De-novo disease-causing variants in CDH: Comparison
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The genetic etiology of congenital diaphragmatic hernia (CDH), a common and severe birth defect, is still incompletely understood. Chromosomal aneuploidies, copy number variations (CNVs), and variants in a large panel of CDH-associated genes, both de novo and inherited, have been described. Due to impaired reproductive fitness, especially of syndromic CDH patients, and still significant mortality rates, the contribution of de novo variants to the genetic background of CDH is assumed to be high. This assumption is supported by the relatively low recurrence rate among siblings. Advantages in high-throughput genome-wide genotyping and sequencing methods have recently facilitated the detection of de novo variants in CDH. 

  • congenital diaphragmatic hernia
  • de novo variants
  • impaired reproductive fitness
  • mortality

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1. Introduction

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect reported to affect 2–3 per 10,000 live births [1]. Due to a high early neonatal and prenatal mortality, the hidden prevalence might be even higher [2]. The term CDH comprises a variety of defects in the diaphragm, ranging from diaphragmatic eventration to localized defects of variable size and locations to diaphragmatic agenesis. The most common type is the so-called “Bochdalek hernia” (dorsolateral) on the left side. CDH leads to herniation of abdominal viscera into the thorax during early embryonic development. Newborn patients typically present with respiratory distress which is, in short, due to hypoplasia of the lungs accompanied by abnormal structure of pulmonary vessels and alveolar septa, and pulmonary hypertension. Advancements in the prenatal diagnosis and postnatal management of CDH have led to reduced but still high mortality rates of 20–30% [3][4][3,4]. Surviving patients often exhibit significant long-term morbidity [5].
The etiology of CDH is incompletely understood. It is suggested that both genetic and environmental factors contribute to CDH, and although associations with different environmental factors have been described, no finding could be replicated to date [6]. From a medical genetics point of view, about 40% of CDH cases present syndromic or non-isolated. These patients present with additional anomalies of other organ systems, mostly cardiac defects, malformations of the central nervous system, urinary tract, and gastrointestinal system [7]. In these cases, a genetic diagnosis can be established more likely than in cases of isolated or non-syndromic CDH. Overall, in about 30% of CDH cases disease-causing genetic aberrations can be identified by chromosomal analysis, molecular karyotyping, and exome/or genome sequencing. Here, it has been shown that about 6% of CDH cases present with chromosomal imbalances detectable by routine chromosomal analysis or molecular karyotyping [8]. Earlier reports describe detection rates of up to 10% [9]. Using a customized array comparative genomic hybridization assay, Zhu et al. reported likely causative CNVs in 13% of a mixed CDH cohort [10]. An additional 3–10% of cases present with known monogenic syndromes. More recent sequencing studies have identified de novo damaging variants in known and novel CDH-associated genes in 10–30% of CDH patients [11][12][13][14][15][16][11,12,13,14,15,16]. Furthermore, is has been shown that the presence of a likely damaging de novo variant in a patient is associated with higher mortality and overall worse clinical outcome [17].
To establish a genetic diagnosis is increasingly important for affected families to provide proper counseling, especially as more CDH survivors reach reproductive age. This review focuses on the role of de novo events in CDH cases.

