_:b595106023 . _:b595105989 "2"^^ . _:b47157718 "We furthermore confirmed the RAD51 variant as the top candidate with two independent variant filtering workflows: ingenuity variant analysis and the family genomics workflow>>12<<." . . _:b47157729 . . _:b595106022 . _:b47157765 . _:b595105988 "2"^^ . _:b47157730 . _:b47157723 . . _:b595106017 . . _:b595105991 "2"^^ . _:b47157731 . _:b595106016 . _:b47157737 . . _:b595105990 "2"^^ . _:b47157740 . _:b595106019 . _:b595105985 "2"^^ . . _:b47157741 . _:b595106018 . _:b595105984 "2"^^ . _:b47157742 . . . _:b595106029 . _:b47157738 _:b47157740 . _:b595105987 "2"^^ . _:b595105884 . _:b595105867 . _:b47157743 . _:b595105885 . _:b47157738 _:b47157741 . _:b47157738 _:b47157742 . _:b595105965 . _:b595105886 . _:b47157738 _:b47157743 . _:b595106028 . _:b595105887 . _:b595105880 . _:b595105986 "2"^^ . _:b595105881 . _:b47157736 . _:b595105882 . _:b595106032 . _:b47157738 _:b47157739 . _:b595106031 . _:b595105883 . _:b595105876 . _:b595105997 "2"^^ . _:b595105877 . _:b47157737 . _:b595105878 . _:b595105997 . . _:b595106030 . _:b595105879 . _:b595105872 . _:b595105996 "2"^^ . _:b595105873 . _:b595105874 . _:b47157717 . _:b47157738 . _:b595105875 . _:b595106025 . _:b595105962 . _:b595105876 . _:b595105999 "2"^^ . _:b595105868 . _:b47157738 _:b47157756 . _:b47157738 _:b47157757 . _:b47157739 . _:b595105869 . _:b47157738 _:b47157758 . _:b595105870 . . _:b47157750 "The parents' genomes were compared using GRAB>>7<<. GRAB predicted \u2018no relationship', indicating that the parents are not likely to have a relationship closer than 9th degree." . _:b595106046 . _:b595106024 . _:b595105871 . _:b47157738 _:b47157752 . _:b595105998 "2"^^ . _:b595105864 . _:b47157738 _:b47157753 . _:b47157748 . _:b595105969 . _:b595105932 . _:b595105865 . . _:b47157738 _:b47157754 . _:b595105866 . _:b47157738 _:b47157755 . _:b595106027 . _:b595105867 . _:b47157738 _:b47157748 . _:b595105993 "2"^^ . _:b595105860 . . _:b47157738 _:b47157749 . _:b47157749 . _:b595105912 . _:b595105861 . _:b595105896 . _:b595105862 . _:b47157738 _:b47157750 . _:b595105880 . _:b595106026 . _:b595105863 . _:b47157738 _:b47157751 . _:b47157738 _:b47157744 . _:b595105992 "2"^^ . . _:b47157750 . _:b47157730 . _:b595105857 . _:b47157738 _:b47157745 . _:b47157738 _:b47157746 . _:b595105858 . _:b47157738 _:b47157747 . _:b595106037 . _:b595105859 . _:b595105916 . _:b595105995 "2"^^ . _:b595105917 . _:b47157751 . _:b595105918 . . _:b595106036 . _:b595105919 . _:b595105912 . _:b595105994 "2"^^ . _:b595105913 . _:b47157744 . _:b595105906 . _:b595105914 . _:b595105915 . _:b595106039 . _:b595105908 . _:b595106005 "2"^^ . _:b595105909 . _:b47157745 . _:b595105910 . _:b595105911 . _:b595106038 . _:b595105904 . _:b595106004 "2"^^ . _:b595105905 . _:b47157746 . _:b595105906 . . _:b595106033 . _:b595105907 . _:b595105900 . _:b595106007 "2"^^ . _:b595105901 . _:b47157747 . _:b595105870 . _:b595105902 . . _:b595105903 . _:b595106058 . _:b595106032 . _:b595105896 . _:b595106006 "2"^^ . _:b595105897 . _:b47157756 . . _:b595105898 . . _:b595105899 . _:b595106035 . _:b595105892 . _:b595106001 "2"^^ . _:b595105893 . _:b47157757 . . _:b595105894 . . _:b47157745 . _:b595106034 . _:b595105895 . _:b595105888 . _:b595106000 "2"^^ . _:b595105889 . _:b47157758 . _:b595105982 . _:b595105890 . _:b595105891 . _:b595106045 . _:b595106003 "2"^^ . _:b47157759 . _:b595106044 . _:b595106002 "2"^^ . _:b47157752 . _:b595106047 . _:b595106013 "2"^^ . _:b47157753 . _:b595106046 . _:b595106012 "2"^^ . . _:b47157754 . . _:b595105868 . _:b595106041 . . _:b595106015 "2"^^ . _:b47157755 . . _:b595106040 . . _:b595106014 "2"^^ . _:b595105951 . . _:b595105911 . _:b595106074 . _:b595106043 . _:b595105935 . _:b595106009 "2"^^ . . _:b595106042 . _:b595106008 "2"^^ . . _:b595106017 . _:b595105938 . _:b595105999 . _:b595106011 "2"^^ . _:b47157741 "(5) Variants were annotated using ANNOVAR>>37<< using the NCBI RefSeq Release 60 and Ensembl Release 74 genome annotations." . . _:b595105947 . _:b595106010 "2"^^ . . . _:b595106021 "2"^^ . . _:b595106020 "2"^^ . _:b595105993 . _:b47157750 . _:b595106023 "2"^^ . _:b595105905 . _:b595105930 . _:b595106022 "2"^^ . . _:b595106017 "2"^^ . . _:b47157751 "Subsequently, cells were dropped onto glass slides and stained with 5% Giemsa (Merck)>>46<<. Per culture, 