CAVC, accounts for ~ 4% of CHD, is a complex cardiac malformation characterized by a variable deficiency of the atrioventricular area in the developing heart [13, 14]. SV, one of the most common forms of severe CHD, comprises a spectrum of congenital cardiac malformations defined by severe underdevelopment of one ventricle [15].
In 262 CAVC patients reported here, 88.6% (39/44) carried the duplication of 21q11.2-21q22.3, which could be diagnosed as trisomy 21, namely Down Syndrome (DS). Based on the results, a striking association of CAVC with DS was found in this study (14.8%, 39/262). All DS patients had the same ~ 3.3 Mb duplication at 21q11.2-21q22.3, and a systematic reanalysis indicated that 21q22.13 was the minimal critical region to the DS phenotype [16]. Additionally, another study detected 57.6% cardiac malformations in 500 patients with DS, and it also suggested CAVC (35.1%) was the most frequent heart anomaly [17]. It is putative that CAVC is the most frequent type of CHD in DS patients, and our study also provide strong evidence for this correlation in Chinese population. Additionally, CAVC also referred to as complete atrioventricular septal defect, and it has been reported that AVSD (atrioventricular septal defects) are more common in the female gender of DS patients [18]. In our study, the female/male ratio of CAVC with DS patients was 1.6 (24:15), which suggest that potential gender differences existed in the prevalence of CAVC in DS patients. Besides, we also noticed that rates of pulmonary hypertension (PH) in DS patients with CAVC was 41% (16/39), which was higher than previous report (28%, 364/1242) [19]. In fact, it was well known that PH is common in children with DS, and our study intensely proved this correlation.
Nowadays, several genes located in the “CHD critical region” on chromosome 21 have been proved to be associated with CAVC, including DSCAM, COL6A1, COL6A2, and DSCR1 [20]. However, there were three DS patients simultaneously had another CNV located at different chromosome in our cohort, and one of the CNVs (3q12.1-3q12.2 dup) has been reported to associate with VSD in Decipher database. Additionally, several DS patients showed not only CAVC (4/27), but also other cardiac anomalies, such as ToF, ASD, patent foramen oval (PFO) and patent ductus arteriosus (PDA). Although the above-mentioned genes can explain partial cardiac phenotypes in DS patients, the genetic causes still were difficult to clarify especially when DS probands accompanied with multiple CNVs and diverse CHD phenotypes.
In our results, two rare CNVs have been identified as main causes of certain syndromes with heart anomalies. The microdeletion on 7q11.23 caused Williams-Beuren Syndrome (WBS; OMIM 194050), which is a multisystemic developmental disorder mostly accompanied with CHD. More than 90% of WBS patients have the ~ 1.55 Mb pair deletion extending from FKBP6 to GTF2I, and it has been widely accepted that the deletion or mutation of an elastin (ELN) allele is a major cause of WBS [21]. One patient in this study (CAVC162) had a ~ 1.52 Mb deletion at 7q11.23 extending from NCF1B to GTF2I, encompassing the ELN gene. The other microdeletion on 8q24.3 have been recognized as associated with Verheij syndrome (OMIM 615583), which is characterized by growth retardation, developmental delay (DD), microcephaly, vertebral anomalies, dysmorphic features, cardiac and renal defects. Poly(U) Binding Splicing Factor 60 (PUF60) were suggested as the main cause for heart defects in the syndrome, since knockdown of Puf60 alone resulted in cardiac structural defects [22]. The patient (CAVC145) reported here had a ~ 2.5 Mb deletion of 8q24.3, representing with growth retardation and heart anomalies.
