Leukemia (2009) 23, 708–711 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE

The isochromosome i(7)(q10) carrying c.258 þ 2t4c mutation of the SBDS does not promote development of myeloid malignancies in patients with Shwachman syndrome

A Minelli1,10, E Maserati2,10, E Nicolis3,10, M Zecca4, L Sainati5, D Longoni6, F Lo Curto2, G Menna7, F Poli8, E De Paoli1, M Cipolli9, F Locatelli4, F Pasquali2 and C Danesino1

1Genetica Medica, Fondazione IRCCS Policlinico San Matteo Universita` di Pavia e, Pavia, Italy; 2Dipartimento Scienze Biomediche Sperimentali e Cliniche, Universita` dell’Insubria, Varese, Italy; 3Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University Hospital of Verona, Verona, Italy; 4Oncoematologia Pediatrica, Fondazione IRCCS Policlinico San Matteo, Universita` di Pavia, Pavia, Italy; 5Clinica di Oncoematologia Pediatrica, Universita` di Padova, Padova, Italy; 6Clinica Pediatrica Universita` Milano-Bicocca, OC San Gerardo, Monza, Italy; 7Dipartimento di Oncologia AORN Santobono Pausilipon, Napoli, Italy; 8Clinica Pediatrica IRCCS Burlo Garofolo, Universita` di Trieste, Trieste, Italy and 9Cystic Fibrosis Center, Verona, Italy

Shwachman–Diamond syndrome (SDS) is an autosomal reces- 20, del(20)(q11). Recently, Shimamura3 reviewed available sive disorder, characterized by exocrine pancreatic insuffi- evidence that patients with the i(7)(q10) should be considered ciency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic as a separate group in terms of prognosis, as, in comparison to syndrome and/or acute myeloid leukaemia (MDS/AML). SDS is patients with different 7 anomalies, such as caused, in nearly 90% of cases, by two common mutations (that monosomy or deletion of the long arms, they have a lower risk is, c.183_184TA4CT and c.258 þ 2T4C) in exon 2 of the SBDS of MDS/AML. gene, localized on . Clonal chromosome anoma- Most SDS patients carry at least one of the two most common lies are often found in the BM of SDS patients; the most mutations of the SBDS gene, in exon 2: c.183_184TA4CT and frequent is an isochromosome for long arms of chromosome 7, þ 4 i(7)(q10). We studied eight patients with SDS carrying the c.258 2T C; the first one introduces an in-frame stop codon i(7)(q10) who were compound heterozygotes for SBDS muta- (p.K62X), whereas the second one disrupts the donor splice site 2 tions. By assessing the parental origin of the i(7)(q10) using of intron 2 and results in a truncated . Other rarer microsatellite analysis, we inferred from the results which mutations were recently reviewed by Costa and Santos.4 The mutation was present in double dose in the isochromosome. SBDS gene, highly conserved throughout evolution, encodes a We demonstrate that in all cases the i(7)(q10) carries a double 250 amino-acid protein with no homology to other of dose of the c.258 þ 2T4C, and we suggest that, as the known function. This protein was shown to be relevant for c.258 þ 2T4C mutation still allows the production of some amount of normal protein, this may contribute to the low biogenesis and, more recently, also for stabilization of 5 incidence of MDS/AML in this subset of SDS patients. the mitotic spindle. A reduced but detectable quantity of the Leukemia (2009) 23, 708–711; doi:10.1038/leu.2008.369; SBDS protein was shown to be present in fibroblasts of two SDS published online 15 January 2009 patients, this finding suggesting that the mutation found in these Keywords: Shwachman–Diamond syndrome; isochromosome 7q; two cases, namely c.258 þ 2T4C, may lead to the presence of ; MDS/AML risk SBDS a small amount of alternatively spliced SBDS mRNA, encoding a functional protein.6 Recently, Nicolis et al.7 demonstrated that wild-type (wt) mRNA can be found in patients carrying this mutation. Introduction To evaluate whether the mutations of the SBDS gene carried by i(7)(q10) have different roles in promoting the development of Shwachman–Diamond syndrome (SDS) is an autosomal reces- MDS/AML, we investigated the parental origin of the i(7)(q10) in sive disorder (OMIM 260400), characterized by exocrine the BM of eight patients, and, as all of them were compound pancreatic insufficiency, skeletal abnormalities and bone heterozygotes for SBDS mutations, we inferred from the result marrow (BM) dysfunction, with a risk, as high as 30%, to which mutation was present in double dose in the isochromosome. develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/AML).1 The SBDS gene (OMIM 607744) is localized on chromosome 7 at the band q11 and mutations of Material and methods this gene are found in 90% of patients.2 Clonal chromosome anomalies are often found in the BM of We retrospectively selected a cohort of eight SDS patients in SDS patients, often involving 7 and 20, the most whom: (i) the clinical diagnosis was confirmed by mutation frequent being an isochromosome for the long arms of analysis and (ii) the cytogenetic studies demonstrated the chromosome 7, i(7)(q10), and a deletion of the long arms of a presence of i(7)(q10) in 20–100% mitoses from BM. Clinical characteristics, as well as essential data on cytogenetics and Correspondence: Professor C Danesino, Genetica Medica, Universita` follow-up, are detailed in Table 1. No patient ever showed signs di Pavia, Via Forlanini 14, 27100 Pavia, Italy. of overt transformation into MDS/AML, including those with the E-mail: [email protected] 10These three authors contributed equally to this work. longest follow-up (nos 1–3 and 5). All patients were compound Received 25 July 2008; revised 20 October 2008; accepted 24 heterozygotes with the c.258 þ 2T4C mutation; the second November 2008; published online 15 January 2009 mutation was either the c.183_184TA4CT or the c.258 þ 2T4C mutation in i(7)(q10) and risk of MDS/AML in SDS A Minelli et al 709 Table 1 Cohort of SDS patients enrolled in the study; none showed signs of overt MDS/AML

