DISCUSSION
The association between congenital anomalies and pediatric cancer has long been recognized 39. Recent studies evidenced an increased risk of pediatric cancer also in birth defects unrelated to chromosomal abnormalities or known genetic syndromes40,41. Hepatoblastomas, in particular, occur in association with a wide variety of congenital abnormalities8,42,43, especially craniosynostosis and renal anomalies 12.
We report here two syndromic patients who developed the rare tumor hepatoblastoma, in addition to Hirschsprung disease, besides other anomalies, such as developmental delay, congenital cataract and nail dysplasia. The only clinical report of an association between hepatoblastoma and Hirschsprung disease corresponds to the Patient 2 of this study 20.
Hirschsprung disease is a multifactorial condition with variable penetrance and expressivity, with several genes already known to cause this pathology when mutated 22,23, however, germline mutations account for only ∼50% of the investigated cases of Hirschsprung disease 44, pointing to the existence of yet unknown genetic mutations and underlying mechanisms contributing to the disease etiology. The gene set with rare coding germline variants identified in these two patients was enriched for pathways related to cancer, FGFR proteins family, Wnt signaling pathway metabolism, cytokine signaling in the immune system, post-translational protein modification, and developmental biology, suggesting they could have a broader role in cancer and congenital abnormalities.
Duplication in segment 14q23.2 have been documented in a few individuals in the control population and was classified as VUS. SYNE2 , encompassed in the germline duplication at 14q23.2 detected in Patient 2, is involved in nuclear migration in retinal photoreceptor progenitors and is required for centrosome migration to the apical cell surface during early ciliogenesis (primary cilia formation). Alterations inSYNE2 have also been associated with retinal defects, the gene has an important role for proper retinal development45,46. This evidence could indicate that alterations in this gene might contribute to patient’s congenital bilateral cataract and sensorineural deafness phenotypes.
In Patient 1, rare germline missense variants were identified in the cancer predisposition genes ERCC2 (c.545C>T) andHRAS (c.75G>A), both in heterozygosity. ERCC2is involved in gene transcription and helps repair damaged DNA47,48. Homozygous variants in this gene are associated with predisposition to childhood solid tumor49, while and other variants in this gene have been related to hypoplastic nails, a clinical characteristic of Patient 1. A variant of maternal inheritance was identified in HRAS , which has been associated with Costello syndrome (OMIM #218040), a rare dominant syndrome which increases the risk of tumors development50. It is not known how HRAS mutations cause the other features of Costello syndrome besides cancer predisposition such as intellectual disability, distinctive facial features, and heart problems, but many of the signs and symptoms probably result from cell overgrowth and abnormal cell division 51–53. Interestingly, a LoF was observed in the ZNF215 , disruptions in this gene were proposed to play a role in the etiology of Beckwith-Wiedemann syndrome.
Germline data of the Patient 2 also revealed four VUS in the cancer-predisposing genes APC , a gene related to FAP, one of the genetic conditions that predisposes to HB 2,54,55;MCC, a regulator of the Wnt signaling pathway56 and a candidate tumor suppressor gene57–59; ODC1 , and BRCA1 . One VUS were also observed in ERBB2 , a gene already associated with the megacolon phenotype 60,61. In addition, eight variants were observed in genes related to cataract (ABCA4, COL4A4, HGSNAT, PCDH15, RPGRIP1, SLC16A12, SON, VAT1 ) 62–64, a clinical condition of this patient.
In this study, we disclosed some potential candidate genes for hepatoblastoma and Hirschsprung disease. Particularly, variants in genesCEP164 and CYP1A1 were interesting because they were previously observed in an HB exome study of somatic mutations14. CEP164 encodes a protein responsible for the repair signaling when DNA damage occurs 65. This gene is involved in the stability of the genome and was previously found to be somatically mutated in two hepatoblastomas 14. A germline variant was identified in the CEP164 of the Patient 2. We also detected, in both patients, germline variants in the geneCYP1A1 . One variant in this gene was previously reported in a case of congenital hepatoblastoma 14. CYP1A1encodes a protein member of the cytochrome P450 superfamily of enzymes66; the expression of this gene is transcriptionally regulated through the AhR receptor, which plays an important role as a mediator of the adaptive response to xenobiotics and also contributes to normal physiology and embryonic development 67.
Interestingly, another cytochrome involved in Hirschsprung disease was disclosed in this study. The germline variant in ALDH1A2(c.1100A>T) is described in COSMIC and ICGC as a somatic mutation with a high score of pathogenicity. The product of this gene is an enzyme that catalyzes the synthesis of retinoic acid from retinaldehyde 68. Retinoic acid, the active derivative of vitamin A, is a hormonal signaling molecule that functions in developing and adult tissues. Studies of a homologous mouse gene suggest that this enzyme and the cytochrome CYP26A1 concurrently establish local embryonic retinoic acid levels, which facilitate later organs development 69.
Hirschsprung disease is a highly heterogeneous genetic condition, and many cases remain idiopathic even after extensive molecular investigation, indicating the existence of underlying genes or genetic mechanism not yet recognized. The disease should be diagnosed early in the neonatal period, the delay in diagnosis and treatment can lead to development failure and other health problems in a considerable proportion of infants 70. In the same way, a growing number of cancer susceptibility genes are being identified and information is available in some public databases. However, for rare pediatric tumors, such as hepatoblastoma, the number of cases and the lack of grouping clinical information, makes it difficult to identify and understand the physiopathology of a causal germline mutations. Our data pointed out novel candidate genes (ALDH1A2, CEP164, CYP1A1 , and SYNE2 ) for Hirschsprung disease biology with a potential functional role in the association with HB tumorigenesis (Figure 3 illustrates the main findings of this study). These results contribute to delineate future studies to understand the biology of Hirschsprung disease and its association with other conditions, like hepatoblastoma.