Mutations of the N-ras Gene in Juvenile Chronic Myelogenous Leukemia By Jun Miyauchi, Minoru Asada, Michiko Sasaki, Yukiko Tsunematsu, Seiji Kojima, and Shuki Mizutani Juvenile chronic myelogenous leukemia (JCML), a myelostitutions involvingcodons 12.13, or 61 in six of 20 patients proliferative disorderofchildhood,isdistinct from adult(30%).Four of six patients with mutations were in chronic (CML) and bears resem- phase andthe other two in blast crisis, indicating no appartype chronic myelogenous leukemia (CMMoL). Since blanceto chronic myelomonocytic leukemia ent correlation with disease stage.Most of the patients with mutations in the N-ras gene have been found at high fremutations were in the older age groupwith poor prognosis, quencies in CMMoL, but only rarely in CML, we analyzed although onepatient in the younger age group also harbored mutations activating the N-ras gene in 20 patients with the mutation. These data suggest that N-res gene mutations JCML. We used the strategy for analysisof gene mutations may be involved in the pathogenesis andlor prognosis of based on in vitro DNA amplification by polymerase chain JCML and provide further evidence that JCML is an entity reaction (PCR) followed by single-strand conformation poly- distinct from CML. morphism (SSCP) analysis andlor direct sequence analysis. 0 1994 by The American Societyof Hematology. Nucleotide sequence analysis showed single nucleotide sub- J UVENILE CHRONIC myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood. JCML apparently arisesfrom multipotenthematopoietic stem cells.”3 Although, as its name indicates, this disorder shares some other clinical findings with adult-type chronic myelogenous leukemia (CML) withPhiladelphia chromosome (Phl) abnormality, it is considered to be a distinct entity because of the many differences and its uniquefeatures’: (1) peripheral blood leukocytosis that is not as severe as in CML andaccompanies monocytosis; (2) elevatedmarrowblast counts at diagnosis; (3) anemia with increased synthesis of hemoglobin F and fetal-type erythropoie~is,~possibly through hypersensitivity to erythropoietin5; (4) thrombocytopenia; (5) elevated serum levels of lysozyme, vitamin B,,, andimmunoglobulins;(6) normal karyotype in mostinstances: and, if present, no consistent chromosomal abnormalitieswitha slight preponderance tomonosomy 7 and trisomy 8,’ but uniformly no Ph’ chromosome;(7) spontaneous macrophage-dominant colony formation posin sibly through autocrine orparacrine production of cytokines, including interleukin (1L)- 1 andgranulocyte-macrophage colony-stimulating factor (GM-CSF),9.’o orhypersensitivity to GM-CSF”; (8) an association with skin rash and neurofibromatosis; (9) rapid progression of disease and poor prognosis; and (10)younger age distribution. These features overlap with those of chronic myelomonocytic leukemia (CMMoL) and suggest that JCML is similar to, and may be a variant form of, CMMOL’ or Ph’-negative CML,” which also overlaps CMMoL. From the Department of Virology, National Children’s Medical Research Center, Clinical Laboratory and Department of Pediatrics, National Children’s Hospital, Tokyo; Urawa Municipal Hospital, Urawa, Saitama; and the Nagoya First Red Cross Hospital, Nagoya. Aichi, Japan. Submitted August 16, 1993; accepted December 8, 1993. Address reprint requests to Jun Miyauchi, MD, Clinical Laboratory, National Children’s Hospital, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8308-0030$3.00/0 2248 Among the oncogenes, the ras genes are the most commonly involved in human neoplasm^.'