The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

Association of radial longitudinal deficiency and thumb hypoplasia: An update using the CoULD registry

BACKGROUND: Deficiency of the radial aspect of the forearm and hand is the most common congenital longitudinal deficiency of the upper limb. Radial longitudinal deficiency is associated with several named syndromes. The purpose of the present study was to explore patterns of radial longitudinal deficiency and thumb hypoplasia in syndromes and to examine the severity of these differences across various syndromes. METHODS: Data were collected from the Congenital Upper Limb Differences (CoULD) registry. Congenital differences are classified in the registry with use of the Oberg-Manske-Tonkin (OMT) classification system. Diagnosis of a syndrome by a physician as noted in the CoULD registry was recorded. Thumb deficiency and radial deficiency were classified according to the modified versions of the Blauth criteria and the Bayne and Klug criteria, respectively. RESULTS: We identified 259 patients with 383 affected limbs with radial deficiency. Eighty-three of these patients had a diagnosed syndrome. The severity of radial deficiency was correlated with the severity of thumb deficiency. The Kendall tau coefficient indicated significant correlation between radial severity and thumb severity (tau = 0.49 [95% confidence interval = 0.40 to 0.57]; p \u3c 0.05). Subjects with a syndrome were twice as likely to have bilateral deficiency and 2.5 times more likely to have both radial and thumb deficiency compared with subjects without a syndrome. Subjects with VACTERL syndrome (vertebral defects, anal atresia, cardiac anomalies, tracheoesophageal fistula, renal anomalies, and limb defects) had patterns of thumb and radial deficiency similar to the general cohort, whereas subjects with Holt-Oram syndrome, TAR (thrombocytopenia absent radius) syndrome, and Fanconi anemia demonstrated varied presentations of thumb and radial deficiency. CONCLUSIONS: The present study investigated the characteristics of patients with radial longitudinal deficiency and thumb hypoplasia. Our results support the findings of previous research correlating the severity of radial deficiency with the severity of thumb deficiency. Furthermore, we identified characteristic features of patients with radial longitudinal deficiency and associated syndromes

BRIP1 (BACH1) variants and familial breast cancer risk: a case-control study

<p>Abstract</p> <p>Background</p> <p>Inactivating and truncating mutations of the nuclear BRCA1-interacting protein 1 (BRIP1) have been shown to be the major cause of Fanconi anaemia and, due to subsequent alterations of BRCA1 function, predispose to breast cancer (BC).</p> <p>Methods</p> <p>We investigated the effect of BRIP1 -64G>A and Pro919Ser on familial BC risk by means of TaqMan allelic discrimination, analysing <it>BRCA1/BRCA2 </it>mutation-negative index patients of 571 German BC families and 712 control individuals.</p> <p>Results</p> <p>No significant differences in genotype frequencies between BC cases and controls for BRIP1 -64G>A and Pro919Ser were observed.</p> <p>Conclusion</p> <p>We found no effect of the putatively functional BRIP1 variants -64G>A and Pro919Ser on the risk of familial BC.</p

Mutation analysis of the Fanconi anaemia A gene in breast tumours with loss of heterozygosity at 16q24.3

The recently identified Fanconi anaemia A (FAA) gene is located on chromosomal band 16q24.3 within a region that has been frequently reported to show loss of heterozygosity (LOH) in breast cancer. FAA mutation analysis of 19 breast tumours with specific LOH at 16q24.3 was performed. Single-stranded conformational polymorphism (SSCP) analysis on cDNA and genomic DNA, and Southern blotting failed to identify any tumour-specific mutations. Five polymorphisms were identified, but frequencies of occurrence did not deviate from those in a normal control population. Therefore, the FAA gene is not the gene targeted by LOH at 16q24.3 in breast cancer. Another tumour suppressor gene in this chromosomal region remains to be identified. © 1999 Cancer Research Campaig

Inhibition of BACH1 (FANCJ) helicase by backbone discontinuity is overcome by increased motor ATPase or length of loading strand

The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5′ tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules

Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links

Cells deficient in the Werner syndrome protein (WRN) or BRCA1 are hypersensitive to DNA interstrand cross-links (ICLs), whose repair requires nucleotide excision repair (NER) and homologous recombination (HR). However, the roles of WRN and BRCA1 in the repair of DNA ICLs are not understood and the molecular mechanisms of ICL repair at the processing stage have not yet been established. This study demonstrates that WRN helicase activity, but not exonuclease activity, is required to process DNA ICLs in cells and that WRN cooperates with BRCA1 in the cellular response to DNA ICLs. BRCA1 interacts directly with WRN and stimulates WRN helicase and exonuclease activities in vitro. The interaction between WRN and BRCA1 increases in cells treated with DNA cross-linking agents. WRN binding to BRCA1 was mapped to BRCA1 452–1079 amino acids. The BRCA1/BARD1 complex also associates with WRN in vivo and stimulates WRN helicase activity on forked and Holliday junction substrates. These findings suggest that WRN and BRCA1 act in a coordinated manner to facilitate repair of DNA ICLs

Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

<p>Abstract</p> <p>Background</p> <p>Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the <it>SLX4 </it>gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of <it>SLX4 </it>in German or Byelorussian familial cases of breast cancer without detected mutations in <it>BRCA1 </it>or <it>BRCA2 </it>has been completed, with globally negative results.</p> <p>Methods</p> <p>The genomic region of <it>SLX4</it>, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of <it>BRCA1 </it>or <it>BRCA2 </it>mutations.</p> <p>Results</p> <p>This mutational analysis revealed extensive genetic variation of <it>SLX4</it>, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them.</p> <p>Conclusions</p> <p>Overall, while the results of this study do not identify clearly pathogenic mutations of <it>SLX4 </it>contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.</p