Cancer Genetics and Cytogenetics 124 (2001) 147–151
Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene Antonella Minellia, Emanuela Maseratib, Giovanni Giudicic, Sabrina Tosid, Carla Olivieria, Livia Bonvinib, Paola De Filippia, Andrea Biondic, Francesco Lo Curtob, Francesco Pasqualib,*, Cesare Danesinoa a Biologia Generale e Genetica Medica, Università di Pavia, C.P. 217, I 27100, Pavia, Italy Sezione di Biologia e Genetica, Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università dell’Insubria, Via J. H. Dunant, 5, I21100, Varese, Italy c Centro “Tettamanti,” Clinica Pediatrica, Università di Milano, Ospedale Nuovo S. Gerardo, I 20052 Monza, Italy d MRC Molecular Haematology Unit, Institute of Molecular Medicine, Oxford, UK Received 20 April 2000; accepted 1 August 2000.
b
Abstract
Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction The familial occurrence of complete or partial monosomy 7 in association with myelodysplastic syndromes (MDS), acute myeloid leukemias (AML), and non-specified myeloproliferative disorders has been reported in 12 pedigrees [1–7]. Data on cytogenetics and on precise clinical diagnoses in these cases are often incomplete, but those available are sufficient to lead to the hypothesis of a germ-line mutation of a tumor-suppressor gene located on chromosome 7 as a first step of carcinogenesis in these patients [8], according to Knudson’s model [9]. Evidence against this hypothesis is available for 2 of the 12 kindreds mentioned [8], and the need of studying additional families to rule in or
* Corresponding author. Tel.: ⫹39-0332-217180; fax: ⫹39-0332217119. E-mail address:
[email protected] (F. Pasquali).
rule out models based on Knudson’s hypothesis was stressed by these authors. We report here two sisters, both with MDS associated with partial monosomy 7, together with molecular data on the parental origin of the chromosomes 7 involved. 2. Case Reports 2.1. Case 1 Female, born in 1976 from unrelated parents. Growth was normal, and since the age of 3 years she suffered from seizures, which disappeared at age 10 after anticonvulsivant therapy. She also suffered from repeated episodes of bronchitis and otitis. In 1993 she was diagnosed as having a refractory anemia (RA) after testing as a possible marrow donor for her younger sister. The diagnosis was based on a blood test (WBC 2,300/mm3, Hb 9.6 g/dl, platelets 130,000/mm3) and
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marrow examination, which showed dyserythropoiesis and myeloid dysplasia. She did clinically well, and the hematological picture remained stable without any need of therapy until 1998, when the anemia and granulocytopenia worsened: in July 1998 blood WBC was 500/mm3 with 60% blasts. A morphological examination of her marrow showed 40% undifferentiated blasts and, together with the cytochemical and immunophenotypic characterization, led to a diagnosis of AML of the M0 FAB type. The therapy started with a protocol including cytosine arabinoside, daunorubicin and etoposide. A severe mycosis forced a change in the therapy, which continued with cytosine arabinoside and 6thioguanine in August 1998, when marrow blasts were 2%. In October 1998 her marrow was aplastic, 6-thioguanine was discontinued, and in December 1998 marrow blasts were 33%. The peripheral blood picture remained stable (since July 1998) with a WBC 400–1500/mm3, Hb 6.5–9.9 g/dl, platelets ⬍50,000/mm3. In February 1999, she underwent a bone marrow transplantation (BMT) from an unrelated donor, but died in March 1999 due to pulmonary aspergillosis. 2.2. Case 2 Female, sister of Case 1, born in 1980. In 1992 she was diagnosed as having RA on the basis of the peripheral blood and marrow pictures. WBC was 1200/mm3, Hb 10.2 g/dl, platelets 64,000/mm3; differential count: neutrophils 20% and lymphocytes 80%. Her marrow showed significant dysplasia of the myeloid and erythroid series, many dysplastic micromegakaryocytes, 5% Perls positive erythroblasts. Only supportive therapy was given, and in 1993 the possibility of a BMT was considered; the sister (case 1) was in fact HLA-identical, but the BMT did not take place due to the diagnosis of MDS made also in the sister. In April 1994, she underwent BMT from an unrelated donor, but died after 11 days due to pneumonia from Candida albicans.
