Genetic etiology analysis of 244 fetal ventricular septal defect in the prenatal setting

doi:10.21203/rs.3.rs-4345913/v1

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Research Article

Genetic etiology analysis of 244 fetal ventricular septal defect in the prenatal setting

https://doi.org/10.21203/rs.3.rs-4345913/v1

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Objective

This study evaluated the application of karyotyping combined with single-nucleotide polymorphism (SNP) array and whole-exome sequencing (WES) of prenatal diagnosis of ventricular septal defect (VSD), and explored the genetic etiology of VSD.

Methods

244 fetuses with VSD diagnosed by prenatal echocardiography were selected, including 59 cases isolated VSD and 185 cases non-isolated VSD, and used for conventional karyotyping and SNP analysis at the same time. Among them, 19 fetuses were used for further Trio-WES detection.

Results

20 chromosomal abnormality were identified by karyotyping/SNP array. Another 21 cases of abnormal copy number variations (CNVs) were identified by SNP array, including 10 cases of pathogenic CNVs and 11 cases of variations of uncertain significance (VUS). 5 cases with (likely) pathogenic genetic variants were identified by Trio-WES. The detection rate of pathogenic chromosomal and gene abnormalities in non-isolated VSD (33/185) was significantly higher than that in isolated VSD (2/59) (17.84% vs 3.39%, p = 0.006). For non-isolated VSD, the detection rate for VSD with extra-cardiac defects (10/20) was significantly higher than that in VSD with cardiac defects (9/45) (50.00% vs 20.00%, p = 0.014) and soft markers (14/116) (50.00% vs 12.07%, p < 0.001). Trisomy 21 and 22q11.2 deletion syndrome were the most common chromosomal abnormalities. Additionally, we found six gene variants might be associated with the causative genetic mechanisms of VSD.

Conclusion

The rational combination of karyotyping, SNP array and Trio-WES can effectively improve the detection rate of chromosomal and gene abnormalities in VSD fetuses. Ultrasound abnormalities, such as VSD with extra-cardiac defects and multiple soft markers added detection of pathogenic abnormalities.

ventricular septal defects

karyotyping

SNP array

Trio-WES

fetal

Congenital heart disease (CHD) was a major cause of infant mortality. About 0.8% of newborns would have CHD [1]. Ventricular septal defects (VSDs) are the most common form of cardiac defects. About 10% of congenital defects have CHD, of them, 30–35% have VSDs [2]. Environmental and genetic factors are considered the main causes of VSDs. Genetic factors mainly include chromosomal aneuploidies, copy number variations (CNVs), and gene variants [3, 4]. Prenatal diagnosis is an effective method of intervention for birth defects. Although prenatal ultrasound could reveal fetal cardiac abnormalities, it cannot identify the reason of the defects. So it is difficult to make a comprehensive assessment of VSDs. Karyotyping has been considered the gold standard in detecting chromosomal abnormalities. But it could not give precise conclusion for submicroscopic chromosomal deletions or duplications in the genome [5]. Single nucleotide polymorphism (SNP) array, as one of a molecular karyotype analysis technique, can detect chromosomal imbalance copy number variations (CNVs) and also triploidies, low-level mosaics, and regions of homozygosity [6, 7]. In prenatal diagnosis, SNP array has become the recommended genetic test in fetuses with ultrasound abnormalities, such as VSDs. Whole exome sequencing (WES), as one of next-generation sequencing technology, focuses on all protein coding regions of the genome can accurately identify point mutations and provide more precise diagnosis. It has been used as an effective complementary methods with normal karyotype and negative SNP array results in the prenatal diagnosis [8–10]. In this study, We compared the prenatal diagnostic rates of VSD fetuses with different methods, including karyotyping, SNP array, and WES, and explored the genetic etiology of VSD to provide the basis for genetic counseling.

