The researchers identified FAAP95 by analysing components of the FA 'core complex', which is responsible for the monoubiquitylation of a key protein involved in the repair of DNA crosslinks, FANCD2. They found that the depletion of FAAP95 reduced the amount of monoubiquitylated FANCD2 produced, prompting the team to examine whether the gene encoding FAAP95 might be one of those responsible for FA. Having obtained the amino-acid sequence of FAAP95, a database search showed that the gene encoding it is localized on the X chromosome. The researchers then looked for a FA complementation group in which all affected individuals were male and had defective monoubiquitylation. Of the 11 identified complementation groups, only one fulfilled these criteria: FA-B. When the researchers sequenced the region encoding FAAP95 in FA-B individuals, they found frameshift mutations that cause premature stop codons. They also transfected FA-B lymphoblasts with cDNA encoding wild-type FAAP95, and found that monoubiquitylation and other phenotypes were restored to normal, confirming that FAAP95 was defective in FA-B individuals. The gene was therefore named
FANCB
.
X-linked inheritance of FA has not previously been reported, and this prompted the researchers to test mothers and grandmothers of FA-B individuals to see if they carried mutant FANCB. Surprisingly, although female carriers were identified, some of the patients' mothers did not seem to carry the mutations. By examining the methylation state of FANCB in these kindreds, the researchers found that mutant FANCB in fact tended to be subject to X-inactivation — whereby most genes on one of the two X-chromosomes are transcriptionally silenced during early embryogenesis. The authors speculate that during development, healthy cells, in which mutant FANCB is inactive, out-compete cells expressing mutant copies of the gene, resulting in carriers with normal FANCB in most of their tissues.
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