Letter to the Editor
1454
Japan E-mail:
[email protected] References
Figure 1 The JAK2 mutation in PV patient. Arrow shows a G-T transversion (V617F) in exon 12.
mutation in any of 22 JMML patients in our series.4 We conclude that the JAK2 V617F in pediatric MPD and leukemia patients is less frequent than that in adults, and that JAK2 mutation is rarely involved in the pathogenesis of pediatric hematologic malignancies.
M-J Park1, A Shimada1, H Asada2, K Koike3, M Tsuchida3 and Y Hayashi1 1 Division of Hematology/Oncology, Gunma Children’s Medical Center, Gunma, Japan; 2 Department of Pediatrics, Iwaki-kyouritsu General Hospital, Iwaki, Japan and 3 Department of Pediatrics, Ibaraki Children’s Hospital, Mito,
1 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 2 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 3 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature 2005; 434: 1144–1148. 4 Tono C, Xu G, Toki T, Takahashi Y, Sasaki S, Terui K et al. JAK2 Val617Phe activating tyrosine kinase mutation in juvenile myelomonocytic leukemia. Leukemia 2005; 19: 1843–1844. 5 Scott LM, Campbell PJ, Baxter EJ, Todd T, Stephens P, Edkins S et al. The V617F JAK2 mutation is uncommon in cancers and in myeloid malignancies other than the classic myeloproliferative disorders. Blood 2005; 106: 2920–2921. 6 Shimada A, Taki T, Tabuchi K, Tawa A, Horibe K, Tsuchida M et al. KIT mutations, and not FLT3 internal tandem duplication, are strongly associated with a poor prognosis in pediatric acute myeloid leukemia with t(8;21): a study of the Japanese Childhood AML Cooperative Study Group. Blood 2006; 107: 1806–1809. 7 James C, Delhommeau F, Marzac C, Teyssandier I, Couedic JP, Giraudier S et al. Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis. Leukemia 2005; 20: 350–353. 8 Levine RL, Loriaux M, Huntly BJ, Loh ML, Beran M, Stoffregen E et al. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 2005; 106: 3377–3379.
Association of the heterozygous germline I171V mutation of the NBS1 gene with childhood acute lymphoblastic leukemia Leukemia (2006) 20, 1454–1456. doi:10.1038/sj.leu.2404285; published online 29 June 2006
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. Leukemia
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.4–6 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 Poznan˜. 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
Letter to the Editor
1455 Table 1
Germline mutations of the NBS1 gene identified among common ALL patients
Mutation
Codon change
Patients
Remarks
283 A4G 511 A4G 511 A4G 511 A4G 511 A4G 511 A4G 628 G4T 643 C/T del ACAAA, FS, X234
D95N I171V I171V I171V I171V I171V V210F R215W 657del5
B B B B B B B B B
Death after ALLO-BMT owing to progression of disease Remission Remission Remission Death after ALLO-BMT owing to progression of disease Remission Second tumor (ependymoma) Remission Remission
32 7 106 120 139 160 60 9 16
Table 2 Statistical analysis of the occurrence of NBS1 gene mutations in ALL patients and controls Mutation
ALL (n)
D95N I171V V210F R215W 657del5 Total
1 5 1 1 1 9
(135) (135) (135) (135) (135) (135)
Controls (n) 1 1 0 0 1 3
(195) (500) (195) (195) (195) (195)
OR (95% CI) 1.45 19.34 4.36 4.36 1.45 4.57
(0.09–23.37) (2.24–167.1) (0.18–107.9) (0.18–107.9) (0.09–23.37) (1.21–17.22)
P 1.000 0.002a 0.409 0.409 1.000 0.014a
Abbreviations: ALL, acute lymphoblastic leukemia; CI, confidence interval; OR, odds ratio. a Result statistically significant (Po0.05).