Subjects

A total of 244 fetuses were diagnosed with VSD using ultrasonography and echocardiography in the Prenatal Diagnosis Center of the General Hospital of Ningxia Medical University, from January 2022 to June 2023. All cases underwent invasive prenatal testing with karyotyping and SNP array analysis.Part of the VSD fetuses with negative karyotyping and SNP array results were further tested by Trio-WES. The study was approved by the ethics committee of the General Hospital of Ningxia Medical University, and informed consent was obtained from the parents. All fetal samples were collected by amniocentesis at the gestational age of mothers was 28.4 years (range,22–42 years) and the amniotic fluid was collected at the gestational age of 23 weeks (range, 18–27 + 6weeks). Among them, 59 fetuses with isolated VSD, the other 185 was non-isolated VSD including other cardiac anomalies, extra cardiac structural anomalies or sonographic soft markers and polyhydramnio. Soft markers included ventricular hyper echoic spot, choroid plexus cysts, tricuspid regurgitation, absent or shortened nasal bone, single umbilical artery, pyelectasis, aberrant right subclavicular artery, increased nuchal translucency, mild ventriculomegaly, echogenic bowel, persistent right umbilical vein, et al.

Karyotyping

All samples of amniotic fluid cells was centrifuged and cultured from 15ml amniotic fluid, and then were collected followed by making sections. For each sample, amniotic fluid cell were analyzed by routine chromosome analysis according to standard procedures using conventional Giemsa banding techniques (450–550 bands resolution). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2020).

SNP array

Genomic DNA was isolated from 10ml uncultured amniotic fluid samples using the QIAamp DNA Blood Mini kits (Qiagen, Hilden, Germany). The DNA concentration were quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). CytoScan® 750K Arrays (Affymetrix, Santa Clara, CA, USA) that cover both polymorphic (SNPs) and non-polymorphic regions of the genome being used to assess DNA copy number variations (CNVs). CNVs can also be filtered based on a minimum size (kb) and marker counts. Gains was setting at > 200 kb in size with a minimum set of 50 marker counts. Loss was setting at > 100 kb in size with a minimum set of 50 marker counts. Regions of allelic homozygosity (ROHs) were reported on a threshold of 10Mb. The detected CNVs were evaluated by Database of Genomic Variants, DECIPHER, UCSC, OMIM and ClinGen. Following the American College of Medical Genetics (ACMG) standards and guideline, the CNVs were classified as Pathogenic (P) CNVs, Likely pathogenic (LP) CNVs, Benign CNVs, Likely benign (LB) CNVs and Variants of uncertain significance (VUS)[6]. Maternal and paternal blood samples were also analyzed by SNP array to determine whether CNVs detected in the fetal samples were inherited or de novo.

Trio-WES

After obtaining informed consent, the extracted fetal amniotic fluid genomic DNA and parental blood genomic DNA were used to construct a whole exon library according to the manufacturer’s instructions of Berrygenomics medical laboratory. High-throughput sequencing (PE150) was performed by Nova Seq 6000 series sequencers (Illumina, San Diego, CA, USA) with sequencing depth of 100×. Single nucleotide site variation (SNV) and small insertion deletion variation (InDels) were analyzed and compared with Public databases such as HPO, OMIM, GHR and Clinvar. Variations were verified by Sanger sequencing.

Statistical analysis

Statistical analyses were performed using Microsoft Office Excel 2013 software and SPSS 23.0. Comparisons between different groups were evaluated by Student’s t-test. P < 0.05 was considered statistically significant.

Sample characteristics

A total of 244 fetuses with VSD were successfully analyzed by conventional karyotyping and SNP analysis. Among them, 41cases had abnormal copy number variations (CNVs). Of the 203 SNP array negative cases, 19 cases underwent Trio-WES testing. The genetic analysis procedure was showed in Fig. 1.

Karyotyping analysis results

244 VSD fetuses were analyzed, including 59 cases VSD and 185 cases non-isolated VSD (Table 1). Among them, 20 (20/244, 8.20%) cases were identified chromosomal abnormalities by karyotyping, include 18 cases with aneuploidy, 1 case with triploid, 1 cases with abnormality of chromosome structure. In the isolated VSD fetuses group, only one chromosomal abnormality associated with trisomy 21 syndrome being identified. In the 185 non-isolated VSD fetuses group, 6 cases were trisomy 18 syndrome, 1 case were triploid, and 11 cases were trisomy 21 syndrome, 1 cases were abnormality of chromosome structure based on karyotyping result (Table 2). Karyotyping and SNP-array results of fetus No. 18 were showed in Fig. 2. Karyotyping and SNP-array results of fetus No. 19 were showed in Fig. 3.

