Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.

BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population.

Involvement of the Fanconi's anemia protein FAC in a pathway that signals to the cyclin B/cdc2 kinase.

Lymphoblastoid cell lines derived from patients with the chromosomal instability disorder Fanconi's anemia (FA) are hyperresponsive to G2 delay and apoptosis induced by cross-linking agents such as mitomycin C (MMC). Here, we investigated whether the protein defective in FA complementation group C (FA-C) cells functions in a pathway that signals to the cdc2 kinase complex, which controls mitotic progression. FA-C lymphoblasts treated with a low dose of MMC (1-5 microM, 1 h) exhibited a protracted G2-M arrest and subsequent apoptosis by 2 days after treatment. This G2-M arrest was mediated by persistent inactivation of the cyclin B1/cdc2 kinase complex characterized by both sustained accumulation of cyclin B1 and tyrosine phosphorylation of cdc2. In phenotypically corrected (wild-type) cells, the same treatment induced only temporal G2-M arrest, associated with a transient inactivation of the cyclin B1/cdc2 kinase complex, after which cells resumed cycling. Treatment with higher dosages (15-30 microM, 1 h) resulted in S-phase arrest and induced a similar high level of apoptosis in FA-C and wild-type cells, accompanied by degradation of cyclin B1 and dephosphorylation of cdc2. In low-dose treated G2-M-arrested FA-C cells, caffeine-dependent activation of cdc2 released the G2-M block but failed to protect against apoptosis, suggesting that apoptosis was not a direct consequence of persistent cdc2 kinase inactivation. Thus, at low doses of MMC, FA-C cells exhibit a unique cyclin B1/cdc2 response that is not observed in wild-type cells treated with an equitoxic high dosage of cross-linker. Although these results do not necessarily implicate a role for FAC in regulating cyclin B/cdc2 kinase activity, available evidence suggests that the FAC protein is involved in a cross-link damage avoidance pathway that signals to this kinase complex.

Integrated human papillomavirus type 16 and loss of heterozygosity at 11q22 and 18q21 in an oral carcinoma and its derivative cell line.

A human papillomavirus (HPV) type 16 containing oral squamous cell carcinoma cell line 93VU147T at early passage was demonstrated to match its primary tumor with regard to HPV status and loss of heterozygosity at loci potentially involved in HPV-mediated carcinogenesis. DNA in situ hybridization of the cell line and the primary tumor revealed the presence of HPV 16 DNA clonally associated with the neoplastic cells. One- and two-dimensional Southern blot hybridization suggested HPV 16 to be integrated in the host genome at over hundred copies/cell. An identical restriction enzyme profile was observed for the tumor and the cell line. Viral DNA integration was confirmed by fluorescence in situ hybridization on metaphase spreads of the cell line, which revealed six stained loci comprising one at 15q14-15 and five at cytogenetically unidentifiable chromosomes. In addition, the tumor and the cell line displayed mRNA expression of the E6/E7 region encoding the viral oncoproteins, as determined by reverse transcription-PCR. Northern blot analysis of the cell line revealed three major and three minor transcripts harboring E6/E7 sequences. Both the primary tumor and cell line showed loss of heterozygosity at the 11q22 (D11S35) and 18q21 (DCC) loci. These data support a role for HPV 16 in the development of a subset of oral cancers, presumably in concert with loss of function of tumor suppressor genes at 11q and 18q.

Inheritance of abnormal expression of SOS-like response in xeroderma pigmentosum and hereditary cancer-prone syndromes.

