Cytogenetic abnormalities and genomic copy number variations in EPO (7q22) and SEC-61(7p11) genes in primary myelodysplastic syndromes

Blood Cells, Molecules and Diseases 59 (2016) 52–57

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Cytogenetic abnormalities and genomic copy number variations in EPO (7q22) and SEC-61(7p11) genes in primary myelodysplastic syndromes Purvi Mohanty a, Seema Korgaonkar a, Chandrakala Shanmukhaiah b, Kanjaksha Ghosh a, Babu Rao Vundinti a,⁎ a b

Department of Cytogenetics, National Institute of Immunohaematology, 13th Floor, New Multistoried Building, KEM Hospital Campus, Parel, Mumbai 400012, India Department of Haematology, 10th Floor, New Multistoried Building, KEM Hospital, Parel, Mumbai 400012, India

a r t i c l e

i n f o

Article history: Submitted 19 February 2016 Accepted 11 April 2016 Available online 13 April 2016 Editor: Mohandas Narla Keywords: Copy number variations EPO SEC-61 Cytogenetics and myelodysplastic syndromes

a b s t r a c t Myelodysplastic syndromes (MDSs) are heterogeneous clonal haematopoeitic stem cell disorders characterized by ineffective haematopoeisis, cytopenias and risk of progression to AML. We studied 150 MDS patients for cytogenetic aberrations and 60 patients with normal karyotype and 40 patients harboring cytogenetic abnormalities for copy number variations (CNVs). Cytogenetic abnormalities were detected in 46% of patients with a majority of patients harboring abnormalities of chromosome 7 and del (20q) at frequencies of 16% and 12% respectively. We explored the potential of quantitative multiplex PCR assay of short fluorescent fragments (QMPSF) to identify CNVs and correlated the findings with cytogenetic data and disease prognosis. CNVs (n = 31) were detected in 28.3% of karyotypically normal and 23% patients with abnormal karyotype. Genetic losses or deletions (n = 26) were more frequent than duplications (n = 5). EPO (7q22) and SEC-61(7p11) emerged as new candidate genes susceptible to genetic losses with 57.7% deletions identified in regions on chromosome 7. The CNVs correlated with International Prognostic Scoring System (IPSS) intermediate disease risk group. Our integrative cytogenetic and copy number variation study suggests that abnormalities of chromosome 7 are predominant in Indian population and that they may play a secondary role in disease progression and should be evaluated further for asserting their clinical significance and influence on disease prognosis. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis occurring predominantly in the elderly, characterized by morphologic dysplasia, ineffective hematopoiesis, peripheral blood (PB) cytopenias and propensity for transformation to acute myeloid leukemia (AML). Cytogenetic abnormalities involving partial or complete loss of chromosomes (monosomy 7/del (7q), monosomy 5/del (5q) and del (20q)) are frequent (~ 20–50%) in MDS and hold prognostic value as they help in prediction of clinical outcome and affect treatment planning [1–4]. However, approximately 50% of primary MDS patients do not harbor an abnormal karyotype suggesting the presence of unknown genetic lesions contributing to the heterogeneous nature of MDS. Copy number variations (CNVs) are consistent structural variations in the human genome wherein a DNA segment ranging from 1 kb to 3 Mb is deleted, inserted, or duplicated, on certain chromosomes thereby creating new genes, affecting gene dosage and modifying gene expression [5–6]. Recent studies utilizing high resolution array-based platforms have identified copy number alterations (deletions and Abbreviations: CNV, copy number variations; QMPSF, quantitative multiplex PCR assay of short fluorescent fragments; IPSS, International Prognostic Scoring System. ⁎ Corresponding author. E-mail address: [email protected] (B.R. Vundinti).

http://dx.doi.org/10.1016/j.bcmd.2016.04.005 1079-9796/© 2016 Elsevier Inc. All rights reserved.

