Genetic and cellular analysis of Italian patients with a Nijmegen breakage-like phenotype

In this work we have studied cells derived from five Italian patients characterised by growth delay, immunodeficiency and microcephaly. Because normal development of the immune system requires the introduction of DNA double strand breaks (DSBs) during antigen receptor gene assembly, defect in the repair of these specialised breaks can lead to profound immunodeficiency. The Mre11/Rad50/Nbs1 (M/R/N) complex is a component of both the nonhomologous end-joining and homologous-recombination pathways and plays a critical role in the repair of DNA double strand breaks. In humans, mutations in the Nbs1 gene lead to the autosomic recessive genetic disorder Nijmegen breakage syndrome (NBS), characterised by a bird-like face phenotype, immunodeficiency, developmental abnormalities and high frequency of malignancies. Cells of NBS patients show spontaneous chromosome instability, high sensitivity to ionising radiation, accelerated shortening of telomeres and abnormal cell-cycle checkpoints. NBS has been shown to be associated with a mutation in Nbs1 gene, which codes for the nibrin protein. About 90% of the NBS patients are of Slavic origin and carry the major founder mutation 657del5 in exon 6. To determine whether defects in factors known to be involved in DSBs repair could contribute to the clinical immunodeficiency observed in our patients, cellular and molecular characteristics of lymphoblastoid cells (LCLs) established from them, were analysed.PCR amplification of exon 6 showed the presence of both copies of the Nbs1 gene in all the investigated individuals. Furthermore, normal levels of proteins involved in the repair of DSBs and in cellular response to DNA damage (NBS1, Mre11, Rad50, ATM, ATR, Lig IV, XRCC4) were detected. Radiosensitivity of some of the LCLs analysed was shown by the induction of chromosome aberrations in a G2-phase assay. The efficiency to rejoin DSBs was evaluated by means of PFGE.In order to ascertain the proficency of DNA-damage response pathways, LCLs have been irradiated with 2-10 Gy of X-rays and proteins subjected to immunoblot with either a p53 antibody, a specific antibody for the phosphorylated form of p53, NBS1 or SMC1. We believe that dissecting the clinical and cellular phenotype of these as well as future NBS-like patients will help to identify a subset of individuals with the NBS clinical phenotype, whose analysis will allow for more clear-cut research of new genes involved in the maintenance of genome integrity.