Nijmegen Breakage Syndrome (NBS) Italian website

Molecular Biology 
Cited literature

Disease Description

• Clinical Phenotype

• Cytogenetics and Cellular Phenotype

• Molecular Biology

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NBS1 gene structure and mutations

NBS is caused by biallelic (homozygous or compound heterozygous) mutations in the NBS1 gene, located on the long arm of chromosome 8 (8q21). The entire gene consists of 16 exons and spans approximately 50 kb of DNA. It encodes a 754 amino acid / 85 kDa protein called nibrin (or p95). 
All known disease-causing mutations in the NBS1 gene have been found within exons 6-10 and all but one of them result in premature truncation of the nibrin protein, leading to an undetectable p95 in NBS cell lines by Western blotting.
A single mutation of Slavic origin, the 657del5, accounts for more than 90% of all mutant alleles in NBS, while nine distinct mutations have been found in patients of different ethnic groups.

Disease-causing mutations in the NBS1 gene identified to date.
(click on the picture below to see a schematic representation)

^#Homozygous/^°compound Heterozygous
*a second family with 900del25 mutation has been identified by Raymonda Varon (personal communication).
^One of the patients, even though not formally tested for NBS1 gene mutations, comes from the same kindred of three other patients diagnosed by molecular sequencing, and shows clinical and cytogenetic signs of NBS.

7 patients from 3 different families of Pakistani origin have been reported by Gennery et al. (2004) and New et al. (2005) with a novel homozygous 1089C>A mutation of the NBS1 gene.
All patients were considered as candidates having Fanconi anemia (FA) before the homozygous mutation of the NBS1 gene was demonstrated. This was due to the fact that their clinical and cellular phenotype was consistent with both NBS and FA; their cell cultures showing sensitivity to both DNA cross-linking agents and ionizing radiation.
One of the patients with a presumed diagnosis of FA underwent Bone Marrow Transplantation (BMT), which represents, in fact, the first described instance of the use of BMT for NBS (see Patient Care and Treatment for more details).

Nibrin structure and function

Nibrin protein has two N-terminal functional domains, a forkhead-associated domain (24-102 a.a.) and a breast cancer carboxy-terminal domain  (108-196 a.a.), and a C-terminal hMre11-binding domain (665-693 a.a.). Several SQ motifs () are found at the central region of the Nbs1 protein, which represent potential targets of phosphorylation..

Click on the picture below to see a schematic representation of the nibrin protein functional domains.

Nbs1 is involved in the maintenance of genome stability, particularly in cellular response to DNA double strand breaks (DSBs). DSBs represent threatening lesions for cell survival and genome integrity that can be induced by exogenous agents (namely ionising radiation) as well as during physiological processes (e.g. immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements). Cellular response to DSBs is carried out through the concerted action of damage sensors, signal transducers and effectors of lesion repair. ATM (ataxia-telangiectasia mutated) protein has a central role in the DSB signaling cascade, phosphorylating several downstream substrates, including histone H2AX and Nbs1.
Nbs1 is recruited to the sites of DSB by the direct interaction of its FHA/BRCT domains with phosphorylated histone H2AX (γ-H2AX). Through interaction with nibrin C-terminal hMre11-binding domain, two other proteins, hMre11 and hRad50, are relocated from cytoplasm to the nucleus to sites of DSBs and the multimeric complex Nbs1/hMre11/hRad50 (N/M/R) forms foci at sites of DNA damage. The complex has DNA binding and nuclease activity, is essential for normal radiation sensitivity and seems to have a role in lesion processing and repair.
At least two different nibrin SQ motifs, at serine residues 278 and 343, are phosphorylated by ATM in response to DSBs. Nbs1 acts in the ATM-dependent cell cycle checkpoint activation cascade, possibly as a signal modifier/adaptor in multiple pathways. Intra-S phase checkpoint is mediated by two parallel routes, one of them involving ATM, NBS1 and SMC1. Nbs1 also seems to modulate ATM phosphorylation of other substrates, such as p53 and Chk2, in G1/S and G2/M transition control.
In NBS cells with biallelic truncating mutations N/M/R foci formation is not observed and defects in DNA DSB repair can result in chromosome abnormalities, such as translocations and inversions. At the same time dysfunction of the cell cycle checkpoint control leads to error-prone repair and damaged-DNA replication, and thus to genomic instability.

Click on the picture to see a schematic representation of the response to DNA damage involving Nbs1.
(click here before opening the picture if you want to have a legend)

Nibrin protein variants in NBS cell lines

Even if full-length p95 is not detectable by Western blotting in NBS cells with biallelic truncating mutations, low expression of abbreviated polypeptides of both N-terminal and C-terminal Nbs1 can be demonstrated in NBS lymphoblastoid cell lines with different mutations. Particularly, C-terminal peptides of lower molecular weight than p95 are detected by means of a co-immunoprecipitation assay, which maintain the ability to interact with hMre11.
A C-terminal 70 kDa protein is produced by internal translation initiation of the 657del5 allele and the same mechanism can be hypothesised also for the 835del4 and 900del 25 alleles which encode for a 60 kDa and a 55 kDa protein respectively.

Recently, the missense mutation R215W (643C>T) has been found at the compound heterozygous state (with the classic 657del5 mutation) in two monozygous twins (Seemanova et al., 2006). The patients presented with a severe form of NBS with neurological abnormalities and without chromosomal instability, and the Authors propose that the compound heterozygosity, 657del5/643C>T(R215W), is the primary cause of the clinical phenotype.
Full length nibrin was present in the lymphoblastoid cell lines of the patients, but with reduced expression when compared with controls and heterozygotes for the 657del5 or 643C>T mutation, as nibrin-Trp215 was demonstrated to be much less abundant than the wildtype nibrin-Arg215. The Authors suggest that this difference may indicate a lower expression level of the 643C>T(R215W) allele, a shorter half life of the mRNA or, more likely, reduced stability of the nibrin-Trp215 protein. This might in turn reflect an inability of the mutant protein to associate correctly with Mre11 and Rad50, and subsequent degradation of non-bound nibrin monomer. In cells with only a truncated nibrin as an alternative (657del5 in these patients), the 643C>T mutation might therefore lead to a reduction in active trimeric complex below a critical level. The missense NBS1 mutation could also interfere with the residual activity of the truncated protein from the 657del5 allele through a dominant negative effect, thus resulting in additional phenotypic effects beyond what might be expected from an absence or reduction of the protein, as normally occurs with truncating mutations (see also Clinical Phenotype and Cytogenetics and Cellular Phenotype).

Page last updated on: 18th May 2007