In this study, we performed NGS targeting JAG1 in a large cohort of 813 subjects with CH, and then identified 10 pathogenic missense mutations (p.V45L, p.V272I, p.P552L, p.G610E, p.G852D, p.A891T, p.E1030K, p.R1060W, p.A1131T, p.P1174L) carried by 25 patients, the mutation rate of JAG1 in CH was 3.08%.
JAG1, widely expressed throughout mammalian development, plays an important role in the development of ALGS-related organ systems and thyroid [7, 10]. In the literature of JAG1 mutations in ALGS, the most common mutation types were frameshift (37%), followed by nonsense (22%), large gene deletions (13%), missense (13%), splice site (12%), and the other types (< 3%). The JAG1 missense variants clustered within the first 6 exons of the gene with a statistically significant, almost a quarter of these missense variants involve the gain or loss of a cysteine within the EGF-like domain [11]. While, in the genetic screening of JAG1 variants in CH cases, Filippis et al identified 7 missense variants (p.P476T, p.F509L, p.Y663C, p.R744Q, p.R937Q, p.I1021T, p.R1094W) [9, 12], and we found 10 missense variants in this study. In summary, all these JAG1 variants detected in CH were missense mutations and were concentrated in exon22-26 significantly (P = 0.049) (Fig. 4), which is quite different from the laws in ALGS [11]. Phenotypic effects of JAG1 mutations are highly penetrant, but with highly variable expressivity, which was consistent with our research, as a high degree of variable expressivity has been observed in patients harboring the same pathogenic variant in this study. Therefore, the hypothesis that a second gene could act as a modifier was put forward, the glycosyltransferases (Lunatic Fringe, Radical Fringe, Manic Fringe, and POGLUT1) and THBS2 were reported as candidate genetic modifier [13–15]. However, whether the hypothesis is applicable for the pathogenic mechanism of JAG1 in CH still needs further verification.
Among these 813 CH patients we enrolled, 224 were classified as TD and the other 589 were DH. Our data showed once again that DH is a more common cause of CH than TD in China. Combined with the genetic screening of 20 genes associated with CH in these 25 JAG1 variants carriers, we further analyzed the correlation between genotype and phenotype, the results revealed that the JAG1 variants confer genetic susceptibility to both TD and DH, but with different inheritance model.
In TD cases, 7 of 8 were monogenic JAG1 variant carriers, the other 1 with heterozygous variant in JAG1 and DUOX2, indicating that JAG1 variants lead to TD mainly through monogenic model. Currently, most knowledge on TD genetics has been accumulated on monogenetic forms of TD, which concern mutations in genes TSHR, NKX2-1, PAX8, FOXE1 and NKX2-5. As causative mutations were identified in less than 5% cases, and genetics of TD are not following simple Mendelian patterns, the hypothesis for the transmission mode in TD remains debated. Beside monogenic, polygenic and epigenetic mechanisms had been proposed [16, 17]. The polygenic origin of TD was initially supported by mouse models, as Nkx2-1 +/− and Pax8 +/− mice displayed normal thyroid gland, while [Nkx2-1, Pax8] +/− mice showed hypoplasia or hemiagenesis [18]. Polygenic variants were also identified in very few TD patients, the combination of variants in genes reported in previous studies were TSHR and TPO/DUOX2/GANS/PAX8/JAG1, PAX8 and NKX2-5, TG and PAX8/TPO/JAG1. Among them, the patient with JAG1 (p.R937Q) and TSHR (p.S526G) variants showed thyroid hypoplasia, and the patient carried JAG1 (p.R744Q) and TSHR biallelic (p.R1250L, p.R1136Q) variants was diagnosed as thyroid ectopy [12, 17, 19]. In addition, JAG1 variant in combination with DUOX2 variant had been found in a CH patient without classification [20]. In this study, only 1 patient with agenesis was detected with digenic variants in JAG1 (p.A1131T) and DUOX2 (p.I967T), the low incidence of polygenic mutations indicating it possess a marginal role in TD.
In DH cases, both monogenic mechanisms (9/17, 52.9%) and oligogenic mechanisms (8/17, 47.1%) play a pivotal role. DH caused by monogenic variants of JAG1 is not inherited in the classic autosomal dominant manner, as all their parents were euthyroid. Although biallelic and monogenic mutations are now considered as the most common causes of DH, oligogenecity has been found in 20-43.5% patients with CH, especially associated with DUOX2 and DUOXA2 [20, 21]. DUOX2 is the main gene responsible for DH, the detection rate of DUOX2 mutations in China is as high as 28%-44% [22], whereas the detection rate obtained in this study was 44%. In our patients with DH, 2 out of 9 cases in monogenic group were severe CH, while for oligogenic group, 7 out of 8 cases were severe CH. Pearson chi-square analysis showed that oligogenic inheritance was significantly associated with severe CH (P = 0.007), and the finding is supported by previous study [23]. To examine the correlation between variant dosage and severity, we compared the fT4 levers in monogenic and oligogenic groups, the results indicated that oligogenicity may contribute to the disease severity of DH, Takeshi et al. had come to the same conclusion [20]. Therefore, the oligogenic model may partly account for the differences in the penetrance and expressivity of CH.
The role of JAG1 in thyroid development had been explored in zebrafish, which affect the proliferation and survival of thyroid follicular cells possibly through a permissive effect on TSH signal. In the biosynthesis of thyroid hormone, JAG1 might play a role by regulating SLC5A5 though Notch effector HES1 [8, 10]. In fact, the existence of an overlap of genetic aetiologies in TD and DH had been identified in many researches, patients with the same mutation demonstrate a broad spectrum of clinical phenotypes [12, 20, 24]. Therefore, the genetics of CH is complex, and the mechanisms involved in its phenotypic heterogeneity remains unclear.
In conclusion, we screened out 25 JAG1 variant carriers in 813 CH patients, the mutation rate is 3.08%. Further analysis revealed that the JAG1 variants confer genetic susceptibility to both TD and DH, but with different inheritance model. The comparation between oligogenic group and monogenic group indicated that CH may exhibit a gene dosage effect. There are two limitations in the present study should be noted. First, patients with other congenital malformations were excluded when collecting samples, as a result, we could not evaluate the effects of these JAG1 variants on other organ systems. Second, we did not carry out functional studies of these JAG1 variants.