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 Poznan˜. 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 751C and resuspension in 0.04 M Tris-HCl. DNA was extracted from Guthrie cards by boiling at 951C 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 951C, cooled and separated on a 7% nondenaturing polyacrylamide gel, with or without the addition of 5% glycerol, at 41C or room temperature. Control samples with known mutations were run in parallel. Silver staining was used for the detection of DNA. The substitution, A4G 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 511A4G 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 BigDyes 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 w2 test. Differences were considered significant below P ¼ 0.05. In the case of polymorphisms, the wild-type genotype/allele served as a reference. In our study, we identified germline NBS1 gene mutations in peripheral blood cells from nine of 135 ALL patients (Table 1) and in three of 195 controls, P ¼ 0.014, OR ¼ 4.57 (95% CI: 1.21–17.22) (Table 2). The analysis of DNA isolated from oral epithelium cells confirmed the germline origin of the observed mutations in all cases. We identified the D95N variant in exon 3 in one c-ALL case and in one control. This variant occurs in the conserved FHA domain of nibrin and was first described by Varon et al.7 in German ALL patients. Here we could extend these results by confirming the germline origin of the mutation. The D95N variant was not observed among 321 British patients with lymphoid malignancies8 nor in 91 North American patients with NHL.4 However, in both studies D95N was detected among healthy controls (1/332 and 2/133, respectively). We also detected the 657del5, R215W and V210F mutations in exon 6 of the NBS1 gene. The association of the 657del5 mutation with increased risk of hematological cancer has been recently shown.3,5 In our study, we observed only one 657del5 mutation in c-ALL patients (1/134) and one in controls (1/195). Similar results were reported by several authors who failed to detect a significant contribution of the 657del5 mutation to lymphoproliferative disease development among both Slavic populations 5 and non-Slavic populations, such as in Germany 5 or Britain.8 The 643C4T mutation was identified in only one c-ALL patient in our study. This transition leads to substitution of the basic amino acid arginine with the nonpolar tryptophan (R215W). Recently, it was shown that heterozygous carriers of germline R215W may have an elevated risk of malignancy, especially of the colon and rectum.3 The reports on the R215W mutation in lymphoproliferative diseases are controversial. This variant was described in one of 47 German patients with T-ALL,7 in four of 231 British leukemia cases (two c-ALL, one T-ALL and one AML) and in one of 51 NHL patients.8 It could be postulated that the R215W mutation in the Polish population does not contribute to the development of ALL. In accordance with our results is the observation of Chrzanowska et al.,5 who Leukemia
Letter to the Editor
1456 found no carriers of the R215W mutation among 545 Polish pediatric patients with lymphoid malignancies. A further mutation in exon 6, identified in a single individual only, was the amino-acid substitution V210F. The same alternation has been reported in one of 60 ALL cases and in one of 91 NHL cases.4,7 Interpretation of these results is limited by the rather low numbers. The most frequent mutation found was the transition 511A4G in exon 5 leading to the amino-acid substitution, I171V. We identified five heterozygous carriers of the I171V mutation among 135 Polish patients with ALL and only one under controls. Because of the high frequency of the I171V mutation the control group was extended to 500 individuals, however, no further carriers were found. The results obtained were statistically significant (P ¼ 0.0020, OR ¼ 19.34 (95% CI: 2.239–167.1)) suggesting that the I171V mutation is indeed associated with ALL (Table 2). Varon et al.7 found four carriers of the I171V mutation in 47 German children with first-relapse ALL, (two – common ALL, one – preB ALL, one – preT ALL). The origin of the mutation was somatic and/or germ line. Taylor et al.8 did not detect this variant in 231 British leukemia patients or controls, but did find it in one patient with hepatic lipase (HL). Thus, ethnic differences and a possible contribution of other variants of the NBS1 gene in different populations could be an explanation for the discrepancy between reported findings. The I171V mutation, in the homozygous state, was described in a Japanese pediatric patient (NCC56) with aplastic anemia (AA).9 Furthermore, results of a cytogenetic analysis in lymphoblastoid cells from the patient and her father, who was heterozygous for the I171V mutation, showed a higher frequency of chromosomal structural aberrations, suggesting genomic instability owing to this mutation. So far, the I171V mutation has been described only among patients with ALL, HL, AA and in control groups. The substitution occurs in the BRCA1 C-terminal domain of nibrin, which is widely conserved and is required for either binding to histone gamma-H2AX or relocalization of the MRE11/RAD50 nuclease complex to the vicinity of DNA damage.10 In addition, we identified novel rare sequence variants in the introns flanking exon 7, IVS6-18G4A and IVS7 þ 36G4A, and two intronic variants, IVS6-29C/T and IVS15 þ 88G4C, recently described in lymphoma patients.4 Each of these variants was observed either in ALL patients or in control samples. Comparisons of the frequencies of rare sequence variants between ALL cases and controls showed no significant differences. One new polymorphism, IVS14-30A/T, and five previously described polymorphisms, 102G4A, 553G4C, IVS9 þ 18C4T, 1197T4C and 2016A4G were detected in both groups. No significant differences in the distributions of genotype and allele frequencies for each of the analyzed polymorphisms were observed in a comparison of ALL cases and controls (data not shown). Our results indicate that heterozygous carriers of the I171V NBS1 gene mutation may exhibit increased susceptibility to childhood ALL. Given the high incidence of heterozygous I171V mutations in patients in remission from ALL (our results) or with relapsed ALL7 and the genomic instability reported in heterozygous cell lines,9 we conclude that the I171V mutation must
indeed be considered as a susceptibility factor for the development of ALL.
