De Novo Copy Number Variants Identify New Genes and Loci in Isolated, Sporadic Tetralogy of Fallot

The combination of a malpositioned aorta that overrides both ventricles, ventricular septal defect, pulmonary stenosis (which obstructs blood flow into the lungs) and right ventricular hypertrophy (Figure 1) defines TOF. The most prevalent form of cyanotic heart disease, TOF occurs in one of 3,000 live births and accounts for 10% of all major congenital heart disease¹. With recent advances in corrective surgery early lethality from TOF is rare but long-term sequelae, including arrhythmia, ventricular dysfunction and often life-long disability, persist.

TOF can arise in the context of prenatal infections, exposure to teratogens, maternal illness, and from dominant mutations that usually alter gene dosage. Haploinsufficiency of cardiac transcription factors genes (NKX2.5, TBX1, TBX5, GATA4) or the transmembrane receptors, NOTCH1 and NOTCH2 and their ligand JAG1 can cause TOF but, more commonly, mutations in these genes produce other heart malformations^2–7. Cytogenetic abnormalities, including deletions of chromosome 22q11.2 (DiGeorge syndrome) or trisomy 21 (Down syndrome), account for 15% and 7% respectively of TOF cases, however these patients usually have multiple non-cardiac abnormalities^8,9. Large de novo CNVs, identified by array CGH, occur with major congenital anomalies^10,11; these typically include congenital heart disease (CHD) in half of all cases¹². Far less is known about genes that cause sporadic and isolated CHD, particularly genes involved in complex malformations.

We hypothesized that de novo mutations that alter the dosage of genes involved in cardiac development might account for isolated TOF. We surveyed the genome of 121 TOF trios, each comprised of one proband and two unaffected parents, using the Affymetrix 6.0 array (Supplementary Figure 1). CNVs identified in TOF cases, but absent from parental samples (putative de novo CNVs), by the algorithm Birdseye¹³ were studied further. CNVs that corresponded to known copy number polymorphisms (CNPs)¹⁴ or that were smaller than 20 kb were discarded. To distinguish between potentially pathogenic variants and unidentified benign CNPs we examined all putative de novo TOF CNVs in 2,265 controls genotyped on the Affymetrix 6.0 array (15 and unpublished) and those TOF CNVs that shared ≥ 50% overlap with CNVs found in ≥ 0.1% of control samples were designated as CNPs and were not studied further. Seven individuals with an excess of rare de novo CNVs were removed from the analysis. Of the 32 remaining putative de novo CNVs, 11 were independently validated using multiplex ligation-dependent probe amplification (MLPA) (Supplementary Table 1) and 21 (66%) were false positives; 12 CNVs were inherited and 9 CNVs could not be confirmed by MLPA (data not shown). In summary, genome-wide analyses and validation identified 11 rare de novo CNVs at 10 unique loci from 114 TOF trios.

We considered whether the frequency of de novo CNVs differed between TOF cases and healthy subjects, by analyzing 98 control trios, including 55 HapMap trios, genotyped using the Affymetrix 6.0 array (Supplementary Figure 1). Using the same computational algorithm, 20 putative de novo CNVs were identified: 12 in HapMap trios and 8 in other control trios. Seven of the CNVs found in HapMap trios have been previously attributed to cell line artifacts (chromosome 14: 105,829,131–106,116,317 in NA10854, NA10838, NA06991, NA18857 and NA19154; chr 22:20,777,493–21,581,602 in NA12707 and NA19154)¹⁶ and two CNVs in HapMap trios and seven CNVs found in the other control trios fulfilled our criteria as CNPs. Four CNVs were validated by MLPA as being de novo in the control trios (Supplementary Table 2).