Case BM cytogeneticsa Haematological follow-up/outcomeb

Date Karyotype % i(7)(q10)+ cells

1 2 December 1997 46,XX,i(7)(q10) [30] 100 16 y/dead when 16-year-old due to BM aplasia 2 10 July 2001 46,XY,i(7)(q10) [20]/46,XY [10] 66 16 y/alive 3 18 November 2004 46,XX,i(7)(q10) [17]/46,XX [4] 81 27 y/alive 4 17 March 2006 46,XX,i(7)(q10) [16]/46,XX [21] 43 3.5 y/alive 5 23 March 2007 46,XY,i(7)(q10) [41]/46,XY [1] 98 21 y/alive 6 30 May 2007 46,XX,i(7)(q10) [15] 100 2.5 y/alive 7 18 September 2007 46,XY,i(7)(q10) [5]/46,XY [25] 20 3 y/alive 8 18 April 2008 46,XX,i(7)(q10) [7]/46,XX [6] 53 1 y/alive Abbreviations: AML, acute myeloid leukaemia; BM, bone marrow; MDS, myelodysplastic syndrome; SDS, Shwachman–Diamond syndrome. aResults of chromosome analyses at the date of sampling also for the molecular study. bFollow-up in years (y) from SDS diagnosis.

Table 2 Results of molecular analysis demonstrating the parental origin of the i(7)(q10)

Case Mutationsa Allele proportionFratiob Parental origin of iso(7)(q10)