^,'^ It is well known that, in hematological malignancies, abnormalities predominate in the N-ras gene, and that these are associated with certain types of disorders. Point mutations of this gene have been found in approximately 15% to 40% of patients with acute myelogenous leukemia (AML),15-2‘)109o to 40% of those with myelodysplastic syndrome (MDS),’6,21-23 and 10% to 20% of those with acute lymphoblastic leukemia (ALL).’6.24.2sIn CMMoL, now categorized as a subtype of MDS in the French-American-British (FAB) classification, or in Ph’-negative CML, mutations of the N-ras gene have been found to be a relatively common abnormality (20% to 6090),12.22,23,26,27while they have been only rarely detected in CML.Ih.28.29 Furthermore, mutations of the N-rus gene have been found to bean extremely rare abnormality in Ph’-positive acute leukemias, including which suggests that activation of the N-ras gene is not a major contributor to the development of Phl-related leukemias. These considerations raise a question whether activation of the N-ras gene might play a role in JCML. Although only smallnumbers of patientswith JCMLhave beenstudied19.27.”.’2 and mutations of this gene reported, indicating a positive role of this gene in JCML, systematic investigations withalargenumberofpatients are lacking,presumably because of the rarity of this disorder. To clarify more precisely the incidence and clinical significance of N-ras gene mutations in JCML and to disclose the pathogenesis of this peculiar disorder, DNA samples collected from 20 patients with JCML, the largest seriesof patients ever studied, were analyzed, and clinical parameters were correlated with abnormalities of the N-vas gene. MATERIALS AND METHODS Patients. Twenty patients with JCML were studied. Clinical data of the patients are listed in Table 1. The diagnosis of JCML was based on a synthesis of data from physical and laboratory examinations,includinghepatosplenomegaly,increasedWBCcountswith all stages of granulocytic differentiation and with monocytosis, increased ratio of hemoglobin F, elevated serum levels of lysozyme and vitamin B,?, and absence of Phi-chromosome, because none of these alone was specific or diagnostic for JCML. Since myeloproliferative disorders associated with monosomy 7 are known to overlap JCML,”~” and since thereare no established criteria for differential diagnosis, we included patients with JCML who had monosomy 7. Blood, Vol 83, No 8 (April 15). 1994: pp 2248-2254 N-RASMUTATIONS IN JCML 2249 Table 1. Summary of Patients and Clinical Data Liver/ Spleen (cm) Outcome (stage) [survival after onset] 3115 4/9 W10 516 312 10115 7112 415 5/3 514 518 415 Alive(CP) I 3 vr. _ .6 mol Alive (CP) 15 yr, 1 mol Alive (CP) I3 yr. 7 mol Alive (CP) 11 yr, 0 mol Dead (BC) I10 mol Dead (CP) l1 yr. 2 mol Dead (EL) 12 mol Dead (CP) I3 yr, 10 mol Dead (CP) I1 yr, 1 mol Dead (BC! I 8 mol Dead (BC) 11 yr. 0 mol Alive (CP) 11 yr. 5 mol 10110 814 Dead (BC) I8 mol Dead (CP) 16 mol 4/7 5f2 215 12/8 210 Dead (BC) l2 yr, 2 mol Dead (BC) I2 yr, 6 mol Dead (BC) 14 mol Dead (BC) I10 mol Dead (CP) 14 mol Peripheral Blood Patient No. Age/Sex (onset) l* 2 3 4 5 6 7 8 9' 11 12 2 mo1M 2 mom 2 mo1F 3 mo1M 7 mo1M 8 mo1F 10 mo1F 1 yr/F 1 yrlF 2 yr/F 2 yr1M 2 yr1M 364 347 304 452 351 259 277 443 140 394 40 1 39 1 40 5131.2 1.1 29 2 43.34.5 10 2100.0 4.9 31 69.1 5.0 18 72.218.4 20 44.0 1.3 13 3.5 36.0 24 7.4 1 12.2 6 64.3 2.4 3 59.42.5 53 5.3 40.2 32 2 45.21.7 13' 14 3 yr/M 3 yrlM 456 343 61 35 15 16* l?* 18 19 20 4 yrlM 4 yrfM 4 yrlM 4 yr1M 5 yr/M 5 vr/F 406 359 254 278 303 226 31 58 89 1 8 3 lo* RBC PLT HbF (%) (xlO') WBC ( ~ 1 0 ' ) (xlO') Mb Mono (%l (%l LAP Score 17 16 14 14 12 13 8 8 17 56 17 7 19.3 94 212 31.0 103 126 282 91 223 70 43.0 171 9 103 40.7 73 1 7 22 5 5 4 7 2 0 0 2 1 14 3 2 9.2 61.6 0 0 9 9.2 224 17 28 59.9 31.3 5.8 10.0 6.0 16.8 1.3 11.8 2.4 4 45.7 2.3 2 32.2 5 3 8 0 15 18 26 14 8 38 5.5 3.5 Bone Marrow Mb(%) 226 12.6 207 35.0 203 ND 24.6 100 213 5 10 4 9 0 8 4 1 5 25 27 2 Serum Lysozyme (pg/mL) 19.2 31.4 57.5 ND 11.3 86.8 ND 20.8 21.1 24.8 10.8 ~ Chromosome Normal Normal Normal Normal 45, XY. -7 ND Normal Normal Normal ND Normal 46, XY, del(7) (q22) Normal 46, XY, inv(4) (~14~16) Normal Normal Normal 45, XY. -7 Normal 