The time course of induction of SOS-like stress responses such as enhanced reactivation (ER) and enhanced mutagenesis (EM) has been investigated in UV-C-irradiated skin fibroblasts from a xeroderma pigmentosum (XP) family, using herpes simplex virus type 1 as a probe. Similar ER studies were performed in a Li-Fraumeni syndrome (LFS) family and in a family with a high incidence of breast, ovarian, and colon cancer. In two XP (complementation group B) patients, with a striking absence of skin tumors even at an age of >40 years, only induction of EM was observed, whereas ER was absent (XPER-). The ER- phenotype was inherited from the father, whereas cells from the mother exhibited normal expression of ER and EM. This suggests that the absence of ER is a hereditary trait that is not correlated with a repair-deficient phenotype. Abnormally high levels of ER were observed in UV-C-exposed skin fibroblasts from rive LFS patients. The inheritance of the ER response was studied in one LFS family. High levels of ER were observed only in cells derived from affected individuals carrying one mutated p53 allele, whereas cells from unaffected family members, carrying two wild-type p53 alleles, exhibited normal ER levels. This result shows that abnormally high levels of ER positively correlate with the occurrence of cancer in affected individuals from a LFS family. Interestingly, abnormally high levels of ER were observed in cells from afflicted as well as from unafflicted members of a family with a high incidence of breast, ovarian, colon, and stomach cancer. This suggests that these latter individuals have inherited a mutated, putative predisposing gene, resulting in abnormal expression of ER, but that cancer had not yet developed. The results indicate that the ER response can possibly be used as a prognostic marker to identify carriers in various hereditary cancer-prone syndromes at an early age.

Synthesis and secretion of the human vitamin B12-binding protein, transcobalamin II, by cultured skin fibroblasts and by bone marrow cells.

Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.

Isolation and sequence analysis of variant forms of human transcobalamin II.

Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5' end and the 3' end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860-7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an approximately 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.

Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance.

Approximately 25% of patients with Fanconi anemia (FA) have evidence of spontaneously occurring mosaicism as manifest by the presence of two subpopulations of lymphocytes, one of which is hypersensitive to cross-linking agents (e.g. mitomycin C) while the other behaves normally in response to these agents. The molecular basis of this phenotypic reversion has not yet been determined. We have investigated 8 FA patients with evidence of mosaicism. Epstein-Barr virus-immortalized lymphoblastoid cell lines established from these patients exhibited an IC50 for mitomycin C of 25 to > 100 nM compared to a mean of 2 +/- 2 nM for 20 nonmosaic FA patients and 49 +/- 11 nM for 8 healthy controls. In 3 patients who were compound heterozygotes for pathogenic FAC gene mutations the molecular mechanism of the mosaicism was investigated by haplotype analysis. The results indicated that an intragenic mitotic recombination must have occurred leading to a segregation of a wild-type allele in the reverted cells and suggested two patterns of recombination. In 1 patient a single intragenic crossover between the maternally and paternally inherited mutations occurred associated with markers located distally to the FAC gene; in the other 2 patients (sibs) the mechanism appears to have been gene conversion resulting in segregants which have lost one pathogenic mutation. In 6 of the 8 patients the hematological symptoms were relatively mild despite an age range of 9-30 years.

Identification of a Glu greater than Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.

Specific activity of human erythrocyte superoxide dismutase as a function of donor age. A brief note.

Superoxide dismutase was assayed both immunochemically and by enzymatic activity in erythrocytes of human donors, 1 to 98 years of age. No change was observed in enzyme activity per unit enzyme antigen as a function of donor age.

Presynaptic nigrostriatal function in genetically tested asymptomatic relatives from the pallido-ponto-nigral degeneration family.

Pallido-ponto-nigral degeneration (PPND) is a dominantly inherited disorder with parkinsonism. We performed PET with [18F]fluorodopa (FD) and, later, gene testing in 12 asymptomatic relatives at risk from a family with PPND and compared the striatal FD uptake constant (Ki) in them with 4 symptomatic individuals and 10 normal control subjects. Four relatives with positive linkage had a significantly reduced Ki from the normal control subjects but to a lesser degree than the symptomatic patients. The mean Ki in the relatives with negative linkage (n = 8) did not differ from normal control subjects. In conclusion, we identified reduced dopaminergic function in asymptomatic relatives with positive genetic linkage from the PPND family. Most of the reduction in this disorder occurs in the fifth decade, when the disease manifests clinically.

Some patients with intracranial aneurysms have a reduced type III/type I collagen ratio. A case-control study.

