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Family Sequencing Study Describes Dominant Negative Form of Fanconi Anemia

NEW YORK (GenomeWeb) – A new Nature Communications study suggests dominant negative mutations in the DNA repair gene RAD51 can cause a distinct Fanconi anemia subtype.

An international team led by investigators at the VU University Medical Center and the Institute for Systems Biology did whole-genome sequencing and exome sequencing on an adult male who displayed some, but not all, Fanconi anemia symptoms and lacked the mutations typically used to diagnose the disease. The man's unaffected parents and sister had their genomes sequenced as well.

The researchers detected a dominant negative mutation in one copy of RAD51 that was absent in the patient's family members. Based on these findings and follow-up cell line and protein experiments, they concluded dominant negative RAD51 mutations may contribute to a subtype of Fanconi anemia dubbed FA-R — results that highlight a neurodevelopmental role for RAD51.

"Different types of mutations in human RAD51 are … associated with different clinical phenotypes," the authors wrote in their paper. "A dominant-negative mutation gives rise to [a Fanconi anemia]-like phenotype, whereas haploinsufficiency of RAD51 has been associated with congenital mirror movement disorder."

Earlier this year, researchers reporting in Molecular Cell characterized a dominant negative alteration affecting another residue of RAD51 in a one-year-old girl with Fanconi anemia-like clinical features but no mutations involving known Fanconi anemia genes.

Fanconi anemia is typically considered a recessively inherited condition, which can be caused by alterations affecting both copies of 17 different genes. In general, the disease causes both developmental and cancer predisposition symptoms, along with blood problems and bone marrow failure.

For the current study, the researchers focused on a 23-year-old man who displayed symptoms such as microcephaly, learning disability, developmental abnormalities, and delayed growth starting when he was around two and a half years old.

Though these symptoms seemed to fit with Fanconi anemia, they explained, the individual has not experienced bone marrow failure, acute myeloid leukemia, head and neck cancer, or any of the other cancers that tend to strike Fanconi anemia sufferers during childhood or adolescence.

To investigate the case further, the team got Complete Genomics to do whole-genome sequencing on the man, his unaffected sister, and their parents using DNA from each individual's lymphoblastoid cell lines.

The researchers also sequenced the affected man's exome using the Illumina GAIIx to assess protein-coding portions of his genome captured with the SureSelect Human All Exon kit.

In an initial search for recessive alterations in the patient's genome, the investigators narrowed in on suspicious changes to PRR12. But with help from a protein interactions-based prioritization method, they ultimately ruled these out, instead turning their attention to de novo mutations that might contribute to the patient's symptoms.

Just one such alteration remained after Sanger validation and prioritization steps: a de novo missense mutation affecting one copy RAD51, a gene coding for a protein that interacts with other Fanconi anemia-related gene products.

Similar to samples from individuals with typical forms of Fanconi anemia, blood cells and immortalized fibroblast cells from the affected male were particularly sensitive to DNA cross-linking compounds. Nevertheless, the team's gene expression and mass spectrometry experiments indicated that his cells contain both typical and mutated versions of RAD51's protein product.

Based on their fibroblast cell line experiments and protein binding assays, meanwhile, the researchers concluded that the mutated RAD51 protein likely exerts a dominant negative effect that alters its protein interactions, DNA binding ability, and enzymatic activity, leading to impaired DNA repair.

"[O]ur data emphasize that dominant mutations can be responsible for diseases usually thought of as (autosomal) recessive," the authors concluded, "and that genome-wide sequence data of family members combined with mechanistic studies may be necessary to pinpoint the responsible gene."