29.6 Patients with mutations of the N-ras gene. Source of DNA. Peripheral blood or bone marrow was obtained after receiving informed consent, and mononuclear cells were isolated by density sedimentation. Total DNA was prepared by lysis of the cells with sodium dodecyl sulfate (SDS) and digestion with proteinase K at 37°C overnight followed by phenollchloroform extraction and ethanol precipitation. Because patients no. 5, 7, 10, 11, 16, and 17 were autopsy cases and fresh cells were not available, DNA was extracted from formalin-fixed paraffin-embedded 10-pm thick tissue sections according to the method described by Goelz et al.'4 Lymph nodes with diffuse leukemic cell infiltration at autopsy and, when available, those obtained by biopsy in chronic phase were used. The DNA extracted from paraffin-embedded tissue sections was purified by using a silica-binding product (Geneclean; Bio 101, La Jolla, CA) before applying it for polymerase chain reaction (PCR). PCR arnpl$cation. Two genomic regions encompassing codons 12/13 and 61 in exons 1 and 2, respectively, were amplified by PCR using two sets of 20-bp synthetic oligodeoxynucleotide primer^.'^ The primers used for these amplifications were sense S'CTGGTGTGAAATGACTGAGT3' and antisense S'GGTGGGATCATATTCATCTA3' for exon 1, and sense 5'GlTATAGATGGTGAAACCTG3' and antisense 5'ATACACAGAGGAAGCClTCG3' for exon 2. Biotinylated primers were also used instead of one of theunlabeled primers. These biotinylated primers, prepared according to the published sequences,35 are sense S'GCTCGCAATTAACCCTGATTAC3' for exon 1 and antisense S'GAClTGCTATTATTGATGGCAA3' for exon 2. A total of 500 ngof genomic DNA was amplified in a 25-pL reaction buffer containing 20 pm01of each primer, 200 pmol/L of deoxynucleotide triphosphate, 1.5 mmoVL of MgCI,, and 1 U of Taq polymerase (AmpliTaq; Takara Biomedicals, Kyoto, Japan). DNA was denatured at 97°C for 7 minutes, then quickly chilled on ice and Taq polymerase added. Reactions were performed for 30 cycles of denaturation at 94°C(30 seconds), annealing at 58°C (30 seconds), and extension at 72°C (1 minute), followed by final elongation at 72°C for 12 minutes in athermal cycler (Perkin Elmer Cetus, Norwalk, CT). Some samples gave undesired reaction products with different molecular sizes in addition to target products. The target PCR products from these samples were gel-purified using a silica-binding product (Geneclean). Approximately 100bp of target DNA sequences thus obtained was used for subsequent single-strand conformation polymorphism (SSCP) and/or direct sequence analyses. SSCP analysis. SSCP analysis was performed according to the method previously described by Orita et al.%One microliter of the amplifiedPCR products was subjected to 18 additional cycles of E R ina 25-pL solution containing 200 pmoVL each of MTP, dGTF', and dTTP, 1 pL of [a-'*P]dCTP (Amersham, Buckinghamshire, UK; 10 mCi/mL), 20 pmol of sense and antisense primers, 10 ~ o U of L Tris-HC1 (pH 8.3), 50 ~ ~ o of U KCl, L 1.5 ~ ~ o of V L MgC12, and 1 U of Taq polymerase in a thermal cycler. PCR was performed under thermal conditions identical to those used for the first PCR. After amplification, 1 p L of the reaction product was mixed with 19 pL of a solution of 0.1% SDS and 10 mmoyL of EDTA. Then, 2 pL of the diluted product was mixed with 2 pL of a solution of formamide, 20 mmoVL of EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol, denatured at 8WC,and directly applied to a 6% polyacrylamide gel containing 90 mmoVL of Trisborate (pH 8.3), 4 m m o a of EDTA, and 10% glycerol at 4°C. Direct sequence analysis. For direct sequencing, 25 cycles of asymmetric E R , using a 50 to 100 reduction of one of the primers MlYAUCHl ET AL 2250 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 N C Fig 1. SSCPanalysisof PCR products amplified for the region encompassing codons12 and 13 ofthe N-ras gene. Samples are from patients no. 6 (lane 11, 15 in chronic phase llane 2). 