Short arm Long arm

1 Propositus: B/C FoMF4M Paternal Father: B Mother: C 2 Propositus: A/B MoFM4F Maternal Father: A Mother: B 3 Propositus: B/C MoFF0.47 (3) M4FF1.6 (3) Maternal Father: C Mother: B 4 Propositus: A/B FoMF0.73 (3) F4MF1.22 (4) Paternal Father: B Mother: A 5 Propositus: A/B FoMF0.68 (1) F4MF1.32 (2) Paternal Father: B Mother: A 6 Propositus: A/B FoMF0.54 (4) F4MF2.00 (3) Paternal Father: B Mother: A 7 Propositus: A/B FoMF0.53 (2) F4MF1.27 (1) Paternal Father: B Mother: A 8 Propositus A/B FoMF0.8 (2) F4MF1.41 (4) Paternal Father: A Mother: B Abbreviations: F, father; M, mother. aMutations are indicated by capital letters: A refers to the c.183_184TA4CT, B refers to c.258+2T4C and C refers to c.183_184TA4CT+258+2T4C. bThe number of STRP analysed is given in brackets. c.183_184TA4CT þ 258 þ 2T4C (Table 2). Some data on or migration time. The GenScanView 1.2/4 software provides cases 1–3 have been previously reported by Maserati et al.,8,9 peak detection (areas and heights) relative to alleles amplified as (unique patient number, UPN1) and Porta et al.10 (UPN25), DNA fragments. After the identification in each patient of respectively. Informed consent to be included in this study was the parental origin of the different STRPs tested, we compared obtained according to the principles of the Declaration of the height of the paternal and maternal alleles and Helsinki from patients or their parents. calculated the ratios between the allele coming from the parent Cytogenetic investigations were performed with routine carrying the c.258 þ 2T4C and that coming from his/her methods. Patients’ DNA was obtained from the same BM spouse, as this mutation is associated with the possibility of specimens used for chromosomal analysis; parental DNA was producing some amount of the wt protein. This method was also available for all cases. DNA extraction was performed with applied to case nos 3–8, whereas case nos 1 and 2 were studied GenElute Blood Genomic kit (Sigma, St Louis, MO, USA) and as reported by Maserati et al.8,9 mutation analysis was performed as reported by Nicolis et al.11 RNA analysis was performed in case nos 4 and 6 as reported We selected the short tandem repeat polymorphisms (STRPs) earlier.7 to be studied depending on their heterozygosity (always above 80%) and chromosomal mapping: D7S3048; D7S1808; D7S1818; D7S1830 (short arm) and D7S1820; D7S796; Results D7S2202; D7S1805 (long arm). Genotyping of STRPs was performed using ABI PRISM multicolour fluorescent dye Essential cytogenetic data are detailed in Table 1. All patients technology based on labelling DNA fragments with different showed the i(7)(q10) as the only anomaly in BM, with colour fluorescent dyes by PCR amplification. The PCR percentages of positive cells ranging from 20 to 100%. amplifications were performed as reported earlier.9 The study of informative STRPs allowed us to determine the The PCR products are displayed as electropherograms, parental origin of the isochromosome. Table 2 shows the mean showing fluorescence intensity as a function of fragment size peak ratios of the STRP alleles derived from the parent carrying

Leukemia c.258 þ 2T4C mutation in i(7)(q10) and risk of MDS/AML in SDS A Minelli et al 710 the c.258 þ 2T4C mutation (indicated as B) and those of his/her The role of cytogenetic abnormalities for MDS/AML develop- spouse: the ratios for the short arm STRP alleles ranged from ment in SDS patients is still under debate. The most frequent 0.47 to 0.80, whereas those for the long arm STRPs alleles chromosomal anomaly found in BM of SDS patients is the ranged from 1.22 to 2.00. These results are in agreement with i(7)(q10), which from literature data may account up to 44% of what we expected: as the i(7)(q10) includes two copies of the the cases with anomalies; other anomalies involving chromo- long arm and no copies of the short arm, a reduced amount of some 7 (monosomy, deletions and translocations) account for a the SRTPs localized on the short arm and