Expression of the testis-specific gene, TSGA10, in Iranian patients with acute lymphoblastic leukemia (ALL)

Leukemia Research 30 (2006) 883–889

Brief communication

Expression of the testis-specific gene, TSGA10, in Iranian patients with acute lymphoblastic leukemia (ALL) Maryam Beigom Mobasheri a , Mohammad Hossein Modarressi a,∗ , Mahdi Shabani b , Hossein Asgarian b , Ramezan-Ali Sharifian c , Parvaneh Vossough d , Fazel Shokri b a Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Department of Hematology and Oncology, Vali Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran d Hematology and Oncology Clinics, Ali Asghar Hospital, Iran University of Medical Sciences, Tehran, Iran b

c

Received 24 September 2005; received in revised form 24 September 2005; accepted 22 November 2005 Available online 6 January 2006

Abstract Testis-specific gene antigen (TSGA10) is expressed in fetus, testis and frequently in human solid cancers and acute leukemias, making it a candidate for immunotherapy and for detection of minimal residual disease (MRD). This gene is considered as a member of cancer-testis (CT) genes. We previously demonstrated TSGA10 expression during spermatogenesis. There is also evidence for potential TSGA10 involvement in cell proliferation. TSGA10 expression has been observed in a wide spectrum of cancers but not in hematopoietic neoplasm. Here we demonstrated expression of TSGA10 by semi-quantitative RT-PCR in 44 (84.6%) out of 52 bone marrow samples and all peripheral blood samples from patients with acute lymphoblastic leukemia (ALL). Twenty-seven (52%) cases had high level of gene expression and 16 (30.7%) cases had a lower expression level of the gene in the patients bone marrow. Presence of TSGA10 expression in ALL may open a window to functional study of mitotic checkpoint proteins in leukemia. RT-PCR of TSGA10 may help in detection of residual clonal cells leading to early diagnosis and better prognostic qualification of the disease. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cancer; Testis; TSGA10 gene; Leukemia

1. Introduction Acute lymphoblastic leukemia (ALL) is a malignant disorder resulting from the clonal proliferation of lymphoid precursors with arrested maturation [1]. The disease can originate from lymphoid cells of different lineages, thus giving rise to B- or T-cell leukemias or sometimes mixed-lineage leukemia. Early diagnosis is critical to achieve appropriate treatment and complete remission. Genes and/or their products that are highly and specifically increased in leukemia Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BM, bone marrow; CR, complete remission; EB, ethidium bromide; MRD, minimal residual disease; PB, peripheral blood; RTPCR, reverse transcriptase polymerase chain reaction ∗ Corresponding author. Tel.: +98 21 8953005; fax: +98 21 6404577. E-mail address: [email protected] (M.H. Modarressi). 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.11.012

could represent useful diagnostic markers for leukemia. They could also be good targets for development of novel therapeutic drugs. Cancer-testis (CT) antigens are a group of proteins expressed in male germinal cells and certain tumor types, but not, or at least 1000-fold less, in other normal tissues [2]. Testis have a so-called blood–testis barrier, which limits contact between testicular and immune cells in the seminiferous tubule supported by the Sertoli cells [3,4]. Due to this barrier and the immune-privileged status of germinal cells, these antigens can be considered functionally tumor-specific. In addition, these tumor-specific markers could represent attractive targets for immunotherapy [5,6]. We previously identified a novel gene designated TSGA10 [7]. This gene could be classified as the member of the cancertestis (CT) gene family. Human TSGA10 is expressed in

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normal testis, but not in a variety of other tissues, nor in testes of infertile patients who suffer from non-obstructive infertility. It is reported that TSGA10 expression was negative for peripheral blood cells obtained from healthy donors using northern analysis [7]. In addition to testicular expression, TSGA10-specific ESTs have been reported from several cancer cells including adenocarcinoma (GeneBank accession nos.: AW057728, AI696619 and AW591313), acute myelogenous leukaemia (GeneBank accession no.: BF243403) and germ cell tumors (GeneBank accession no.: BE047007). It has been demonstrated that TSGA10 is also expressed in actively dividing and fetal differentiating tissues and in various primary tumors [8]. Mouse homologue (Mtsga10) mRNA is first detected in the post-meiotic phase of spermatogenesis. We previously reported that Mtsga10 mRNA is translated to a 65 kDa protein, which appears to be processed to a major fibrous sheath protein of sperm tail [9]. Assays that test for cancerous cells have increased our ability to detect residual disease at levels below the sensitivity of morphologic evaluation. A new concept called minimal residual disease (MRD) is becoming an important prognostic factor in ALL [10]. Molecular techniques can help in detection of residual gene expression in clonal cells and this may result in early diagnosis and better prognosis. In the present study TSGA10 expression was investigated in leukemia cells from ALL patients to get future insights into its potential role as a broad tumor-associated antigen (TAA) implicated in tumorogenesis of ALL.

