Background
Congenital heart disease (CHD) is one of the most common birth defects. The incidence of CHD in the neonate is 8-9/1000, and nearly 1.35 million CHD neonates were born every year in the whole world [
1]. Despite improvement of various treatment measures, CHD is still one of the major causes of children mortality.
The causes of CHD include non-genetic factors and genetic factors. Non-genetic factors include: environmental factors, maternal exposure and infection. Chromosomal causes of CHD include chromosome aneuploidies, like trisomy 21, and copy number variations (CNVs). Chromosomal aneuploidies represent 12.5% of CHD causes [
2]. The spectrum of CHD-related CNVs ranges from recurrent microdeletion and microduplication syndromes, like DiGeorge syndrome (22q11 deletion syndrome) and Williams-Beuren syndrome (7q11.23 deletion syndrome), which are associated with a distinct clinical recognizable phenotype, to rare CNVs, flanked by unique breakpoints [
3‐
5].
The resolution of conventional karyotype analysis is limited to 5 Mb or larger genomic imbalances [
6]. The drawback of fluorescence in situ hybridization (FISH) lies in its a targeted approach to detect chromosomal defects, rather than a genome-wide screening method like microarrays or MLPA [
7]. Chromosome Microarray Analysis (CMA) is a routine technique in clinical molecular testing nowadays, which contains two types of arrays: oligonucleotide arrays and Single Nucleotide Polymorphism arrays (SNP arrays). Both the arrays could detect genome-wide CNVs. Moreover, SNP arrays can detect the mosaicism, triploid, loss of heterozygosity (LOH) and uniparental disomy. In 2010, the American College of Medical Genetics issued practice guidelines for CMA, and pointed out that CMA was recommended as a first-tier test for postnatal patients with multiple congenital anomalies (MCA), intellectual disabilities/developmental delay (ID/DD) and autism spectrum disorders [
8]. Recently, CMA has been successfully applied to detect CNVs in patients with CHD, which confirmed the relationship between chromosome microdeletion/microduplication and CHD [
9‐
19].
In this study, we present the results of genome-wide high resolution SNP array analysis in 106 children with CHD in the Chinese cohort, to explore the clinical implication of CMA in genetically etiological diagnosis of CHD.
Results
CMA was performed in 104 children (67 males and 37 females) with CHD aged from 5 days to 8 years old. CNVs were identified in 96.2% (100/104) of the children. The size of CNVs ranged from 102 kb to 13.8 Mb. CNVs were interpreted as benign or likely benign in 69.2% (72/104) children. The detection rate for PCNVs was 27.9% (29/104), and the VOUS rate was 2.9% (3/104) after parental analysis.
Detailed CHD classification and demographic data of the children were listed in Table
1. The percentages of children with simple CHD and complex CHD were 58.7% (61/104) and 41.3% (43/104), respectively. PCNVs were identified in 31.1% (19/61) children with simple CHD and in 23.2% (10/43) children with complex CHD. Pearson Chi-square test showed that there was no significant difference between these two groups (
P > 0.05). PCNVs were detected in 23.3% (10/43) of septal defect, 0 (0/3) of AVSD, 12.5% (2/16) of conotruncal defects, 35.7% (5/14) of LVOTO, 45.5% (5/11) of RVOTO, 75% (3/4) of LVOTO + RVOTO, 0 (0/1) of SV, and 33.3% (4/12) of PDA.
The detection rates of PCNVs in isolated CHD and syndromic CHD were 17.9% (7/39) and 33.8% (22/65), respectively. There was no significant difference between these two groups (
P > 0.05). The detection rates for PCNVs were 20% (5/25) in CHD plus MCA, 63.2% (12/19) in CHD plus ID/DD, and 23.8% (5/21) in CHD plus MCA and ID/DD, respectively (Table
2). The PCNVs rate of CHD plus ID/DD was significantly higher than that of isolated CHD (63.2 vs 17.9%,
P = 0.001) and CHD plus MCA (63.2 vs 20%,
P = 0.004).