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 Bogumi"a Erenz-Surowy from Screening Research Laboratory in Poznan˜ for collecting Guthrie cards. This work was supported by the Ministry of Sciences and Informatics grant no PBZ-KBN-09.0/P05/2003
M Mosor1, I Zio´"kowska1, M Pernak-Schwarz1, D Januszkiewicz-Lewandowska1,2 and J Nowak1 1 Institute of Human Genetics, Polish Academy of Sciences, Poznan˜, Poland and 2 Medical University School in Poznan˜, Oncology and Hematology Clinic, Poznan˜, Poland E-mail:
[email protected] References 1 Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrzanowska KH, Saar K et al. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 1998; 93: 467–476. 2 Seemanova E. An increased risk for malignant neoplasms in heterozygotes for a syndrome of microcephaly, normal intelligence, growth retardation, remarkable facies, immunodeficiency and chromosomal instability. Mutat Res 1990; 238: 321–324. 3 Steffen J, Varon R, Mosor M, Maneva G, Maurer M, Stumm M et al. Increased cancer risk of heterozygotes with NBS1 germline mutations in Poland. Int J Cancer 2004; 111: 67–71. 4 Cerosaletti KM, Morrison VA, Sabath DE, Willerford DM, Concannon P. Mutations and molecular variants of the NBS1 gene in non-Hodgkin lymphoma. Genes Chromosomes Cancer 2002; 35: 282–286. 5 Chrzanowska KH, Piekutowska-Abramczuk D, Popowska E, Gladkowska-Dura M, Maldyk J, Syczewska M et al. Carrier frequency of mutation 657del5 in the NBS1 gene in a population of Polish pediatric patients with sporadic lymphoid malignancies. Int J Cancer 2006; 118: 1269–1274. 6 Rischewski J, Bismarck P, Kabisch H, Janka-Schaub G, Obser T, Schneppenheim R. The common deletion 657del5 in the Nibrin gene is not a major risk factor for B or T cell non-Hodgkin lymphoma in a pediatric population. Leukemia 2000; 14: 1528–1529. 7 Varon R, Reis A, Henze G, von Einsiedel HG, Sperling K, Seeger K. Mutations in the Nijmegen Breakage Syndrome gene (NBS1) in childhood acute lymphoblastic leukemia (ALL). Cancer Res 2001; 61: 3570–3572. 8 Taylor GM, O’Brien HP, Greaves MF, Ravetto PF, Eden OB. Correspondence re: R. Varon et al., Mutations in the Nijmegen breakage syndrome gene (NBS1) in childhood acute lymphoblastic leukemia. Cancer Res 2001; 61: 3570–3572, Cancer Res. 2003 Oct 1;63(19):6563-4; author reply 6565. 9 Shimada H, Shimizu K, Mimaki S, Sakiyama T, Mori T, Shimasaki N et al. First case of aplastic anemia in a Japanese child with a homozygous missense mutation in the NBS1 gene (I171V) associated with genomic instability. Hum Genet 2004; 115: 372–376, E-pub 2004 August 24. 10 Kobayashi J, Tauchi H, Sakamoto S, Nakamura A, Morishima K, Matsuura S et al. NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr Biol 2002; 12: 1846–1851.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Leukemia