2. Known Genetic Factors

2.1. Associated Microscopic and Submicroscopic Anomalies

Except for the theoretical possibility of a trisomy 21 due to parental balanced translocation of chromosome 21 (not reported/investigated by most papers), all aneuploidies associated with CDH to date have been described to occur de novo. Aneuploidies (rarely) associated with CDH include trisomy 13, 18, 21, and triple X [17]. Furthermore it has been described in females with 45,X karyotype [18]. More frequently CDH has been described in patients with mosaic tetrasomy 12p (Pallister-Killian syndrome) [19], which always occurs de novo.
Other frequently detected CNVs include 15q26 deletion [20], comprising the CDH-associated gene NR2F2 [21]; 8p23.1 deletion [22], comprising the CDH-associated gene ZFPM2 [23]; 11q23 duplication typically resulting from parental balanced translocations [24], and 1q41–42 deletion [25], which includes the CDH-associated genes HLX and DISP1 [26][27][26,27].
Less frequently described in association with CDH 4p16 deletions (Wolf-Hirschhorn syndrome) [28][29][28,29], comprising the CDH-associated gene FGFRL1 [30]; 22q11.2 deletion [31]; deletion and duplication of 17q12 [32][33][32,33], and 1q12 duplication [34]. Very rare CNVs in CDH patients have been described and comprehensively been reviewed by Wynn et al. [18].
Among the CNVs found in CDH cases are, as expected, many de novo events. Other CNVs are caused by unbalanced translocations from a parental balanced translocation. Few CNVs are reported to be inherited [32][35][32,35]. The genome-wide de novo CNV rate in general is estimated to be 0.5–3% [36][37][36,37], about 2–12 times lower than the rate of de novo CNVs in CDH patients. CNVs are more likely to be detected in non-isolated cases of CDH than in isolated cases [8] and in general, more deletions (with a pathomechanism of haploinsufficency for CDH-associated genes) have been reported. Overall, de novo CNVs have been shown to be a major contributor to the formation of CDH.

2.2. de novo Variants in Monogenic CDH Syndromes

More than 20 syndromes with known genetic causes have been associated with the occurrence of CDH. Among these are dominant, recessive, and X-linked inherited syndromes. de novo events commonly play a role in autosomal dominant or X-linked syndromes. The rare occurrence of de novo events leading to a recessive CDH-associated syndrome is described for Cutis laxa Type 1C [38]. Some well-known monogenic syndromes caused by de novo events and featuring CDH are Cornelia de Lange syndrome (NIPBL) [39][40][39,40]; Craniofrontonasal syndrome (EFNB1) [41]; Focal dermal hypoplasia (PORCN) [42]; and Kabuki syndrome (KMT2D; MLL2) [14][43][44][14,43,44]. A full list of monogenic syndromes in which de novo events are reported is provided in Table 1. It has to be noted that for many described variants in other CDH-related autosomal dominant inherited syndromes, the inheritance pattern is not investigated or reported, but appears to be likely dominant de novo.
Table 1. Monogenic syndromes with associated CDH caused by de novo events.
Syndrome OMIM Gene Chromosomal Location Genomic Coordinates (GRCh38/hg38) Additional Malformations References
PDAC syndrome #615524 RARB 3p24.3 chr3: 25,428,263–25,597,932 Micro-/Anophtalmia, pulmonary hypoplasia, cardiac abnormalities [45]
Cornelia de Lange syndrome #122470 NIPBL 5p13.2 chr5: 36,876,769–37,066,413 Hypertelorism, synophrys, low anterior hairline, upper limb malformations [40][46][47][40,46,47]
Coffin-Siris syndrome #135900, #614609 ARID1B, SMARCA4 6q25.3 chr6: 156,776,020–157,210,779

chr19: 10,961,001–11,062,256
Growth retardation, long eyelashes, frequent respiratory tract infections, hypotonia, developmental delay [14][48][14,48]
Congenital heart defects and skeletal malformations syndrome (CHDSKM) #617602 ABL1 9q34.12 chr9: 130,713,016–130,885,683 Dysmorphic facial features, congenital heart disease, skeletal abnormalities, joint laxity, failure to thrive, gastrointestinal problems, male genital anomalies [14][49][14,49]