3. Materials and methods
then repeated during the MDS phase and the transformation into AML. Chromosome analysis of both parents was performed on lymphocyte cultures. QFQ- and GTG-banding techniques were applied. The following fluorescence in situ hybridizations (FISH) were performed: whole chromosome paint (Cambio, UK) for chromosome 7 in both cases, and for chromosome 1 in case 1; interphase nuclei analysis with a chromosome 7 specific alphoid centromeric probe (Appligene-Oncor, UK), in both cases; FISH with a commercially available 7q subtelomeric probe (Appligene-Oncor-Lifescreen, UK), and with a cosmid clone cos10.1 specific for the subtelomeric 7p region in case 1. 3.2. Molecular studies Molecular studies were performed to gather information on the parental origin of the chromosomes 7 and 8 involved in anomalies; this regarded the partial monosomy 7 of both sisters and the trisomy 8 of case 1. The loci tested, scattered along the whole chromosome, were seven for chromosome 7 and six for chromosome 8, and three more loci on different chromosomes were tested to confirm paternity. We extracted DNA with routine techniques from bone marrow cells sampled on 26.10.1995 and 23.07.1998 in case 1 and on 12.10.1992 in case 2, and from their parents’ blood samples. Genotyping of STRPs (Research Genetics) was performed by use of standard procedures. PCR amplifications were performed in 8 l reaction mixtures containing 20 ng genomic DNA; 330 nM of each primer; 200 M of dCTP, dGTP, dTTP and dATP; 50 mM KCl; 10 mM TRIS pH 9; 1.5 mM MgCl2; 0.1% Triton X-100; 0.01% gelatine and 0.2 U of Taq Polymerase. The PCR conditions consisted of an initial denaturation step followed by 30 cycles of 94⬚C for 40 s, 57⬚C for 40 s, 72⬚C for 40 s and a final extension at 72⬚C for 5 min, using a PTC-100 thermal cycler (MJ Research). Four microliters of each PCR product were resolved by electrophoresis on denaturing (7M urea) 7% polyacrylamide gels for 1–3 h at 30 W. Gels were fixed in 10% methanol/acetic acid, and stained with silver nitrate 0.012 M.
3.1. Cytogenetic studies Chromosome analyses were performed on direct preparations of bone marrow and cultures of both sisters at onset of MDS by routine techniques. In case 1, the analysis was
4. Results Table 1 summarizes the results of chromosome analyses performed at various dates in both sisters. Case 1 showed a
Table 1 Results of chromosome analyses Patient
Date
Material
Karyotype
Case 1
08.07.1993 03.09.1993 09.11.1993 26.10.1995
BM BM BM BM
23.07.1998 12.10.1992
BM BM
46,XX[19].nuc ish 7cen(D7Z1⫻2) 46,XX[8].nuc ish 7cen(D7Z1⫻2) 46,XX[50] 47,XX,⫹1,der(1;7)(q10;p10),⫹8[12].ish der(1;7)(7pter→7p11)(wcp7⫹)der (1;7)(1q11→1qter)(wcp7⫺).ish der(1;7)(Tel7p⫹).ish der(1;7)(Tel7q⫺).nuc ish 7cen(D7Z1⫻2) 47,XX,⫹1,der(1;7)(q10;p10),⫹8[25].ish der(1;7)(1q10→1qter)(wcp1⫹) 45,XX,add(2)(q32),⫺7,add(13)(q32)[18].ish add(13)(q32)(wcp7⫹).nuc ish 7cen(D7Z1⫻1)[85%]
Case 2
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Fig. 1. Case 1: cut-out of relevant chromosomes from BM analysis showing the t(1;7) and the trisomy 8 (Table 1).
normal karyotype at MDS onset. However, monosomy 7 was found by FISH on interphase nuclei, in view of finding this anomaly in case 2. In 1995, an abnormal clone appeared with trisomy 8 and an unbalanced whole-arm t(1;7), resulting in chromosome 1 long-arm trisomy and chromosome 7 long-arm monosomy (Fig. 1). FISH results confirmed this interpretation (Table 1), and showed that alphoid sequences from chromosome 7 were maintained at the centromere of the structurally abnormal chromosome. Nevertheless, on metaphases this signal was less intense than in the normal 7, suggesting a breakpoint within the centromeric alphoid sequences. The abnormal clone persisted during transformation into AML, in 1998. Chromosome results in case 2 are limited to the RA onset, when one chromosome 7 was missing and additional material of unknown origin was present on the long arms of chromosomes 2 and 13. The quality of these preparations was suboptimal, but painting with a chromosome 7 library showed that only a small portion of 7 was in fact transposed onto the der(13) chromosome.
