Diagnostic value of CMA in different phenotypes of fetal congenital heart defects

doi:10.21203/rs.3.rs-2044674/v1

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Research Article

Diagnostic value of CMA in different phenotypes of fetal congenital heart defects

https://doi.org/10.21203/rs.3.rs-2044674/v1

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Objective

To evaluate the detection rate of fetal chromosomal abnormalities in congenital heart defects (CHD), further dig the potential diagnostic value of Chromosomal Microarray Analysis (CMA) technology for different phenotypes, and explore the possible genetic pathogenic factors of CHD.

Methods

We analyzed the CMA of 427 cases of CHD fetuses, and divided CHD into different groups according to two dimensions. According to whether they were combined with ECA, they were divided into isolated CHD and non-isolated CHD; According to the cardiac phenotype, they were divided into ten groups. The correlation between numerical chromosomal abnormalities (NCA), and copy number variations (CNVs, except likely benign and benign CNVs) with CHD was analyzed by Mantel test.

Results

In general, CHD with ECAs were more likely to have a genetic abnormality than those without ECAs (39.3% vs. 14.8%, P<0.05). The genetic abnormality rate of 427 fetuses was 21.8% (93/427), the detection rate of NCA was 12.9% (55/427), the most relevant were skeletal, Craniofacial, VSD, AVSD (P<0.05); and the detection rate of pCNVs was 8.9% (38/427), the most relevant were IAA, A, IAA, B, RAA, TAPVC, CoA, TOF and thymic abnormality. 22q11.2DS and had the highest detection, the detection rate of 22q11.2DS in the subgroups were IAA, B, RAA, PS, CoA, TOF and thymic abnormality. Overall analysis of cases, the CNV deletion fragment larger than 1 Mb may be pathogenic. In CNV, deletion was more likely to be pathogenic than duplication.

In addition, we detected 12 CNV syndromes; among of them, 9 syndromes that may be related to CHD. gene LIMK1 and MYH11 have been identified as part of a common pathway between cardiovascular and neurological development; candidate genes are considered to be related to heart and/or involved in embryonic development, FLI1, NIPBL, DLL1, PTPN11, TBX5.

Conclusion

Increased risk of genetic abnormalities in non-isolated CHD; CHD phenotype most related to NCA, pCNV and 22q11.2DS was found; and 12 CNV syndromes and 7 meaningful candidate genes were detected.

congenital heart defects

chromosomal microarray analysis

copy number variations

numerical chromosomal abnormalities

Ultrasound

The occurrence of CHD is related to genetic factors.

Increased risk of genetic abnormality in non-isolated CHD, found the ultrasonic diagnostic clues of NCA, pCNVs and 22q11.2DS in CHD, explored the distribution of CNV with different fragment sizes and the next research direction. some candidate genes noticed us to find a common pathway for the development of heart and nervous system.

congenital heart defects (CHD), It is a birth defect that cardiovascular system is affected by many factors during embryonic development, resulting in local structural abnormalities and actual or potential impact on cardiac function [1]. Its incidence rate is 0.4%-0.8% among infants at birth, ranking first among birth defects [2].Although progress has been made in pediatric cardiac diagnosis and health care, CHD remains the leading cause of perinatal and infant death [3]. The pathogenesis of CHD has not been clarified yet. Most scholars believe that it may be caused by genetic and environmental factors and their interactions in embryonic stage. When combined with chromosomal abnormalities, the prognosis of CHD fetuses is not satisfactory due to severe extra-cardiac abnormalities (ECAs) or postnatal neurological development disorders. Therefore, prenatal genetic diagnosis of fetus with CHD is recommended.

Chromosomal microarray analysis (CMA) can simultaneously detect numerical chromosomal abnormalities (NCA) and chromosomal micro abnormality at the whole gene level. This technique can detect copy number variations (CNVs).