11 llane 3). 13 (lane 41.1 (lane 5),10 (lane 6). 8 (lane 71.2 (lane 8). 3 (lane 9). 18 (lane 101. 16 in chronic phase (lane 11). 14 (lane 121, and 19 (lane 13). N. normal control; C, DNA double-strand control without heat denaturation; ds, double strand; ss, single strand. and 1 pL of the initial PCR products,wereperformedunderthe same thermal conditions as described earlier. The resulting singlestranded DNA was purified and sequenced by the dideoxynucleotide terminator method described by Sanger et aL3' using a sequencing kit (United States Biochemical, Cleveland, OH) and analyzed on a polyacrylamidegelcontaining 7 moVLurea.Electrophoresis was performed at 35 W for 2 hours at roomtemperature, then thegel wasdriedonfilterpaper and autoradiography was performed. For the PCR products amplified usinga set of biotinylated and unlabeled primers, the single-stranded biotinylated PCR products were separatedfromtheunlabeled PCR productsusingstreptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin: Dynal, Oslo, Norway).'* These single-stranded templates were sequenced by the dideoxy chain termination method using a Bca-BEST kit (Takara Biomedicals) and analyzed after polyacrylamide gel electrophoresisand autoradiography as described earlier. RESULTS SSCP analysis of point mutations. For screening of mutations in exons 1 and 2 of the N-rus gene, amplified PCR products were subjected to SSCP analysis. Figure 1 shows the results of SSCP analysis on exon 1. A mobility shift of the single-stranded PCR products was seen with samples from patients no. IO and 13: normal bands were not evident in either of these patients. The remaining patients showed a migration pattern identical to that of normal controls. However, as described later, direct sequencing showed point mutations at codon 13 of the N-rus gene in patient no. 1, who showed a normal migration pattern in SSCP analysis (Fig l). This result in SSCP analysis was therefore considered to be false-negative. Similar false-negativity was also present in one (no. 17) of 14 patients examined for exon 2 (data not shown). For this reason, direct sequence analysis was performed for all samples. Direct sequence analysis. Direct sequence analysis showed point mutations at codons 12, 13, or 61 of the N ras gene in six patients. In patient no. I , single nucleotide substitution at codon 13 from GGT to GAT (amino acid substitution from glycine to aspartic acid) was detected with concomitant normal sequences (Fig 2). although SSCP analysis did not disclose this mutation, as described earlier. Peripheral blood cells of this patient 3 years after the initial examination had the same mutation at codon 13 (data not shown), although the patient's hematologicalfindings remained well controlled within, or nearly within, the normal limits during this period. In patient no. 13,a single nucleotide substitution at codon 12 from GGT to TGT (amino acid substitution from glycine to cystein) was identified withconcomitant normal sequences (Fig 2). The identical point mutation was also detected in patient no. IO (data not shown). Two simultaneous point mutations were present at codons 12 and 13 in patient no. 9 (Fig 2). Codon 61 mutation was identified in patient no. 17 as a single nucleotide substitution from CAA to CTA (amino acid substitution from glutamic acid to leucine) with concomitant normal sequences (Fig 3). The same point mutation atcodon 61 was also identified in patient no. 16 (data not shown). Frequency and distribution of N-ras gene mutations in JCML. The results of sequence analysis are listed in Table 2. The mutations were present in four patients in chronic phase (no. l , 9, 13, and 16) and in two patients at blast crisis (no. IO and 17). indicating no particular preponderance of mutations at certain disease stages. Three patients (no. 7, 15, and 16) were examined at both chronic-phase andblastcrisis stages. Among these three, onlypatientno. 