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Association of the heterozygous germline I171V mutation of the NBS1 gene with childhood acute lymphoblastic leukemia

Leukemia volume 20, pages 1454–1456 (2006)Cite this article

Biallelic mutations of the NBS1 gene, localized on chromosome band 8q21.3, are responsible for Nijmegen breakage syndrome (NBS).1 The most frequent mutation of the NBS1 gene is a five base pair deletion, 657del5, which is often termed the Slavic mutation and for which over 90% of NBS patients are homozygous. Nibrin, the product of the NBS1 gene is a member of the MRE11/RAD50/Nibrin (M/R/N) protein complex involved in the repair of DNA double-strand breaks, immunoglobulin class switching, meiotic recombination, telomere maintenance and the response of the cell cycle to DNA damage. About 40% of NBS patients develop a malignancy before the age of 21 years, of these, approximately 70% have non-Hodgkin's lymphomas (NHLs). The unusually high incidence of malignancies among close relatives of NBS patients 2 suggests that heterozygous mutation of the NBS1 gene may lead to an increased cancer risk. Several studies have focused on searching for an association between mutations in the NBS1 gene and cancer incidence, especially of hematological malignancies. Although heterozygous carriers of the NBS1 657del5 mutation have been shown to have an increased risk for breast cancer, melanoma, colon and rectum cancer,3 other studies have found no association between NBS1 gene mutations and Hodgkin's lymphoma or NHL.4, 5, 6 In contrast, Varon et al.7 detected several point mutations in the NBS1 gene among 47 patients with childhood acute lymphoblastic leukemia (ALL). These point mutations all lead to amino acid substitutions, S93L, D95N, I171V and R215W. The mutation, I171V, of somatic and/or germline origin, was found in four ALL cases, but was not observed in normal controls, indicating a possible involvement of the NBS1 gene in that disease. The aim of the present study was to extend the analysis of this association by examining the NBS1 gene in Polish children with ALL.

Blood samples were collected from 135 children aged between 1 and 18 years of age, who were diagnosed with ALL and were under treatment at the Oncology and Hematology Clinic of the Medical University School in Poznañ. The selection criterion for the diagnosis of ALL was more than 95% lymphoblasts in bone marrow as demonstrated by fluorescence-activated cell sorter analysis. Using the French–American–British (FAB) classification, 100 of the patients had L1 lymphoblast morphology, 19 had L1/L2 morphology, 11 had L2 morphology and five were of L3 morphology. Approximately 94% of the cases were of the B-cell precursor type, of these 117 patients expressed the CD10 antigen, six had a pre-B immunophenotype, five patients had B-cell leukemia and five patients T-cell leukemia. The research protocol was approved by the Ethics Committee of the Medical University School in Poznañ. Anonymous blood samples collected on Guthrie cards drawn from the newborn screening program of the Wielkopolska voivodship were used as controls. Genomic DNA was isolated from whole blood using a DNA extraction kit (A&A Biotechnology, Gdynia, Poland). To confirm the germline origin of the mutations detected, DNA was also isolated from buccal swabs by incubation in 0.2 M NaOH for 5 min at 75°C and resuspension in 0.04 M Tris-HCl. DNA was extracted from Guthrie cards by boiling at 95°C for 90 min. Polymerase chain reaction (PCR) was used to amplify sequences from all 16 exons of the NBS1 gene. A set of 20 specific intronic primers was designed to amplify the exons as PCR products of 143–328 bp (Supplementary Table 1). The PCR mixture contained Taq DNA polymerase, buffer with 15 mM MgCl2 (Eppendorf, Hamburg, Germany), deoxyribonucleotide triphosphates (Sigma-Aldrich, Steinheim, Germany), primers (Oligo, Warszawa, Poland) and genomic DNA. Mutations and polymorphisms were detected by single-strand conformation polymorphism (SSCP). Briefly, the PCR product and loading buffer were mixed in a ratio of 1:3 and denatured for 5 min at 95°C, cooled and separated on a 7% non-denaturing polyacrylamide gel, with or without the addition of 5% glycerol, at 4°C or room temperature. Control samples with known mutations were run in parallel. Silver staining was used for the detection of DNA. The substitution, A>G at position 511 in exon 5 (I171V), was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The 328 bp PCR product of exon 5 was digested with MunI according to the manufacturer's instruction (Fermentas, Vilnius, Lithuania). Mun1 cleaves the PCR product from wild-type DNA to generate fragments of 130 and 198 bp, but does not cut products containing the 511A>G mutation. PCR-digests were analyzed on 2.5% agarose gels. PCR products showing shifts in SSCP or PCR-RFLP were purified and directly sequenced in the forward and reverse direction using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in the automated ABI PRISM 310 sequencer. The differences in the incidence of mutations and polymorphisms between the ALL group and controls were statistically evaluated by the two-sided Fisher's exact or the χ2 test. Differences were considered significant below P=0.05. In the case of polymorphisms, the wild-type genotype/allele served as a reference.

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Acknowledgements

We are grateful to Professor Dr Martin Digweed from the Institute of Human Genetics, Humboldt University in Berlin for the revision of the manuscript. We thank mgr Bogumiła Erenz-Surowy from Screening Research Laboratory in Poznañ for collecting Guthrie cards. This work was supported by the Ministry of Sciences and Informatics grant no PBZ-KBN-09.0/P05/2003

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  1. Institute of Human Genetics, Polish Academy of Sciences, Poznañ, Poland

    M Mosor, I Ziółkowska, M Pernak-Schwarz, D Januszkiewicz-Lewandowska & J Nowak

  2. Medical University School in Poznañ, Oncology and Hematology Clinic, Poznañ, Poland

    D Januszkiewicz-Lewandowska

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Mosor, M., Ziółkowska, I., Pernak-Schwarz, M. et al. Association of the heterozygous germline I171V mutation of the NBS1 gene with childhood acute lymphoblastic leukemia. Leukemia 20, 1454–1456 (2006). https://doi.org/10.1038/sj.leu.2404285

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