TY - JOUR
T1 - A syngeneic variance library for functional annotation of human variation
T2 - Application to BRCA2
AU - Hucl, Tomas
AU - Rago, Carlo
AU - Gallmeier, Eike
AU - Brody, Jonathan R.
AU - Gorospe, Myriam
AU - Kern, Scott E.
PY - 2008/7/1
Y1 - 2008/7/1
N2 - The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Δexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2Δex11/Dex11 and BRCA2Δex11/Y3308X were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2 Δex11/Y3308Y, BRCA2Δex11/P3292L, and BRCA2Δex11/P3280H had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2 Δex11/S3291E) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2 Δex11/S3291A) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.
AB - The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Δexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2Δex11/Dex11 and BRCA2Δex11/Y3308X were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2 Δex11/Y3308Y, BRCA2Δex11/P3292L, and BRCA2Δex11/P3280H had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2 Δex11/S3291E) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2 Δex11/S3291A) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.
UR - http://www.scopus.com/inward/record.url?scp=48549084778&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=48549084778&partnerID=8YFLogxK
U2 - 10.1158/0008-5472.CAN-07-6189
DO - 10.1158/0008-5472.CAN-07-6189
M3 - Article
C2 - 18593900
AN - SCOPUS:48549084778
SN - 0008-5472
VL - 68
SP - 5023
EP - 5030
JO - Cancer Research
JF - Cancer Research
IS - 13
ER -