In our study, we aimed to use CMA to comprehensively evaluate the detection rate of chromosomal abnormalities in CHD fetuses. It will also evaluate the potential diagnostic value for different types of CHD and explore the possible genetic pathogenic factors of CHD. Because there are variety of CHD, and the relationship between CHD and genetic factors is not exclusive, we adopt a new method to explore the distribution of chromosome variation, and try to find the relationship with CHD as much as possible, so as to contribute to the prevention and treatment of CHD.

Subjects

We collected the fetuses diagnosed with CHD by echocardiography in Beijing Obstetrics and Gynecology hospital from January 2012 to December 2021, and the fetuses tested by CMA were taken as the research objects. NCA and CNVs were mainly analyzed. A total of 427 fetuses were included in the study, the gestational age range was 15–27+6 weeks and the maternal age range was 20–47 years. This work was approved by the Medicine Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital (No. 2018-KY-003-03). and patients provided informed consent.

Classification of cardiac phenotypes and ECAs

427 cases of CHD were divided into isolated CHD and non-isolated CHD according to whether they were complicated with ECAs. Major ECAs were defined as abnormalities predicted to have surgical, medical or important cosmetic implications for the newborn. ECAs were categorized according to the affected organ system (including central nervous, pulmonary, renal, musculoskeletal, gastrointestinal, craniofacial, abdominal wall defect, etc).

Another dimension of classification is based on cardiac phenotype. The classification of cardiac phenotype was independently verified by two doctors according to the echocardiographic report and classified according to the National Birth Defect Prevention Study (NBDPS; botto et al.[4]). According to the phenotype of CHD, 427 cases of CHD were divided into ten groups, including Conotruncal defects, Septal defect, left ventricular outflow tract obstruction (LVOTO), right ventricular outflow tract obstruction (RVOTO), atrioventricular septal defect (AVSD), total anomalous pulmonary venous connection (TAPVC), vascular abnormality, Complex CHD, Heterotaxy, other CHD. All fetal samples were obtained by amniocentesis to draw amniotic fluid or cord blood, or by retaining skin and muscle tissue after termination of pregnancy. Once fetal pathogenic CNVs were confirmed, approximately 2.0 mL of peripheral venous blood was col-lected from their parents.

Chromosomal microarray analysis and data analysis

The extracted DNA samples passed the quality control test. The cytoscan TM 750k array chip (Thermo Fisher, USA) was used for CMA test in strict accordance with the manufacturer's operating instructions. The chip includes 200000 SNP probes and 550000 non-polymorphic probes. According to the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resource (ClinGen) standards and guidelines. CNVs results are divided into five categories: pathogenic CNVs (pCNVs), likely pathogenic CNVs (likely pCNVs), variation of uncertain significance (VOUS), likely benign and benign CNVs. The internationally recognized databases for reference mainly include UCSC Genome Browser, OMIM, decipher, DGV, PubMed, ClinGen, etc.

Statistical analysis

R studio 4.2 and IBM SPSS Statistics, version 26 (IBM Corp, Armonk, N.Y, USA) were used for statistical analyses. Chi-square test was used for comparison between groups for categorical variables. Mantel test is used to analyze the correlation between CHD group and genetic results, r value >0.2 and P value <0.05 was defined as statistically significant in the tests.

Results of CMA

Genetic diagnosis was made in 427 cases. There were 55 cases (12.9%) of NCA, 38 cases (8.9%) of pCNVs, 86 cases (20.1%) of VOUS, 233 cases (54.6%) of benign CNVs and 15 cases (3.5%) of other (including chromosomal structural abnormalities, low proportion chimera).

The phenotype and ECAs in fetal CHD

Among 427 CHD fetuses, 305 (71.4%) were isolated CHD,122 (28.6%) were non-isolated, Those with ECAs were more likely to have a genetic abnormality than those without ECAs (39.3% [48/122] vs. 14.8% [45/305], P<0.05) (Table 1). The NCA, pCNVs and VOUS which were considered as variations of chromosome classified by chromosome number, and they were included in the Mantel test of cardiac phenotype, and ECAs.

