The clinical manifestation of a defective response to DNA double-strand breaks as exemplified by Nijmegen breakage syndrome.

The autosomal recessive genetic disorder Nijmegen breakage syndrome (NBS) was first described in 1981 in patients living in Nijmegen, Holland. NBS patients display a characteristic facial appearance, microcephaly and a range of symptoms including immunodeficiency, increased cancer risk and growth retardation. In addition, NBS patient cells were found to have elevated levels of chromosomal damage and to be sensitive to ionizing irradiation (IR). This radiosensitivity had fatal consequences in some undiagnosed patients. The most dangerous DNA lesion caused by IR is considered to be the double-strand break (DSB) and indeed, NBS patient cells are sensitive to all mutagens that produce DSBs directly or indirectly. We discuss here our current understanding of how a deficiency in DSB repair manifests as the particular symptom complex of NBS.

Nijmegen breakage syndrome (NBS) with neurological abnormalities and without chromosomal instability.

BACKGROUND: Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability disorder with hypersensitivity to ionising radiation. The clinical phenotype is characterised by congenital microcephaly, mild dysmorphic facial appearance, growth retardation, immunodeficiency, and greatly increased risk for lymphoreticular malignancy. Most NBS patients are of Slavic origin and homozygous for the founder mutation 657del5. The frequency of 657del5 heterozygotes in the Czech population is 1:150. Recently, another NBS1 mutation, 643C>T(R215W), with uncertain pathogenicity was found to have higher frequency among tumour patients of Slavic origin than in controls. This alteration results in the substitution of the basic amino acid arginine with the non-polar tryptophan and thus could potentially interfere with the function of the NBS1 protein, nibrin. METHODS AND RESULTS: Children with congenital microcephaly are routinely tested for the 657del5 mutation in the Czech and Slovak Republics. Here, we describe for the first time a severe form of NBS without chromosomal instability in monozygotic twin brothers with profound congenital microcephaly and developmental delay who are compound heterozygotes for the 657del5 and 643C>T(R215W) NBS1 mutations. Both children showed reduced expression of full length nibrin when compared with a control and a heterozygote for the 657del5 mutation. Radiation response processes such as phosphorylation of ATM and phosphorylation/stabilisation of p53, which are promoted by NBS1, are strongly reduced in cells from these patients. CONCLUSIONS: Interestingly, the patients are more severely affected than classical NBS patients. Consequently, we postulate that homozygosity for the 643C>T(R215W) mutation will also lead to a, possibly very, severe disease phenotype.

Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group.

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder. Clinical care is complicated by variable age at onset and severity of hematologic symptoms. Recent advances in the molecular biology of FA have allowed us to investigate the relationship between FA genotype and the nature and severity of the clinical phenotype. Two hundred forty-five patients from all 7 known complementation groups (FA-A to FA-G) were studied. Mutations were detected in one of the cloned FANC genes in 169 patients; in the remainder the complementation group was assigned by cell fusion or Western blotting. A range of qualitative and quantitative clinical parameters was compared for each complementation group and for different classes of mutation. Significant phenotypic differences were found. FA-G patients had more severe cytopenia and a higher incidence of leukemia. Somatic abnormalities were less prevalent in FA-C, but more common in the rare groups FA-D, FA-E, and FA-F. In FA-A, patients homozygous for null mutations had an earlier onset of anemia and a higher incidence of leukemia than those with mutations producing an altered protein. In FA-C, there was a later age of onset of aplastic anemia and fewer somatic abnormalities in patients with the 322delG mutation, but there were more somatic abnormalities in patients with IVS4 + 4A --> T. This study indicates that FA patients with mutations in the FANCG gene and patients homozygous for null mutations in FANCA are high-risk groups with a poor hematologic outcome and should be considered as candidates both for frequent monitoring and early therapeutic intervention. (Blood. 2000;96:4064-4070)

Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex. (Blood. 2000;96:1625-1632)

Overexpression of cDNA encoding FANCC, SPHAR, MPG, SNM1 or HA 3611 does not render CHO cells more resistant to DNA crosslinking agents.