100 metaphases were scored from at least two coded slides (blind)." . _:b595105923 . . _:b595106016 "2"^^ . . . _:b595106012 . _:b595106019 "2"^^ . _:b595106013 . . _:b595106014 . _:b595106015 . _:b595106008 . _:b595106018 "2"^^ . _:b595106009 . _:b595106010 . _:b595106011 . _:b595106004 . _:b595106029 "2"^^ . _:b595106005 . _:b595106006 . _:b595106007 . . _:b595106028 "2"^^ . _:b595106000 . _:b595106001 . _:b595106075 . . _:b595106002 . _:b595106003 . _:b595105996 . _:b595106031 "2"^^ . _:b595105997 . _:b595105998 . _:b595105999 . _:b595105941 . _:b47157709 . _:b595105992 . _:b595106030 "2"^^ . _:b595105993 . _:b47157761 . _:b595105994 . _:b595105995 . . _:b595105988 . _:b595106025 "2"^^ . . _:b595105989 . _:b595105990 . . _:b595106022 . _:b595105991 . _:b595105984 . _:b595106024 "2"^^ . _:b595105985 . _:b595105986 . _:b47157711 "Relationship estimation>>7<< based on the parents' genomes confirmed paternity and excluded consanguinity." . . _:b595105987 . _:b595106044 . _:b595106027 "2"^^ . _:b595106045 . _:b595105990 . _:b595106046 . _:b595106047 . _:b595106040 . _:b47157725 . _:b595106026 "2"^^ . . _:b595106041 . _:b595106068 . _:b595106042 . _:b595106043 . _:b595106036 . _:b595106037 "2"^^ . _:b595106037 . _:b595106038 . _:b595106039 . _:b595106032 . _:b595106036 "2"^^ . _:b595106033 . _:b595106034 . _:b47157759 "discussion" . _:b595106035 . _:b595106028 . _:b595106039 "2"^^ . _:b595106029 . . _:b595106030 . _:b595105984 . . _:b595106031 . . _:b595106024 . _:b595106038 "2"^^ . . _:b595106033 . _:b595106025 . _:b595106026 . _:b595106027 . _:b595105922 . _:b595106020 . _:b595106033 "2"^^ . _:b47157738 . _:b595106021 . _:b595105936 . _:b595106022 . _:b595106023 . _:b595106001 . _:b595106016 . _:b595106032 "2"^^ . _:b595105958 . _:b595106017 . _:b47157720 . _:b595106018 . _:b595106019 . _:b595105948 . _:b595106035 "2"^^ . _:b595105949 . _:b595105950 . _:b595105951 . _:b595105883 . _:b595105944 . _:b595106034 "2"^^ . _:b595105948 . _:b595105945 . _:b47157744 . _:b47157759 . _:b595105946 . . _:b595105947 . _:b595105940 . _:b595106045 "2"^^ . _:b595105941 . _:b595105942 . _:b595105943 . _:b595105936 . _:b47157716 "10) and has a high score (ranking 65, scoring 6.377) with the FA-ranking tool>>8<<. RAD51 is a member of the RAD51 family of related genes, of which RAD51C had already been recognized as an FA gene11." . _:b595106044 "2"^^ . _:b595105937 . . _:b595105938 . _:b595105939 . _:b47157710 "To identify the disease-causing mutation(s), we performed whole-genome sequencing (WGS)>>6<< on DNA from the proband, his parents and his unaffected sister. Relationship estimation7 based on the parents' genomes confirmed paternity and excluded consanguinity." . _:b595105932 . _:b595106047 "2"^^ . _:b595105933 . _:b595105987 . . _:b595105934 . _:b595105935 . _:b595105928 . _:b595106046 "2"^^ . _:b595105921 . _:b595105929 . . _:b595105930 . _:b595105931 . _:b595105924 . _:b595106041 "2"^^ . _:b595105925 . . . _:b595105926 . _:b595105927 . _:b595105920 . _:b47157764 . _:b595106040 "2"^^ . _:b595105921 . _:b595105922 . _:b595105923 . _:b595105980 . _:b595106043 "2"^^ . _:b595105981 . . _:b595105982 . _:b595105983 . _:b595105976 . _:b595106042 "2"^^ . _:b595105977 . _:b595105978 . _:b595105979 . _:b595105972 . _:b595106053 "2"^^ . . _:b595105973 . _:b595105974 . _:b595105975 . . _:b595105968 . _:b595106052 "2"^^ . _:b595105969 . _:b595105970 . . _:b595105971 . _:b595105964 . _:b595106055 "2"^^ . _:b595106035 . _:b595105954 . _:b595105965 . _:b595105966 . _:b595105967 . . _:b595106054 "2"^^ . _:b595105960 . . _:b595105961 . . _:b47157745 "The remaining variants were then assessed using SIFT38, Polyphen39, MutationTaster40 and CADD>>41<< for pathogenicity." . . _:b595105962 . _:b595105963 . . _:b595105956 . _:b595106049 "2"^^ . _:b595105957 . _:b595105958 . . _:b595105959 . _:b595105952 . _:b595106048 "2"^^ . _:b595105953 . _:b595105954 . . _:b595105955 . _:b595106051 "2"^^ . _:b47157747 "Subsequently, SNVs and small indels were called using SAMtools>>43<< and VarScan44. The resulting list of variants was annotated with ANNOVAR." . . _:b47157762 . _:b595106050 "2"^^ . _:b595105949 . . _:b595106061 "2"^^ . _:b47157755 "Reactions were performed in a final volume of 10\u2009\u03BCl containing 5\u2032-end-labelled (Alexa Fluor 488) 90-nt ssDNA (SK3 