Table 1

The abnormal findings of 244 VSD fetuses
Classification		Chromosomal abnormalities	SNP-array results		WES results
Classification		Chromosomal abnormalities	pCNVs	VUS	P/LP
Isolated VSD		1/59	1/59	2/59	0
Non- isolated-VSD	VSD with extra-cardiac defects	7/20	1/20	2/20	2/10
	VSD with cardiac defects	2/45	4/45	3/45	3/9
	VSD with soft markers	10/116	4/116	4/116	0
	VSD with polyhydramnios	0/4	0/4	0/4	0
Total		20/244	10/244	11/244	5/19

Table 2

Abnormal karyotyping results of VSD fetuses
Case	Karyotype	SNP-array	Prenatal ultrasound	Postnatal outcome
1	47, XN, + 18	arr(18) × 3	Skull dysplasia, VSD, ASD, Overlapping fingers, Acromphalus	TP
2	47, XN, + 18	arr(18) × 3	Skull dysplasia, TOF	TP
3	47, XN, + 18	arr(18) × 3	VSD, Absent or shortened nasal bone, Choroid plexus cysts	TP
4	47, XN, + 18	arr(18) × 3	VSD, Overlapping fingers	TP
5	47, XN, + 18	arr(18) × 3	VSD, Choroid plexus cysts, Ventricular hyperechoic spot	TP
6	47, XN, + 18	arr(18) × 3	Overlapping fingers, VSD, Increased nuchal translucency, Coarctation of the aorta	TP
7	47, XN, + 21	arr(21) × 3	VSD	TP
8	47, XN, + 21	arr(21) × 3	VSD, Coarctation of the aorta	TP
9	47, XN, + 21	arr(21) × 3	Vascular circle, VSD, Micrognathia	TP
10	47, XN, + 21	arr(21) × 3	Absent or shortened nasal bone, Increased nuchal translucency, VSD, Tricuspid regurgitation	TP
11	47, XN, + 21	arr(21) × 3	Absent or shortened nasal bone, VSD, Ventricular hyperechoic spot	TP
12	47, XN, + 21	arr(21) × 3	VSD, Tricuspid regurgitation	TP
13	47, XN, + 21	arr(21) × 3	VSD, Absent or shortened nasal bone	TP
14	47, XN, + 21	arr(21) × 3	Increased nuchal translucency, VSD, Choroid plexus cysts	TP
15	47, XN, + 21	arr(21) × 3	VSD, Absent or shortened nasal bone	TP
16	47, XN, + 21	arr(21) × 3	VSD, ASD	TP
17	47, XN, inv(9) (p12q13), + 21	arr(21) × 3	VSD, Tricuspid regurgitation	TP
18	46XN, der(21;21) (q10; q10), + 21	arr(21) × 3	Increased nuchal translucency, VSD, Tricuspid regurgitation	TP
19	46XN, psu dic(9;9) (q34:q12-13)	arr[hg19]9p24.3q13(208,454 − 68,324,147)×3,9q34.3(139,268,640 − 141,018,648)×1	VSD, Persistent left superior vena cava, Strephenopodia	TP
20	69, XXX	arr(1–22, X)× 3	Hydrocephalus, VSD, Tricuspid regurgitation	TP
TD term delivery, TP termination of pregnancy

SNP array results

244 fetuses were also performed with SNP array hybridization, the results proved that 41 (41/244, 16.8%) cases have abnormal chromosome, which including 20 cases identified by karyotyping and another 21 cases did not verified by karyotyping. The 21 cases identified by SNP assay including 10 pathogenic CNVs, 11 VUS. The pathogenic CNVs ranged from size of 0.56Mb to 8.22Mb involved deletions of 22q11.2, 17q12, 1q21.1, 1p36, 16p13.1; duplications of 22q11.2, 16p11.2, 17p11.2 (Table 3). Combined with ultrasound and SNP assay analysis, we only found 3 cases isolated-VSD, which including 1 fetal with pathogenic CNVs and 2 fetal with VUS. The SNP-array results of fetus No. 27 was showed in Fig. 4.

Table 3

SNP array analysis of VSD fetuses with normal karyotyping results
Case	results	Size	Prenatal ultrasound	Classification	Parental material available	Known syndrome/gene	Obstetrical outcomes
21	arr[hg19]22q11.21(18,631,364 − 21,800,471)×1	3.1Mb	Coarctation of the aorta, VSD	Pathogenic	Not available	22q11.2 deletion syndrome	TP
22	arr[hg19]22q11.21(18,648,855 − 21,800,471)×1	3.1Mb	VSD, Vascular circle	Pathogenic	Not available	22q11.2 deletion syndrome	TP
23	arr[hg19] 22q11.21(18,636,749 − 21,800,471)x1	3.2Mb	VSD, Omphalocele, Echogenic bowel	Pathogenic	Not available	22q11.2 deletion syndrome	TP
24	arr[hg19]22q11.21(18,648,855 − 21,800,471)x3	3.15Mb	VSD, Tricuspid regurgitation, Increased nuchal translucency	Pathogenic	Materal inherited	22q11.2 duplication syndrome	TD
25	arr[hg19]1q21.1q21.2(145,895,746 − 147,885,600)×1	1.99Mb	VSD, Ventricular hyperechoic spot	Pathogenic	de novo	1q21.1deletion syndrome	TP
26	arr[hg19]17p11.2(16,500,000–20,080,000)x3	3.58Mb	VSD	Pathogenic	Not available	17p 11.2duplication syndrome	TP
27	arr[hg19]16p13.11p13.11(15,040,000–16,300,000)×1	1.26Mb	VSD, Single umbilical artery, Tricuspid regurgitation	Pathogenic	Materal inherited	16p13.11recurrent region(includes MYH11)	TD
28	arr[hg19]17q12(34,822,465 − 36,418,529)×1	1.5Mb	VSD, Coarctation of the aorta	Pathogenic	Not available	17q12 deletion syndrome	TD
29	arr[hg19] 16p11.2(29,640,000–30,200,000)x3	0.56Mb	VSD, Aortico-pulmonary septal defect	Pathogenic	de novo	16p11.2 duplication syndrome	TP
30	arr[hg19]1p36.33p36.23(820,000–9,040,000)×1	8.22Mb	VSD, Ventricular hyperechoic spot	Pathogenic	de novo	1p36 deletion syndrome	TP
TD term delivery, TP termination of pregnancy

Trio-WES results

19 fetuses with negative SNP-array results and their parents were further requested voluntarily for Trio-WES testing, the results showed that 5 cases (5/19, 26.3%) have likely pathogenic/pathogenic gene variants which were confirmed by Sanger sequencing (Table 4). Combined with ultrasound and Trio-WES results, we found 5 cases were all identified in the non-isolated VSD fetuses group, of which 2 cases were VSD with extra cardiac structural anomalies and 3 cases were VSD with other cardiac anomalies. The Trio-WES results of fetus No. 32 was showed in Fig. 5.