2.1 the correlation between variations of chromosome with ECAs

NCA and chr18 were associated with skeletal abnormality (r=0.37, r=0.35, respectively, P<0.05). NCA and chr13 were associated with Craniofacial abnormality (r=0.18, r=0.36, respectively, P<0.05). pCNVs and chr22 were associated with thymic abnormality (r=0.35, r=0.35, respectively, P<0.05),. Other correlations show lower Pearson’s r. (Fig 1A).

2.2 the correlation between variations of chromosome and phenotype of CHD

According to the phenotype of CHD, 427 cases of CHD were divided into ten groups. Table 1 and table 2 for details. NCA and chr18 were associated with septal defect (r=0.19, r=0.15, respectively, P<0.05). NCA and chr21 were associated with AVSD (r=0.18, r=0.13, respectively, P<0.05). Deletion of CNVs was associated with conotruncal defects (r=0.19, P<0.05). Variations of chr22 was associated with vascular abnormality (r=0.17, P<0.05). The group of RVOTO, complex CHD and TAPVC also showed correlation with chromosomal variation, but no significant correlation with pathogenicity was found in Table 2, that may require further genetic testing. There was no significant distribution of pCNVs in each group (Fig 1B).

In Table 2, the detection rate of Trisomy 18 in septal defect group was 23% (14/60), Trisomy 21 in AVSD group was 12.8% (5/39). the detection rate of NCA and pCNVs in conotruncal defects group was similar (8.0%[11/138] vs10.9%[15/138]), but in the subgroups IAA, B and TOF, the detection rate of pCNVs were higher than NCA (8.0%[1/3] vs 0%[0/3], 14.7%[10/68] vs 4.4%[3/68]), while in DORV subgroups, the detection rate of NCA was higher than pCNVs (20.0%[7/35] vs 2.9%[1/35]); In the vascular abnormality group, the RAA subgroup with the highest detection rate of pCNVs, combined with the conclusion in Fig 1, pCNVs was mainly 22q11.2DS. Therefore, the detection rate of pCNVs in the subgroups were IAA, A (50%,1/2), IAA, B (33.3%,1/3), RAA (28.6%,4/14), TAPVC (25.0%,1/4), CoA (22.2%,2/9), TOF (14.7%,10/68). Mantel test combined with tables can better reflect the correlation.

It was worth mentioned that the detection rate of NCA and pCNVs in the Heterotaxy were 0%, and the pCNVs in AVSD was also 0%.

Figure1 A shows CHD with ECA, B shows the cardiac phenotype of all CHD.

Firstly check the column and then check the row, the right column in the figure 1, regarded as many factors that may affect the occurrence of CHD. determining a column factor, then check the row factor which was the largest and lightest color box was most associated with the column factor. Factor in the columns of figure 1 A -B, orange and bold lines represent those associated with specific phenotypes.

CHD, congenital heart defects; Del, deletion of CNVs; dup, duplication of CNVs; NCA, numerical chromosomal abnormalities; pCNVs, pathogenic copy number variations; chr, chromosome; AVSD, atrioventricular septal defect; LVOTO, left ventricular outflow tract obstruction; RVOTO, right ventricular outflow tract obstruction; TAPVC, total anomalous pulmonary venous connection; other*, rhabdomyoma, hydropericardium, abnormal heart rhythm and cardiac function.

Table 1. Incidence of extracardiac abnormalities (ECAs) and genetic abnormalities (GA) found in 427 fetuses diagnosed with congenital heart defects (CHD).