16 had the mutation, which was present in the chronic phasebut disappeared at blast crisis. A comparison of the gene analysis with clinical information showed no particular clinical parameters thatwere closely relatedtothepresence of the N-ras gene mutations (Table l). Regarding prognosis, our patients could be categorized into two groups with relatively poor or good prognosis, with a demarcating line in age between those older and younger than 6 months old, respectively: 15 patients excluding one in the older age group died 13 months on average after the onset, whereas four patients in the younger age group are all alive at an average of 40 months after the onset. Although most (five of six) of the patients with N-rus gene mutations were in the older age group, one of the four patients in the younger age group also harbored the mutation. The number ofpatients examined was insufficient to allow a statistical analysis of the difference in frequency of the mutations between the two groups, and the relevance of the mutations to prognosis remains uncertain. DISCUSSION Mutations of the N-rus gene were detected in six of 20 patients (30%) with JCML in the present study. This frequency is similar to the incidence reported for AMLI5-l9or C M M O L , ~ 'and . ~ ~is clearly different from that reported for CML, in which mutations have been detected only r a r e l ~ . ' ~ This . ~ " ~result ~ indicates that abnormalities of this gene are involved in a substantial proportion of patients with JCML, and supports the view thatJCML is a disorder distinct from CML and similar to, and possibly a variant of, CMMoL or Ph'-negative CML. Regarding leukemias, mutations of the N-ras gene have been reported to occur preferentially in those that involve monocytic lineage, ie,M4andM5 of 2251 N-RASMUTATIONS IN JCML Patient I Normal Patient 13 Patient 9 G A T C G A T C G A T C C A T C 3' T codon 13 G Fig 2. Direct sequence analysis of PCR products corresponding t o codons 12 and 13 of the N-rssgene. Arrowheads indicate mutated sequences. (Results shown in the figure were allobtained usinga set of biotinylated and unlabeled primers). ~ G! T codon 12 G G A C I G S AML in the FAB c l a s s i f i c a t i ~ n ' ~ ~ 'and ~ ' CMMOL.'"'~ ~.'~~~~ Our present data on JCML are consistent with this observation, since JCML is a myeloproliferative disorder affecting primarily the cells of myelomonocytic lineage; spontaneous macrophage-dominant colony formation in vitro'."9is one of the most characteristic and unique features of JCML. It has been suggested that this in vitro spontaneous growth of leukemia cells is caused by IL-Io or GM-CSF"' secreted in autocrine or paracrine fashion and, more recently, hypersensitivity to GM-CSF has been suggested to playthe major role in the pathogenesis of this disorder.'' On the other hand, the rus genes are known to code for proteins that are associated with plasma membrane, have GTPase activity, and participate in the transduction of signals, including those induced by growth factors."." Taken together, thepoint mutations in the N-rus gene might affect signal transduction through GM-CSF and be involved in the establishment of this unique feature of JCML, although activation of other oncogenes that also affects GM-CSF signal transduction may be involved in other patients without abnormalities in the N-rus gene. Mutations of the N-rus gene were detected in four patients PB in chronic phase and two patients at blast crisis, suggesting 3' Normal G A T C Patient 17 G A T C A A G codon 61 A A C[ PB CP G G T C G 5' Fig 3. Codon 61 point mutation of the N-ms gene in patient no. 17 detected by directsequence analysis with PCR-amplifiedproducts. Arrowhead indicates the mutated sequence. (Results shown in the figure were obtained using unlabeled primers.) that apparently