A reduced production of type III collagen has been reported in previous studies to be associated with intracranial aneurysms. The purpose of this prospective case-control study was to assess the possible role of a reduced type III collagen production as a risk factor for having an intracranial aneurysm. The study group consisted of 41 consecutively admitted patients with intracranial aneurysms. Intracranial aneurysms were demonstrated by intraarterial digital subtraction cerebral angiography or during operation. The control group consisted of 41 healthy volunteers matched for age and sex. Fibroblasts were cultured from skin biopsies from patients and control subjects, and the type III/type I collagen ratios were determined. The type III/type I collagen ratios in the controls ranged from 5.5 to 19.8%, with a median ratio of 10%, and none had a ratio below 5.5%. The type III/type I collagen ratios in patients ranged from 1.1 to 25.1%, with a median ratio of 10.5%, and eight patients (19.5%) had a low (< 5.5%) ratio (p = 0.005, Fisher's exact test). Our findings support the hypothesis that a reduced production of type III collagen may contribute to the formation of intracranial aneurysms in some patients.

An atypical case of Fanconi anemia in elderly sibs.

We describe a 56-year-old woman suspected of Fanconi anemia on the basis of the following clinical findings: microcephaly, short stature, congenital deafness, and the clinical findings in her deceased brother. Hematologic or other signs of malignancy were absent. The diagnosis was confirmed by demonstrating hypersensitivity of her lymphocytes to mitomycin C (MMC). Cell fusion experiments indicated that the patient belongs to complementation group A. The patient's brother died at the age of 50 of heart and renal failure, and anemia. He had clinical findings similar to those of his sister, and a horseshoe kidney. From 31 years on he had thrombocytopenia and leucopenia. Both patients had insulin-dependent diabetes mellitus. A chromosomal breakage test carried out elsewhere before his death failed to demonstrate MMC hypersensitivity of his lymphocytes, which led to the investigation of his sister. To our knowledge these two cases are the oldest Fanconi anemia patients reported thus far.

Cytogenetic characteristics of oral squamous cell carcinomas in Fanconi anemia.

Fanconi anemia (FA) is an autosomal recessive syndrome with a marked predisposition to malignancies, in particular acute myeloid leukemia and squamous cell carcinoma of the oral cavity. We examined oral squamous cell carcinoma tissue from two FA patients (FA-A and FA-C) by comparative genomic hybridization. Both tumors, which were negative for human papilloma as well as Epstein-Barr viral sequences, showed multiple alterations with a high proportion of whole-arm chromosomal gains and losses. This combination of features as well as the sites involved in chromosomal breakage are very similar to what is typically observed in non-FA oral tumors. These results suggest that the process leading to early occurrence of oral cancer in FA patients follows a similar pathway as in non-FA cancer patients, which would support a caretaker function for FA genes in the protection against oral carcinogenesis. Since FA patients are uniquely hypersensitive to DNA cross-linking agents, while oral cancer in the general population is thought to be environmentally induced, these results also suggest that environmental DNA cross-linkers may be causally involved in oral carcinogenesis.

Cytogenetic toxicity of paraquat and streptonigrin in Fanconi's anemia.

We reported earlier that the frequency of chromosomal aberrations observed in Fanconi's anemia lymphocyte cultures depends on the oxygen tension during growth of the cultures, suggesting that "activated oxygen species" (superoxide, O2-; hydrogen peroxide, H2O2; hydoxyl radical, OH; and singlet oxygen, 1O2) or other reactive products generated during oxygen metabolism may be involved in the production of chromosomal damage in this syndrome. Paraquat and streptonigrin, agents that have been proposed as model compounds exerting cellular toxicity through overproduction of superoxide, were tested for their clastogenic potency in lymphocyte cultures from healthy controls and patients with Fanconi's anemia. Paraquat, at concentrations that severely affected mitotic activity (100-200 micrograms/ml), appeared to be a weak clastogen in human lymphocytes, whereas a clastogenic effect of streptonigrin was demonstrable already at a concentration as low as 0.005 microgram/ml. The results indicate that Fanconi's anemia lymphocytes fail to exhibit an increased sensitivity to the antimitotic and clastogenic effects of paraquat and streptonigrin. This suggests that intracellular superoxide is not critically involved in the generation of spontaneous chromosomal aberrations in Fanconi's anemia. We infer from these and previous data that singlet oxygen (1O2) may be a critical contributor to the chromosomal breakage in this disorder.