WES and validation by Sanger sequencing
WES and validation by Sanger sequencing in PNGS-252 revealed an apparent homozygous c.4C>G missense alteration (GenBank: NM_014176.3), resulting in the amino Brilliant Blue G substitution p.Gln2Glu (Figure 1A). This mutation must be very rare, because this is not listed in the NHLBI Exome Sequencing Project or the Human Genetic Variation Browser databases. The glutamine residue (Gln2) is highly conserved in the homologs found from vertebrates to worms excluding plants (Figure 1E) and the mutation is rated as “damaging” by both SIFT and PolyPhen predictions. The Gln2 is located in the N-terminal helix of UBE2T, which constitutes part of the hydrophobic E3-E2 interaction surface, near the conserved E2 UBC fold (Figures 1C and 1F). Copy-number analysis using WES data suggested that there was a heterozygous deletion across the UBE2T locus in the PNGS-252 sample (data not shown). Indeed, our targeted array comparative genome hybridization (array CGH) revealed an area of reduced hybridization signal encompassing almost the entire UBE2T (Figure 1A). The deletion junction carried 3 bp of microhomology (Figures S2A–S2D), suggesting that the junction arose from microhomology-mediated repair. This person’s father carried the genomic deletion, and the mother had the heterozygous c.4C>G mutation (Figure S3). There was no family history of malformations, hematological abnormalities, or cancer predisposition. In the individual PNGS-255, WES revealed the c.4C>G mutation as well as a splice donor site mutation (c.180+5G>A) (Figures 1B and 1C). Both alterations were heterozygous and on different chromosomes (Figure S4). Thus, this individual was compound heterozygous for the UBE2T mutations. In bone marrow fibroblasts, we found a small fraction of UBE2T transcripts with skipped exon 2, resulting in a frameshift and premature stop codon (p.Gln37Argfs∗47) (Figure S5). Family members of this person were not available for further evaluation. However, the results of SNP array analysis using the HumanOMni5 v.1.0 array (Illumina) suggested that a haplotype containing the c.4C>G mutation was shared by PNGS-252, her mother, and PNGS-255 (not shown). Thus, they might have a common ancestral origin. We extended WES to AP65P FA fibroblasts provided by the JCRB Cell Bank and found the same UBE2T c.4C>G mutation. Moreover, 99.9% of the SNPs listed in dbSNP131 and identified in AP65P were identical to those in PNGS-252 (2,244 out of 2,247), demonstrating that AP65P was derived from PNGS-252 (Table 1). The AP65P individual has been reported as carrying no mutations in FANCA, FANCG, and FANCC. We transformed the cells with human TERT (hTERT) and termed them AP65P-hTERT. Unfortunately, we were unable to immortalize bone marrow fibroblasts from PNGS-255. Interestingly, AP65P-hTERT cells displayed roughly similar protein levels of UBE2T as normal control cells (48BR), indicating that the p.Gln2Glu substitution does not significantly destabilize UBE2T protein (Figure 2A). We also detected the auto-monoubiquitinated form of UBE2T as previously described,7, 8 suggesting that the mutant protein is able to receive activated ubiquitin from the E1 enzyme (Figure 2A). However, only faint amounts of long-form ID proteins were observed, even after MMC stimulation (Figure 2A). As expected, AP65P-hTERT cells transduced with lentivirus encoding normal UBE2T, but not with the mutant, clearly restored the MMC-induced long form of FANCD2 (Figure 2A) as well as FANCD2 foci formation (Figure 2B). Furthermore, both the increased levels of MMC-induced chromosome breakage (Figure 2C) and the MMC sensitivity (Figure 3A) in AP65P-hTERT cells were suppressed by exogenous wild-type UBE2T but not with UBE2T carrying p.Gln2Glu. Taken together, these results firmly established that the FA phenotype in these individuals is caused by the UBE2T mutations. How, exactly, does the UBE2T alteration affect the activity of the protein in promoting monoubiquitination of the ID complex? We hypothesized that the p.Gln2Glu substitution might disrupt the FANCL-UBE2T interaction. Indeed, the p.Gln2Glu alteration drastically reduced the signal intensity in a mammalian two-hybrid assay (Figure 4A). This was confirmed by a GST pull-down experiment using purified recombinant human or chicken GST-FANCL and wild-type or mutant UBE2T proteins (Figure 4B, 4C, and S6A). In an in vitro monoubiquitination assay, the mutated UBE2T protein displayed ∼3-fold less efficiency in promoting FANCD2 monoubiquitination in the presence (Figure 4D) or absence (Figure S6B) of stimulator DNA, whereas auto-ubiquitination was normal compared to control proteins (Figure S6C). The p.Gln2Glu substitution abrogated FANCL monoubiquitination in vitro (Figures 4D, S6B, and S6D); however, the FANCL-independent FANCI monoubiquitination was not affected (Figure S6D). These results are well explained by the specific disruption of the FANCL-UBE2T interaction by the p.Gln2Glu substitution.