there is no correlation between the presence of N-rus gene mutations and progression to blast crisis. In patient no. 16, a mutation was present at codon I3 in chronic phase, but disappeared after blast crisis. The disappearance of initially detected r n u t a t i ~ n s ' or ~ ~appearance ' ~ ~ ~ ~ ~of~ ~new ~~~ mutations at relapse'"'' has been reported in AML or ALL. The recent studies reported by Bashey et aL3' who detected mutations of the N-rus gene in varying proportions of, but not all, leukemic colonies in vitro, and that reportedby Kubo et al."" who found polyclonality of rus gene mutations in AML, suggest that the whole population of cells in a leukemic clone does not harbor the mutations and, therefore, that the mutations of the N-rus gene are not involved in the initial step of leukemogenesis. In the case of patient no. 16, it is Table 2. Summary of N-rssGene Mutations in JCML Patient No. 1 2 3 4 5 6 7 8 9 BC 10 11 PB 12 13 14 15 16 17 18 19 PB20 Stage PB CP CP CP CP BC CP CP EL CP CP BC CP PB CP CP BC CP BC BC CP CP CP Material N-ras Mutation Codon 13, GGT1Glyl-t GATIAspl BM BM LN PB LN LN BM BM LN LN - - - Codon 12, GGTIGlyl Codon 13, GGTIGlyl Codon 12, GGTIGlyl - Codon 12, GGTIGlyl BM PB LN LN LN BM BM - Codon 61, CAAIGlnl Codon 61, CAAIGlnl - -- TGTICysl GATIAspl TGTlCysl -. TGTlCysl - CTAlLeul CTAlLeul - Abbreviations: CP. chronic phase; BC, blast crisis; EL, erythroleukemic phase; PB, peripheral blood; BM, bone marrow; LN, lymph node. 2252 possible that the disappearance of the cells with the mutation at blast crisis was caused by the overgrowth of subclones that did not harbor the mutation. In the case of patient no. 1, in contrast, the same point mutation at codon 13 of the N-rus gene was detected 3 years after the initial examination, during which the patient’s hematological features remained well controlled. Therefore, the presence of mutations of the N-rus gene alone may not be sufficient to cause aggressive malignant disease. Evidence for this possibility has been provided by Finney and Bishop:’ who showed that the activated H-rusl allele is not by itself dominant over the normal allele, and requires other events, such as amplification of the mutant allele, to transform cells, and by Cohen and Levinson:’ who demonstrated thatthe transforming activity of the H-rus gene is regulated by a second single nucleotide alteration in the last intron. Clinical information from the patients was compared between those with and without mutations of the N-ras gene (Table l), but no particular correlations between the presence of mutations and clinical parameters were noted. Concerning the prognosis of JCML, data have been provided by other investigators that there are two subgroups of patients with JCML, which differ in mortality rates43;these investigators found that short-term survivors could be predicted based on initial clinical characteristics, including older age ( 2 2 years). Another group of investigators also reported a different prognosis between two groups of patients, namely, infants (< 1 year old) with good prognosis and children ( 21 year old) with poor pr0gnosis.4~Regarding our patients, a clear distinctionwas made between those older and youngerthan 6 months of age, who showed poor and good prognosis, respectively, a finding in agreement with the data described earlier that older patients have a poor prognosis. Althoygh most (five of six) of the patients with N-rus gene mutations were in the older age group, the number of patients was insufficient for statistical analysis and did not allow a clearcut conclusion as to the prognostic role of N-ras gene mutations in JCML. Nevertheless, it remains possible that the mutations might play a role in the establishment of a more malignant nature of the disease in the older age group. On the other hand, the younger age group may be heterogeneous, as has been suggested by others: and