Table 2
Classification of children with CHD and/or other diagnoses (MCA, ID/DD)
Isolated CHD | 3 | 15 | 20.0 | 4 | 24 | 16.7 | 7 | 39 | 17.9 |
Syndromic CHD | 16 | 46 | 34.8 | 6 | 19 | 31.6 | 22 | 65 | 33.8 |
CHD + MCA | 4 | 18 | 22.2 | 1 | 7 | 14.3 | 5 | 25 | 20 |
CHD+ ID/DD | 8 | 13 | 61.5 | 4 | 6 | 66.7 | 12 | 19 | 63.2 |
CHD + MCA+ ID/DD | 4 | 15 | 26.7 | 1 | 6 | 16.7 | 5 | 21 | 23.8 |
Total | 19 | 61 | 31.1 | 10 | 43 | 23.2 | 29 | 104 | 27.9 |
PCNVs were identified in 29 children (Table
3, including 22q11 deletion syndrome (
n = 6), 22q11 duplication syndrome (
n = 1), Williams-Beuren syndrome (
n = 6), Angleman/Prader-Willi syndrome (
n = 1), Wolf-Hirschhorn syndrome (
n = 2), Schinzel-Giedion midface retraction syndrome (
n = 1), 15q24 recurrent microdeletion syndrome (
n = 1), 1p36 microdeletion syndrome (
n = 1), Cornelia de Lange syndrome 4(
n = 1), Marfan syndrome (
n = 1), Opitz G/BBB syndrome (
n = 1), 6q24 LOH (
n = 1), and CNVs overlapped with DECIPHER patient entries (
n = 6).
Table 3
Pathogenic copy number variants and variants of unknown significance detected by Chromosome Microarray Analysis in children with CHD
Pathogenic CNVs |
1 | 8 y | ASD | ID | Dup 11q24.2-q25 (8.5 Mb) | Decipher number 255590 | None |
Del 1q43-q44 (6.2 Mb) | Decipher number 284767 | None |
2 | 7 m | PS + ASD | None | Del 22q11.21 (3.2 Mb) | 22q11 deletion syndrome |
TBX1
|
3 | 10 m | PDA | Congenital anal atresia + DD | Dup 3p26.1-p24.3 (13.8 Mb) | Decipher number 260758 |
CRELD1
;
RAF1
|
Del 6q13-q14.1 (5.2 Mb) | Decipher number 249539 | None |
Dup 17q12 (1.4 Mb) | Decipher number 278456 | None |
4 | 4 y | PDA | Leukodystrophy | Del 1p36.33-p36.31(4.8 Mb) | 1p36 microdeletion syndrome |
DVL1;SKI
|
5 | 13 m | ASD | DD | Del 15q24.1-q24.2 (3.1 Mb) | 15q24 recurrent microdeletion syndrome |
STRA6
|
6 | 5 y | PS | ID | Del 15q11.2-q13.1 (4.9 Mb) | Angelman/Prader-Willi syndrome | None |
7 | 5 m | VSD | DD | Del 4p16.3-p16.2 (5.7 Mb) | Wolf-Hirschhorn syndrome |
EVC2; EVC
|
8 | 2 m | ASD | Laryngeal cartilage dysplasia | Del 22q11.21 (2.4 Mb) | 22q11 deletion syndrome |
TBX1
|
9 | 2 y | VSD | ID | Del 22q11.21 (3.2 Mb) | 22q11 deletion syndrome |
TBX1
|
10 | 14 m | ASD | None | Del 22q11.21 (1.4 Mb) | 22q11 deletion syndrome |
TBX1
|
11 | 2 y | ASD | Cleft palate + ID | Dup 18q12.3 (0.64 Mb) | Schinzel-Giedion midface retraction syndrome |
SETBP1
|
12 | 2 y | AS + PS | DD | Del 7q11.23 (1.4 Mb) | Williams-Beuren syndrome |
ELN
|
13 | 3 y | TOF + PLSVC + Pericardial defect | Diaphragmatic hernia + ID | Dup 2q12.3 (0.42 Mb) | Decipher number 287980 | None |