Groups	Total				Isolated CHD				ECAs
Groups	n	GA (n(%))	NCA	pCNVs	n	GA (n(%))	NCA	pCNVs	n	GA (n(%))	NCA	pCNVs
Conotruncal defects	138	25(18.1)	11	14	96	13(13.5)	5	8	34	12(35.3)	6	6
Septal defect (VSD)	60	25(41.7)	19	6	27	8(29.6)	7	1	33	17(51.5)	12	5
LVOTO	47	8(17.0)	2	6	35	4(11.4)	1	3	12	4(33.3)	1	3
RVOTO	42	8(19.0)	4	4	32	5(15.6)	3	2	10	3(30.0)	1	2
AVSD	39	13(33.3)	13	0	22	5(22.7)	5	0	17	8(47.1)	8	0
TAPVC	4	1(25.0)	0	1	3	0(0)	0	0	1	1(100)	0	1
vascular abnormality	22	6(27.3)	2	4	19	5(26.3)	1	4	3	1(33.3)	1	0
Heterotaxy (complex CHD)	25	0(0)	0	0	23	0(0)	0	0	2	0(0)	0	0
Complex CHD (Multiple, Single ventricle)	33	5(15.2)	2	3	28	4(14.3)	1	3	5	1(20.0)	1	0
other	17	2(11.8)	2	0	12	1(8.3)	1	0	5	1(20.0)	1	0
Total	427	93(21.8)	55	38	305	45(14.8)	24	21	122	48(39.3)	31	17

Table 2. Rate of genetic anomalies in 427 fetuses diagnosed with CHD.

Groups and Subgroups	n	n of total GA	(%) of total GA	(%) of NCA	(%) of pCNVs	T21	T18	T13	45, X	Other NCA*	22q11.2 DS	other CNV syndrome	Other pCNVs
Conotruncal defects	138	26	18.8	10.9	8.0	1	8	0	0	2	10	3	2
TOF	68	13	19.1	14.7	4.4	0	3	0	0	0	7	2	1
common arterial trunk	16	3	18.8	12.5	6.3	0	1	0	0	0	0	1	1
d-TGA	16	1	6.3	6.3	0	0	0	0	0	0	1	0	0
DORV	35	8	22.9	2.9	20.0	1	4	0	0	2	1	0	0
IAA, type B	3	1	33.3	33.3	0	0	0	0	0	0	1	0	0
Septal defect (VSD)	60	24	40.0	8.3	31.7	4	14	1	0	0	0	4	1
LVOTO	47	8	17.0	6.4	10.6	0	0	0	2	0	3	1	2
Aortic stenosis	14	2	14.3	14.3	0	0	0	0	0	0	1	0	1
Coarctation of aorta (CoA)	9	4	44.4	22.2	22.2	0	0	0	2	0	1	1	0
HLHS	20	1	5.0	5.0	0	0	0	0	0	0	0	0	1
Mitral valve dysplasia	2	0	0	0	0	0	0	0	0	0	0	0	0
IAA, type A	2	1	50.0	50.0	0	0	0	0	0	0	1	0	0
RVOTO	42	8	19.0	4.8	14.3	3	1	0	0	0	2	0	2
HRHS	9	1	11.1	11.1	0	0	0	0	0	0	0	0	1
Ebstein's anomaly，Tricuspid valve dysplasia	8	2	25.0	0	25.0	2	0	0	0	0	0	0	0
PA-IVS	8	0	0	0	0	0	0	0	0	0	0	0	0
PS	17	5	29.4	17.6	11.8	1	1	0	0	0	2	0	1
AVSD	39	13	33.3	0	33.3	5	5	1	1	1	0	0	0
TAPVC	4	1	25.0	25.0	0	0	0	0	0	0	0	1	0
vascular abnormality	22	6	27.3	18.2	9.0	2	0	0	0	0	3	0	1
RAA	14	4	28.6	28.6	0	0	0	0	0	0	3	0	1
Double aortic arch	2	0	0	0	0	0	0	0	0	0	0	0	0
left superior vena cava	6	2	33.3	0	33.3	2	0	0	0	0	0	0	0
Heterotaxy (complex CHD)	25	0	0	0	0	0	0	0	0	0	0	0	0
Complex CHD (Multiple, Single ventricle)	33	5	15.2	9.1	6.1	1	1	0	0	0	1	1	1
other	17	2	11.8	0	11.8	0	0	0	0	0	0	0	0
Rhabdomyoma	8	0	0	0	0	0	0	0	0	0	0	0	0
hydropericardium	5	2	40.0	0	40.0	1	0	1	0	0	0	0	0
Abnormal heart rhythm And cardiac function	4	0	0	0	0	0	0	0	0	0	0	0	0
Total	427	93	21.8	12.9	8.9	17	29	3	3	3	19	10	9