Table 2 Results of polymorphic loci analyses Case 1
D7S2514 D7S507 D7S673 D7S1820 D7S796 D7S495 D7S2462 D8S349 D8S1469 D8S1130 NEFL D8S279 D8S198
Case 2
F
BM(95)
BM(98)
BM
M
BC AB AB CD AB BD AB CD AC AB BC AB CD
BC AB BC AD AB BC BC AD AC AA CC AB AC
BC AB BC AD AB BC BC AD AC AA CC AB AC
B B B C A B B BC AB AB AC AA BC
AB AC CC AB BC AC CD AB AB AA AC AA AB
A–D refer to the alleles in decreasing molecular size. Bigger or smaller font size indicate higher or lower band intensity; in bold informative results.
Fig. 2. Examples of informative loci (Table 2) of chromosome 7 short arm (top row), long arm (middle row), and chromosome 8 (bottom row). Lane 1: father, Lane 2 and 3: case 1 BM sampled in 1995 and 1998, respectively (see Table 1), Lane 4: case 2 BM, Lane 5: mother.
The parent’s karyotypes were normal in 500 mitoses scored. The results of STRPs analysis are shown in Table 2 and illustrated in Fig. 2. In the marrow sample of case 1 in 1995, all the informative loci from chromosomes 7 and 8 tested showed alleles of similar intensity, whereas in the 1998 sample four 7q loci consistently gave an uneven pattern of amplification, with the paternal alleles less intense. Paternal and maternal alleles of 7p loci were of similar intensities. In the same 1998 material, four of the six chromosome 8 loci tested showed a higher intensity of the maternal allele. This result was confirmed by a densitometric analysis performed on two loci: the ratio of maternal to paternal alleles was 1.43 for D8S1469 and 1.39 for D8S198. In case 2, all chromosome 7 loci showed the absence or lesser intensity of the maternal allele. No differences were seen for chromosome 8 loci (Fig. 2, Table 2). 5. Discussion Twelve pedigrees with familial MDS/AML associated with monosomy 7 have been reported; a different parental origin of the lost chromosome 7 was demonstrated in two of them [5]. This argues against the hypothesis of the germline mutation of a possible tumor-suppressor gene located
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on chromosome 7. Shannon et al. [5] stressed the need of additional data to definitely rule out this hypothesis. In the sisters reported here, quoted without details as case 229 and her sister in the review of Hasle et al. [6], we now demonstrate that the chromosome 7 lost was paternal in case 1, and maternal in case 2. The FISH study performed in case 2 showed that monosomy 7 was not complete, as a small fragment of chromosome 7 was present and transposed to a der(13) chromosome (Table 1). This led to change the first interpretation of the anomaly and supports the view expressed by Sessarego et al. [10], who found hidden material from chromosome 7, overlooked by conventional techniques, in 8 out of 12 cases, and confirmed the poor prognostic relevance of complex karyotypes with chromosome 7 anomalies. A t(1;7), similar to that of our case 1, has been frequently reported in MDS, and in two cases in association with hypereosinophilia [11]. In these, it was shown that the eosinophils were part of the dysplastic clone; trisomy 8 was present in one of these patients [12], as in our case 1, who, however, had no sign of eosinophil involvement. We previously reported on the maternal origin of concurrent constitutional trisomy 21 and acquired monosomy 7, suggesting that in that case maternal chromosomes might be prone to nondisjunction [13]. In case 1 of the present report, the additional chromosome 8 was of maternal origin, opposite to the 7 involved in partial monosomy (Table 2). The occurrence of a disease uncommon in young age, such as MDS, in two sisters along with the same chromosome involved in a structural anomaly can hardly be interpreted as random. Most of the reported cases with familial monosomy 7 share a young age at MDS/AML onset (20 out of 24 cases were younger than 18 years of age). Noteworthy is the fact that in each family the interval between the onset age of the affected members was 4 years or less in 10 out of 12 families. In three families, the myeloid disorder with familial monosomy 7 was associated with a disease that was (or was supposed to be, lacking a precise diagnosis) a mendelian one. They are