Germline variants
Chromosome microarray analysis excluded the presence of pathogenic germline CNVs in both patients. The Patient 2 carries an interrupted duplication encompassing a total segment of ~364 kb at 14q23.2, not inherited from his father (arr[GRCh37] 14q23.2(64374657_64435014)x3; arr[GRCh37] 14q23.2(64474837_64738458)x3; Figure 1); maternal material was not available for segregation analysis. This CNV partially duplicates the genomic sequence of two Morbid OMIM genes (SYNE2 #608442 andESR2 #601663) and was classified as VUS. Rare duplications overlapping this region were reported in the Database of Genomic Variants (DGV - http://dgv.tcag.ca)
In Patient 1, quality controls of the exome data revealed 97% of 10x median coverage on target, with 98.03% of Q>30; in Patient 2, 98% of 10x median coverage on target, with 71.28% of Q>30. It was identified six regions of homozygosity in Patient 2 using exome data (Supporting Information Table S1 and Supporting Information Figure S2), result in accordance with the reported parental consanguinity.
An active evaluation of a set of known genes for hepatoblastoma and Hirschsprung disease (Supporting Information Table S2) was performed by visual analysis of the BAM files to confirm that the exonic sequences of these genes were properly covered. Considering the data of both patients, a total of 333 rare coding non-synonymous variants were detected, mapping to 317 genes.
No homozygous variant was detected in Patient 1 after filtering. A total of 72 rare heterozygous variants fulfilled the analysis criteria (seeMethodology ), 24 of them were inherited from her mother, also heterozygous for these variants. Eleven out of the 72 variants were LoF affecting the genes C5orf47, CWC22, FRMPD2, GGCT, HAO2, KIR2DL4,LHX8, P2RX6, SPAG8, TMPRSS7 , and ZNF215. Particularly, missense variants in the cancer predisposition genes ERCC2(c.545C>T rs142936491) and HRAS(c.75G>A rs142218590) were identified, and one variant related to Hirschsprung disease in the ALDH1A2(c.1100A>T) gene.
After filtering, 261 rare variants were prioritized for further analysis in Patient 2, 243 of them in heterozygosity, comprising thirteen LoF affecting the genes APOB/PLCD4, CEP57L1, CNBD2, DENND6A, ELMOD3, FRG1B, GCA, FAM3A, LSM14A, PCDHGA1, RFPL1, SBK2, and SLC19A1;and 18 variants in homozygosity, including one LoF in the geneFAM3A. Fifty-six variants were also present in heterozygosity in the father. Variants were detected in the cancer-predisposing genesAPC (c.3895G>C - rs1801166), MCC(c.862G>T - rs34696815), ODC1 (rs116522452 - c.568G>A), BRCA1 (c.4039A>G - rs28897689), and ERBB2 (c.170A>G - rs140441229). Eight variants were mapped in genes related to cataract (ABCA4, COL4A4, HGSNAT, PCDH15, RPGRIP1, SLC16A12, SON, and VAT1 ), another clinical condition of the Patient 2. Details of the missense variants classified as damaging for at least five algorithms and the detected LoF variants are shown in Supporting Information Table S3 (57 for Patient 1 and 62 for Patient 2).
The list of 317 genes with rare coding non-synonymous variants from both patients was submitted to the prioritization tool VarElect33 using the phenotypes hepatoblastoma and congenital megacolon. This analysis revealed one gene directly related to Hirschsprung disease (ALDH1A2 ), and seventeen genes directly or indirectly (related with liver cancer, liver functions/structure, cancer, or syndromes that predispose to hepatoblastoma development) associated with hepatoblastoma (APC, BRCA1, CEP164, CYP1A1, ERBB2, ERCC2, FASN, HGS, HRAS, KMT2D, ODC1, PLCD4, SERPINA6, SLC25A47, SLC6A6, MCC, and ZNF215 ). Table 1 presents the germline VUS associated with the specific phenotypes of each patient.
Different CYP1A1 variants were identified in Patients 1 (c.1390C>A) and 2 (c.877C>G), both classified as VUS; additionally, one variant was detected in the Patient 2 affecting CEP164 (c.1429C>T), somatic mutations in this gene were previously reported in HB samples14. The variants in the CYP1A1 and CEP164 genes were validated by Sanger sequencing (Figure 2).
To explore the pathways in which the genes with rare germline coding non-synonymous variants are involved and their biological roles, we used Reactome Pathway Database (https://reactome.org/ - Version 71 Released). The gene set 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.