The frequency of rare de novo CNVs was greater in TOF trios than in control trios but the difference was not statistically significant (11/114 vs. 4/98, p = 0.18). Although de novo CNVs and pathogenesis appears to be related in schizophrenia^17,18 and autism¹⁹, our trio study may be underpowered to detect a similar relationship in TOF. Alternatively, TOF mutations may be incompletely penetrant; a consideration that prompted assessment of whether inherited CNVs occurred at loci discovered by de novo CNVs analyses. In three TOF trios we identified CNVs at the 1q21.1, 3p25.1 and 7p21.3 loci that were inherited from unaffected parents (Table 1), a finding that supports the role of additional genetic or environmental interactions in TOF.

To further evaluate the pathogenicity of CNVs, we used MLPA to assess nine loci in a second cohort of sporadic, non-syndromic TOF cases (n = 398). Because the cases in this validation cohort had prior chromosome 22q11.2 analyses, this locus was excluded from further study. At least two unique synthetic oligonucleotide MLPA probes (Supplementary Table 3) were designed to hybridize within each of the nine loci. MLPA studies demonstrated four additional TOF patients with 1q21.1 CNVs (three duplications, one deletion, Supplementary Table 1). The boundaries of these CNVs were delineated by Affymetrix 6.0 array analyses. In combination with our initial genome-wide studies a total of 17 CNVs were found at 10 loci in 512 TOF cases (Table 1). CNVs at each of these loci were absent or very rare in 2,265 controls. CNVs at four loci (1q21.1, 3p25.1, 7p21.3 and 22q11.2) were found in at least two TOF cases. Since small CNVs would be predicted to escape detection by the array platform and detection algorithm used here, our data defines a minimum estimate, approximately 10% (11/114), for the frequency of de novo CNVs in sporadic, isolated TOF. This is lower than the 25–30% frequency of de novo events seen in individuals with syndromic heart malformations that occur with additional birth defects^10,11 but importantly, identified causes for isolated TOF.

At chromosome 1q21.1 CNVs were found in five TOF cases (Figures 2a and 2b) that are structurally complex (Supplementary Figure 2). The shared duplicated segment in four TOF cases spans a small interval on chromosome 1q21.1 where seven validated genes are encoded: PRKAB2, PDIA3P, FMO5, CHD1L, BCL9, ACP6 and GJA5. Transcriptional analyses of human right ventricular outflow tract (RVOT), which is malformed in TOF, demonstrated six of these genes are expressed. Among these six genes, PRKAB2, CHD1L, BCL9 and GJA5 had enriched expression in RVOT (Table 2), a finding that increases their candidacy as disease genes. In 96 independent sporadic TOF cases we sequenced exons and flanking splice sites in GJA5 and CHD1L, which encode connexin 40 and chromodomain helicase DNA binding protein-1 respectively. No non-synonymous changes at conserved residues or small insertions/deletions were identified (data not shown).

Chromosome 1q21.1 has been previously implicated in CHD²⁰ but more recently CNVs at this locus were identified in subjects with neuro-cognitive, psychiatric, and developmental phenotypes ^{17,18,21–26}. Notably, there is no perfect correlation between 1q21 dosage and phenotype in all studies to date (including ours). Two studies described mild (7 cases) or severe (5 cases, not including TOF) cardiovascular malformations, but 9 of these cases had additional phenotypes: developmental or intellectual disabilities, dysmorphic features or other congenital anomalies^22,23. In contrast, all TOF cases with 1q21.1 CNVs identified in our study had normal cognition, social behavior, and neurologic function (Table 3). The remarkable combination of structural heart malformations and functional (non-structural) brain deficits resembles the DiGeorge phenotype, due to chromosome 22q11.2 deletions that disrupt TBX1^8,9. While the variable length of 1q21.1 CNVs may imply that these mutations alter contiguous genes with distinct roles in cognition and heart development, we speculate that this locus contains a single causal gene which, like TBX1, functions in progenitor cells (perhaps neural crest cells), critical for both cardiovascular and brain development. Regardless of whether these or another explanation accounts for both clinical phenotypes, we note that less than 0.2% of patients with neuro-psychiatric disorders and 0.02% of controls had chromosome 1q21.1 duplications²³. In contrast, 1q21.1 duplications were significantly (p = 0.007) more common in TOF, occurring in 1% of patients studied here.