amplifications) in a proportion (18–68%) of cytogenetically normal MDS cases having prognostic significance [7–11]. However, the role of CNVs in haematological malignancies is unclear because their random distribution and their evaluation in heterogeneous disorder of MDS are of interest as they could provide potential clues for the discovery of new candidate genes with implications in their pathogenesis and identify variants having prognostic significance. We modified a singleplex quantitative multiplex PCR assay of short fluorescent fragments (QMPSF) established previously [14] to identify CNVs in 20 genes located in several regions involved in pathogenesis of MDS involving chromosomes 7, 8 and on the 5q, 12p, 17p and 20q regions and studied cytogenetic abnormalities in primary MDS patients correlating the findings with disease prognosis. 2. Material and methods 2.1. Subjects The study was carried out in 150 primary MDS (78 males and 72 females) patients belonging to the age-group of 15 to 89 years. The diagnosis of MDS was established and patients were subgrouped according to revised clinical and pathomorphological criteria of World Health Organization [15]. The patients were also stratified based on cytogenetic status, number of cytopenias, and blast percentage according International

P. Mohanty et al. / Blood Cells, Molecules and Diseases 59 (2016) 52–57

Prognostic Scoring System (IPSS, 1997) [4]. The haematological parameters and other details including age, sex, occupation, living environment, past illness and habits were recorded at the time of diagnosis. Bone marrow aspirate (BMA)/peripheral blood (PB) was collected from the patients in heparin (2 ml) and EDTA (3 ml) vacutainers for cytogenetic and molecular study. Fifty (50) age-matched healthy controls with no family history of haematological malignancies were recruited for QMPSF study. Written informed consent was obtained from all patients and healthy controls participating in the study, and the study protocols were approved by the Institutional Ethics Committee. 2.2. Cytogenetic study The BMA in heparin vacutainer (2 ml) was cultured in F-10 nutrient media (Sigma, USA) with 20% fetal bovine serum for 24 to 72 h or directly harvested after arresting with colchicine (50 μg/ml). The cultures treated with 0.075 M hypotonic solution (KCl) were fixed with Carnoy's fixative (methanol: acetic acid (3:1 v/v)). The chromosomal preparations obtained by dropping on pre chilled slides were subjected to GTG banding for karyotyping. The chromosomal analysis was done from at least 20 metaphases from an individual and karyotyping was done according to International System for Human Cytogenetic Nomenclature (ISCN) 2013 [15]. Fluorescence in-situ hybridization (FISH) analysis was carried out using centromeric probe (Vysis, Abbott Molecular, Illinois, USA) for chromosome 8 and locus specific probes (Vysis, Abbott Molecular, Illinois, USA) LSI 5q31 EGR1, LSI 7q31 and LSI 20q12 according to standard procedure. 2.3. QMPSF assay QMPSF is a sensitive method for detecting genomic deletions or duplications based on the simultaneous amplification of short genomic fragments using dye labeled primers under quantitative conditions (patent FR 020924) [16]. Genomic DNA was extracted from PB of 100 patients (60 patients with normal karyotype and 40 patients harboring cytogenetic abnormalities) and 50 controls using Qiagen Blood Mini Kit according to manufacturer's protocol and quantified thereby. We modified a singleplex QMPSF assay previously described [14] into two distinct multiplex PCR assays each of which contain the following target genes: Assay 1 — PTPRT (20q12), TP53 (17p13), CDKN1B (12p13.1), CD69 (12p13.1), SPOCK (5q31.2), SEC61 (7p11), HIPK2 (7q34), ATAD2 (8q24), EPO (7q22), EGFR (7p11) and EGR1 (5q31); Assay2: NRF1 (7q32), POP7 (7q22), MLL3 (7q36), MET (7q31), CCNG1 (5q34), UNC5D (8p12), CYP24A1 (20q13), ETV6 (12p13) and CSF1R (5q33). The CECR1 gene, located at 22q11was chosen as a reference gene as it appears uncommonly affected by aneuploidy or focal gains or losses [14]. Primer pairs published previously were utilized for the assay [14]. PCR were run from 100 ng of genomic DNA in a final volume of 25 μl using Platinum® Multiplex PCR Master Mix and 0.1 to 0.3 μmol/l of each primer, one primer of each pair carrying a 6-FAM label. After initial denaturation for 2 min at 95 °C, 26 cycles were performed consisting of denaturation, 95 °C for 30 s, annealing 56 °C for 90 s and extension 72 °C, 30 s (ramping3°C/s, followed by a final extension step for 30 min at 60 °C). Polymerase chain reaction (PCR) products were analyzed on a sequencing platform used in the fragment analysis mode using Gene mapper software, in which both peaks heights and areas are proportional to the quantity of template present for each target sequence. We used two different methods to calculate allele dosage in this study: visual sample-to-control comparison and numerical sample-to-control comparison as described previously [16]. The normalized peak height ratio (R) was calculated using the formula R = (P/Pr) / (Wt/Wtr) = (Peak Height of patient for each gene) / (Peak Height of patient for reference) / (Control Peak height / Control Peak height of reference gene). The visual sample to control and numerical estimation was done in each of the subjects using at least 5 controls per patient and a 2 fold change in peak height indicated deletion or

