmitting balanced translocations and X-linked causes of CDH. Hence, accurate recurrence risk estimations for both isolated and non-isolated CDH requires an understanding of the underlying molecular cause.

Genetic Testing as a Prerequisite for Fetal Therapy

Fetal therapy, specifically fetal endoluminal tracheal occlusion (FETO) offered in specialized centers, is becoming more common in the care of fetuses with CDH (77, 78). At the centers where these procedures are being performed, most protocols require participants to have isolated CDH and some level of negative genetic testing. However, the level of genetic testing required by protocols can vary significantly. If genetic testing is to be a prerequisite for fetal intervention, it seems reasonable to perform the type of genetic testing that is most likely to identify an underlying genetic diagnosis. Future studies will be needed to determine which interventions provide advantages to fetuses with non-isolated CDH and/or fetuses with CDH caused by specific genetic disorders.

Currently, fetal therapy for CDH is focused on surgical interventions. In the future, gene- or mutation-specific fetal therapies may be developed to ameliorate the effects of CDH. When such therapies become available, their applicability will be determined, at least in part, by the results of genetic testing.

Conclusions

An increased understanding of the genetic basis of CDH, coupled with advances in genetic testing, have made it possible to identify a molecular diagnosis in a significant percentage of individuals with isolated and non-isolated CDH. Understanding the capabilities and limitations of the various genetic tests available will allow the selection of the most effective testing strategy in each case. Array-based CNV analysis, with or without G-banded chromosome analysis, is the optimal first-line test for identifying chromosomal abnormalities in fetuses with CDH. To identify causative sequence variants, WES is the most comprehensive strategy currently available. WGS with CNV analysis has the potential to become the most efficient and effective means of identifying an underlying diagnosis but is not yet routinely available during the prenatal period. As with any prenatal test, individuals and families should be offered detailed pre-test counseling so that they can make decisions based on a clear understanding of the associated benefits and risks. Obtaining an accurate molecular diagnosis will improve counseling regarding prognosis and recurrence risk. It may also inform medical management and help to determine which candidates are most likely to benefit from fetal interventions.

Bulleted statements:

What’s already known about this topic?

• Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that is often identified prenatally. A deeper understanding of the genetic causes of CDH, coupled with advances in genetic testing, have made it possible to obtain a molecular diagnosis in an increasing percentage of fetuses with CDH. Practitioners must be prepared to help individuals and families make informed decisions regarding genetic testing and to request the most appropriate genetic tests.

What does this review add?

• We provide a review of chromosomal abnormalities, copy number variants, and genes that are clearly associated with the development of CDH in humans. We describe current genetic testing modalities so that practitioners can select the most appropriate genetic test(s). To aid practitioners, we also provide a review of topics that should be discussed with individuals and families so that they can make informed decisions regarding genetic testing.

Data Availability Statement:

N/A