oligonucleotide>>48<<, 5\u2032-AATTCTCATTTTACTTACCGGACGCTATTAGCAGTGGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGT-3\u2032) at 3.6\u2009\u03BCM (nucleotides), 1.2\u2009\u03BCM RAD51 (wild type or A293T mutant), 50\u2009mM Tris-HCl (pH 7.5), 1\u2009mM dithiothreitol (DTT), 0.1\u2009mg\u2009ml\u22121" . _:b595106060 "2"^^ . _:b47157728 . _:b595105919 . _:b595106063 "2"^^ . _:b47157747 . _:b595105967 . _:b47157744 "The remaining variants were then assessed using SIFT38, Polyphen39, MutationTaster>>40<< and CADD41 for pathogenicity." . _:b595106059 . _:b595106062 "2"^^ . _:b595106067 . . . . _:b595106057 "2"^^ . _:b595105892 . _:b595105901 . _:b595106056 "2"^^ . _:b595106059 "2"^^ . _:b47157768 "Intriguingly, FA cells are also exquisitely sensitive towards acetaldehyde, a metabolite of ethanol linked to fetal alcohol syndrome, which is an important cause of learning disability>>3435<<." . . _:b595106058 "2"^^ . _:b595106069 "2"^^ . . _:b595106068 "2"^^ . _:b47157740 . _:b595105944 . _:b595106062 . _:b595105908 . _:b595106071 "2"^^ . . _:b47157733 "Ala293Thr mutation is located in a highly conserved region of RAD51, which is involved in RAD51 monomer\u2013monomer interactions>>2425<< (Fig. 5). The mutated aa is directly adjacent to the highly conserved so-called L2 loop, which is involved in DNA binding26 (Fig. 5a). The protein\u2013interaction interface displays different conformations associated with ATP- or ADP-binding" . . _:b595106070 "2"^^ . _:b47157719 . _:b595105862 . . . _:b47157710 . . _:b595106065 "2"^^ . _:b595105877 . _:b595106064 "2"^^ . . _:b595105998 . _:b595106076 . _:b595106067 "2"^^ . _:b595106077 . _:b595105920 . _:b595106078 . . _:b595105943 . . _:b595106066 "2"^^ . _:b595106072 . _:b595106073 . _:b595106074 . _:b595106075 . _:b47157743 "The remaining variants were then assessed using SIFT38, Polyphen>>39<<, MutationTaster40 and CADD41 for pathogenicity." . _:b595106068 . _:b595106077 "2"^^ . _:b595106043 . _:b595106069 . _:b47157764 "In early reviews, a link between FA and mental retardation/central nervous system has been established, albeit at a low frequency>>31<<3233. Also the two cases with biallelic BRCA1 mutations were found to be associated with developmental delay, while for one of the patients, mental retardation was mentioned explicitly. Thus, it could be hypothesized that absence of" . _:b595106070 . _:b595106071 . _:b47157729 "Binding of RAD51 to dsDNA changes the helical pitch such that the DNA molecule extends to 1.5-fold its B-form length>>22<<23. If filament formation is not complete, DNA will be less extended and the filaments shorter. The wild-type RAD51 nucleoprotein filaments on the 3.9-kb DNA were 2,025\u00B167\u2009nm long, while filaments formed on the same-length DNA with" . _:b595106064 . _:b595106076 "2"^^ . _:b595106065 . _:b595106066 . _:b595106067 . _:b595105970 . _:b595105890 . _:b595106060 . _:b595106061 . _:b595106062 . _:b595106063 . _:b595106056 . _:b595106078 "2"^^ . _:b595106057 . _:b595106058 . _:b595106059 . _:b595105983 . _:b595106052 . _:b595106073 "2"^^ . _:b595106053 . _:b595106013 . _:b595106054 . "10.1038%2Fncomms9829" . _:b595106055 . _:b595106048 . _:b47157748 . _:b595106072 "2"^^ . . _:b595106049 . _:b595106050 . . _:b595106051 . _:b595106075 "2"^^ . . _:b595106074 "2"^^ . . _:b595105898 . _:b595105976 . . _:b47157766 . _:b595105915 . . . _:b595106030 . _:b47157758 "The analysis of nucleoprotein filaments was done using SFMetrics>>51<<. The contours of filaments were traced manually." . _:b47157762 "Intriguingly also the recently described cases of biallelic carriers of BRCA1 mutations>>4<<30, had reached adulthood without bone marrow failure." . _:b47157712 "We tested this candidate gene with a prioritization approach tailored for the FA/BRCA pathway>>8<<. This tool considers the entire proteome exploiting the basic FA protein properties, protein\u2013protein interactions (Molecular Interactions workflow of EnVision 2), protein function prediction (FuncNet) and literature mining (Nermal). With" . _:b595105952 . _:b47157736 . _:b595105979 . _:b595106007 . _:b47157720 "wild-type and mutant RAD51 proteins was in the same range, excluding the possibility that the observed negative effects on cell growth could be attributed to higher expression levels of the