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duplication and a normalized peak height ratio (R) b 0.5 indicated deletion and N 1.5 indicated duplication (Fig. 4,Table 3). The QMPSF assay was replicated thrice using multiple controls each time to confirm the presence of deletion or duplication identified. 3. Results 3.1. Patients characteristics The study was carried out in 150 primary MDS including 78 (52%) men and 72 (48%) women and patient's clinical and haematological parameters are reported in Table 1. The age group of MDS patients ranging from 15 to 89 years and median age at diagnosis was 52 years. The majority of patients in our cohort presented with anemia or pancytopenia and hypercellular erythroid preponderant marrow (51%). Patients were classified into WHO subtypes as follows; 53 (36%) cases of refractory anemia (RA); 6 (4%) cases of refractory anemia with ring sideroblasts (RARS); 47 (31%) cases of refractory cytopenia with multilineage dysplasia (RCMD); 30 (20%) cases of refractory anemia with excess of blasts-1 (RAEB-1); and 14 (9%) cases of refractory anemia with excess blasts-2 (RAEB-2). The frequency of low-risk MDS subgroups (RA, RCMD and RARS) was higher (71%) compared to high-risk subgroups (RAEB1 and RAEB2) (29%) in our cohort (Table 1). The prognostic risk stratification based on IPSS score identified a vast majority (82%) of patients belonging to low 76 (51%) and intermediate-1, 48(32%) prognostic groups. 3.2. Cytogenetics The clonal chromosome abnormalities were detected in 46% of a total of 150 primary MDS patients studied (Table 2). Identified chromosome

Table 1 Clinical characteristics of MDS patients. Clinical characteristics

MDS patients (n = 150)

Age (yrs) Median Range

52 15–89

Sex Male, n (%) Female, n (%)

78 (52) 72 (48)

Haemoglobin (g/l) Median Range

6.6 2–12.4

Total leucocyte count (×109/l) Median Range

3.5 1.2–9.5

Platelet count (×109/l) Median Range

76 6–430

WHO subtype RA, n (%) RARS, n (%) RCMD, n (%) RAEB1, n (%) RAEB2, n (%)

53 (36) 6 (4) 47 (31) 30 (20) 14 (9)

IPSS risk group Low, n (%) Intermediate-1, n (%) Intermediate-2, n (%) Poor, n (%) Transfusion dependence, n (%)

76 (51) 48 (32) 18 (12) 8 (5) 138 (92)

BM cellularity Normocellular, n (%) Hypercellular, n (%) Hypocellular, n (%)

48 (32) 76 (51) 26 (17)

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P. Mohanty et al. / Blood Cells, Molecules and Diseases 59 (2016) 52–57 Table 2 Cytogenetic features of MDS patients. Characteristics

No of patients (%)

Total patients, n = 150 Karyotype Normal Abnormal

81 (54) 69 (46)

Chromosomal aberrations del (5q31) del (7q31) Monosomy 7 del (20q12) Trisomy 8 del (5q) + del (20q12) del (5q) + trisomy 8 del (7q) + del (20q) del (7q) + trisomy 8 del (20q) + trisomy 8

9 (5) 15 (10) 9 (6) 18 (12) 10 (6.6) 1 (0.6) 1 (0.6) 4 (2.6) 1 (0.6) 1 (0.6)

