We screened a cohort with right-sided cardiac defects for mutations in JAG1, and identified functionally significant sequence variants in 3% (2/94) of TOF cases, and 4% (2/50) of PS/PPS/PA cases. These cases did not meet criteria for a diagnosis of AGS, nor did they have overt findings of any other known syndrome. These findings suggest that JAG1 mutations are present in a small percentage of seemingly non-syndromic cases of TOF or PS/PPS and add to the growing list of disease genes for specific types of cardiac defects.
The low incidence of JAG1
mutations the cohorts we studied, may be consistent with the high degree of heterogeneity for these phenotypes. A study of 114 patients with apparently isolated sporadic TOF using an array designed to detect deletions or duplications revealed that 1/114 individuals had a JAG1
deletion (Greenway, et al., 2009
). On analysis of 230 patients with both syndromic and non syndromic TOF, genetic abnormalities were identified in 18% (42/230) (Rauch, et al., 2009
). The most common genetic alteration was the 22q11.2 deletion associated with DiGeorge/VCFS syndrome, seen in 7.4%. Single gene mutations in JAG1
(MIM# 600584), and TBX1
(MIM# 602054) were found in 1.3%, 0.9%, and 0.4% of the cohort, respectively, however, the JAG1 mutations were all in patients with Alagille Syndrome (Rauch, et al., 2009
). Clearly, TOF is characterized by heterogeneity, with small contributions from various loci. Nonetheless, these studies further suggest that careful scrutiny for a family history of right-sided heart defects and careful examination for sub-clinical features of AGS is warranted in patients with right-sided heart defects, particularly those with isolated PPS. The clinical and reproductive implications of finding a JAG1
mutation carrier are significant, as identifying the patient with a JAG1
mutation may help identify the unsuspecting mutation-bearing parent with subclinical features of AGS and allow for more informed genetic counseling.
In this study six different JAG1
sequence variants were identified: two protein truncating and four missense changes (p.C664S, p.P810L, p.R937Q, p.H1104Q). We also investigated the functional significance of a previously reported missense variant, p.C234Y (Le Caignec, et al., 2002
). The protein truncating mutations are predicted to lead to the creation of premature termination codon, and the transcripts of these alleles should undergo nonsense-mediated decay leading to 50% normal JAG1
expression. These mutations are very similar to nonsense mutations seen in AGS, which is caused by haploinsufficiency of JAG1
. We therefore predict that these mutations would lead to JAG1
Three of the missense variants occurred in the epidermal growth factor-like (EGF) repeats of the JAG1
protein. These repeats are known to be important in ligand-receptor interaction (Kopan and Ilagan, 2009
). Residue 234 (p.C234Y) is located in the first EGF repeat, while 664 (p.C664S) is in the eleventh, and 810 (p.P810L) is in the fifteenth (). Cysteine residues are required for proper folding of EGF repeats due to their role in the formation of disulfide bridges. A partial crystal structure of JAGGED1 was recently produced which encompassed the DSL domain and first 3 EGF repeats (Cordle, et al., 2008
). This region of the protein was shown to be crucial for receptor-ligand interactions. Residue 234 lies in the first EGF repeat within this critical region, suggesting that loss of this cysteine residue would have deleterious effects on Notch signaling. Unfortunately, no available crystal structures of JAGGED1 include the other residues found altered in this study. Both affected cysteine residues show conservation not only through all vertebrates, but also in both the Delta and Serrate ligands in Drosophila
(). The proline at position 810 is conserved in vertebrates, but not in the Drosophila
ligands. The p.R937 residue lies within a region of the JAG1
protein whose significance is unknown, and this residue showed far less conservation (). While conserved in mammals such as chimp, mouse, and dog, it was not present in lower vertebrates like zebrafish. The p.H1104 residue resides in the intracellular portion of JAG1
, and exhibits the least conservation of the five missense variants (). No functionally significant variants in the intracellular domain of JAG1 have been reported in AGS patients to date. Residue p.H1104 also does not lie in any of the intracellular regions known to be required for proper function of JAG1 (Glittenberg, et al., 2006
) A missense variant in the intracellular region of JAG1 was reported in a patient with Biliary Atresia, but the functional significance of this variant was never investigated (Kohsaka, et al., 2002
). The low conservation of this residue combined with the lack of precedent for functionally significant intracellular missense variants suggests that p.H1104Q is likely a benign rare variant.
Figure 4 A: The locations of the five sequence variants indentified in the JAG1 protein are shown, as is the domain structure of JAG1. Domains are labeled as such; SP, Signal Peptide; NT, N-Terminal Region; DSL, Delta-Serrate-Lag2 conserved region; EGF, Epidermal (more ...)