mutant protein (Fig. 3e; Supplementary Fig. 4)>>14<<. Furthermore, the data support the notion that the mutant protein is not destabilized by the mutation (see Fig." . _:b595106038 . _:b47157727 . . _:b595105909 . . _:b595105893 . _:b47157726 . . _:b595106054 . _:b595106039 . _:b47157764 . . _:b47157765 . . . . _:b47157766 . _:b595106053 . _:b47157767 . . _:b595106052 . . _:b47157760 . . _:b595106055 . _:b47157749 . _:b47157761 . _:b595105937 . _:b595106054 . _:b47157709 "results" . _:b47157762 . _:b595106049 . _:b47157763 . _:b595106048 . . _:b595105895 . _:b595106051 . . _:b595106050 . _:b595106061 . . . _:b595105953 . _:b595106060 . . _:b47157768 . _:b47157742 "The remaining variants were then assessed using SIFT>>38<<, Polyphen39, MutationTaster40 and CADD41 for pathogenicity." . . _:b595106050 . _:b595106063 . _:b47157739 . _:b595106062 . . _:b595105871 . _:b595106057 . _:b595105910 . _:b595106056 . _:b595106059 . . _:b595106058 . _:b595106026 . . . _:b595106069 . . _:b595106068 . . _:b47157753 "The expression construct for untagged RAD51A293T was generated by the QuikChange site-directed mutagenesis method by using the wild-type RAD51 pET11d as template>>16<<. The construct was sequenced verified. Protein expression and purification were performed using 12-l batches of Rosetta2/pLysS cells containing either expression construct grown at 37\u2009\u00B0C until an OD 600\u2009nm reached 0.5." . . _:b595106071 . . . . _:b595106070 . _:b595106029 . . _:b595106065 . _:b595105887 . . . _:b595106064 . . _:b595106067 . _:b595106066 . . _:b47157725 "A key structural intermediate in DNA repair through HR is the RAD51 nucleoprotein filament>>19<<. Although EMSA experiments allow quantification of protein\u2013DNA complexes, they do not reveal any information on their architectural arrangement." . _:b595106077 . _:b47157758 . _:b595105861 . _:b47157752 "Growth inhibition assays on lymphoblasts and fibroblasts were performed in tissue culture flasks (25\u2009cm2)>>47<<, which were grown in increasing concentrations of MMC, CPT or PARP inhibitor (KU58948)." . _:b595106076 . . _:b595105860 . _:b595106023 . _:b595105863 . _:b595106078 . _:b595105862 . _:b47157717 "RAD51 is a member of the RAD51 family of related genes, of which RAD51C had already been recognized as an FA gene>>11<<. We furthermore confirmed the RAD51 variant as the top candidate with two independent variant filtering workflows: ingenuity variant analysis and the family genomics workflow12." . _:b595106073 . _:b47157722 "4a, left panel)>>16<<. Pivotal steps in HR, which are catalysed by RAD51, are recognition of homology and strand exchange between homologous DNA partners to form a joint molecule between a processed DNA break and the repair template." . _:b595105857 . _:b47157713 "In keeping with this, short interfering RNA knockdown experiments did not result in an FA-like phenotype>>8<<. Altogether, the causal genetic defect underlying the FA-like phenotype in the current patient does not seem to be associated with a recessive mode of inheritance." . . _:b47157721 . _:b595106072 . . _:b595106075 . _:b595106027 . _:b595105902 . _:b47157712 . . _:b595105859 . _:b595106074 . _:b595105980 . _:b47157713 . . . _:b595105858 . . _:b595105882 . _:b595105869 . _:b595105868 . . . . . _:b47157716 . _:b595106065 . _:b595105871 . _:b595105870 . _:b595106066 . _:b595105942 . _:b595105977 . . _:b595105865 . _:b595106069 . _:b595106048 . . . _:b595105864 . _:b595105955 . _:b595106070 . . . _:b595105867 . _:b595105986 . _:b595105866 . . _:b595105878 . . _:b47157715 "FA proteins and (iii) FA protein-like properties. Considering these criteria, RAD51 turned out to be the prime candidate since RAD51 mediates DNA strand exchange, the key step in HR repair9, binds the FA proteins BRCA2 and PALB2 (ref. >>10<<) and has a high score (ranking 65, scoring 6.377) with the FA-ranking tool8. RAD51 is a member of the RAD51 family of related genes, of which RAD51C had already been recognized as an FA gene11." . _:b595105877 . . _:b595105964 . . _:b595105876 . . _:b595105996 . _:b47157732 "Ala293Thr mutation is located in a highly conserved region of RAD51, which is involved in RAD51 monomer\u2013monomer interactions>>24<<25 (Fig. 5). The mutated aa is directly adjacent to the highly conserved so-called