Chromosome aberrations in WHO MDS subtypes RA, n = 53 RARS, n = 6 RCMD, n = 47 RAEB1, n = 30 RAEB2, n = 14

19 (12.6) 3 (2) 16 (10.6) 16 (10.6) 11 (7.3)

IPSS cytogenetic risk groups Good, n (%) Intermediate, n (%) Poor, n (%)

106 (71) 20 (13) 24 (16)

aberrations included del (5q) (5%), del (7q) (10%), monosomy 7 (6%), del (20q) (12%), trisomy 8 (6.6%) and complex chromosome aberrations (5.7%) (Table 2). Of the total cytogenetic aberrations (n = 69), the abnormalities involving chromosome 7 were the most frequent i.e. 24 (34.7%) and del (20q12) was the second most frequent 18 (26%) cytogenetic abnormality, identified in our study group (Fig. 1). The chromosomal abnormalities were identified at a higher frequency (66%) i.e. in 29 of 44 highrisk MDS subgroups (RAEB1 and RAEB2) as compared to low-risk MDS subgroups (RA, RARS and RCMD) (38%) (Fig. 2). The del (5q) was associated with RA with or without excess blasts i.e. in 8 out 9 patients, monosomy 7 was identified majorly in RCMD subgroup i.e. in 4 out of 10 patients while del (20q) was associated with low-risk MDS subgroups (RA and RCMD) in 11 out of 18 patients (Fig. 2). In our study, a high frequency of patients belonged to IPSS-good cytogenetic risk group 106 (71%) while 20 (13%) patients belonged to intermediate and 24 (16%) belonged to poor risk groups (Table 2).

Fig. 2. Frequency of cytogenetic abnormalities in MDS.

QMPSF enabled detection of deletions in 17/60 (28.3%) karyotypically normal MDS patients and in 9/40 (23%) patients harboring known cytogenetic aberrations while duplications were detected in 2/60 (3.3%) of patients with normal karyotype and in 3/40 (7.5%) patients with cytogenetic abnormalities (Fig. 3). 73% (19/26) of genetic losses or deletions were identified in regions on chromosome 7 namely 7q22, 7q31 and 7p11 each harboring genes EPO (34.6%), MET (15.4%) and SEC-61 (23.1%) (Fig. 5). 9 patients showed deletion at 7q loci gene EPO while 1 patient showed duplication; 6 patients with deletion at 7p loci gene SEC61 while 1 patient showed duplication and 4 patients showed deletion at 7q loci gene MET (Table 3). The other regions harboring CNVs in our study group included 12p13.1 (16%) and 5q31 (11%) (Fig. 5). The CNVs were uniformly distributed across MDS subtypes (Table 2). The genetic losses were commonly associated with intermediate-1(n = 10) and intermediate-2(n = 7) IPSS prognostic groups (Fig. 6). 13/60 (21.6%) karyotypically normal MDS patients harboring CNVs belonged to intermediate-poor prognostic groups Table 3. EPO and SEC-61 deletions were associated with IPSS intermediate prognostic groups in 6 out 9 patients and in 4 out of 7 patients respectively (Table 3). 4. Discussion

We studied CNVs in 100 primary MDS patients (60 patients with normal karyotype and 40 patients harboring cytogenetic abnormalities).

MDS are heterogeneous clonal haematopoeitic stem cell disorders characterized by ineffective haematopoeisis, cytopenias and risk of progression to AML. We studied 150 primary MDS patients having median age at diagnosis of ~ 52 yrs which is in agreement with reports from India/Asia where a younger age at diagnosis is observed and therefore, contradicting reports from the Western countries wherein MDS is primarily an elderly disease with median age at diagnosis of ~ 66 years [17–19]. A majority (58%) of patients in our cohort were N 50 yrs of age the predominant age group being 50–65 yrs (n = 46) which was in similar to the trend seen in Japan and other studies from India [18–20]. The trend towards development of MDS in younger age in

Fig. 1. Distribution of cytogenetic aberrations in MDS subtypes. The X axis denotes cytogenetic abnormalities in MDS patients and the Y-axis denotes the number of patients.

Fig. 3. Frequency of CNVs in MDS according to the presence or absence of cytogenetic abnormality.

