DISCUSSION
We explored the clinical features and spectrum of rare germline variants possibly associated with cancer risk in 30 children who developed hepatoblastoma. We report here that 37% of HB patients presented with birth defects, mainly craniofacial (17%, including craniosynostosis) and kidney (7%) anomalies, as well as Hirschsprung disease (7%) and nail dysplasia (7%). There is a growing robust bulk of evidence emphasizing that various birth defects occur in association with a significant increase in the risk of developing childhood cancer [48–50]. Recent studies also supported an increased risk of pediatric cancer in individuals with birth defects unrelated to chromosomal abnormalities or known genetic syndromes [51, 52]. Hepatoblastoma, in particular, occurs in association with a wide variety of congenital abnormalities [53–55], especially craniosynostosis and renal anomalies [56], which we also observed in this study. In addition, a large case-control study confirmed the association between hepatoblastoma and kidney and bladder abnormalities [57]. In this study, we also described the cases of two patients with Hirschsprung disease and HB, a condition with extensive genetic heterogeneity [58, 59]; most Hirschsprung disease cases are sporadic (approximately 70%), while a smaller portion of patients have other associated congenital anomalies. However, pathogenic or likely pathogenic variants mapped to morbid OMIM genes that could explain the syndromic phenotypes of our patients were not detected, reinforcing that, despite the long-standing knowledge about the intersection between biological pathways of cancer and development, the etiology of most of these associations in specific cases remains unknown.
There is a recognized sexual dimorphism in hepatoblastoma, with increased prevalence in males [42, 60], and this study also reflected this tendency. We investigated whether this sex bias could be explained by an increased burden of rare damaging variants in one of the sexes; however, significant differences were not detected, suggesting that most likely environmental factors modulate the sexual dimorphism in HB prevalence.
In addition to SNV/indel variants, germline CNVs were already reported as causally related to cancer [61], but pathogenic CNVs affecting known/candidate CPGs were not detected in this study. One patient carried a gain of an entire Y chromosome, which is associated with an increased risk of learning disabilities, behavioral disturbances, and other clinical signs [62, 63]. Moreover, two 15q11.2 CNVs, considered risk factors for neurodevelopmental disorders [64, 65], were identified in two HB patients with global delay.
Germline mutations have been detected in 8-18% of children and adolescents with cancer [3, 8–10], and the most prevalent CPGs reported to be mutated are TP53, APC, BRCA2, NF1, PMS2, RB1, andRUNX1 [3]. In a very recent work about the spectrum of germline mutations in childhood cancer, the majority of the mutations (55%) were mapped to genes not previously associated with the patient’s tumor type [10]; this work included a small number of HB patients (3 patients) with no pathogenic or likely pathogenic variants. Our findings revealed a high burden of damaging germline variants mapped to CPGs, with only 40% of these patients presenting a family history of cancer. Most of the variants affected autosomal-dominant CPGs not previously linked to HB, since only one mutation was detected in a known gene of HB development (APC ). Interestingly, our data showed a clear predominance of CPGs related to gastrointestinal/colorectal cancer risk, such as familial adenomatous polyposis 1 and 2 (APC andMUTYH ), nonpolyposis colorectal cancer hereditary types 1 (MSH2 ) and 6 (TGFBR2 ), Li-Fraumeni syndrome 2 (CHEK2 ), and Diamond-Blackfan anemia 1 (RPS19 ). Furthermore, the CPGs DROSHA and VHL , which showed P/LP variants, are known to be associated with renal cancer. VHL is a known predisposing gene for renal cell carcinoma, as well as other cancers, such as pheochromocytoma, hemangioblastoma, hypernephroma, pancreatic cancer, paraganglioma, and adenocarcinoma of the ampulla of Vater. Finally, a LoF variant was identified in the DNA repair geneERCC5 , in which homozygous mutations increase the skin cancer risk, and a damaging missense variant was mapped to FAH and causes an autosomal-recessive disorder characterized by progressive liver disease with increased liver cancer risk. Compared to previous pan-cancer studies, although not in HB patients, the findings of germline variants in APC, CHEK2, ERCC5, MSH2, MUTYH, andVHL were also detected in our work [3, 5, 10, 66].