L2 loop, which is involved in DNA binding26 (Fig. 5a). The protein\u2013interaction interface displays different conformations associated with ATP- or" . _:b595105879 . . _:b595105866 . _:b595105878 . . . . _:b595105879 . _:b595106056 . . _:b595105933 . _:b595105873 . _:b595105946 . _:b47157738 "methods" . _:b595105872 . _:b595105925 . . . _:b595105875 . _:b595105975 . _:b595106009 . _:b47157743 . _:b595105874 . _:b595105989 . . _:b595105885 . _:b47157730 "Binding of RAD51 to dsDNA changes the helical pitch such that the DNA molecule extends to 1.5-fold its B-form length>>2223<<. If filament formation is not complete, DNA will be less extended and the filaments shorter. The wild-type RAD51 nucleoprotein filaments on the 3.9-kb DNA were 2,025\u00B167\u2009nm long, while filaments formed on the same-length DNA with" . _:b595105884 . . _:b595105887 . _:b595105885 . . _:b595105886 . _:b595106014 . _:b595105881 . . _:b595105960 . . _:b595105899 . _:b595106051 . _:b595105880 . _:b595106052 . _:b47157759 _:b47157768 . . _:b595105881 . _:b595105883 . _:b47157767 "Intriguingly, FA cells are also exquisitely sensitive towards acetaldehyde, a metabolite of ethanol linked to fetal alcohol syndrome, which is an important cause of learning disability>>34<<35." . . . _:b595105882 . _:b47157759 _:b47157760 . _:b47157759 _:b47157761 . _:b595106015 . _:b47157759 _:b47157762 . _:b47157759 _:b47157763 . _:b47157735 . _:b595106005 . _:b595105893 . _:b47157759 _:b47157764 . . _:b47157759 _:b47157765 . . _:b47157759 _:b47157766 . _:b47157759 _:b47157767 . _:b595105859 . _:b595105892 . _:b595105957 . _:b47157714 . _:b595105939 . _:b595105895 . _:b47157715 . _:b595105861 "5"^^ . _:b595105894 . _:b47157753 . _:b595105860 "5"^^ . _:b595105889 . _:b47157754 . _:b595105863 "5"^^ . _:b47157763 "Intriguingly also the recently described cases of biallelic carriers of BRCA1 mutations>>430<<, had reached adulthood without bone marrow failure." . _:b595105888 . . _:b595105874 . . _:b595105891 . _:b595105862 "5"^^ . _:b595105858 "6"^^ . _:b595105974 . . . _:b595105981 . _:b595105890 . _:b595105859 "6"^^ . _:b595106028 . . _:b595105901 . _:b595105857 "13"^^ . . _:b595105900 . . . _:b595105861 . _:b595105903 . . _:b595105869 "4"^^ . _:b595105902 . _:b47157751 . _:b595105858 . _:b595106057 . _:b595105868 "4"^^ . _:b595105897 . . _:b595105871 "4"^^ . _:b47157727 "Under these conditions, RAD51 forms stable highly regular nucleoprotein filaments of defined length that completely cover the DNA>>20<<, (Fig. 4f, i). With the same conditions, some of the observed DNA did have bound RAD51A293T protein (examples shown in Fig. 4f, ii), but the SFM analysis revealed that these DNA\u2013RAD51 complexes differed markedly from the regular wild-type" . . _:b595105896 . _:b595106037 . _:b47157755 . _:b47157767 . _:b595105870 "4"^^ . _:b595105899 . . _:b595105934 . _:b595105865 "5"^^ . . _:b595105898 . _:b595105860 . _:b595105864 "5"^^ . _:b595106041 . _:b595105909 . . _:b595105867 "4"^^ . _:b595105908 . . _:b595105866 "5"^^ . _:b595105911 . _:b595105877 "4"^^ . . _:b595105910 . . _:b595105876 "4"^^ . _:b595105905 . . _:b595105879 "4"^^ . _:b595105904 . _:b595106061 . _:b595106072 . _:b595105878 "4"^^ . _:b595105907 . _:b47157768 . _:b595105886 . _:b47157733 . _:b595105873 "4"^^ . _:b595105940 . _:b595105906 . _:b595105872 "4"^^ . _:b595105897 . _:b595105917 . . . _:b595105875 "4"^^ . _:b595105916 . _:b595106004 . _:b595105874 "4"^^ . . _:b595105919 . _:b47157741 . _:b595105885 "3"^^ . _:b47157719 "Ala351Thr) of RAD51 has been demonstrated to yield a semi-dominant-negative phenotype>>13<<. We thus hypothesized that the human counterpart would have a similar effect. To test this, we generated a fibroblast cell line with inducible expression of the human mutant RAD51, and a cell line with the wild-type gene as control." . _:b595105918 . _:b47157724 "to its role in HR. We tested whether the mutation affected the ATPase activity of RAD51. As previously reported, wild-type RAD51 displayed a low constitutive ATPase activity that is stimulated approximately twofold by ssDNA (Fig. 4b)>>18<<. In contrast, RAD51A293T had a somewhat lower baseline ATPase activity that could not be stimulated by DNA (Fig." . _:b595105950 . _:b595105884 . _:b595105884 "4"^^ . _:b595105913 . _:b595105887 "3"^^ . _:b595105912 . _:b595106012 . _:b595105886 "3"^^ . _:b595105924 . _:b595105915 . _:b595106049 . _:b595105881 "4"^^ . _:b595105914 . _:b595105995 . _:b595105863 . _:b595105880 "4"^^ . _:b595105925 . _:b595105883 "4"^^ . _:b595105924 . _:b595105891 . _:b595105913 . _:b595105882 "4"^^ . _:b595106016 . _:b595105927 . _:b595106025 . _:b595105893 "3"^^ . . _:b47157746 "To detect and evaluate SNVs and small indels, reads were mapped to the GRCh37 reference genome (hg19) by the Burrows\u2013Wheeler Aligner>>42<<. Subsequently, SNVs and small indels were called using SAMtools43 and VarScan44. The resulting list of variants was annotated with ANNOVAR. Finally, manual inspection of relevant variants was carried out by visualizing the mapped reads in" . . _:b595105926 . _:b595105892 "3"^^ . _:b595105907 . . . _:b595105921 . _:b595105895 "3"^^ . _:b47157766 "In early reviews, a link between FA and mental retardation/central nervous system has been established, albeit at a low frequency>>313233<<. Also the two cases with biallelic BRCA1 mutations were found to be associated with developmental delay, while for one of the patients, mental retardation was mentioned explicitly. Thus, it could be hypothesized that absence of paediatric" . _:b47157722 . _:b595106021 . _:b595106044 . _:b595105920 . _:b595106064 . _:b595105894 "3"^^ . _:b595105904 . _:b595105873 . _:b595105923 . _:b595105889 "3"^^ . _:b47157756 "at 3.6\u2009\u03BCM (nucleotides), 1.2\u2009\u03BCM RAD51 (wild type or A293T mutant), 50\u2009mM Tris-HCl (pH 7.5), 1\u2009mM dithiothreitol (DTT), 0.1\u2009mg\u2009ml\u22121 acetylated BSA, 30\u2009mM KCl, 2\u2009mM CaCl2 and 1\u2009mM ATP>>49<<. After 5\u2009min incubation at 37\u2009\u00B0C, 2\u2009\u03BCl of supercoiled pUC19 plasmid DNA at 0.8\u2009mg\u2009ml\u22121 (prepared by detergent lysis and purified by double CsCl density-gradient centrifugation) was added and further incubated for 20\u2009min at 37\u2009\u00B0C." . _:b47157761 "29) and RAD51C>>11<< who, together with the proband of the current study, seem to compose a distinct subtype." . _:b595105922 . _:b595105888 "3"^^ . _:b595106020 . "PMC0" . _:b47157757 "The DNA used in the SFM experiments was made by linearizing the 3,901-bp plasmid pDR5 with XmnI>>50<<. RAD51\u2013DNA complexes were formed in 20-\u03BCl reactions containing 3.75\u2009\u03BCM DNA (concentration in bp), 1.25\u2009\u03BCM RAD51 (wild type or the A293T mutant), 50\u2009mM Tris-HCl (pH 7.5), 2\u2009mM CaCl2, 1\u2009mM ATP, 1\u2009mM DTT and 30 or 100\u2009mM KCl as indicated." . _:b595105933 . . _:b595105891 "3"^^ . _:b595105894 . . _:b595105932 . . _:b595105890 "3"^^ . . . _:b47157752 . _:b595105935 . . _:b595105901 "3"^^ . . . _:b595105985 . _:b595105934 . _:b595105900 "3"^^ . . _:b595105929 . . _:b595105903 "3"^^ . . _:b595105928 . _:b595105902 "3"^^ . _:b595105927 . _:b595105931 . . . _:b595105897 "3"^^ . . _:b595106002 . _:b595105930 . _:b595106047 . _:b595105896 "3"^^ . . _:b47157728 "Ala293Thr mutation on RAD51 nucleoprotein filaments in more detail, we assembled RAD51\u2013DNA complexes in conditions expected to stabilize otherwise unstable filaments>>21<< at 30\u2009mM KCl instead of the 100\u2009mM used above." . _:b595105941 . _:b595105899 "3"^^ . _:b595105929 . _:b47157742 . _:b595105940 . _:b595105918 . _:b595105898 "3"^^ . . _:b47157734 "The mutated aa is directly adjacent to the highly conserved so-called L2 loop, which is involved in DNA binding>>26<< (Fig. 5a). The protein\u2013interaction interface displays different conformations associated with ATP- or ADP-binding status (Fig. 5b right panel), which gives rise to high-affinity or low-affinity ssDNA binding, respectively. The region" . . _:b595105943 . . _:b595105909 "3"^^ . _:b595105942 . . . _:b595105908 "3"^^ . . _:b595105937 . _:b47157735 "A dominant-negative mutation gives rise to an FA-like phenotype, whereas haploinsufficiency of RAD51 has been associated with congenital mirror movement disorder>>27<<. Importantly, the FA-like patient has not displayed mirror movements. So far, no hereditary disorders have been linked to overexpression of RAD51." . _:b47157714 "Considering these criteria, RAD51 turned out to be the prime candidate since RAD51 mediates DNA strand exchange, the key step in HR repair>>9<<, binds the FA proteins