3.3. Copy number variations in MDS

P. Mohanty et al. / Blood Cells, Molecules and Diseases 59 (2016) 52–57

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Table 3 The normalized peak height ratios as detected by QMPSF in different MDS subgroups and its correlation with cytogenetic abnormalities and IPSS prognostic risk group. Gene

Normalized peak Height ratio R = (P/Pr) / (Wt/Wtr)

Result Deletion-R = b0.5 Duplication-R= N 1.5

Cytogenetic abnormality

MDS-subgroup

Age (yrs)/sex

IPSS risk group

EPO

0.43 0.40 0.44 0.40 0.33 0.43 0.36 1.97 0.41 0.39 1.56 0.43 0.3 0.35 0.39 0.41 2.91 0.44 0.45 2.27 0.40 0.43 0.37 0.43 0.43 0.44 3.49 0.33 0.39 0.44 0.32

Deletion

NIL TRISOMY 8 del (20q) NIL NIL NIL del (20q) del (7q), del (20q) NIL NIL NIL NIL del (5q) del (5q) NIL del (20q) NIL NIL NIL del (7q) NIL NIL NIL NIL del (20q) NIL del (7q) + trisomy 8 NIL del (7q) NIL Monosomy 7

RA RA RCMD RAEB1 RA RA RAEB2 RAEB1 RAEB2 RAEB2 RCMD RAEB2 RAEB1 RAEB1 RA RAEB2 RCMD RAEB2 RA RA RA RCMD RAEB1 RA RAEB2 RAEB2 RCMD RA RAEB2 RCMD RAEB1

24/F 48/F 75/F 48/M 35/F 40/F 60/M 56/M 50/M 42/F 55/M 40/F 55/F 60/M 56/M 70/F 50/M 52/F 43/M 60/M 58/M 45/F 50/F 34/M 67/F 62/M 50/F 48/F 73/M 56/F 49/M

Int-1 Low Int-1 Int-1 Low Int-1 Poor Poor Int-2 Int-2 Low Poor Int-1 Int-1 Int-1 Int-2 Int-1 Int-2 Low Low Low Int-1 Int-1 Low Int-2 Int-2 Int-1 Low Poor Int-1 Int-2

EGR1

CDKN1B

CD69

SEC61

MET

Duplication Deletion Duplication Deletion

Deletion Duplication Deletion Duplication Deletion

Duplication Deletion

India may be due to environmental, occupational and lifestyle (habits) factors. In our study group most patients belonged to low-risk MDS subgroups RA and RCMD subgroups (n = 53, n = 47) compared to high risk subgroups RAEB1 and RAEB2 (n = 30 and n = 14) suggesting that the occurrence of MDS is an early event associated with later progression, which is similar to the trend observed in studies from Asia although a reverse trend is observed in Western countries (Table 1) [18–21]. Cytogenetic status at diagnosis and during the course of the disease plays a decisive role in defining disease prognosis and treatment planning and its importance can be asserted from its inclusion in most international prognostic scoring systems in MDS [22]. Our present study identified 46% chromosomal aberrations in a larger cohort of 150 Indian MDS patients using karyotyping and FISH which is in accordance with studies reported in India as well as worldwide [18–22]. Of the total cytogenetic aberrations (n = 69) identified, the abnormalities involving chromosome 7 were the most frequent i.e. 24 (34.7%) and del (20q12) was the second most frequent i.e. 18 (26%) cytogenetic abnormality identified in 16% and 12% primary MDS patients respectively (Fig. 1,Table 2). Chaubey et.al [19] and Haase et.al [22] identified monosomy 7 as the most common abnormality in MDS while deletions of chromosome 7 at 7q31 loci were frequent in our study group identified in 10% of 150 patients studied. Hasse et.al [22] suggests abnormalities of chromosome 7 are identified in poor prognostic groups, however, in our study chromosome 7 deletions were isolated in intermediate-poor prognostic groups. Interestingly, 6/9 patients with monosomy 7 showed normocellular marrow with mild dysmyelopoeisis at presentation all of which were associated with IPSS poor prognostic group. In our series, 20q deletion was the second most common cytogenetic aberration associated with RA and RCMD (n = 11) subgroups which is consistent with the frequencies seen in studies from Asia [18–20]. Although del (20q) is associated with good prognosis [22], in our series it was found to be clustered in IPSS intermediate prognostic group. Therefore, the abnormalities of chromosome 7 and 20 particularly deletions were

frequently identified in our population with uniform distribution across MDS subgroups and associated with intermediate to poor IPSS prognostic groups. In the present study we modified a previously published QMPSF [14] assay which enabled detection of CNVs in 28.3% karyotypically normal patients and 23% patients with abnormal karyotype. Previous studies on detection of CNVs reported a similar frequency utilizing expensive, high resolution array based platforms [7–13,25]. QMPSF is a sensitive, non-invasive technique to detect submicroscopic deletions and duplications and should be utilized for detection of CNVs in MDS. Barring a single study no other study utilized QMPSF as a tool for detection of CNVs in MDS although its sensitivity and specificity has been explored in other cancers [14,16]. Deletions (26 of 31 CNVs) were common as compared to duplications (5 of 31 CNVs) in our study which is consistent with previous reports [14,25] on CNVs in MDS (Fig. 3). Additionally, QMPSF could enable detection of deletion and duplications in patients already harboring cytogenetic abnormalities (n = 12) suggesting it could complement FISH and CC in detection of cytogenetic abnormalities (Table 3). 13/60 (21.6%) karyotypically normal MDS patients harboring CNVs belonged to intermediate-poor prognostic groups (Table 3) indicating the role of cryptic CNVs in disease outcome in patients without cytogenetic aberrations. Patients harboring cytogenetic abnormalities and CNVs both together showed random distribution across different IPSS prognostic groups (Table 3). The genetic losses at regions on chromosome 7 namely, 7q22 (EPO), 7q31 (MET) and 7p11 (SEC-61) by QMPSF were frequent (73%) in our study and a similar frequency has been identified in our previous study by Kawankar et.al [23] while contradicting a recent study by Zhang et.al [26] which identified other regions prone to copy number alterations in MDS and Acute leukemia patients. The highlight of our study was that EPO (7q22) and SEC-61 (7p11) emerged as new candidate genes susceptible to genetic losses with 9 of 26 and 6 of 26 deletions respectively present in these regions. These regions should be evaluated in future studies on CNVs in

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P. Mohanty et al. / Blood Cells, Molecules and Diseases 59 (2016) 52–57

Fig. 4. Representative image depicting duplication (upper panel) at gene loci 12p13.1 and 7p11 and deletion (lower panel) at gene loci 7q22 and 5q31 by visual sample to control comparison of peak height by QMPSF.

MDS to assert their clinical significance. SEC-61 is associated with apoptosis and tumor cell survival in glioblastomas and could account for mature cell death in BM of MDS patients [24]. Copy number variants in EPO gene could account for dyserythropoiesis in bone marrow of MDS as this gene is involved in proliferation of erythrocytes and previously 7q22 has been associated with deletions in solid tumors [25].The prognostic impact of the non-invasive and inexpensive approach of QMPSF should be explored in MDS in future to complement conventional cytogenetics and or FISH in a clinical setting.

5. Conclusions Cytogenetic abnormalities including deletions of chromosome 7 and chromosome 20 are frequently identified in Indian subjects. EPO (7q22) and SEC-61(7p11) emerged as new candidate genes susceptible to genetic losses associated with intermediate IPSS prognostic groups and future studies should focus on delineating the clinical impact of these regions and their role in disease progression. QMPSF could complement conventional cytogenetics and FISH in a clinical setting for accurate prediction of diagnosis/prognosis in MDS. Conflict of interest None.

Fig. 5. Frequency and distribution of deletions at regions 7q22, 7q31, 7p11, 5q31 and 12p13.1 harboring genes EPO, MET, SEC-61, EGR1, CDKN1B and CD69 across MDS subgroups.

Fig. 6. Distribution of deletions and duplications according to IPSS risk groups. The X-axis denotes IPSS prognostic risk groups and Y-axis denotes the number of patients.

P. Mohanty et al. / Blood Cells, Molecules and Diseases 59 (2016) 52–57

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