other NCA*，47,XXY, Trisomy 9 and Triploid. TOF, tetralogy of Fallot; d-TGA, d-transposition of great arteries; DORV, double outlet right ventricle; IAA, interrupted aortic arch; VSD, ventricular septal defect; LVOTO, left ventricular outflow tract obstruction; HLHS, Hypoplastic left heart syndrome; RVOTO, right ventricular outflow tract obstruction HRHS, Hypoplastic right heart syndrome; PA-IVS, Pulmonary atresia with intact ventricular septum; PS, pulmonary stenosis; AVSD, atrioventricular septal defect; TAPVC, total anomalous pulmonary venous connection; RAA, right aortic arch.

Figure 2A CNVs deletion and NCA of chr1-22 and ChrX; Figure 2B CNVs duplication and NCA of chr1-22 and ChrX&Y.

Fig 2A-2B, the abscissa represents chromosomes, and the ordinate represents the proportion of NCA, pCNVs and VOUS. the number in the figure represent the cases with different sizes of fragments. The red boxes in the figure represent pCNVs. in fig 2A, the position of deletion were 1p36, 5p13, 6q26q27, 7q11, 8p23, 11q14q22, 11q24q25, 17p11, 17p12, 17p13, 21q22, 22q11.2, 22q13, Xp22, respectively; in fig 2B the position of duplication were 10q24, 12q23q25, 16p13, 22q11.2, CES, Xp11q28, Xq28, respectively. T* triploid.

Detection of chromosomal variations

In Fig 1, The results directly perceived that pCNVs accounted for the most in deletion of CNVs (41.6%, 32/77); variations of chr18 and chr21 accounted for the most in NCA (29(29/427, 6.8%) trisomy 18, 17(17/427, 4.0%) trisomy 21), and variations of chr22 accounted for the most in pCNVs (57.9%, 22/38) (Fig 1).

Chr1-22, ChrX and chrY had different degrees and proportions of chromosomal variations, except chr20. The fragment sizes in pCNVs were as follows: in deletion, 1-3Mb accounted for 43.8% (14/32), 3-5Mb for 40.6% (13/32), and > 5Mb for 15.6% (5/32) (Fig 2A); in duplication, 1-3Mb accounted for 42.9% (3/7), and > 5Mb for 57.1% (4/7) (Fig 2B). Therefore, the CNV deletion fragment larger than 1 Mb may be pathogenic. In CNV, deletion was more likely to be pathogenic than duplication.

3.1 Detection of CNV syndrome

Among the 38 cases of pCNVs, 29 (29/38, 76.3%) cases were related to CNV syndromes, and 9 (9/38, 23.7%) cases contained the pathogenic gene.

The proportion of 22q11.2 deletion syndrome (22q11.2DS, OMIM #192430) was 4.4% (19/427), and the proportion of 22q11.2 duplication syndrome (OMIM # 608363) was 0.2% (1/427). One of the 22q11.2DS was associated with a 1.1 Mb deletion on 17p13.3 deletion syndrome. The five cases of 22q11.2DS were all denovo. The detection rate of 22q11.2DS in the subgroups were IAA,B (33.3%,1/3), RAA (21.4%,3/14), PS (11.8%,2/7), CoA (11.1%,1/9), TOF (10.3%,7/68).

Except for 22q11.2 syndrome, A total of 10 CNV syndromes were found in 9 fetuses. Table 3 shows the details of pCNVs cases. CNV syndromes were 1p36 monosomy syndrome (OMIM #607872), 7q11.23 deletion syndrome (Williams-Beuren Syndrome, OMIM #194050), 16p13.11 recurrent microduplication, 17p11.2 deletion syndrome (Smith-Magenis syndrome, SMS，OMIM #182290), Hereditary Liability to Pressure Palsies (HNPP, OMIM #162500), 17p13.3 deletion syndrome (Miller-Dieker syndrome, OMIM #247200), 22q13 deletion syndrome (Phelan-Mcdermid syndrome, #606232), Cat eye syndrome (CES, OMIM #115470), Leri-Weill dyschondrosteosis (LWD, OMIM #127300) and Xq28 duplication syndrome (MECP2 duplication syndrome, OMIM #300260).