BRCA2 and PALB2 (ref." . _:b595105911 "3"^^ . _:b595105936 . . _:b595106077 . _:b595105910 "3"^^ . . _:b595105939 . . . _:b595105905 "3"^^ . _:b595105938 . _:b595105904 "3"^^ . . _:b595105949 . _:b595105907 "3"^^ . _:b595105948 . . _:b47157726 "By contrast, several structural features of RAD51\u2013DNA complexes can be quantified by scanning force microscopy (SFM)>>20<<. Wild-type RAD51 was incubated with 3.9-kbp dsDNA in the presence of Ca2+ and ATP, and the reaction mixture was analysed by SFM. Under these conditions, RAD51 forms stable highly regular nucleoprotein filaments of defined length that" . _:b595105906 "3"^^ . _:b595105857 . _:b595105951 . _:b595105926 . _:b595105917 "3"^^ . _:b595105950 . . _:b595105916 "3"^^ . _:b47157731 "As RAD51 functions in the context of a helical nucleoprotein filament that requires repeated monomer\u2013monomer contacts>>22<<, it is possible that defective DNA binding of the mutant could impose a dominant-negative effect on the function of wild-type RAD51." . . _:b595105945 . _:b595105869 . . _:b595105919 "3"^^ . _:b595105914 . _:b47157737 "Somatically acquired altered expression of RAD51 or absence of RAD51 foci in cancers has been linked to therapy response>>1428<<. However, our data call attention to the potentially clinically relevant finding that cells displaying RAD51 foci may still harbor subtle dominant mutations in RAD51, and could be sensitive to drugs, such as PARP inhibitors." . _:b595105944 . _:b595105918 "3"^^ . _:b595106042 . . _:b595105947 . _:b595105913 "3"^^ . . _:b47157757 . . _:b595106076 . _:b595105946 . _:b47157721 "This is in line with the observed embryonic lethality associated with Rad51 knockout in mice>>15<<." . _:b595105912 "3"^^ . _:b595106018 . _:b595106010 . _:b595105957 . _:b595105931 . _:b595105915 "3"^^ . . . _:b595105956 . _:b595105914 "3"^^ . _:b595106045 . _:b595105959 . . _:b47157723 "A biochemical assay reporting on these activities is D-loop formation, which measures homology-dependent joint molecule formation between a single-strand oligonucleotide and a supercoiled plasmid>>17<<. The p.Ala293Thr mutation in RAD51 reduced the formation of D-loop intermediates by ninefold (Fig. 4a, right panel). The RAD51A293T protein is thus severely defective in joint molecule formation, a key intermediate step in DNA repair by" . _:b595105994 . _:b595105925 "3"^^ . _:b595105958 . _:b595105916 . _:b595105924 "3"^^ . _:b595105953 . _:b595105927 "3"^^ . . _:b595105952 . _:b595106011 . _:b595105926 "3"^^ . . _:b595105955 . _:b47157732 . _:b595105872 . _:b595105921 "3"^^ . _:b595105954 . _:b47157760 . _:b595105920 "3"^^ . _:b595105965 . . . _:b595105923 "3"^^ . . _:b595105964 . . _:b595105922 "3"^^ . _:b595105966 . _:b595105967 . _:b595105933 "2"^^ . _:b47157756 . _:b595105966 . _:b595105932 "3"^^ . _:b595105961 . _:b595105935 "2"^^ . . _:b595105960 . . _:b595106034 . _:b595105934 "2"^^ . _:b595105961 . _:b595105963 . _:b595106006 . _:b47157763 . _:b595105929 "3"^^ . . . _:b595105962 . . . _:b595105928 "3"^^ . _:b595105973 . _:b595105931 "3"^^ . _:b595105972 . _:b595105978 . _:b595105930 "3"^^ . _:b47157746 . _:b47157740 "(2) Next, we filtered for rare variants having a minor allele frequency equal or smaller than 1% in the European American population of the 1000genomes project>>36<< and the NHLBI ESP exomes (https:" . . _:b47157718 . . _:b47157754 "Final fractions of the purified proteins were stored at \u221280\u2009\u00B0C in 25\u2009mM Tris-HCl (pH 7.5), 0.5\u2009mM EDTA, 5\u2009mM \u03B2-mercaptoethanol, 500\u2009mM NaCl and 5% glycerol>>16<<." . _:b595105975 . _:b595105941 "2"^^ . _:b595105864 . . _:b595105974 . _:b595105940 "2"^^ . _:b595105888 . _:b595105969 . _:b595105943 "2"^^ . _:b595105968 . . _:b595105942 "2"^^ . _:b595105875 . _:b595106008 . _:b595105971 . . _:b595105937 "2"^^ . _:b595105970 . _:b595105936 "2"^^ . _:b595106071 . _:b595105981 . _:b595105939 "2"^^ . _:b595105980 . . _:b595105938 "2"^^ . _:b595105983 . . _:b595105949 "2"^^ . _:b595106000 . _:b595105982 . _:b595105948 "2"^^ . _:b47157739 "WGS was performed at Complete Genomics Inc.