14 | 1 m | IAA,A + VSD | fingers of both hands and left toe deformity | Dup Xp22.2 (0.72 Mb) | Opitz G/BBB syndrome |
MID1
|
15 | 1 y | PS + VSD | ID | Del 22q11.21 (3.2 Mb) | 22q11 deletion syndrome |
TBX1
|
16 | 15 m | PDA | DD | Del 1p36.33 (0.35 Mb) | Decipher number 106 | None |
Dup 17q25.1-q25.3 (6.4 Mb) | Decipher number 249584 | None |
17 | 18 m | ASD | Cleft palate + DD | Del 4p16.3-p16.1 (7.6 Mb) | Wolf-Hirschhorn Syndrome |
EVC2; EVC
|
18 | 11 d | IAA, A + VSD | None | Del 7q11.23 (1.4 Mb) | Williams-Beuren syndrome |
ELN
|
19 | 8 m | AS + PS | DD | Del 7q11.23 (1.4 Mb) | Williams-Beuren syndrome |
ELN
|
20 | 2 y | PS | None | Dup 15q21.1 (1.58 Mb) | Marfan syndrome |
FBN1
|
21 | 3 y | TOF | Absence of corpus callosum + cerebellar vermis hypoplasia + ID | Del 1q43-q44 (7.6 Mb) | Decipher number 249647 | None |
Dup 10p15.3-p14 (6.7 Mb) | Decipher number 278831 | None |
22 | 3 y | COA + VSD + ASD | ID | LOH 6q24.1-q24.2 (5.2 Mb) | Decipher number 290225 |
CITED2
|
23 | 1 m | AS | Hemivertebra + Adduction deformity of thumb + Polydactyly + Funnel chest | Del 8q23.3-q24.11(1.24 Mb) | Cornelia de Lange syndrome 4 |
RAD21
|
Dup 11p15.3-15.2 (0.75 Mb) | None | None |
24 | 2 y | VSD | ID | Del 22q11.21 (3.2 Mb) | 22q11 deletion syndrome |
TBX1
|
25 | 2 y | PS | ID | Del 7q11.23 (1.5 Mb) | Williams-Beuren syndrome |
ELN
|
26 | 2 m | AS | None | Del 7q11.23 (1.5 Mb) | Williams-Beuren syndrome |
ELN
|
27 | 2 m | AS + PS | None | Del 7q11.23 (1.4 Mb) | Williams-Beuren syndrome |
ELN
|
28 | 7 m | PDA | Cleft palate | Dup 22q11.21 (2.5 Mb) | 22q11 duplication syndrome |
TBX1
|
29 | 6 m | ASD + VSD | None | Dup 17q25.1-q25.3 (8.5 Mb) | Decipher number 254723 | None |
Del 20q13.33 (1.3 Mb) | Decipher number 2615 | None |
Variants of unknown significance |
30 | 1 m | CoA + Heart Enlargement | None | Dup 11p15.4 (0.18 Mb) | None |
STIM1
|
31 | 1 m | AS + VSD | None | Dup 10q21.3 (0.4 Mb) | None |
CTNNA3
|
32 | 1 m | D-TGA + VSD + ASD | None | Dup 6q22.31 (0.36 Mb) | None |
PLN
|
In this study, PCNVs in 79.3% (23/29) of the children contained genes contributing to CHD (Table
3). The genes responsible for syndromic CHD included
TBX1 (22q11 deletion syndrome),
ELN (Williams-Beuren syndrome),
EVC2 and
EVC (Wolf-Hirschhorn syndrome),
STRA6 (15q24 recurrent microdeletion syndrome),
FBN1 (Marfan syndrome),
MID1 (Opitz G/BBB syndrome),
RAD21 (Cornelia de Lange syndrome 4) and
SETBP1 (Schinzel-Giedion midface retraction). In addition, the genes contributing to non-syndromic CHD included
CRELD1, RAF1 and
CITED2. D
VL1 and
SKI were identified as candidate genes for CHD in the current study.