2.3. de novo Variants in Non-Isolated CDH

Several genes harboring de novo variants in non-isolated CDH cases have been identified, most of them by whole exome (WES)/whole genome (WGS) sequencing techniques. Among these are some well-known CDH-associated genes. De novo variants in GATA4 have been described in non-isolated [17][22][56][17,22,56] and isolated CDH [57]. GATA4 is known to be associated with congenital heart defects in humans and is further supported by a mouse model [58]. It encodes a transcription factor that is part of the retinoic acid signaling pathway, which has been implicated in diaphragm development [59].
Repeatedly, non-isolated CDH cases were found to carry de novo variants in NR2F2 [16][17][21][57][16,17,21,57], an interaction partner of ZFPM2, a gene commonly affected by the deletion of 8p23.1 observed in CDH patients. The role of NR2F2 in diaphragm development is further supported by its expression pattern and a mouse model [60]. More recently, de novo variants in MYRF, a membrane associated transcription factor, have been described in non-isolated CDH cases, also showing cardiac and genitourinary malformations [12][17][61][62][63][12,17,61,62,63].
Other genes with described de novo variants in non-isolated CDH cases are listed in Table 2. Clinical features of patients are available in Table S1. In very few genes, variants in more than one case could be detected. This illustrates the heterogeneity of the genetic background of CDH. The largest WES/WGS study on family trios could identify de novo likely gene-disrupting (LGD) or deleterious missense (D-mis) variants in 21% of non-isolated CDH cases [12]. Another family trio study also showed an increased burden of de novo D-mis and LGD variants in a mixed cohort of isolated and non-isolated CDH [13]. Recently a WES study established a genetic diagnosis in 28/76 (37%) non-isolated CDH patients, of which 15/76 (20%) were attributable to de novo variants [14]. These findings further strongly support a major role of de novo variants in CDH.
Table 2. Genes with de novo variants in non-isolated CDH cases.
Gene Chromosomal Location Genomic Coordinates (GRCh38/hg38) Number of Cases with de novo Variants References Design/Method of Studies
PRKACB 1p31.1 chr1: 84,078,062–84,238,498 1 [14] trio WES
targeted sanger sequencing
[12][23][68][12,23,68], GATA4 [57], and PTPN11 [12][16][17][12,16,17]. As in non-isolated CDH, variants in very few genes could be implicated in more than one case. A list of genes with de novo variants in isolated CDH is provided in Table 3. Notably, some genes are reported to carry de novo variants in non-isolated and isolated CDH cases.
Table 3. Genes with de novo variants in isolated CDH cases.
Gene Chromosomal Location Genomic Coordinates (GRCh38/hg38) Number of Cases with de novo Variants References Design/Method of Studies
HSPG2 1p36.12 chr1: 21,822,244–21,937,310 2 [13][14][13,14] trio WES
SLC5A9 1p33 chr1: 48,222,716–48,248,638 1 [14] trio WES
ATAD3A 1p36.33 chr1: 1,512,175–1,534,685 1 [12] trio WES/WGS ZNF362 1p35.1 chr1: 33,256,492–33,300,719 1 [17] trio WES/WGS
POGZ 1q21.3 chr1: 151,402,724–151,459,494 1 [12] trio WES/WGS HSPG2 1p36.12 chr1: 21,822,244–21,937,310 1 ° [17] trio WES