We also observed numerous rare damaging VUS in known or candidate CPGs. Variants mapped to the ATM, BRCA2, COL7A1, DHCR7, DOCK8, FANCD2, FANCM, and GLI3 genes were observed in more than one HB patient. In particular, the same DHCR7 VUS (c.988G>A) was spotted in two patients (P09, born with only one functional kidney, and P26), both diagnosed with low-risk epithelial fetal hepatoblastoma; this gene encodes an enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol [67] and is a candidate for familial breast cancer in BRCA1- and BRCA2 -negative breast cancer families [68], in a recessive mode of action. As in our study, VUS mapped to MET, ATM, SLX4 , and FANCA were also reported in HB patients in a recently published large study [10].
Strong support exists for the hypothesis that monoallelic CPG mutations confer an increased risk of cancer for adult carriers, while biallelic carriers would have a high risk of childhood cancer [69]. More recently, large studies have shown that variants in heterozygosity affecting recessive genes can also increase the predisposition to pediatric cancer [70], and a second hit in the tumor, such as a loss of heterozygosity or an inactivation of the second allele, has been observed in some of these patients (4). A highlight has been given to the role of germline monoallelic variants in cancer predisposition in genes involved in the recognition and repair of DNA damage, such as theATM , PALB2 , and Fanconi anemia genes [71–77]. Since 1971 [78], it was proposed that individuals who were heterozygous for the Fanconi anemia genes might be at increased risk of cancer, and the premise would be that a modest reduction in the DNA repair efficiency could lead to tumor development. In our work, we detected P/LP monoallelic variants in CPGs associated with recessive clinical conditions (FAH, ERCC5, and MUTYH ) and in DNA repair genes (MSH2, CHEK2, ERCC5, and MUTYH ). In addition, we observed a general enrichment of heterozygous germline damaging VUS affecting DNA repair genes related to Fanconi anemia, nucleotide and base excision repair, and homologous recombination repair. Sequencing the tumors of the carriers could contribute to changing the classification of such variants [10] and clarifying the role of these variants in cancer predisposition.
One of the most interesting findings of this study was the disclosure of rare germline damaging variants affecting genes linked to liver differentiation and function, such as FAH , that cause a recessive disorder related to hepatocellular carcinoma risk. Several rare damaging variants were observed in genes of the cytochrome P450 (CYP) family, including one pathogenic heterozygous variant in CYP21A2 (OMIM #201910 ADRENAL HYPERPLASIA, CONGENITAL, DUE TO 21-HYDROXYLASE DEFICIENCY, a recessive condition associated with testicular neoplasia in adults – P22) and two heterozygous pathogenic CYP1B1 variants (OMIM #617315, #231300; recessive conditions) detected in different patients (P03 and P30). Ten rare variants mapped to CYP1A1 (one LoF and nine missense variants), which is associated with primary liver metabolism, were present in eight patients (26%). CYP1A1 encodes a xenobiotic-metabolizing enzyme acting in the placenta [79–81], as well as in several drugs and compounds widely used in pharmacotherapy [82, 83] or present in the diet [84]. CYP1A1 expression is transcriptionally regulated through the AhR receptor [85–87] and various exogenous AhR binders, such as nitrosamines, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and halogenated dioxins, can be found in products from combustion processes, such as chimney soot, grilled food, and cigarette smoke, or as the product of incineration waste [88–91]. Elevated CYP1A1 activity through AhR activation in the placentas of female smokers has been associated with pregnancy complications, including low birth weight [92], a known risk factor for HB. In our previous study (14), in which we investigated the mutational burden of hepatoblastoma, a mutational signature similar to the COSMIC 29 signature was detected, which was initially observed only in oral gingivo-buccal squamous cell carcinoma, which develops in individuals with the habit of chewing tobacco. Currently, this mutational signature has also been detected in patients with lung, thyroid, bladder, biliary, breast, pancreas, liver, and kidney cancer and can be linked to nitrosamine exposure [93]. Therefore, we can speculate that a defective response during pregnancy to xenobiotic exposure, such as nitrosamines, could be linked toCYP1A1- damaging germline variants, increasing the risk for HB development and resulting in the mutational signature we previously found in HB.
In conclusion, our major findings in HB patients were the detection of germline variants in CPGs associated with gastrointestinal/colorectal and renal cancer risk, not always linked to a familial history of cancer; the enrichment of monoallelic variants in CPGs and DNA repair genes; and a high frequency of birth defects. Most studies of pediatric tumors are from North America, Europe, and Asia, and our study on HB is pioneering in South America and contributes to elucidating the genetic architecture of HB risk. Further validation of the genes highlighted as HB germline risk factors in other cohorts can provide new insights regarding HB development.