CNVs detected in 9 children were classified as VOUS initially, further parental microarray analysis showed that CNVs in 6 children were inherited. The remaining CNVs in the other 3 children (Table
3, child 30-32) were de novo and the clinical significance was still unknown. Therefore, the VOUS rate was 2.8% in this study. The three VOUS were 11p15.4 duplication (chr11:3,923,985-4,242,111, 180 kb), 10q21.3 duplication (chr10:69,026,332-69,430,434, 400 kb) and 6q22.31 duplication (chr6:118,693,553-119,050,523, 360 kb). Three genes,
STIM1,
CTNNA3 and
PLN relevant with CHD, located in these fragments respectively. For the other children with benign or pathogenic CNVs, their parents rejected further parental analysis by CMA.
Discussion
In the past few years, several studies have investigated postnatal cases with syndromic CHD by array CGH (aCGH) (Table
4) [
9,
11,
13,
14,
16‐
19]. Different arrays were used in these studies, and the PCNVs detection rate ranged from 10.9 to 25.5%. Bachman’s study showed that the lowest PCNVs detection rate was 10.9% (5/46) using Roche NimbleGen 135 K arrays [
18]. The highest detection rate was 25.5% (5/20) by Agilent 244 K array from Syrmou’s study [
19]. In our study, the total detection rate for PCNVs reached 27.9% (29/104) by CytoScan HD array, including 1,950,000 oligonucleotide probes and 750,000 SNP probes. Our results demonstrated further that denser arrays with high resolution will lead to a proportional increase in number of PCNVs [
13,
24].
Table 4
The comparison of studies in postnatal patients with isolate/syndromic CHD
Thienpont et al. 2007 [ 9] | 1 Mb BAC | 60 | MCA, ID | 16.7% | 11.7% |
Richards et al. 2008 [ 11] | 385 K oligo arrays | 20 | MCA, DD/ID | 25% | 9.8% |
20 | None | 0 |
| 1 Mb BAC or 1 × 244 K Agilent arrays | 105 | None | 4.7% | Unknow |
Greenway et al. 2009 [ 12] | Affymetrix SNP 6.0 | 114 | None | 5.3% | Unknow |
Breckpot J et al. 2010 [ 13] | 1 Mb array | 150 | MCA | 17.3% | Unknow |
| 100 K Affymetrix | 19 | MCA | 21% | Unknow |
Goldmuntz et al. 2011 [ 16] | 100 K Oligo array | 58 | MCA | 20.7% | 3.4% |
Breckpot et al. 2011 [ 15] | Affymetrix SNP 6.0 | 46 | None | 4.3% | Unknow |
Derwinska et al. 2012 [ 17] | 180 K Oligo | 150 | MCA | 14% | Unknow |
| 1 × 244 K or 4 × 180 K Agilent arrays | 55 | MCA | 25.5% | Unknow |
| Roche NimbleGen 135 K arrays | 46 | MCA | 10.9% | Unknow |
Our study | Affymetrix CytoScan HD arrays | 104 | MCA, ID/DD | 27.9% | 2.9% |
CMA has also been applied in children with isolated CHD previously [
10‐
12,
15]. The PCNVs detection rates ranged from 0 to 5.3% (Table
4). Richards et al. studied 20 children with isolated CHD and 20 children with CHD plus MCA, the results showed that PCNVs detection rate in isolated CHD was 0 and that in CHD plus MCA was 25%, and the highest PCNVs detection rate was 45% in children with CHD plus neurologic defects [
11]. Therefore, CMA was not recommended by Richards for children with isolated CHD. In our study, PCNVs detection rate was 17.9% (7/39) in isolated CHD. The PCNVs detection rates for CHD plus MCA, CHD plus ID/DD and CHD plus MCA and ID/DD were 20, 63.2 and 23.8%, respectively. PCNVs detection rate in CHD plus ID/DD was significantly higher than that of isolated CHD (
P = 0.001) and CHD plus MCA (
P = 0.004). Our data demonstrated that CMA is the most useful for genetic diagnosis in children with CHD plus ID/DD. In addition, we also recommended CMA investigation for children with isolated CHD.