KDM5B 1q32.1 chr1: 202,724,495–202,808,421 1 [12] trio WES/WGS Apert syndrome #101200 FGFR2 10q26.13
UBAP2L 1q21.3 chr1: 154,220,955–154,270,847 1chr10: 121,479,857–121,598,403 [17Acrocephaly, micrognathia, limb malformations ] trio WGS[50]
trio WES/WGS Denys-Drash syndrome, Meacham syndrome #194080, #608978 WT1 11p13 chr11: 32,389,058–32,435,360 Male pseudohermaphroditism, cardiac abnormalities [51][52][51,52]
POGZ 1q21.3 chr1: 151,402,724–151,459,494 1 [12]
MYT1L 2p25.3 chr2: 1,789,124–2,331,348clinical WES 1 [12] trio WES/WGS Kabuki syndrome #147920 KMT2D 12q13.12 chr12: 49,018,978–49,060,794 Mental retardation, short stature, eversion of eyelids, finger pads [
DISP1 1q4114 chr1: 222,815,039–223,005,995][43][44][14[,43 153 [27],44]
FOXP1 3p13targeted sanger sequencing chr3: 70,954,708–71,583,978,53 1]
[12] trio WES/WGS Marfan syndrome Type 1 #154700 FBN1 15q21.1 chr15: 48,408,313–48,645,709 Congenital contractures, arachnodactyly, aortic dilatation, cardiac valve insufficiency [14
SRGAP32q14.2 chr2: 120,346,136–120,351,803 1 [14] trio WES][54][14,54]
3p25.3 chr3: 8,980,594–9,249,213 1 [12] trio WES/WGS Geleophysic dysplasia 2 #614185 FBN1 15q21.1 chr15: 48,408,313–48,645,709 Short stature, cardiac valvular thickening, skin thickening, joint problems
TTC21B 2q24.3[17]
chr2: 165,873,362–165,953,776 1 [17] trio WGS Rubinstein-Taybi syndrome 2
KPNA1 3q21.1 chr3: 122,421,902–122,514,939 1 [17] trio WGS #613684 EP300 22q13.2 chr22: 41,092,592–41,180,077 Failure to thrive, cardiovascular abnormalities, motor and speech delays, dysmorphic facial features [
ROBO1 3p12.3 chr3: 78,598,688–79,019,01514 1 [17]][55][14,55]
targeted panel sequencing
NAA15 4q31.1 chr4: 139,301,505–139,391,384 1 [12] trio WES/WGS Focal dermal hypoplasia #305600 PORCN Xp11.23 chrX: 48,508,992–48,520,808 Sparse hair, anophtalmia, limb malformations, Pentalogy of Cantrell
FOXP1 3p13 chr3: 70,954,708–71,583,978 1 [15][42]
clinical WES
SMO 7q32.1 chr7: 129,188,633–129,213,545 Craniofrontonasal syndrome #304110 EFNB1 Xq13.1 chrX: 68,829,021–68,842,160 Coronal craniosynostosis, duplex thumb, partial agenesis of corpus callosum
1 [12] RAF1 3p25.2[41]
trio WES/WGS
chr3: 12,583,601–12,664,117
trio WES/WGS
chr14: 74,881,916–74,903,743
GATA4 8p23.1 chr8: 11,704,202–11,760,002 1 [57] targeted panel sequencing FAT4 4q28.1 chr4: 125,314,955–125,492,932 1 [
ZFPM217] 8q23.1 chr8: 105,318,438–105,804,539 3 [12][23][68]trio WGS
[12,23,68] WES, trio WES/WGS, targeted sanger sequencing CDO1 5q22.3 chr5: 115,804,733–115,816,659 1 [14] trio WES
FOXP4 6p21.1 chr6: 41,546,426–41,602,384
EMX2 10q26.11 chr10: 117,542,746–117,549,546 1 [121 [12] trio WES/WGS
PTPN12 7q11.23 chr7: 77,537,295–77,640,069 1 [14] trio WES
] trio WES/WGS
WT1 11p13 chr11: 32,389,058–32,435,360 3 [12][16][12,16] trio WES/WGS
PTPN11 12q24.13 chr12: 112,419,112–112,504,764 3 [12][16][17][12,16,17] trio WES/WGS BRAF 7q34 chr7: 140,719,327–140,924,810 1 [12] trio WES/WGS
trio WES
MEIS2 15q14 chr15: 36,889,204–37,100,549 1 [12] trio WES/WGS GATA4 8p23.1 chr8: 11,704,202–11,760,002 3 [17][22][56][17,22,56] targeted sanger sequencing, trio WGS
TBX6 16p11.2 chr16: 30,085,793–30,091,924 1 [11] WES EYA1 8q13.3 chr8: 71,197,511–71,548,061 1 [11][57][11,57] WES, targeted panel sequencing
CTCF 16q22.1 chr16: 67,562,467–67,639,176 1 [17] trio WGS TLN1 9p13.3 chr9: 35,696,948–35,732,195 1 ° [17] trio WES
AP1G1 16q22.2 chr16: 71,729,000–71,808,834 1 PLPP6 9p24.1 chr9: 4,662,294–4,665,258 1 [14] trio WES
[12] trio WES/WGS
MYH10 17p13.1 chr17: 8,474,207–8,630,761 1 [17] NOTCH1 9q34.3 chr9: 136,494,433–136,546,048 1 [17] trio WGS
targeted panel sequencxing
SRSF1 17q22 chr17: 58,000,919–58,007,246 1 [17] trio WGS CTR9 11p15.3 chr11: 10,751,246–10,779,746 1 * [16] trio WES
MYRF 11q12.2 chr11: 61,752,636–61,788,518 11 [12][17]][12[61,17][62][63,61,62,63] trio WES/WGS, clinical WES, trio WGS
PTPN11 12q24.13 chr12: 112,419,112–112,504,764 1 [12] trio WES/WGS
HNRNPC 14q11.2 chr14: 21,210,613–21,269,421 1 [17] trio WGS
BMP4 14q22.2 TCF12 15q21.3 chr15: 56,918,644–57,289,853 1 [15] clinical WES
SIN3A 15q24.2 chr15: 75,370,933–75,455,783 1 [14] trio WES
NR2F2 15q26.2 chr15: 96,330,700–96,340,258 4 [16][17][21]
LONP1 19p13.3 chr19: 5,691,835–5,720,572 2 [17] trio WGS [
CIC 19q13.2 chr19: 42,268,537–42,295,796 1 [12] trio WES/WGS
LAMA5 20q13.33 chr20: 62,309,065–62,367,312 1 [12] trio WES/WGS
DIDO1 20q13.33 chr20: 62,877,738–62,937,952 1 [12] trio WES/WGS chr14: 53,949,736–53,956,825 1
HSD17B10 Xp11.22[ chrX: 53,431,261–53,434,37064] 1 [12] trio WES/WGS
FLNA Xq28 chrX: 154,348,529–154,371,283 1 [17] 57][65][16,17,21,57,65] clinical WES, targeted panel sequencing, trio WES, trio WGS
TRAF7 16p13.3 chr16: 2,155,782–2,178,129 1 [15] clinical WES
trio WGS ANKRD11 16q24.3 chr16: 89,285,175–89,490,318 1 [17] trio WGS
MYH10 17p13.1 chr17: 8,474,207–8,630,761 1 [66] clinical WES
TP53 17p13.1 chr17: 7,668,421–7,687,490 1 * [16] trio WES
NLK 17q11.2 chr17: 28,042,677–28,196,381 1 [17] trio WGS
FZD2 17q21.31 chr17: 44,557,484–44,561,262 1 [32] aCGH
ATXN7L3 17q21.31 chr17: 44,191,805–44,198,070 1 [17] trio WGS
ALYREF 17q25.3 chr17: 81,887,835–81,891,586 1 [12] trio WES/WGS
GATA6 18q11.2 chr18: 22,169,589–22,202,528 1 [67] trio WES
NACC1 19p13.13 chr19: 13,118,264–13,141,147 1 [12] trio WES/WGS
LONP1 19p13.3 chr19: 5,691,835–5,720,572 1 [14] trio WES
LTBP4 19q13.2 chr19: 40,601,369–40,629,818 1 [38] targeted sanger sequencing
ZC3H4 19q13.32 chr19: 47,064,187–47,113,776 1 [12] trio WES/WGS
PCNA 20p12.3 chr20: 5,114,953–5,126,626 1 [12] trio WES/WGS
chr20: 36,092,712–36,230,343 1 [12] trio WES/WGS
ZBTB18 1q44 20q13.13 chr20: 48,921,711–49,036,693 1 [14] trio WES
chr1: 244,051,283–244,057,476 1 [12] EPB41L1 20q11.23 ADNP 20q13.13 chr20: 50,888,918–50,931,437 1 [17] trio WGS
SCAF4 21q22.11 chr21: 31,671,000–31,732,118 1 [17] trio WGS
DDX3X Xp11.4 chrX: 41,333,348–41,350,287 1 [15] clinical WES
USP9X Xp11.4 chrX: 41,085,445–41,236,579 1 ° [17] trio WES/WGS
CLCN4 Xp22.2 chrX: 10,156,975–10,237,660 1 [14] trio WES
HCCS Xp22.2 chrX: 11,111,301–11,123,078 1 [15] clinical WES
STAG2 Xq25 chrX: 123,961,314–124,102,656 1 [14] trio WES

2.4. de novo Variants in Isolated CDH

In patients with isolated CDH a genetic cause is less likely to be established by current genotyping or sequencing techniques. The above-mentioned study on case-parent-trios could identify de novo likely gene-disrupting or deleterious missense variants in only 12% of isolated CDH cases [12]. Among the described de novo variants in isolated CDH are variants in the already mentioned genes ZFPM2
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