may include patients with nonleukemic perturbed hematopoiesis of unknown cause. However, the presence of a patient with N-ras gene mutation in the younger age group suggests that these two age groups are not distinct and that the presence of mutations of the N-rus gene by itself does not necessarily indicate poor prognosis. JCML may affect a spectrum of patients ranging from infants to older children, the former group having neoplasms of less developed malignant nature, possibly because of a less frequent chance of a second hit to the genes that cause more aggressive biological behavior. It is well known that myeloproliferative disorders associated with monosomy7 closely resemble JCML and that there is substantial overlap between the two.32,33 Although some investigators suggested the usefulness of elevated fetal hemoglobin as a diagnostic criterion for JCML versus monosomy 7, there have been no widely accepted ways of distin- MlYAUCHl ET AL guishing between the two. We therefore includedthree patients with JCML associated withmonosomy 7 in our series. Although a slightly lower incidence of mutations of the N-rus gene inmonosomy 7 syndrome hasbeen reported,” and none of our patients with chromosomal abnormalities including monosomy 7 and a related aberrant karyotype (deletion 7q22) harbored mutations, the significance of the difference in the frequency of mutations of the N-rus gene needs to be further clarified. SSCP analysis detects nucleotide sequence alterations as electrophoretic mobility shifts of single-stranded nucleic acids caused by changes in their three-dimensional structure. Although it has been considered that alterations in nucleotide sequence may notnecessarily affect electrophoretic mobility, a perfect concordance with the results of direct sequence analysis has been d e m o n ~ t r a t e d . ” . However, ~ ~ . ~ ~ we found false-negativity in SSCP analysis in two of 27 patient samples inthis study. The sensitivity of SSCP and direct sequence analyses, namely, the minimal proportion of abnormal cells detectable by these methods, has been estimated to be similar, ie, less than 10%.’7”6A5”7”8 In patient no. 1, a substantial proportion of abnormal cells was present as judged from the density of mutated and wild-type bands in direct sequence analysis (Fig 2), indicating that falsenegativity in SSCP analysis is unlikely to be ascribable to sensitivity. It is known that the conformational changes of a single-stranded DNA molecule are influenced by the physical environment in the gel, including temperature, concentrations of ions, and solvent^.^' Since we examined different temperature conditions (room temperature versus 4°C) in our preliminary experiments, and demonstrated higher resolution under thelatter condition with our PCR products, electrophoresis was performed at 4°C in the subsequent experiments. Other physical conditions of electrophoresis that we used, including 6% acrylamide gel and 10% glycerol, have been widelyusedby other investigator^.^^^^^.^^ These considerations suggest that the false-negativity that we found in SSCP analysis is unlikely to be due to technical problems. On the other hand, the fact that only positive cases in SSCP analysis have been used for sequence analyses in most studies performed by other investigators suggests that, even if positive cases yielded results concordant with sequence analyses, they might have missed false-negative cases in SSCP analysis. In light of our present findings, false-negativity in SSCP analysis maynot be as rare a phenomenon, as has been considered, and the determination of nucleotide changes will be necessary for a precise analysis of gene abnormalities. ACKNOWLEDGMENT TheauthorsthankDrs M. Ichikawa, R. Hanada, Y . Kaneko, Y. Nasu, K. Horio, K. Hattori, and H. Hara for providing patient samples and clinical information. 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