the possible Noonan syndrome diagnosed in 2 or 3 members of the family described by Chitambar et al. [3]; cerebellar ataxia/atrophy present in 6 members of the family reported by Li et al. [2]; and Fanconi’s anemia in 2 sisters studied by Stivrins et al. [4]. However, nonfamilial monosomy 7 in myeloid disorders is found sometimes in association with a variety of hereditary disorders, including Fanconi anemia, Kostmann agranulocytosis, cyclic neutropenia, Shwachman syndrome (SS), type-1-neurofibromatosis [14], as well as with Silver-Russell syndrome, macrocephalus, hydrocephalus, facial dysmorphisms, mental retardation, deafness, blindness, ptosis, vermis cerebelli agenesis, and atrial sept defect [6]. We recently postulated a mutator effect of the mutation causing SS, which by pleiotropy may induce karyotype instability, and, in turn, anomalies of chromosome 7 in the bone marrow leading to myeloid dysplasia or neoplasia [15]. We also suggest that in familial total or partial mono-
somy 7 the first step in MDS/AML pathogenesis is the mutator effect of an inherited gene mutation. This could be responsible for numerical and structural anomalies, as a possible link between them, with the latter following the former, was reported by Schinzel et al. [16] for a constitutional complex chromosomal aneuploidy. This putative mutator gene would cause a diagnosed mendelian disease in some cases, as in the three families mentioned, but it would be in any case responsible of a form of karyotype instability leading to marrow chromosome 7 anomalies and, in turn, to the development of MDS/AML. Acknowledgments Partially supported by MURST, Cofin 1998. DNA from the patient was stored in the DNA and cell bank supported by Telethon, Project C30. We thank Prof. M. Fraccaro for critically reading the manuscript. References [1] Kamiyama R, Shibata T, Mori W. Two autopsy cases of atypical myeloproliferative disorder with group C monosomy occurring in siblings. Acta Path Jap 1973;23:815–21. [2] Li FP, Hecht F, Kaiser-McCaw B, Baranko PV, Potter NU. Ataxiapancytopenia: syndrome of cerebellar ataxia, hypoplastic anemia, monosomy 7, and acute myelogenous leukemia. Cancer Genet Cytogenet 1981;4:189–96. [3] Chitambar CR, Robinson WA, Glode LM. Familial leukemia and aplastic anemia associated with monosomy 7. Am J Med 1983;75: 756–62. [4] Stivrins TJ, Davis RB, Sanger W, Fritz J, Purtilo DT. Transformation of Fanconi’s anemia to acute nonlymphocytic leukemia associated with emergence of monosomy 7. Blood 1984;64:173–6. [5] Shannon KM, Turhan AG, Rogers PCJ, Kan YW. Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7. Genomics 1992;14: 121–5. [6] Hasle H, Aricò M, Basso G, Biondi A, Cantù-Rajnoldi A, Creutzig U, Fenu S, Fonatsch C, Haas OA, Harbott J, Kardos G, Kerdrup G, Mann G, Niemeyer CM, Ptoszkova H, Ritter J, Slater R, Stary` J, Stolmann-Gibbels B, Testi AM, van Wering ER, Zimmermann M for the European Working Group on MDS in Childhood (EWOG-MDS). Myelodysplastic syndrome, juvenile myelomonocytic leukemia, and acute myeloid leukemia associated with complete or partial monosomy 7. Leukemia 1999;13:376–85. [7] Kwong YL, Ng MH, Ma SK. Familial acute myeloid leukemia with monosomy 7: late onset and involvement of a multipotential progenitor cell. Cancer Genet Cytogenet 2000;116:170–3. [8] Shannon KM, Turhan AG, Chang SSY, Bowcock AM, Rogers PCJ, Carroll WL, Cowan MJ, Glader BE, Eaves CJ, Eaves AC, Kan YW. Familial bone marrow monosomy 7. J Clin Invest 1989;84:984–9. [9] Knudson AG. Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Res 1985;45:1437–1443. [10] Sessarego M, Fugazza G, Gobbi M, Bruzzone R, Bisio R, Ghio R, Patrone F. Complex structural involvement of chromosome 7 in primary myelodysplastic syndromes determined by fluorescence in situ hybridization. Cancer Genet Cytogenet 1998;106:110–15. [11] Forrest DL, Horsman DE, Jensen CL, Berry BR, Dalal BI, Barnett MJ, Nantel SH. Myelodysplastic syndrome with hypereosinophilia and a nonrandom chromosomal abnormality dic(1;7): confirmation of eosinophil clonal involvement by fluorescence in situ hybridization. Cancer Genet Cytogenet 1998;107:65–8.
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