Two TOF cases shared overlapping duplications at the 3p25.1 locus. Whereas one duplication spanned 12 Mb, the other duplication affected only two genes, RAF1 and TMEM40 (Figure 2c). RAF1 is expressed at approximately 100-fold higher levels than TMEM40 in the RVOT (Table 2). Gain-of-function point mutations of RAF1 cause Noonan syndrome, a multisystem disorder with cardiac manifestations, that rarely causes TOF but moreover produces one of its components: hypertrophy, atrial or ventricular septal defects, or pulmonary stenosis^27,28. Identification of CNVs at 3p25.1 in TOF cases prompted re-evaluation for signs of Noonan syndrome (Table 3). Subtle craniofacial abnormalities were identified in one case (patient 756), suggesting some phenotypic overlap between RAF1 gain-of-function mutations and increases in RAF1 copy number produced by the 12 Mb duplication. The smaller 3p25.1 CNV truncates and duplicates RAF1 and further study is necessary to determine which alteration causes the TOF phenotype.

Reciprocal CNVs were found at 7p21.3 in two TOF cases but since no known genes, mRNAs, microRNAs or ESTs are encoded at this locus (Supplementary Figure 3) ongoing RVOT transcript analyses and re-sequencing may help identify the target of these CNVs.

Chromosome 22q11.2 deletions were identified in two TOF patients who lacked any extra-cardiac phenotype that accompanies the DiGeorge syndrome²⁹. A large 22q11.2 deletion was also found in one control subject. These data confirm substantial incomplete penetrance of 22q11.2 deletions³⁰. Whether genetic mechanisms that compensate for the deleterious consequence of 22q11.2 deletions also influence other CNVs is unknown.

Of six de novo CNVs found in only one TOF subject two altered previously described congenital heart disease genes, NOTCH1 and JAG1. NOTCH1 null mutations primarily cause familial bicuspid aortic valve and less commonly other malformations⁷. Mice engineered to lack components of the NOTCH1 signaling pathway have TOF-like phenotypes³¹. Our findings provide direct evidence for NOTCH1 mutations in TOF. JAG1 mutations are known to cause TOF in the context of Alagille syndrome and in isolation^4,32. The patient with the 4 Mb de novo deletion of JAG1 identified here has no clinical features of Alagille syndrome. The finding of CNVs that altered NOTCH1 and JAG1 underscores the need for assessing gene dosage in mutation analyses of congenital heart disease genes

Four other loci were altered by CNVs in individual TOF patients. CNVs at these candidate TOF loci occurred at a similar low frequency to NOTCH1 and JAG1 mutations, emphasizing the genetic heterogeneity of TOF. Three genes are encoded at the 2p23.3 locus: RAB10, KIF3C and ASXL2. Although none were previously implicated in cardiac development, each is expressed in the RVOT (Table 2). The expression of RAB10, which encodes a GTPase, is highest. Because KRAS, another GTPase, is activated by Noonan syndrome mutations³³, RAB10 is a promising candidate gene in TOF.

Pathogenicity of a 1.8 Mb de novo duplication at 2p15 found in one TOF case is likely given its size and the absence of CNVs at this locus in all controls. Of 12 genes encoded in the duplicated interval nine are expressed in the human RVOT (Table 2) and none are previously implicated in cardiogenesis. The 4q22.1 deletion encompasses PPM1K and transcripts encoding this phosphatase were present in RVOT tissues. The deletion at 10q11.21 contains no known genes, coding or non-coding RNAs.

In summary, our studies defined seven new loci that substantially increase risk (odds ratio ≥ 8.9) for sporadic, non-syndromic TOF. While some loci are large (>100 kb) and three loci exhibited incomplete penetrance, expression data of human RVOT implicates an important subset of genes, prioritizing further investigation. Moreover these data indicate de novo CNVs at 10 loci accounted for 10% of TOF, which explains sporadic presentation and defines genetic heterogeneity in this serious heart malformation.