Among the rest rare CNVs identified in the CAVC patients, CNVs located at 8q21.13-8q21.2, 9p24.3-9p13.1 and 3q12.1-3q12.2 have been seldom reported. The detected rare deletion CNV, 8q21.13-8q21.2, encompasses Zinc Finger and BTB Domain Containing 10 (ZBTB10), which has been known as a CHD gene. ZBTB10 encodes a telomere-associated protein [23]. Lately, a GWAS involving 4,000 unrelated Caucasian patients diagnosed with CHD indicated that ZBTB10 was associated with TGA, since two highly significant SNPs (rs148563140 and rs143638934) closely located to this gene [24]. Furthermore, they suggested strong cell-type specificity in murine cardiac development for Zbtb10. Except the known CHD gene ZBTB10, this CNV region in the patient (CAVC102) also included STMN2 related to abnormality of the cardiovascular system. STMN2 encodes a member of the stathmin family of phosphoproteins, functioning in microtubule dynamics and signal transduction [25]. Compared with controls, methylation of STMN2 significantly increased (FDR p-value = 4.27 × 10− 51) in VSD cases [26]. Besides, It has been shown that Stmn2 expresses in atrioventricular node, endocardium and outflow tract in mouse according to the LifeMap Discovery database. Among the rest 2 duplication CNVs, duplication of 9p24.3-9p13.1 (CAVC207) has been reported as VSD or TOF in DECIPHER. In this region, only Rfx3 gene was in “ventricular septal defect” derived from the MGI (mouse genome informatics) database. For the duplicated region of 3q12.1-3q12.2, a report had shown a VSD patient had a ~ 116 kb duplication of this region, and TBC1D23 has been identified as the major candidate gene [27]. In our study, the patient (CAVC274) had a ~ 0.8 Mb duplication at 3q12.1-3q12.2, encompassing this CHD candidate gene TBC1D23.
In 259 SV patients, 66.7% CNVs (12/18) has been identified as common CNVs. Among them, we found three duplication CNVs (Xq28, 2q13, Xp22.2) had already been reported as novel CNVs in another studies with 223 SV patients by microarray analysis [28]. Compared with this previous report, we noticed that only several genes overlapped in regions of these three CNVs, namely HSFX1, MAGEA11, TMEM185A, CXorf40B for Xq28, BUB1 for 2q13, and MID1 for Xp22.2. In our study, these CNVs were all identified as common CNVs.
The rest 6 CNVs (8q21.13-8q21.3, 1q42.12-1q42.13, Xq22.1, 5q34-5q35.1, 2p13.2, 7p14.1) were rarely reported, and the deleted region of 8q21.13-8q21.3 (SV007) overlapped with the above-mentioned CNVs in the CAVC patient (CAVC102). Furthermore, we also noticed that duplication of 1q42.12-1q42.13 (SV163) included 2 known CHD risk genes, LEFTY1 and LEFTY2 [29]. Especially, It has been reported that the SNP rs2295418 in the LEFTY2 gene is associated with CHD in Chinese Han populations[30]. For other detected CNVs, only several genes were included in the deleted or duplicated regions, namely Xq22.1 (SV147; PCDH19, TNMD), 2p13.1 (SV195; DYSF, CYP26B1, EXOC6B) and 7p14.1 (SV176, INHBA and SUGCT). There were no related reports between CHD and these detected CNVs, so we focused on three genes (PCDH19, CYP26B1 and INHBA) with a significantly higher pLI score, which reflects the intolerance to the loss of function mutations. PCDH19 (Protocadherin 19) is a member of the delta-2 protocadherin subclass of the cadherin superfamily. CYP26B1 (cytochrome P450 family 26 subfamily B member 1) involves in limiting retinoic acid (RA) levels within vertebrate embryos, which facilitate RA degradation [31]. It has been well-known that RA is important for the development of the heart. INHBA (Inhibin Subunit Beta A) encodes a member of the TGF-beta (transforming growth factor-beta) superfamily of proteins, and it has been shown as a candidate gene for cardiac development [32].
As for the proband (SV143) with a ~ 1.6 Mb duplication at 5q34-5q35.1, we found that this region encompasses 2 genes (SLIT3 and TENM2) related to septal defects of heart. SLIT3 (Slit Guidance Ligand 3) expressed in cardiomyocyte-like progenitor cells [33], and membranous ventricular septum defects as well as atrioventricular and aortic valve abnormalities are exhibited in SLIT3-mutant mice [34]. Recently, SLIT3 variants in humans has shown association with CHD involving in ToF and septal and outflow tract defects [35]. TENM2 (Teneurin Transmembrane Protein 2) expresses abundantly in human fetal heart. Moreover, patients with loss of TENM2 presented ASD in Decipher database, but gain of TENM2 didn’t show any type of CHD yet.