increased amounts of further 33%, whereas the del(20)(q11) is found in around 16% of the long arms (as determined from the height of the peaks) patients.1 Although in patients without SDS affected by myeloid should be found in the patients’ BM, in relation to the parental malignancies the del(20)(q11) and anomalies of chromosome 7 origin of the chromosome 7 involved in the formation of the are frequent,14 the i(7)(q10) was reported in a limited number of i(7)(q10). patients, such as a case with a transient picture suggestive of In case nos 1 and 2, the parental origin of the i(7)(q10), MDS,15 a Down syndrome patient with AML16 and few other paternal and maternal, respectively, was determined earlier,8,9 MDS and AML cases.14,17 Therefore, the i(7)(q10) may be and in both cases all the markers consistently showed that the considered a fairly specific marker of SDS. abnormal chromosome carried the c.258 þ 2T4C alleles. As in Alter12 reviewed 510 reported SDS cases, and, it is other cases, the variations in peak height observed (Figure 1) noteworthy, no patient carrying the i(7)(q10) had developed consistently indicated that the i(7)(q10) originated from the MDS/AML, whereas in patients with overt evolution into MDS/ parent carrying the mutation c.258 þ 2T4C, the mother in case AML, other chromosome changes, including complex karyo- no. 3, and the father in case nos 4–8. Thus, the i(7)(q10) types, were observed. The difference as to the risk of MDS/AML originated from the parent carrying the c.258 þ 2T4C allele in between SDS cases with the i(7)(q10) and those with other all the eight cases studied. acquired chromosome changes has been recently emphasized The SBDS wt-mRNA level was investigated in case nos 4 and up to the point that haematopoietic stem cell transplantation is 6 and expressed as (wt/wt þ mutated) peak height ratio; the ratio no longer considered indicated in patients with the i(7)(q10).18 observed, 0.25 for case no. 4 and 0.20 for case no. 6, indicates However, some exceptions to the rule that SDS patients with that wt SBDS mRNA is present in both patients, at a level i(7)(q10) do not develop MDS/AML may exist, as suggested by decreased by about 80% with respect to healthy controls (data two old reports of patients carrying this cytogenetic abnormality not shown). The strength of the normal, mutated and cryptic who developed refractory anaemia.19,20 splicing sites was determined by the Analyzer Splice Tools The c.258 þ 2T4C mutation modifies the usual splice site at the software (http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.htm) and donor site of intron 2 and, in turn, a cryptic splice site present at the results for splice sites score/number of base pairs/DG were position 251–252 in exon 2 becomes active, resulting in an 8-bp as follows: normal site 84.90/9/–9; mutated site 67.0/7/–5.3 and deletion in mRNA sequence, which also causes a premature cryptic site 68.40/5/–2.6. truncation of the encoded protein by frameshift (p.84Cfs3).2 However, the c.258 þ 2T4C mutation, either in homozygous condition or in association with a rare mutation such as c.505C4T (p.R169C), still allows the production of some amount of normal Discussion protein.6 This observation was recently confirmed by Nicolis et al.7 who demonstrated the presence of some amount of normal mRNA SDS is a complex disorder caused by mutations of the SBDS in patients carrying the c.258 þ 2T4C mutation both as com- gene, in which MDS/AML may develop in a variable number of pound heterozygotes (258 þ 2T4C/183_184TA4CT) and as patients, 14–30%.1,12 No clear-cut correlation between certain homozygotes (258 þ 2T4C/258 þ 2T4C).Infact,whenthe cytogenetic anomalies found in BM (as well as specific strength of the mutated and cryptic splice sites is compared, the mutations of the SBDS gene) and evolution into MDS/AML of scores obtained support the available evidence that the mutated SDS patients has been recognized in the literature.1,13 splice site might be partially active.