3.2 Other related pathogenic genes

In addition, in other pCNVs we identified five genes that expressed in the heart and/or involved in embryonic development in four cases: FLI1, NIPBL, DLL1, PTPN11, TBX5. The other five cases contained pathogenic genes for other reasons，At present, there is no research to prove that it is related to cardiac abnormalities, table 3.

Outcomes

326 fetuses terminated pregnancy, including 91 cases of chromosomal abnormalities, 69 cases of CNV VOUS, 157 cases of CNV benign and 9 cases of other chromosomal abnormalities; 90 cases of termination of pregnancy were confirmed by autopsy (some cases were shown in Fig 3-10, Fig 3-5 were two cases of VOUS and one case of trisomy 18, respectively; Fig 6-10 were the autopsy of pCNVs in table 3), other cases returned to the local hospital without autopsy. 25 cases were born, 2 cases of pCNVs, 6 cases of CNV VOUS, 14 cases of CNV benign and 3 cases of other chromosomal abnormalities. and 76 cases lost follow-up, including 11 cases of CNV VOUS, 62 cases of CNV benign and 3 cases of other chromosomal abnormalities.

CHD is one of the most common birth defects, with a wide variety and complex etiology, it is always a difficult problem to explore the etiology of CHD. As the primary means of prenatal detection of CHD, CMA is not only cheap, but also can detect aneuploidy and pCNVs, which provides important clues for prenatal diagnosis of CHD fetus. Therefore, we need to expand its potential diagnostic efficiency as much as possible and provide a reliable plan for prenatal consultation at this time.

Although the occurrence of CHD is related to genetic factors, it is not an exclusive relationship. One CHD phenotype can have multiple genetic results, and one CNV result may be related to several CHD phenotypes, the phenotype of CHD is always compound. The above reasons were easy to cause wrong judgment when analyzing the correlation. A Mantel test measures the correlation between two matrices typically containing measures of distance, it is one way of testing for spatial autocorrelation, Its advantage is that it can also find the most relevant relationship when the CHD phenotype complex occurs at the same time and directly perceived in Fig. Therefore, this study can better analyze how genetic factors affect the linear spatial structure of CHD composition by using Mantel test.

Our study showed that the incidence of the ECAs was 28.6%, which was only the result of prenatal diagnosis, similar to the studies of Rishav et al[5]. The chromosomal abnormalities rate of CHD fetus was 21.8 % and the detection rate of NCA was 12.9%. Wang et al[6].found that the incidence of chromosomal abnormalities was 20.8%, and more than half of them were NCA, and our study is consistent with its result.

After grouping the cardiac phenotypes in our study, the highest detection rate was trisomy 18, and the most relevant cardiac phenotype was septal defect (Mainly VSD); The number of trisomy 21 is the second, and the most relevant cardiac phenotype was AVSD. The results of our study were similar to those of the previous studies[7–8] .

Our study believes that Conotruncal defects, RVOTO, complex CHD, vascular abnormality and TAPVC, these CHD groups were related to chromosomal variation; while Heterotaxy, LVOTO, other CHD groups were not found to be related to chromosomal variation. Does this suggest that some CHD phenotypes are suitable for further examination? Wang et al[6]. Through the CMA combined with exome sequencing (ES), the author believes that CHD phenotypes such as complex CHD, RVOTO, Conotruncal defects, etc, were slightly higher incremental yields of pathological and like pathological sequence variations, which makes us more confidence that these phenotypes of CHD were more necessary for further detection, and there may be some correlation. Next, we want to further test the Heterotaxy and the AVSD which with negative results, exploring new genetic factors.

CHD with ECAs was more prone to occur genetic abnormality, Therefore, we used Mantel test to explore their correlation. Among the ECA, trisomy 18 has been known to be associated with skeletal system abnormality. Trisomy 13 was associated with facial abnormality. thymic abnormality was associated with 22q11.2DS combined with table 3. Variations of chr13, chr16, chr18 and chrX which show lower Pearson’s r in Fig. 1 can be further detected by ES to verify the prediction of our study.