DNA crosslinking agents (DCA) are commonly used cytostatic drugs, whose efficiency in tumor therapy is limited due to the appearance of drug resistant tumor cells. In an effort to modulate the resistance of cells to DCA, we transfected into Chinese hamster cells various cDNAs whose loss of function was previously shown to render cells more sensitive to crosslinking agents. We show that overexpression of FANCC, SPHAR, MPG, SNM1 or HA 3611 (a human homologue of the yeast crosslink DNA repair gene SNM1) does not alter the level of resistance of CHO cells to clinically relevant DCA, such as mafosfamide, melphalan and mitomycin C. Therefore, DCA resistance frequently observed in tumor cells is not likely to be the result of up-regulation of either one of these genes, but a more complex phenomenon. Also, the data suggest that protection of normal cells from toxic side effects of DCA cannot easily be accomplished by transfer of either one of these genes.

[Molecular basis of Fanconi's anemia]. / Molekulare Grundlagen der Fanconi-Anämie.

Fanconi anaemia (FA) is an autosomal recessive genetic disorder characterised clinically by progressive bone marrow failure, skeletal deformities and a predisposition to neoplasia. Patient cells manifest an extreme chromosomal instability and hypersensitivity to polyfunctional alkylating agents. It is assumed that the basic defect is related to the repair of DNA damage, in particular that of so-called DNA crosslinks. Currently there are eight complementation groups in FA (FA-A-FA-H) which indicates that at least eight independent genes can lead to FA. Three of these genes have been identified: FANCA, FANCC and FANCG. In this review, the molecular biology and genetics of FA are presented and possible functions of the FANC proteins are discussed.

Nijmegen breakage syndrome: consequences of defective DNA double strand break repair.

The autosomal recessive genetic disorder, Nijmegen Breakage Syndrome, is characterised by an excessively high risk for the development of lymphatic tumours and an extreme sensitivity towards ionising radiation. The most likely explanation for these characteristics, a deficiency in the repair of DNA lesions, has been greatly substantiated by the recent cloning of the gene mutated in Nijmegen Breakage Syndrome patients and the analysis of its protein product, nibrin. The direct involvement of this protein in the processing of DNA double strand breaks caused by ionising radiation and those also necessary for normal DNA metabolism can be correlated with many of the cellular and clinical aspects of the disease, including the cancer predisposition of patients and their heterozygous relatives.

The Fanconi anemia group E gene, FANCE, maps to chromosome 6p.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disease with bone marrow failure and predisposition to cancer as major features, often accompanied by developmental anomalies. The cells of patients with FA are hypersensitive to DNA cross-linking agents in terms of cell survival and chromosomal breakage. Of the eight complementation groups (FA-A to FA-H) distinguished thus far by cell fusion studies, the genes for three-FANCA, FANCC, and FANCG-have been identified, and the FANCD gene has been localized to chromosome 3p22-26. We report here the use of homozygosity mapping and genetic linkage analysis to map a fifth distinct genetic locus for FA. DNA from three families was assigned to group FA-E by cell fusion and complementation analysis and was then used to localize the FANCE gene to chromosome 6p21-22 in an 18.2-cM region flanked by markers D6S422 and D6S1610. This study shows that data from even a small number of families can be successfully used to map a gene for a genetically heterogeneous disorder.

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.

Assessment of mitomycin C sensitivity in Fanconi anemia complementation group C gene (Fac) knock-out mouse cells.

Fanconi anemia (FA) is a genetic disorder defined by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). MMC causes increased FA cell death, chromosome breakage, and accumulation in the G2 phase of the cell cycle. Recently, Fanconi anemia complementation group C (fac) gene knock-out mice have been developed, and SV40-transformed fibroblasts were established from fac homozygous knock-out (-/-), heterozygous (+/-), and wild-type mice (+/+). MMC sensitivity of these cell lines was assessed by three methods: colony-formation assay in the presence of MMC, chromosome breakage, and cell cycle analysis to detect G2 phase arrest. The fac knock-out fibroblasts (-/-) showed a significantly higher sensitivity to MMC than did fibroblasts from wild-type (+/+) or heterozygous (+/-) mice (three experiments). In addition, we analyzed hematopoietic progenitor colony assays of bone marrow cells from fac knock-out (-/-) and heterozygous (+/-) mice. CFU-E, BFU-E, and CFU-GM colony formation from fac nullizygous mouse progenitors was markedly diminished by MMC when compared to growth of progenitors from heterozygous mice. These results show that fac knock-out mouse cells mimic the behavior of human FA-C patient cells in terms of MMC hypersensitivity. The fac knock-out mouse may be used to model some aspects of human FA and should be useful for understanding the function of the FAC protein.

Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. We describe the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks.

Retroviral gene transfer for the assignment of Fanconi anemia (FA) patients to a FA complementation group.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure, cancer susceptibility, and a variety of developmental defects. The disease is clinically heterogeneous; eight different complementation groups (FA A-H) and, thus, genetic loci have been discovered. Two genes, FAA and FAC, have been cloned. Disease-associated mutations have been detected and rapid mutation screening makes possible the assignment of patients without resorting to time-consuming cell fusion and complementation analysis. Amplification of specific cDNAs from RNA followed by direct or indirect sequence analysis is a standard method for mutation detection. During the course of such examinations of the FAC gene, we have noted that frequently only one of the expressed alleles is successfully amplified. This can lead to false assignment of patients to a complementation group. As we report here, such cases can be rapidly clarified by retroviral gene transfer and complementation analysis.

Irreversible repression of DNA synthesis in Fanconi anemia cells is alleviated by the product of a novel cyclin-related gene.

Primary fibroblasts from patients with the genetic disease Fanconi anemia, which are hypersensitive to cross-linking agents, were used to screen a cDNA library for sequences involved in their abnormal cellular response to a cross-linking challenge. By using library partition and microinjection of in vitro-transcribed RNA, a cDNA clone, pSPHAR (S-phase response), which is able to correct the permanent repression of semiconservative DNA synthesis rates characteristic of these cells, was isolated. Wild-type SPHAR mRNA is expressed in all fibroblasts so far analyzed, including those of Fanconi anemia patients. Correction of the abnormal response in these cells appears therefore to be due to overexpression after cDNA transfer rather than to genetic complementation. The cDNA contains an open reading frame coding for a polypeptide of 7.5 kDa. Rabbit antiserum directed against a SPHAR peptide detects a protein of 7.9 kDa in Western blots (immunoblots) of whole-cell extracts from proliferating, but not resting, fibroblasts. The deduced amino acid sequence of SPHAR contains a motif found in the cyclins, and it is proposed that SPHAR acts within the injected cell by interfering with the cyclin-controlled maintenance of S phase. In agreement with this proposal, normal cells transfected with an antisense SPHAR expression vector have a significantly reduced rate of DNA synthesis during S phase and a prolonged G2 phase, reflecting the need for postreplicative DNA processing before entry into mitosis.

Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).

We have isolated the gene for human type I keratin 9 (KRT9) and localised it to chromosome 17q21. Patients with epidermolytic palmoplantar keratoderma (EPPK), an autosomal dominant skin disease, were investigated. Three KRT9 mutations, N160K, R162Q, and R162W, were identified. All the mutations are in the highly conserved coil 1A of the rod domain, thought to be important for heterodimerisation. R162W was detected in five unrelated families and affects the corresponding residue in the keratin 14 and keratin 10 genes that is also altered in cases of epidermolysis bullosa simplex and generalised epidermolytic hyperkeratosis, respectively. These findings provide further evidence that mutations in keratin genes may cause epidermolysis and hyperkeratosis and that hyperkeratosis of palms and soles may be caused by different mutations in the KRT9 gene.

Human genetic instability syndromes: single gene defects with increased risk of cancer.

The experimental findings of the last 5 years are reviewed for the genetic instability syndromes: Xeroderma pigmentosum, Fanconi's anaemia, Ataxia telangiectasia and Bloom's syndrome. In these autosomal recessive genetic diseases, single gene defects lead to genetic instability, increased mutation rates and cancer. Deficiencies in the ability to effectively repair DNA lesions have been suggested for all of these syndromes. The status of characterization of these DNA repair defects is presented and the possible mechanisms of lesion fixation as mutation are discussed. The four known human genes whose mutation leads to inherited genetic instability are described.

Routine applications of DNA fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5.

The use of DNA fingerprinting with synthetic oligonucleotides is illustrated for practical applications familiar in clinical diagnostics: pre- and postnatal zygosity determination and the monitoring of bone marrow transplantation. A simple method for non-radioactive detection is described which may be interesting for many diagnostic laboratories.

Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts.

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures.

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then structurally characterized. This RNP was compared to the 5S-rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However, besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5-5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1-3) of 5'-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/P3 by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5-5S-RNA complex. For the sarcoplasmic L5-5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated.