Novel recurrent chromosome anomalies in Shwachman–Diamond syndrome

Two chromosome anomalies are frequent in the bone marrow (BM) of patients with Shwachman–Diamond syndrome (SDS): an isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q). These anomalies are associated with a lower risk of developing myelodysplasia (MDS) and/or acute myeloid leukemia. The chromosome anomalies may be due to an SDS‐specific karyotype instability, reflected also by anomalies that are not clonal, but found in single cells in the BM or in peripheral blood (PB).


BACKGROUND
Two chromosome anomalies are frequently acquired in the bone marrow (BM) of patients with Shwachman-Diamond syndrome (SDS): isochromosome of the long arm of chromosome 7, i(7)(q10), and interstitial deletion of the long arm of chromosome 20, del(20)(q). 1 The role of these clonal changes in relation to the risk of these patients to develop myelodysplasia (MDS) and/or acute myeloid leukemia (AML) has been investigated, and both anomalies have been shown to be Abbreviations: a-CGH, microarray-based comparative genomic hybridization; AML, acute myeloid leukemia; BM, bone marrow; MDS, myelodysplastic syndrome; PB, peripheral blood; SDS, Shwachman-Diamond syndrome; UPN, unique patient number associated with a low risk. 2,3 Starting in 1999, we monitored the cytogenetic picture (in BM and peripheral blood [PB]) in a cohort of Italian patients with SDS (in total 91 subjects). We report here clonal chromosome anomalies found in these patients different from the more frequent ones, associated or not with the i(7)(q10) or the del(20)(q). Some of these chromosome changes are recurrent, and we tried to infer their possible relevance in prognostic evaluations.
We have already postulated that clonal chromosome anomalies are due to an SDS-specific karyotype instability, which may be reflected by anomalies that are not clonal, but found in single cells both in the BM and PB. 4,5 Therefore, we also report on the chromosome anomalies that we found in single cells in BM or PB of the same patients.

Patients
For the analysis, we took into account the results of all cytogenetic analyses performed since 1999 in the follow-up of our cohort of 91 Italian patients with SDS, all with ascertained biallelic mutations of the SBDS gene. The cohort included 57 males and 34 females, with a median age of 20 years, excluding six patients who died during the study (three males and three females). More details on gender and age are presented in Supplementary File S1. A portion of the cytogenetic results has already been reported. 3,[5][6][7] The patients are identified by their unique patient number (UPN), both in the text and in the tables, as in our previous publications.
Informed consent to this study was obtained according to the principles of the Declaration of Helsinki from the patients or from patients' parents.

Methods
Chromosome analyses were performed using routine methods and the Q-banding by fluorescence using quinacrine technique on BM direct preparations and 24-48 hr cultures, and on unstimulated and phytohaemagglutinin-stimulated PB cultures.
FISH on metaphases and interphase nuclei was carried out with standard procedures utilizing the probes and libraries described previously 5,6 ; these are able to detect the most frequent chromosome anomalies and monitor abnormal clones with less frequently identified anomalies.
The microarray-based comparative genomic hybridization (a-CGH) was performed with the 244 K genome-wide system (Agilent Technologies Inc., Santa Clara, CA, USA), according to the manufacturer's instruction on DNA from BM and PB. The DNA was extracted using the Qiagen Flexigene kit (QIAGEN GmbH, Hilden, Germany), and competitor DNA was either purchased from Promega (Promega Corporation, Madison, WI, USA) or from Agilent as part of the labeling kit.
Slides were scanned using Agilent's microarray scanner G2565CA and microarray images were analyzed using Agilent's Feature Extraction

RESULTS
We monitored all available patients at least once a year. Therefore, these longitudinal cytogenetic data provided a chromosome picture of the BM varying over time, specifically the proportion of cells with anomalies, the appearance of novel clonal anomalies, variation in their percentage, and even anomalies that disappeared. 7 Clonal chromosome anomalies in BM were present in 41 out of 91 patients. In 19 of these, the anomaly was already present when they were enrolled into the cohort, whereas in 22 it was acquired during the follow-up. The i(7)(q10) was observed in 17 patients, and the del(20)(q) in 15, both of these changes were found in three, but in independent clones, as already mentioned. 7 So, in total, the most frequently observed clonal anomalies were found in 35 patients. Other, different, clonal anomalies were found in the BM of 13 patients, in eight cases in the absence of i(7)(q10) or del(20)(q) ( Table 1), and in five cases in association with one of these changes ( Table 2). We defined these anomalies as clonal also when detected in very few cells, in some cases even only one or two cells, but observed repeatedly in analyses made during subsequent years. Tables 1 and 2 list the results obtained from the analyses in which informative results were found, and give the proportion of cells with an infrequent anomaly from each analysis. All structural anomalies were confirmed by FISH with libraries for painting of the involved chromosomes and with informative probes, also on interphase nuclei in some patients (UPN 53 and 58). Five patients with a clone bearing one of the more common changes also developed a clone with an infrequent anomaly ( Table 2). We have already demonstrated that in all cases the additional anomaly is acquired in an independent clone. 7 In Table 2 Table 2). The rearrangement was almost identical in both patients, and has already been reported by our group, 6,7 the a-CGH profiles have been reported as well in Valli et al. 3 The different percentages observed in the deleted and duplicated regions in these two patients ( Table 2) Table 3, as in Tables 1 and   2, only the results of analyses that disclosed nonclonal anomalies are presented.

DISCUSSION
In the clonal anomalies (different from i (7) is associated with a lower risk of developing MDS/AML. However, the presence of one or more independent clones with other anomalies could be linked to a higher risk: in our cohort, MDS/AML developed in two out of five such cases (Table 2).  (UPN 17 and 20). Their a-CGH profiles astonished us, as they were almost identical 3 (Tables 2 and 3 Nonclonal chromosome changes, found in single cells (Table 3), were present in 15 of 91 patients in our cohort, with a frequency higher than control subjects. In particular, a translocation t(7;14) was found in five cases, with a much higher frequency as compared to controls. 13,14 These data support our former hypothesis of a peculiar SDS-associated karyotype instability, which may be linked to the acquisition of clonal anomalies in the BM and to variations in the risk of progression to MDS/AML (higher or lower according to the chromosome anomaly acquired). 5 Patients with SDS and rare, infrequent acquired chromosome anomalies in BM need more frequent cytogenetic monitoring due to a possible higher risk of developing MDS/AML; this monitoring should include standard cytogenetics, FISH, and more sophisticated methods to detect chromosome imbalances, such as array-CGH.