In our study, the detection rate of pCNVs was 8.9%, which was similar to the results of previous studies[9]. In the deletion, the fragment size of pCNVs was mainly 1-3Mb and 3-5Mb; on the contrary, duplication was dominated by larger fragments (> 5Mb). Therefore, the CNV deletion fragment larger than 1 Mb may be pathogenic. In CNV, deletion was more likely to be pathogenic than duplication. However, in the normal population, then deletion should, on average, be both less frequent and smaller than duplication in the general population, this may just be the deletion of its own characteristics. Some studies on CHD also found that the length and number of deletions were more than those of duplication, the reason is that both are generally believed to involve non-allelic homologous recombination (NAHR) [10]. It may be that the size of duplication in our cases are too large, which makes the results biased.

22q11.2 syndrome was the CNV syndrome most related to CHD. In our study, the detection rate of 22q11.2DS were 4.4%, and 22q11.2 duplication syndrome was 0.2%. 22q11.2DS was associated with thymic abnormalitiy, RAA, Conotruncal defects, PS and CoA. The core clinical phenotype of 22q11.2 syndrome was still characterized by a conotruncal defects, aplasia/hypoplasia of the thymus and parathyroidglands. Now, the vascular ring has also been extended to the spectrum of associated clinical features, of course, RAA also belongs to a kind of vascular ring. In addition, through our study, we should note that when CHD was found, ultrasound should also observe the thymus and repeatedly check whether there are any abnormality in the diameter and shape of aorta and pulmonary artery.

Except for 22q11DS and 22q11.2 duplication syndrome, There were many genetic factors leading to CHD. Our study found that the following genetic disorders were related to CHD, include 1p36 monosomy, Williams–Beuren syndrome, SMS, Miller–Dieker syndrome[11]. Some CNV syndromes only have a few studies have shown that they were related to CHD. such as 16p13.11 recurrent microduplication, CES and LWD. Other syndromes, such as HNPP, 22q13 deletion syndrome, and Xq28 duplication syndrome, which no studies have shown to be associated with the development of cardiac, but only exhibit neurodevelopmental delay[12–13]. It was worth mentioned that our case 4 in table 3 dominated by CoA, 7q11.23 region encompassing LIMK1. Recent studies have shown that a couple of genes have been identified as part of a common pathway between cardiovascular and neurological development (via the Rho GTPases pathway), and LIMK1 was one of them [10].Our case 10 in table 3, 16p13.11 region encompassing MYH11. Studies have shown that MYH11 as a smooth muscle MHC gene can affect arterial duct closure[14–15]. As mentioned above, another gene in the common pathway between cardiovascular and neurological development (via the Rho GTPases pathway) was MYH11[10].

Among other pathogenic genes in pCNV, the following genes were considered to be related to heart and / or involved in embryonic development: FLI1, NIPBL, DLL1, PTPN11, TBX5. (1) Hart et al[16]found the expression level of FLI1 was very high in the development of vasculature and endocardium in mice. (2) Northern blot analysis showed that NIPBL was strongly expressed in fetal and adult heart and other tissues[17]. NIPBL were enriched for rare variants in AVSD[18]. (3) The DLL1 gene plays an important role in the developing nervous system and somites[19]. (4) There were also reports[20]concluded that duplication of PTPN11 represents an uncommon cause of Noonan syndrome. (5) The mutation of TBX5 gene causes Holt- Oram syndrome. Several studies have shown that TBX5 associates with other genes interactions affect heart development[21–23].

Our study regrets to find that most pregnant women choose to terminate pregnancy, because this research center is a well-known consultation center in China, so the situation of CHD was more complex and critical. Similarly, in the past decade, the development of surgery has also been under exploration, many pregnant women are unwilling to bear the high cost of surgery and follow-up care, which also brings difficulties to our follow-up research. We cannot predict the outcome of those VOUS and benign results.