In this study, PCNVs were detected in 31.1% (19/61) children with simple CHD and 23.2% (10/43) children with complex CHD by CMA. There was no significant difference between the two groups (
P > 0.05). Detection rates in various types of CHD were different. Shaffer et al. reviewed 580 fetuses with CHD and normal karyotype by aCGH [
25], and revealed the detection rates of PCNVs as follows: 16.2% (11/68) in LVOTO, 11.6% (5/43) in conotruncal defect and 10.6% (14/132) in septal defect. The above three types of CHD in fetuses were the most frequent. In our study, the PCNVs detection rates in different types of CHD in isolated or with additional anomalies were as follows in turn: 75% (3/4) in LVOTO + RVOTO, 45.5% (5/11) in RVOTO, 35.7% (5/14) in LVOTO, 33.3% (4/12) in PDA, 23.3% (10/43) in septal defect, 12.5% (2/16) in conotruncal defects. Our data demonstrate that LVOTO and/or RVOTO were most probably related to microdeletion/microduplication. Of the 29 children with PCNVs, 22 (75.9%) were complicated with MCA and/or DD/ID. High detection rate in children with PDA (33.3%) was obtained in this study, and we noticed that all the 12 children with PDA were complicated with MCA and/or ID/DD. There was no PCNVs detected in children with AVSD (
n = 2) and SV (
n = 1), this may be due to the small sample size in our study.
Gijsbers et al. suggested that as the rising of aCGH resolution, there will be more VOUS identified [
26]. In the previous studies in children with CHD by microarray, the VOUS detection rates ranged from 3.4 to 11.7% [
9,
11,
16]. In this study, CNVs detected in 9 children were unknown of clinical significance. After parental microarray analysis, CNVs in 6 children were inherited, the remaining CNVs in the other 3 children (Table
3, child 30-32) were de novo and the clinical significance was still unknown. Finally, the VOUS rate was 2.9% in our study, which did not increase obviously as the resolution rise compared with previous studies. Therefore, parental analysis could assist in interpreting CNVs and reducing VOUS rate.
The clinical features of 1p36 microdeletion syndrome include microcephaly, brachycephaly, developmental delay with hypotonia, seizures and cardiac defects [
27]. In our study, CMA revealed a 4.8 Mb deletion at 1p36.33-p36.31 (chr1:849,466-5,685,789) in child 4 with PDA and leukodystrophy, which contained the
SKI and
DVL1 genes.
SKI morphant embryos showed severe cardiac anomalies, especial complete failure in cardiac looping and malformations of the outflow tract [
28]. Researchers have studied
DVL1 null mice and reported that
DVL1-mediated planar cell polarity signal was crucially for cardiac outflow tract development [
29]. Based on the above data,
SKI and
DVL1 could be the main genes responsible for CHD phenotypes in 1p36 deletion syndrome.
A 4.9 Mb deletion at 15q11.2-q13.1 (chr15: 23,620,191- 28,540,345) was detected in child 6 (Table
3) with pulmonary stenosis plus ID. He was diagnosed as Angelman/Prader-Willi syndrome, the clinical features include ID, microcephaly, seizures, truncal ataxia, feeding difficulties in infancy and muscular hypotonia. Soemedi et al. first reported the association of 15q11.2 deletion with CHD, in their study the phenotypes contained left-side malformations, coarctation of the aorta, septal defect and TOF [
30]. Geng et al. have detected four patients with 15q11.2 deletion (size range 245-2703 kb) in 502 CHD patients, their result also showed the 15q11.2 deletion has low penetrance in CHD patients [
31]. These data indicated that 15q11.2 deletion, containing the Angelman/Prader-Willi syndrome region, represents a critical CHD locus. However, further studies are need to ascertain the genes responsible for CHD.
