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.