Cell and molecular biology

Cells from FA patients show an abnormally high frequency of spontaneous chromosomal breakage and hypersensitivity to the clastogenic effect of DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C (MMC). A laboratory test is available for FA. This is based on the increased chromosomal breakage seen in FA cells compared with normal control subjects after exposure to low concentrations of DEB or MMC ('DEB/MMC stress test') (Figure 12.2). Other features of the FA cell phenotype include abnormal cell cycle kinetics (prolonged G2 phase), hypersensitivity to oxygen, increased apoptosis and accelerated telomere shortening.

There is considerable genetic heterogeneity in FA. Complementation analysis of somatic cell hybrids has provided evidence for eight complementation groups (FA-A, FA-B, FA-C, FA-D1,

FA-D2, FA-E, FA-F and FA-G), suggesting that there are several FA genes. Table 12.4 shows the approximate prevalence of the different FA subgroups and shows that a large number of FA mutations have been identified. Thus, although it is now possible to undertake molecular analysis in FA patients (including the use of retroviral complementation with cloned FA cDNAs and immunoblots for FA proteins), the considerable molecular heterogeneity means that the DEB/MMC stress test remains the front-line diagnostic test.

Identification of the first five FA genes (FANCA, FANCC, FANCE, FANCF and FANCG) provided no immediate clues to their function as they showed no significant homology to each other or to other known genes in the databases. Subsequent studies have demonstrated that the FANCA, FANCC, FANCE, FANCF and FANCG proteins encoded by these genes interact with each other and are part of a larger nuclear complex. The

Table 12.4 FA complementation groups/genetic subtypes.

Complementation

Approximate percentage

Chromosome

Protein(amino acids)

Mutations

group/gene

of FA patients

location

identified

A (FANCA)

65-70

16q24.3

1455

> 120

B (FANCB*)

< 2

?

?

?

C (FANCC)

5-10

9q22.3

558

10

D1 (FANCD1*)

<2

13q12-13

3417

9

D2 (FANCD2)

<2

3p25.3

1451

5

E (FANCE)

2-5

6p21.3

536

3

F (FANCF)

<2

11p15

374

6

G (FANCG)

10-15

9p13

622

21

*FANCD1, and possibly FANCB, were recently shown to be BRCA2.

The percentages of the different subgroups refer to the EUFAR (European Fanconi Anaemia Registry) data.

*FANCD1, and possibly FANCB, were recently shown to be BRCA2.

The percentages of the different subgroups refer to the EUFAR (European Fanconi Anaemia Registry) data.

identification of the FANCD2 gene provided an important link between the FA proteins and DNA repair. The nuclear complex containing the FA proteins (A/C/E/F/G) is required for the activation of the FANCD2 protein to a mono-ubiquitinated (a post-translational modification of a protein by the addition of a single ubiquitin molecule) isoform (FANCD2-Ub). In normal (non-FA) cells, FANCD2 is mono-ubiquitinated at lysine 561 in response to DNA damage (e.g. MMC) and is targeted to discrete nuclear foci.

Activated FANCD2 protein collocalizes and co-purifies with the breast cancer susceptibility protein, BRCA1, a protein that is important in many DNA damage-response pathways. These nuclear foci appear in cells after DNA damage and in those cells undergoing DNA replication. Proteins such as the recombination molecule RAD51, and those in the RAD50-NBS1-MRE11 DNA repair pathway are also present in BRCA1-containing foci (NBS1, complex Nijmegen breakage syndrome 1; MRE11, meiotic recombination 11). In cells from A, C, E, F or G patients, FANCD2 ubiquitination is not observed and it is not targeted to nuclear foci. ATM (ataxia telangiectasia mutated) can phos-phorylate both FANCD2 and BRCA1. The ATM-dependent phosphorylation of FANCD2 (at serine 222) occurs in response to ionizing radiation. Therefore, this puts FANCD2 in a central position in signalling DNA damage; double-strand DNA breaks caused by ionizing radiation result in ATM-dependent phos-phorylation of FANCD2 at serine 222, whereas interstrand DNA cross-links caused by MMC produce mono-ubiquitination of FANCD2 at lysine 561 via the FA nuclear complex.

Recently, it has been shown that cell lines derived from FA-D1 (and possibly FA-B) patients have biallelic mutations in BRCA2. This finding now links the cloned FA genes (FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG) with BRCA1 and BRCA2 in a common pathway. BRCA2 protein is important in the repair of DNA damage by homologous recombination, in part by regulating the activity of RAD51. Cells lacking BRCA2 inaccurately repair damaged DNA and are hypersensitive to DNA cross-linking agents. These recent scientific developments therefore suggest that BRCA2 and other FA proteins cooperate in a common DNA damage response pathway, 'the FA/BRCA pathway'. A schematic representation of the FA/BRCA pathway is given in Figure 12.3. The precise functions of BRCA1 and BRCA2 in this pathway remain to be elucidated. It is also possible that the FA proteins have other functions in addition to their role in this pathway.

In vitro gene transfer studies have demonstrated that introduction of the appropriate wild-type FA gene into FA human lymphoid and haemopoietic cells markedly enhances their growth and normalizes their response to MMC; and in lymphoid lines, cell kinetics (G2 phase) and chromosomal breakage are normalized. Thus, the transfer of the wild-type FA genes corrects the extreme sensitivity to DNA cross-linking agents, the hallmark of the FA cell phenotype. These studies provide the rationale for haemopoietic gene therapy (discussed below).

Nucleus damage

Ionizing radiation

D2 Nuclear foci

Nucleus

D2 Nuclear foci

BRCA2

damage

Ionizing radiation

Figure 12.3 Schematic representation of the FA/BRCA pathway. In response to DNA damage (MMC) the FA protein complex (A/C/E/F/G complex) is formed and results in mono-ubiquitination of FANCD2 (D2). Mono-ubiquitination targets D2 to DNA repair nuclear foci containing BRCA1, BRCA2, and RAD51, which are important in maintaining genomic stability by promoting homologous recombination repair. D2 phosphorylation by ATM in response to DNA damage induced by ionizing radiation is important in S-phase checkpoint response.

BRCA2

Figure 12.3 Schematic representation of the FA/BRCA pathway. In response to DNA damage (MMC) the FA protein complex (A/C/E/F/G complex) is formed and results in mono-ubiquitination of FANCD2 (D2). Mono-ubiquitination targets D2 to DNA repair nuclear foci containing BRCA1, BRCA2, and RAD51, which are important in maintaining genomic stability by promoting homologous recombination repair. D2 phosphorylation by ATM in response to DNA damage induced by ionizing radiation is important in S-phase checkpoint response.

Murine mouse models of FA have shown that haemopoietic progenitors are hypersensitive to tumour necrosis factor a (TNF-a) and interferon-y. This differential hypersensitivity to interferon-y is thought to be mediated by /as-induced apoptosis and may turn out to be an important mechanism in the development of progressive BM failure in FA. It is noteworthy that patients with idiopathic AA usually have raised interferon-y levels, thus providing a possible link in the pathophysiology of BM failure in both idiopathic and FA-associated AA. The presence of short telomeres in cells from both FA and idiopathic AA patients can also be expected to be important in the pathophy-siology of BM failure in both diseases. This issue is discussed further in relation to DC.

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