Table 4

WES results of VSD fetuses with a negative result on karyotyping and SNP array
Case	Gene	Inheritance pattern	Prenatal ultrasound	Variant(s)	Classification	Parental material available	Obstetrical outcomes
31	RIT1	Noonan syndrome 8, AD	VSD, Ventricular hyperechoic spot, Mild stenosis of aortic and pulmonary valves。	NM_006912.6: c.244T > G: p.F82V	P	de novo	TP
32	NR2F2	46,XX sex reversal 5, AD; Congenital heart defects, multiple types, 4, AD	VSD, Increased nuchal translucency	NM_021005.4: c.679G > A: p.A227T	LP	Paternally Inherited Het	TD
33	PTPN11	LEOPARD syndrome 1, AD;Metachondromatosis, AD;Noonan syndrome 1 , AD	VSD, Native aortic coarctation	NM_002834.5: c.1510A > G: p.M504V	P	de novo	TP
34	TBX1	DiGeorge syndrome, AD; Tetralogy of Fallot, AD; Velocardiofacial syndrome, AD	VSD, Right aortic arch with mirror branch, umbilical hernia	NM_080647.1: c.840 + 2T > G:	P	de novo	TP
35	GATA6	Atrioventricular septal defect 5, AD; Pancreatic agenesis and congenital heart defects, AD;Tetralogy of Fallot, AD	VSD, Double outlet right ventricle	NM_005257.5: c.613del: p.L205Cfs*89	P	de novo	TP
35	GATA5	Congenital heart defects, multiple types, 5, AD/AR	VSD, Double outlet right ventricle	NM_080473.5: c.712C > T: p.R238C	LP	Materal inherited het	TP
TD term delivery, TP termination of pregnancy

Comparison of detection rates

244 fetuses with VSD underwent karyotyping and SNP array analyses, among them, 19 fetuses were tested by WES. The results showed that the detection rate of chromosomal abnormality with SNP array was significantly higher than that with Karyotyping analysis (16.80% vs 8.20%, p = 0.004). Compared with isolated VSD fetus (2/59), non-isolated VSD fetus (33/185) have high detection rate of pathogenic chromosomal and gene abnormalities (17.84% vs 3.39%, p = 0.006). In the non-isolated VSD fetus, the detection rate for VSD with extra-cardiac anomalies was 50.00% (10/20), the detection rate of VSD with other cardiac anomalies was 20.00% (9/45), and the detection rate of VSD with sonographic soft markers was 12.07% (14/116).

Pregnancy Outcomes

Based on karyotyping, SNP assay and Trio-WES results, combined with genetic counseling, 31 pregnant women finally chose to terminate the pregnancy, including 20 with VSD and chromosomal abnormality, 7 with pathogenic CNVs and 4 with P/LP genetic variation. One case of 22q11.2 duplication syndrome, one case of 16p13.11 microdeletion syndrome and one case of 17q12 microdeletion syndrome were identied with SNP-array chose to continue the pregnancy due to the variable expression and incomplete penetrance. One cases with likely pathogenic genetic variants of NR2F2 gene was identified by Trio-WES, which originated from the father, chose to continue the pregnancy and deliver to term. All of the 4 cases with normal postpartum examination at 1 year of age. 11 cases of VUS were identified with SNP-array chose to continue the pregnancy. Among them, 10 cases with normal postpartum examination at 1 year of age, one case was lost to follow-up.

VSDs are openings in the ventricular septum, were associated with genetic factors. In the prenatal setting, about 13–23% of fetuses with VSD have chromosome aneuploidy and CNVs, most of them are trisomy 21, trisomy18, and 22q11 microdeletions [11–13]. WES as an additional diagnostic technique beyond traditional methods can added diagnostic yield from 5–20% for CHD [14, 15]. But only few studies focused specifically on VSD anomalies, with a diagnostic yield ranging from 13–20% [16, 17]. In this study, We identified 20 (8.20%) cases chromosomal abnormalities, including 6 cases of trisomy 18, 12 cases of trisomy 21, 1 case of triploid and 1 case of chromosome structural abnormalities by karyotyping. We also identified 41 (16.80%) cases abnormal chromosome with SNP array, including 20 cases identified by karyotyping and another 21 cases did not verified by karyotyping. Here we found karyotyping combined with SNP array could increase the detection rate (16.80% vs 8.20%, p = 0.004). Overall, the diagnostic yield of 16.8% (41/244) for conventional tests (karyotyping and SNP array) in our study is consisted with the previous research [11, 16]. Furthermore, In our study, among 203 cases with negative SNP-array results, only 19 pregnant women volunteered to undergo Trio-WES, and 5 cases with likely pathogenic/pathogenic gene variants were detected. The detection rate was 26.32% (5/19), higher than previous research [16, 17], which may be related to not all cases had undergone WES and selection bias. Here in this study, we identified prenatal use of WES can provide more genetic diagnoses beyond traditional methods.

