ERCC1 is required for FANCD2 focus formation.

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

A novel lamin A/C mutation in a family with dilated cardiomyopathy, prominent conduction system disease, and need for permanent pacemaker implantation.

BACKGROUND: The LMNA gene, which encodes the nuclear envelope protein lamin A/C, is thought to be the most common of 8 autosomal disease genes implicated in familial dilated cardiomyopathy (FDC). Each family reported to date has a unique mutation and variable degrees of cardiac conduction system, dilated cardiomyopathy, or skeletal muscle disease. METHODS AND RESULTS: Coding regions of the LMNA gene were screened in 12 biological members of a family with dilated cardiomyopathy and conduction system disease. A novel missense mutation (Leu215Pro) in exon 4 was identified in 8 subjects. Disease was manifested as brady- and tachyarrhythmias, often necessitating permanent pacemaker implantation, and later onset of dilated cardiomyopathy and heart failure. No features of skeletal muscle disease were noted. The high percentage of affected individuals who needed pacemaker therapy (88%) was a unique characteristic of this family compared with other FDC families with LMNA mutations. CONCLUSIONS: Careful examination of clinical data in families with FDC and LMNA mutations may reveal subtle genotype-phenotype correlations. Knowledge of such correlations may help to further define the mechanisms of disease in LMNA-associated FDC and can assist in the monitoring of disease for at-risk family members.

Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links.

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N(2)-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was approximately 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ' and 3 ' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N(2)-N(2)-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N(2)-N(2)-guanine ICLs.

Mutations of presenilin genes in dilated cardiomyopathy and heart failure.

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.

Cardiac troponin T lysine 210 deletion in a family with dilated cardiomyopathy.

BACKGROUND: The gene for cardiac troponin T (TNNT2) is 1 of 7 autosomal disease genes implicated in familial dilated cardiomyopathy (FDC). Identical deletions in exon 13 of TNNT2 have been reported in 2 families with FDC, but little is known about the frequency of this deletion among patients with FDC and idiopathic dilated cardiomyopathy (IDC) and the associated phenotype. METHODS AND RESULTS: Exon 13 of the cardiac troponin T gene was sequenced in 61 subjects with FDC and 53 subjects with IDC. A 3-base pair deletion (DeltaLys210), identified in 1 family with at least 7 clinically affected family members, is reported. Age of disease onset and disease severity varied widely among affected individuals; phenotypic findings included dilated cardiomyopathy, sudden cardiac death, conduction system disease including atrial fibrillation and atrioventricular block, and heart failure. Sudden-onset, rapidly progressive disease was observed in younger individuals. CONCLUSIONS: Cardiac troponin T exon 13 lysine deletions can cause FDC of varying severity and are an important but uncommon cause of FDC.

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia.

Myelodysplastic and leukemic stem cell clones that evolve in children and adults with Fanconi anemia universally bear complex cytogenetic abnormalities. The abnormalities are generally recurring deletions or chromosomal loss and involve precisely the same chromosomes with the same frequency as has been described in marrow cells from patients with secondary acute leukemia induced by alkylating agents. Reasoning that acquired Fanconi anemia protein dysfunction might contribute to cytogenetic instability in secondary acute myelogenous leukemia (AML) cells, we analyzed leukemic cells bearing characteristic complex cytogenetic defects obtained from a 68-year-old man whose lymphoblasts showed no evidence of Fanconi anemia. Unlike the lymphoblasts, this myeloid leukemia cell line (UoC-M1) was hypersensitive to mitomycin-C (MMC) and diepoxybutane (DEB) and exhibited a marked decrease in nuclear FANCA, FANCG, and FANCD2-L. Retroviral transduction of FANCA significantly reduced MMC sensitivity but FANCF, FANCG, and FANCC did not. Overexpression of FANCA restored levels of both FANCA and FANCG, whereas overexpression of FANCG or FANCC did not restore FANCA levels. The molecular mass of cytoplasmic FANCA, FANCG, FANCC, and nuclear FANCD2 were normal. All exons of FANCA and FANCG were sequenced, and no mutations were found. We conclude that perturbations of as yet unidentified factors that govern the binding activity or intracellular localization of FANCA may promote cytogenetic instability and clonal progression in patients with AML who do not have Fanconi anemia.