Genome wide association studies (GWAS) have provided evidence suggesting that CNVs are one of the significant contributors to increased risk for congenital heart disease in conjunction with nervous system abnormalities[24–26] But we all know, syndromic disease associated features may be nonspecific, or erro-neously attributed to the cardiac lesion. The clinical manifestations of genetic syndrome are variable,even, f well-defined disorders. Summarizing the clinical characteristics and gene functions of these CNV syndromes and pathogenic genes, we found that the known clinical phenotypes are always accompanied by nervous system abnormalities, our study also concluded that pCNVs showed correlation with neurologic abnormality, which often had no obvious ultrasonic signs, that would weaken the correlation between them.

In the human fetus a large part of the heart and brain development occurs in a similar critical window and the presence of genetic alteration can impact the brain and heart development[27]. Prenatal ultrasound evaluated the nervous system can only be assessed from structural abnormalities and soft-markers signs. It is difficult to identify many ultrasonic signs of neurodevelopmental retardation, some of which can only be found after birth. This is a common challenge now. Genetic testing seems to open up this situation, and it has become a new idea to study the nervous system development of CHD fetuses. Given that CHDs are a lifelong condition, it is very important to deeply understand its relationship with neurodevelopmental and neurocognitive impairment, because it is meaningful to evaluate the condition and guide the prognosis.

Our study not only explores the genetic factors of CHD, but also puts forward a direction for thinking the diagnosis management of CHD. And we point out the direction for further research, in order to find some correlation, expand the diagnostic value of CMA technology, and provide help for clinical consultation; CNVs still make a significant contribution to the etiology of CHD. We hope to expand the number of cases and explore more useful information in the future.

Our study identified the increased risk of genetic abnormality in non-isolated CHD. Found the ultrasonic diagnostic clues of NCA, pCNVs and 22q11.2DS, especially the diagnostic clues of different CHD phenotypes. Explored the distribution of CNV with different fragment sizes, the CNV deletion fragment larger than 1 Mb may be pathogenic. In CNV, deletion was more likely to be pathogenic than duplication. We also found that CNVs still make a significant contribution to the etiology of CHD In this study, twelve CNV syndromes were detected, Among them six CNV syndromes were relatively related to CHD, three CNV syndromes have a few studies shown that they were related to CHD, including the emergence of candidate LIMK1and MYH11, noticed us to find a common pathway for the development of heart and nervous system, provided a new idea for prenatal diagnosis, genetic counseling and etiology discussion.

CHD congenital heart defects; CMA Chromosomal microarray analysis; ECAs extra-cardiac abnormalities; NCA numerical chromosomal abnormalities; CNVs copy number variations ; pCNVs pathogenic copy number variations; VOUS variation of uncertain significance; LVOTO left ventricular outflow tract obstruction; RVOTO right ventricular outflow tract obstruction; AVSD atrioventricular septal defect; TAPVC total anomalous pulmonary venous connection; TOF tetralogy of Fallot; DORV double outlet right ventricle; IAA interrupted aortic arch.

Authors’ contributions All authors contributed to the study conception and design. Material preparation and data collection were performed by Simin Zhang, Yan Pei, Jijing Han, Yani Yan, Juan zhang, Yan Liu, Fangfei Su, Jinyu Xu. Statistics analysis were performed by Jingjing Wang and Xiaowei Xiong. The first draft of the manuscript was written by Simin Zhang. Qingqing Wu checked and revised manuscript. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding This work was supported by a grant from the National Key Research and Development Program of China (No.2016YFC1000104).

Availability of data and material The data that support the findings of this study are available on request from the corresponding author.

Code availability No code.

Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital (14th December, 2020/No. 2018-KY-003-03).

Consent to participate Informed consent was obtained from all parents of the fetus.

Consent for publication Consents for publication were obtained from the participants.

Conflict of interest Authors declare no competing interests.

Competing Interests The authors have no relevant financial or non-financial interests to disclose.

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No competing interests reported.

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Diagnostic value of CMA in different phenotypes of fetal congenital heart defects

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