To further analysis the cases, we found that there are 2 cases of chromosome rearrangements in trisomy 21 syndrome which was identified by G-banding karyotyping rather than SNP array. One type of trisomy 21 syndrome karyotype was designated as 46XN, der (21;21)(q10༛q10), + 21, that had de novo isochromosome of long arm of the chromosome 21 (Table 2, cases 18). Another type of trisomy 21 syndrome karyotype have one pericentric (9)(p12q13) inversion (Table 2, cases 17). Balanced chromosomal rearrangement would not influence the current pregnancy, but it relevant to future reproductive counseling. For aberration on chromosome 9, karyotyping showed a fragment was added to the end of the long arm, resulting in trisomy of this segment. But SNP-array showed that there were 68.1Mb duplication in the 9p24.3q13 region and 1.7Mb deletion in 9q34.3 region, which was determined to be pCNVs (Table 2, cases 19). The result showed that compared with standard karyotyping, SNP array could be more informative but it cannot replace karyotyping because of their inability to detect balanced chromosomal translocations or inversions, which consisted with previous reports [18, 19].

Previous clinical studies indicated that SNP array could be used as the first-line genetic diagnostic test for VSDs because of it had high ranges in prenatal evaluation of VSDs and can provide more information for clinical fertility decisions [20, 21]. In this study 10 cases of pathogenic CNVs being verified by SNP array. Of them, 3 cases of 22q11.2 deletion syndrome, 1 cases of 22q11.2 duplication syndrome. The results consisted with the previous research that 22q11.2 deletion syndrome was the most common CNVs for VSD fetuses [12]. We also found 1 case of 1q21.1 deletion syndrome, 1 case of 17q12 deletion syndrome, 1 case of 16p11.2 duplication syndrome, 1 case of 1p36 deletion syndrome, 1 case of 17p11.2duplication syndrome and 1 case of 16p13.11 deletion region. These syndromes were all connected with pathogenic variations, and the clinical phenotypes were variable and incomplete penetrance [22, 23]. Studies have shown that the clinical phenotype of 22q11.2 duplication syndrome varies from normal or almost normal to severe congenital structural malformations, with a penetrance of about 21.9% [24]. These variations were mostly inherited from parents and new mutations in proximal classical microrepeats account for about 25.5–30% [25]. In this study, ultrasound of the 22q11.2 duplication syndrome (inherited from the mother) showed VSD, tricuspid regurgitation, and NT thickening. After clinical genetic counseling, parents of the case 24 (Table 3) finally selected to continue pregnancy, and the clinical phenotype was normal after birth. Studies have reported that 16p13.11 deletion region was related to mental retardation and a variety of congenital abnormalities include VSD, with a penetrance of about 13.1% [26]. In our study, ultrasound of the 16p13.11 deletion (1.26Mb, inherited from the mother) showed VSD, single umbilical artery, tricuspid regurgitation. Parents of the case 27 (Table 3) also selected to continue pregnancy after genetic counseling and the clinical phenotype was normal after birth. 17q12 deletion syndrome was reported with structural or functional abnormalities of kidney and urinary system, also manifested as cardiac abnormalities, with penetrance of about 34.4% [27]. In this study, ultrasound showed VSD and aortic arch narrowing in case 28, but no phenotypic abnormality was found after birth. Because some chromosomal microdeletions and microduplications could not accurately predict the clinical phenotype in prenatal, which would bring challenges for genetic counseling. Therefore, we suggest that pregnant women and their families should be fully informed about the scope and limitations of the technology in clinical work. When pCNVs were detected in fetus, pregnancy should not be terminated blindly. The comprehensive judgment should be made based on ultrasound phenotype, family analysis and inherited or de novo, which would provide more accurate and reliable information for clinical diagnosis and fertility guidance.