CD19+ , CD 10+ , CD20+ ) (n = 12), and B-ALL (HLA-DR+ , CD 19+ , CD 10− , CD20+ ) (n = 2). The main criteria for inclusion in this study were that the patients were at presentation stage and have not taken any therapeutic medication at the time of sampling. The adult and child patients were selected from patients attending the Hematology and Oncology Clinics of Vali-Asr and AliAsghar hospitals, affiliated to Tehran University of Medical Sciences and Iran University of Medical Sciences, respectively. Heparinized PB samples collected from 10 normal healthy donors were used as controls to determine TSGA10 expression. This study was approved by the ethical committee of Tehran University of Medical Sciences, and informed consent was obtained from patients or their parents. 2.2. Extraction of RNA and cDNA preparation Peripheral blood or bone marrow was collected in EDTA. White blood cells were immediately isolated using Ficoll density centrifugation, washed twice with phosphatebuffered saline (PBS, PH 7.4, 0.15 M), and the cell pellet was lysed in Trizol Reagent (Invitrogen, USA). Total RNA was extracted following the manufacturer’s instructions with minor modifications. Single-stranded cDNA was prepared from 1–5 ␮g total RNA of various samples using MMLV reverse transcriptase (Invitrogen) and random primer (Pharmacia, Sweden).

2. Materials and methods 2.3. Semi-quantitative RT-PCR and nested PCR 2.1. Patients and controls The study included 52 bone marrow and 14 peripheral blood of patients with ALL and 10 healthy controls, who were diagnosed on the basis of clinical and laboratory findings and surface markers studied by flow cytometry using a panel of monoclonal antibodies specific for B-lineage markers (CD 19 and CD20), T-lineage markers (CD3, CD4, CD5 and CD8), myeloid markers (CD 13, CD33 and CD 14), common acute lymphoblastic leukemia antigen (CD 10), the class II major histocompatibility complex antigen (HLA-DR), terminal deoxynucleotidyltransferase (TdT) and the common leukocyte antigen CD45. Major clinical and hematological features of the patients are summarized in Table 1. Patients older than 14 years were considered adults (14 cases with mean age of 22 years) and those younger than or equal to 14 years were considered children (43 cases, with mean age of 6 years). Forty-six (60%) patients belonged to B-lineage and 11 (14%) belonged to T-lineage ALL. One case (1.2%) was diagnosed as bilineal lymphoid leukemia (T and B-ALL). Based on immunophenotypic profile, the B-ALL patients were further classified into Pro-B (HLADR+ , CD19+ , CD10− , CD20− ) (n =6), early Pre-B (HLADR+ , CD19+ , CD 10+ , CD20− ) (n = 27), Pre-B (HLA-DR+ ,

Human cDNAs were checked for cDNA quality using primers designed from exon 10 5 -TCCGACTGAGCGGCACTGGGAGTGC-3 and exon 11 5 -GCCCGCAGGTCCTCTTTCCCTCACA-3 of the housekeeping gene phosphoglucomutase-1 (PGM1). The RT-PCR amplification was performed using 1/20 of the cDNA. The PCR amplification for (PGM1) was performed with 30 cycles of 45 s at 94 ◦ C, 45 s at 64 ◦ C, and 50 s at 72 ◦ C. A pair of specific primers (forward—GSP9: 5 -CAACGGCACATGCTATTCTC-3 , reverse—Rll: 5 -ATTGATCTCATTTGCCAGC-3 ), were designed to amplify 635 bp and 679bp fragments of TSGA10 (GeneBank accession no.: AF254756). The primers were positioned in different exons of TSGA10 to avoid falsepositive results caused by genomic DNA contamination of the RNA preparation. The total reaction volume was 25 ␮l containing 1 ␮l cDNA, PCR set and Taq enzyme (GIBCO). The TSGA10 amplification reactions consisted of 30 cycles of denaturation at 94 ◦ C for 45 s, annealing at 55 ◦ C for 45 s, extension at 72 ◦ C for 45 s and a 7 min final extension at 72 ◦ C. In order to identify low amount of TSGA10 expression, semi-nested PCRs were performed using 1 ␮l of the first PCR reactions. Negative control reaction of the first PCR was used for the semi-nested PCR to identify