>>6<< (CG) using their standard sequencing pipeline, version 2.0.2.26." . _:b595105977 . _:b595105951 "2"^^ . _:b595106078 . _:b595105976 . _:b595105950 "2"^^ . _:b595105956 . _:b595105979 . _:b595105945 "2"^^ . . _:b595106055 . _:b595105978 . _:b595105944 "2"^^ . _:b595105989 . _:b47157749 "Finally, manual inspection of relevant variants was carried out by visualizing the mapped reads in the Integrative Genome Browser (IGV)>>45<<." . _:b47157709 _:b47157712 . _:b595105947 "2"^^ . _:b47157709 _:b47157713 . _:b47157709 _:b47157714 . _:b47157748 "Subsequently, SNVs and small indels were called using SAMtools43 and VarScan>>44<<. The resulting list of variants was annotated with ANNOVAR." . _:b47157709 _:b47157715 . _:b595105988 . . _:b595105865 . _:b595105946 "2"^^ . _:b47157709 _:b47157716 . _:b47157709 _:b47157717 . _:b47157709 _:b47157718 . _:b47157709 _:b47157719 . _:b595105991 . _:b47157709 _:b47157720 . _:b595105957 "2"^^ . _:b47157709 _:b47157721 . _:b47157709 _:b47157722 . _:b47157709 _:b47157723 . _:b595105990 . _:b595105889 . _:b595105956 "2"^^ . _:b47157709 _:b47157724 . _:b47157709 _:b47157725 . _:b595106040 . _:b47157709 _:b47157726 . _:b595106073 . _:b47157709 _:b47157727 . _:b595105985 . . _:b595105959 "2"^^ . _:b595105903 . _:b595105984 . . _:b595105958 "2"^^ . _:b595105987 . _:b595105953 "2"^^ . _:b47157709 . _:b595106024 . _:b595105986 . _:b595105952 "2"^^ . _:b595106060 . _:b47157710 . _:b47157709 _:b47157710 . _:b595106063 . _:b47157709 _:b47157711 . _:b595105997 . _:b595105988 . _:b595105945 . _:b595106036 . _:b595105955 "2"^^ . _:b47157711 . _:b595105996 . _:b595105954 "2"^^ . . _:b595105999 . . _:b595105965 "2"^^ . . _:b595105998 . . _:b595105964 "2"^^ . . _:b47157724 . _:b595105993 . _:b47157709 _:b47157728 . _:b595105967 "2"^^ . _:b47157709 _:b47157729 . . _:b47157709 _:b47157730 . . _:b595105992 . _:b47157709 _:b47157731 . _:b47157709 _:b47157732 . _:b595105966 "2"^^ . _:b47157709 _:b47157733 . _:b47157716 . _:b47157709 _:b47157734 . . _:b595105995 . _:b595105968 . _:b47157709 _:b47157735 . . _:b47157709 _:b47157736 . _:b595105961 "2"^^ . _:b47157709 _:b47157737 . _:b47157717 . . _:b595105994 . _:b595105917 . _:b595105960 "2"^^ . _:b47157718 . . _:b595106005 . _:b595105963 "2"^^ . _:b47157719 . _:b47157765 "In early reviews, a link between FA and mental retardation/central nervous system has been established, albeit at a low frequency>>3132<<33. Also the two cases with biallelic BRCA1 mutations were found to be associated with developmental delay, while for one of the patients, mental retardation was mentioned explicitly. Thus, it could be hypothesized that absence of" . _:b595106004 . . _:b595105962 "2"^^ . . _:b47157712 . _:b595106007 . _:b47157729 . _:b595105973 "2"^^ . . _:b47157713 . _:b595105972 . _:b595106006 . _:b595105972 "2"^^ . . _:b47157714 . . _:b47157731 . _:b595106001 . . _:b595105975 "2"^^ . _:b595106031 . _:b47157715 . . _:b595105959 . _:b595106000 . _:b595106003 . _:b595105974 "2"^^ . . _:b47157724 . . . _:b595106003 . _:b595105992 . _:b595105969 "2"^^ . . _:b47157725 . _:b595105973 . . . _:b595106002 . . _:b595105968 "2"^^ . _:b47157726 . _:b47157736 "Somatically acquired altered expression of RAD51 or absence of RAD51 foci in cancers has been linked to therapy response>>14<<28. However, our data call attention to the potentially clinically relevant finding that cells displaying RAD51 foci may still harbor subtle dominant mutations in RAD51, and could be sensitive to drugs, such as PARP inhibitors." . _:b595105900 . _:b595106013 . . _:b595105971 "2"^^ . _:b47157727 . _:b595106012 . . _:b595105970 "2"^^ . . _:b47157720 . _:b595105991 . _:b595106015 . . _:b595105981 "2"^^ . _:b47157711 . _:b47157721 . _:b595106014 . _:b595105928 . _:b595105980 "2"^^ . _:b47157722 . _:b595106053 . _:b595106009 . . _:b47157734 . _:b595105983 "2"^^ . . _:b47157723 . _:b595106008 . _:b595105982 "2"^^ . _:b47157732 . . _:b595106011 . _:b595105977 "2"^^ . . _:b47157733 . _:b595106010 . . _:b595105976 "2"^^ . _:b47157734 . . _:b595106021 . _:b595106019 . _:b595105963 . _:b595105979 "2"^^ . _:b47157735 . _:b595105971 . _:b595106020 . _:b595105978 "2"^^ . _:b47157728 . _:b47157760 "marrow failure, which constitute the prime life-threatening symptoms. Notably, not all FA patients acquire bone marrow failure. Indeed, this phenotype has not been associated with patients affected by biallelic mutations in BRCA2 (ref. >>29<<) and RAD51C11 who, together with the proband of the current study, seem to compose a distinct subtype." .