In this study, gene pathogenic variants in RIT1, NR2F2, PTPN11, TBX1, GATA6 and GATA5 were detected by Trio-WES and sanger sequencing, which have been reported to be associated with abnormal cardiac development [28, 29]. In case 32, a heterozygous variant of NR2F2 gene c.679G > A was detected, which was inherited from the father who has normal clinical phenotype. Previous studies reported that Nuclear receptor subfamily 2 group F member 2 (NR2F2) encodes a transcription factor which was highly expressed during mammalian development. But the phenotype spectrum associated with the pathogenic variation of NR2F2 still lacks specificity and clinical features were variable, including cardiac malformation, developmental delays and genital abnormalities [30]. After clinical genetic counseling, parents of the case 32 (Table 4) finally selected to continue pregnancy, and no abnormalities were found after birth. Another 4 case with P/LP genetic variation, which were de novo, finally chose to terminate the pregnancy. This study elucidated prenatal WES maybe provide more precise genetic diagnosis for VSD.

It has been reported that when VSD is accompanied with additional structural anomalies, the incidence of chromosomal abnormality would increase [16, 31]. We compared the detection rate of pathogenic chromosomal and gene abnormalities in fetuses with isolated VSDs and non-isolated VSD. The result proved that the detection rate in non-isolated VSDs (19 chromosomal abnormality, 9 pCNVs, 5 P/LP genetic variation; 33/185) was significantly higher than that of isolated VSDs (1 chromosomal abnormality, 1 pCNVs; 2/59) (17.84% vs 3.39%, p = 0.006). This outcome confirmed that VSD with additional structural anomalies tend to be at high risk for genetic abnormalities. It is well accepted that ultrasonic soft markers and polyhydramnios are associated with an increased risk of fetal aneuploidy and pathogenic CNVs [32]. Our study showed that the detection rate for VSD with extra-cardiac defects (7 chromosomal abnormality, 1 pCNVs, 2 P/LP genetic variation; 10/20) was significantly higher than that in VSD with cardiac defects (2 chromosomal abnormality, 4 pCNVs, 3 P/LP genetic variation; 9/45) (50.00% vs 20.00%, p = 0.014) and soft markers (10 chromosomal abnormality, 4 pCNVs; 14/116) (50.00% vs 12.07%, p < 0.001). This study suggests that when ultrasound indicates the presence of VSD, detailed ultrasound scanning and prenatal genetic diagnosis are necessary.

One of the limitations of this study was that we did not get the information of the parents who have CNVs. So we could not do more heredity analysis for the disease. Another limitation was that the cases we collected in the study were too little, more clinical cases are need for further analysis to verify our results.

In summary, our study illustrates that karyotyping combined with SNP array and WES are valuable tools for identifying chromosomal and gene abnormality in prenatal diagnosing with VSDs. The combination of ultrasonic detection and genetic testing can offered more information and gave better guideline for clinical counseling.

VSD ventricular septal defect

SNP single-nucleotide polymorphism

WES whole-exome sequencing

CNVs copy number variations

VUS variations of uncertain significance

CHD congenital heart disease

ROHs regions of allelic homozygosity

SNV single nucleotide site variation

InDels insertion deletion variation

Acknowledgements

We thank all of the families for their participation and members of Prenatal Diagnosis Center，Medical experiment center.

Authors’ contributions

BW and FSZ designed and planned the study. BW, WM, and XYY performed experiments and analyzed the data. MJL and YJM contributed many helpful suggestions about data processing and manuscript. BW and FSZ wrote the first and final draft of the manuscript. All authors read and approved the final manuscript.

Funding statement

This work was supported by the Ningxia Natural Science Foundation, Grant/Award Number: 2022AAC03543.

Data Availability

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This study was conducted according to the Declaration of Helsinki and approved by the Ethics Committee of General Hospital of Ningxia Medical University (Ethics No. 2020—0520B). Written informed consents were obtained from all participants involved in the study.

Consent for publcation

Not applicable.

Competing interests

None of the authors have any competing interests to declare.

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Genetic etiology analysis of 244 fetal ventricular septal defect in the prenatal setting

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Subjects

Karyotyping

SNP array

Trio-WES

Statistical analysis

Results

Sample characteristics

Karyotyping analysis results

SNP array results

Trio-WES results

Comparison of detection rates

Pregnancy Outcomes

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1