A protein prioritization approach tailored for the FA/BRCA pathway.

Fanconi anemia (FA) is a heterogeneous recessive disorder associated with a markedly elevated risk to develop cancer. To date sixteen FA genes have been identified, three of which predispose heterozygous mutation carriers to breast cancer. The FA proteins work together in a genome maintenance pathway, the so-called FA/BRCA pathway which is important during the S phase of the cell cycle. Since not all FA patients can be linked to (one of) the sixteen known complementation groups, new FA genes remain to be identified. In addition the complex FA network remains to be further unravelled. One of the FA genes, FANCI, has been identified via a combination of bioinformatic techniques exploiting FA protein properties and genetic linkage. The aim of this study was to develop a prioritization approach for proteins of the entire human proteome that potentially interact with the FA/BRCA pathway or are novel candidate FA genes. To this end, we combined the original bioinformatics approach based on the properties of the first thirteen FA proteins identified with publicly available tools for protein-protein interactions, literature mining (Nermal) and a protein function prediction tool (FuncNet). Importantly, the three newest FA proteins FANCO/RAD51C, FANCP/SLX4, and XRCC2 displayed scores in the range of the already known FA proteins. Likewise, a prime candidate FA gene based on next generation sequencing and having a very low score was subsequently disproven by functional studies for the FA phenotype. Furthermore, the approach strongly enriches for GO terms such as DNA repair, response to DNA damage stimulus, and cell cycle-regulated genes. Additionally, overlaying the top 150 with a haploinsufficiency probability score, renders the approach more tailored for identifying breast cancer related genes. This approach may be useful for prioritization of putative novel FA or breast cancer genes from next generation sequencing efforts

Identification of the Fanconi Anemia Complementation Group I Gene, FANCI

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592

The Fanconi anemia pathway.

<p>For further explanation see main text (newest FA member XRCC2 not shown).</p

Overview of the main intrinsic protein properties of sixteen FA proteins.

<p>NLS score: Nuclear Localization Signal score. pI: iso-electric point. Cellular localization: N = Nucleus; N/C = Nucleus and Cytoplasm. ^*These percentages have been calculated using RefSeq sequences as the orthologs assignment in EnsEMBL were incorrect. FANCE: EnsEMBL’s ortholog mouse protein incorrect; FANCE: NP_068741 (human: 100% identical with ENSP00000229769) and NP_001157291 (mouse). FANCF: no mouse ortholog available in EnsEMBL. FANCF: NP_073562.1 (human: 100% identical with ENSP00000330875) and NP_001108559.1 (mouse).</p

Top 150 genes from the combined ranking.

<p>Top 150 genes from the combined ranking.</p

Overview genes involved in “Cell cycle process”, Ranked vs Nermal.

<p>Analysis of the overlap between our combined approach and Nermal for the GO biological process term “Cell cycle process” (GO:0022402). Top 150 of either our combined ranking or Nermal alone were analyzed for GO term “DNA repair” (GO:0006281) and these genes were discarded. The remaining lists (Ranking combination scheme: 89 genes and Nermal: 88 genes) were further compared. In total, 7 genes were found in common (<i>BACH1</i>, <i>NFE2L3</i>, <i>DDX11</i>, <i>CHEK2</i>, <i>MAPT</i>, <i>BUB1B</i>, <i>UBASH3A</i>). A combined list of the remaining genes (Ranking: 81 genes and Nermal: 82 genes; total 163 genes) was analyzed with the Genomatix Pathway System (GePS). The biological process term “Cell cycle process” was the most enriched (P-value: 4.83E−25). The relation between the different cell cycle proteins is depicted (red: candidates combined ranking scheme, blue: candidates Nermal).</p

GO enrichment top 150 of Ranked, Nermal, and FuncNet.

<p>Number of genes observed with the GO terms “response to DNA damage stimulus” (GO:0006974) and “DNA repair” (GO:0006281) in the top 150 of our combined ranking approach “Ranked”, the literature mining tool “Nermal”, and the protein function prediction tool “FuncNet”. Number of genes observed, number of genes expected (P-value) for GO term “response to DNA damage stimulus” (GO:0006974): Ranked 71, 5 (5.50E−64); Nermal 65, 5 (7.10E−56); FuncNet 53, 5 (4.27E−42). GO term “DNA repair” (GO:0006281): Ranked 61, 4 (1.28E−60); Nermal 62, 3 (1.24E−62); FuncNet 47, 3 (1.54E−42).</p

Scoring scheme.

*<p>The FuncNet and Nermal scores were integrated and a new Fisher score was calculated that includes all FuncNet P-values as well as a P-value for Nermal. The “−1000” score is merely to clearly separate entries with Nermal/FuncNet scoring from those without. The maximum score possible with this scheme is 8.5.</p