We performed functional analysis on four of these missense variants to determine the extent to which these residue substitutions affect normal cell-surface localization, post-translational modification, and signaling ability. As mentioned, the p.R937Q variant protein appears to function like wild-type, in that it is expressed at the cell surface, properly post-translationally modified, and is able to activate Notch signaling. Furthermore, the residue is not as highly conserved as the other residues in question, suggesting it is of less importance for correct ligand function. This variant has been previously reported in the literature as an AGS mutation, although parental samples were not screened (Ropke, et al., 2003
). The results of our functional analysis of p.R937Q raise some questions as to its pathogenicity. Although the p.R937Q variant clearly has no affect on the ligand’s function in canonical Notch signaling, this does not rule out the potential for a secondary effect of the substitution on a non-canonical JAG1 function that we did not test.
The p.C234Y and p.P810L mutations were not present at the cell surface, were not properly post-translationally modified, and could not initiate Notch signaling. We would therefore predict that these mutations lead to JAG1
haploinsufficiency, with only the wild-type allele in carriers of this dominant mutation appearing on the cell surface. The p.P810L and p.C234Y missense mutations and the two nonsense mutations are therefore all similar to those that have been studied in cases with Alagille syndrome. It is worth noting that the p.P810L missense mutation is in fact a maternally inherited mutation, although the mother did not have any cardiac defects and she was not available for evaluation of other sub-clinical features of AGS. The p.C234Y variant is a familial mutation segregating with cardiac defects, deafness, and posterior embryotoxon, yet all carriers have normal liver function (Le Caignec, et al., 2002
). Familial JAG1
mutations commonly exhibit variable expressivity in AGS, with very mildly affected parents harboring a disease causing mutation (Kamath, et al., 2003
). It is not completely surprising to us to see the same scenario in the case of these two CHD-related mutations, although the lack of any liver dysfunction in the nine family members studied by Le Caignec et al. is unusual.
In contrast, the missense mutation p.C664S appears to have a “leaky” phenotype, with some of the protein appearing on the cell surface and some being improperly trafficked. This mutation is similar in that regard to the previously reported p.G274D mutation seen in a large family segregating isolated cardiac disease in the absence of other features of Alagille syndrome (Eldadah, et al., 2001
; Lu, et al., 2003
). We have previously shown via reporter assays that p.G274D cannot initiate Notch signaling (Lu, et al., 2003
). However, p.C664S appears to be able to initiate Notch signaling, suggesting that the mutation may only be affecting ligand trafficking, not receptor binding and activation. This finding implies that p.C664S is not a null allele, and that complete haploinsufficiency is not required for manifestation of a cardiac phenotype. Since we have only examined two “leaky” mutations, further studies will be required to understand the extent and impact of this “leaky” signaling.
TOF and PS/PPS are two relatively common structural cardiac defects that are heterogeneous in etiology. Both of these disorders can be seen as isolated findings, or in the context of multiple different congenital anomalies or genetic syndromes. For example, TOF is commonly seen in patients with 22q11.2 deletions and/or AGS, and PS is commonly seen in patients with Noonan syndrome (MIM# 163950), LEOPARD syndrome (MIM# 151100), and AGS, while PPS is rather uniquely found in patients with AGS and Williams syndrome (MIM# 194050). Our functional analysis of JAG1
missense mutations clearly confirms that the activity of most mutation-bearing proteins differs from wild-type JAG1
in signaling ability, subcellular localization, and post-translational modification suggesting that these mutations are indeed responsible for the cardiac manifestations seen in these cases. What remains unknown is why these cases present with only a cardiac phenotype and not the full clinical features of AGS, particularly hepatic disease. The first mutation we examined, p.G274D, was identified in a family with apparently isolated cardiac disease (Eldadah, et al., 2001
; Lu, et al., 2003
). The p.G274D mutant clearly displayed the “leaky” phenotype, similar to p.C664S. We hypothesized at the time that the peculiar nature of these mutations could result in hypomorphic activity and not true haploinsufficiency, and that this increased level of functionality was sufficient to prevent the typical liver manifestations seen in AGS, but the developing heart was too sensitive to slightly decreased Notch signaling and was still affected. However, the p.C234Y and p.P810L mutations appear to be completely haploinsufficient, yet the patients with these mutations do not have the full spectrum of AGS, consistent with the presence of additional modifying factors. In an era of increasing whole genome analysis, future studies may elucidate what these modifying factors are and allow for better predictive diagnosis in humans with JAG1