In the present study, we identified two children with 1q43-q44 deletion. Child 1 manifested atrial septal defect (ASD) and ID. Child 21 manifested TOF and absence of corpus callosum. Our study showed significantly higher frequency of 1q43-q44 deletion (2/104) than that of the 9170 control cases (4/9170) (
P = 0.002) [
30,
32]. Therefore, 1q43-q44 should be considered as a locus associated with cardiac development. The clinical features of 1q43-44 deletion syndrome include ID, language retardation, characteristic facial features, abnormalities of the corpus callosum and seizures [
33]. Summarizing the literature, patients with 1q43-q44 deletion manifesting ID/DD or structural anomaly of central nervous system (69/83, 83.1%) more frequently than CHD (19/76, 25%) (
P < 0.001, Table
5). These data suggested 1q43-q44 deletion may have low penetrance in CHD children.
Table 5
1q43-q44 deletion in our study compared with other studies
ID/DD or CNS abnormal | 2/2 | 22/27 | 24/24 | 15/24 | 6/6 | 69/83 |
Congenital heart defects | 2/2 | 8/24 | 5/22 | 2/24 | 2/4 | 19/76 |
The child 11, a girl with ASD and cleft palate which had been corrected surgically. She was diagnosed with mental retardation at 2 years old by pediatric doctor. CMA test revealed chromosome 18q12.3 duplication (chr18: 41,814,626- 42,453,303).
SETBP1 gene located in the fragment which was responsible for Schinzel-Giedion midface retraction syndrome, and point mutation is the most common type. The clinical features included severe mental retardation, distinctive facial features, and multiple congenital malformations such as skeletal abnormalities and cardiac defects. A dominant-negative or gain-of-function mechanism was proposed to underlie this syndrome [
34]. We sequenced the 5 exons of
SETBP1 gene, and no mutation was detected. The
SETBP1 gene duplication was in accordance with gain-of-function mechanism, and the child’s manifestations were similar with the phenotypes of Schinzel-Giedion midface retraction syndrome, Therefore,
SETBP1 should be the main responsible gene for this patient.
The child 22 had coarctation of aorta, VSD, ASD (cardiac surgery was performed when he was 1 month old) and ID. Brain MRI showed normal result. CMA test revealed a 5.2 Mb LOH on chromosome 6q24.1-q24.2 (chr6:139,184,381-144,411,648). This LOH fragment included
PLAG1 and
HYMAI genes, which were associated with imprinting disorder intrauterine growth retardation and neonatal hyperglycemia [
35]. Another gene
CITED2 also located in the LOH, which has been identified associated with cardiac malformations, including atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle and right-side aortic arches [
36]. Therefore, this fragment was classified as PCNVs.
Of the 29 children with PCNVs, 7 (24.1%) were with 22q11 deletion (n = 6) or duplication (n = 1) syndrome, which was the most common type. The incidence of 22q11 deletion/duplication was 6.7% (7/104) in the children with CHD in the present study. These data indicate that in addition to CMA it could be more cost-effective to exclude 22q11 deletion/duplication firstly by targeted technique such as MLPA in children with CHD.
In the 3 children with VOUS, we identified CHD candidate genes such as
STIM1, CTNNA3 and
PLN.
STIM1 gene mutation could cause cardiomyocyte hypertrophy [
37].
PLN gene mutation was associated with cardiomyopathy [
38].
CTNNA3 gene mutation could cause arrhythmogenic right ventricular dysplasia, compound heterozygous deletion was related to ASD [
39,
40]. However, the children’s symptoms in this study were inconsistency with genotypes in the database. Further study of these genes are still needed to evaluate the clinical implication.