Case 3 Case 6

father father

mother mother

D7S1830, chrom 7p D7S1805, chrom7q

Figure 1 Examples of short tandem repeat polymorphism (STRP) analysis with parental origin of the i(7)(q10).

Leukemia c.258 þ 2T4C mutation in i(7)(q10) and risk of MDS/AML in SDS A Minelli et al 711 As the SBDS gene is located on 7q11, BM cells with the 5 Austin KM, Gupta ML, Coats SA, Tulpule A, Mostoslavsky G, i(7)(q10) have three copies of the gene, and we demonstrated Balazs AB et al. Mitotic spindle destabilization and genomic that in all the eight cases studied the i(7)(q10) carries two copies instability in Shwachman–Diamond syndrome. J Clin Invest 2008; of the c.258 þ 2T4C mutation (258-iso7). 118: 1511–1518. 6 Austin KM, Leary RJ, Shimamura A. The Shwachman–Diamond We did not find any i(7)(q10) carrying the c.183_184TA4CT SBDS protein localizes to the nucleolus. Blood 2005; 106: mutation, and no evidence leads to postulate that the 1253–1258. c.258 þ 2T4C (and not the c.183_184TA4CT) might induce 7 Nicolis E, Durie PR, Ip WF, Rommens J, Shimamura A, Sainati L the formation of the i(7)(q10). A possible interpretation is et al. SBDS mRNA Expression in Shwachman–Diamond Syndrome that a cell with the genotype (258 þ 2T4C/258 þ 2T4 Patients Carrying the 258+2T4C Mutation. Proceedings of the 4th C/183_184TA4CT) may have a selective advantage over a cell International Congress on Shwachman–Diamond Syndrome; 10–12 June 2007; Boston, MA. p 67. with a possible genotype (183_184TA4CT/183_184TA4CT/ 8 Maserati E, Minelli A, Olivieri C, Bonvini L, Marchi A, Bozzola M 258 þ 2T4C) as it may have a still small but larger amount of et al. Isochromosome (7)(q10) in Shwachman syndrome the wt protein. Therefore, only clones carrying the genotype without MDS/AML and role of chromosome 7 anomalies in (258 þ 2T4C/258 þ 2T4C/183_184TA4CT) would survive myeloproliferative disorders. Cancer Genet Cytogenet 2000; 12: and would become identifiable. 167–171. Austin et al.5 demonstrated that the SBDS protein stabilizes 9 Maserati E, Minelli A, Pressato B, Valli R, Crescenzi B, Stefanelli M et al. Shwachman syndrome as mutator phenotype responsible for the mitotic spindle preventing genomic instability; this observa- myeloid dysplasia/neoplasia through karyotype instability and tion may suggest that cells with the 258-iso7 are less prone to chromosomes 7 and 20 anomalies. Chromosomes Cancer develop additional chromosome abnormalities, which in turn 2006; 45: 375–382. may lead to MDS/AML, in keeping with the observation 10 Porta G, Mattarucchi E, Maserati E, Pressato B, Valli R, reviewed above that the i(7)(q10) was rarely seen in SDS Morerio C et al. Monitoring the isochromosome i(7)(q10) patients with MDS/AML. in the bone marrow of patients with Shwachman syndrome by real-time quantitative PCR. J Pediatr Hematol Oncol 2007; 29: Our results provide the explanation for a biological advantage 163–165. of BM cells with 258-iso7 (with respect to cells with other 11 Nicolis E, Bonizzato A, Assael BM, Cipolli M. Identification of chromosome changes), and a convincing biological basis for the novel mutations in patients with Shwachman–Diamond syndrome. clinical observation of a reduced risk of developing MDS/AML Hum Mutat 2005; 25: 410. in these SDS patients, which is of course relevant for their 12 Alter BP. Malignancies in Shwachman–Diamond Syndrome: Data clinical follow-up and management. from Literature. Proceedings of the 4th International Congress on Shwachman–Diamond Syndrome; 10–12 June 2007; Boston, MA. p9. 13 Ellis L, Corey M, Morrison J, Richards M, Dror Y, Freedman M Acknowledgements et al. Myelodysplasia and Clonal Marrow Cytogenetic Abnormal- ities (CMCA) and Shwachman Diamond Syndrome Genotype. This study was partially supported by grants from AISS (Associa- Proceedings of the 2nd International Congress on Shwachman– zione Italiana Sindrome di Shwachman) and from MIUR Diamond Syndrome; 16–17 June 2003; Toronto, Canada. p 8. (Ministero dell’Istruzione, Universita` e della Ricerca)––PRIN 14 Mitelman F, Johansson B, Mertens F (eds). Mitelman Database of 2006065440. This study was carried out at Universita` di Pavia, Chromosome Aberrations in Cancer 2008 (database on the Internet). Available from: http://cgap.nci.nih.gov/Chromosomes/ Universita` di Varese, University Hospital of Verona. AM Mitelman. performed mutation analysis and worked on establishing parental 15 Leung EW, Woodman RC, Roland B, Abdelhaleem M, Freedman origin, EM performed cytogenetics and EN performed mutation MH, Dror Y. Transient myelodysplastic syndrome associated with analysis and RNA studies. isochromosome 7q abnormality. Pediatr Hematol Oncol 2003; 20: 539–545. 16 Wong KF, Lam SC, Leung JNS. Isochromosome 7q in Down References syndrome. Cancer Genet Cytogenet 2006; 164: 152–154. 17 Mertens F, Johansson B, Mitelman F. Isochromosomes in 1 Dror Y. Shwachman–Diamond syndrome. Pediatr Blood Cancer neoplasia. Genes Chromosomes Cancer 1994; 10: 221–230. 2005; 45: 892–901. 18 Hall GW, Dale P, Dodge JA. Shwachman–Diamond syndrome: 2 Boocock GR, Morrison JA, Popovic M, Richards N, Ellis L, UK perspective. Arch Dis Child 2006; 91: 521–524. Durie PR et al. Mutations in SBDS are associated with Shwach- 19 Smith OP, Hann IM, Chessells JM, Reeves BR, Milla P. man–Diamond syndrome. Nat Genet 2003; 33: 97–101. Haematological abnormalities in Shwachman–Diamond 3 Shimamura A. Shwachman–Diamond syndrome. Semin Hematol syndrome. Br J Haematol 1996; 94: 279–284. 2006; 43: 178–188. 20 Dror Y, Squire J, Durie P, Freedman MH. Malignant myeloid 4 Costa E, Santos R. Hematologically important mutations: Shwachman– transformation with isochromosome 7q in Shwachman–Diamond Diamond syndrome. Blood Cells Mol Dis 2008; 40: 183–184. syndrome. Leukemia 1998; 12: 1591–1595.

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