Table 1 Classification of data from ALL patients WBC × 103

Lym (%)

RBC × 106 cell/ml

HB (g/dl)

PLT × 103

Cellularity (BM)

Blast (%) (BM)

FAB

Immunophenotype (BM)

TSGA10 expresstion first PCR

Nested PCR

Alternative splicing

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

ND2 ND3 ND7 ND8 ND9 ND10 ND14 ND15 ND16 ND17 ND19 ND21 ND22 ND24 ND25 ND26 ND28 ND29 ND32 ND33 ND35 ND36 ND59* ND40 ND41 ND42* ND43 ND44* ND45* ND47 ND48* ND49 ND52 ND53 ND54* ND55 ND56 ND57* ND58* ND60* ND61* ND62 ND65* ND67* ND68*

9.9 8.4 90 7.9 222 14.2 53.7 3.1 NA 5.3 4.8 70.2 45.7 14.8 6.4 9.6 8.4 3.1 20.4 2.1 3.1 8.6 22.9 482 3.2 63.5 23.1 46.9 10 5.8 47.6 11.9 31.1 4.4 145 3.1 13.7 16.7 22.7 38.9 34.5 2 4.1 245 91.7

90 92 73 60 40 95 83 NA NA NA 34 87 90 0 83 91 91 88 25 78 90 94 NA NA 64 58 NA NA 92 89 NA 93 90 NA NA NA 19 81 83 85 98 86 NA NA NA

2.5 2.8 5.1 3.2 2.6 2.9 3.7 3.5 NA 3.8 4 2.3 3.2 3.8 NA 3.2 2.3 2.7 3.7 4.1 2.9 4.1 4.7 3.3 3.3 NA 3.5 NA 4.1 2.7 2.8 3.8 3 3.5 3.4 3.9 2.6 2.8 2.2 3.1 NA 3 4.2 1.6 NA

8.8 8.7 13.1 8.2 7.2 6.2 9.9 10.2 NA 10.3 11 5.3 8.8 9.5 6.4 11.5 7.2 8.8 10.3 11.2 8.4 6.3 13.6 10.1 9.5 13.3 11.8 8 11.5 8 7.6 10.6 8.1 9.1 9.5 10.4 6.8 8.2 6.2 8.6 5.6 8.9 12.2 6.9 5.5

69 57 24 145 105 59 82 107 NA 22 641 23 147 13 120 15 75 15 180 <10 33 <10 103 40 158 7 149 75 19 23 13 <10 9 19 46 86 56 32 <10 23 104 88 70 23 32

Hyper. Hyper. Hyper. Hyper. Hyper. Hyper. Hyper. Hyper. NA NA NA Hyper. Hyper. NA Hyper. Hyper. Hyper. NA Hyper. Normal Hyper. Hyper. NA Hyper. Hyper. Hyper. NA Hyper. Hyper. Hyper. NA Normal Normal NA Hyper. Normal NA Hypo. Hypo. Hyper. Hyper. Normal NA Hyper. Hyper.

40 60 50 70 60 32 80 NA NA 90 80 40 60 98 80 65 90 30 75 50 52 65 NA 90 65 70 90 60 50 20 NA 80 55 NA 60 >90 80 30 60 50 40 10 NA 90 90

L1 L1 L1 L1 L1 L3 L1 LI NA NA L1 L1 L1 L2 L1 LI L1 L1 L3 L1 L2 L1 NA mixed L1 L1 L2 L1 L1 L1 L1-L2 L1 L1 L1 L2 L2 L2 L2 L1 L1 L1 L1 L2 L1 L2

Early Pre-B Early Pre-B Early Pre-B Early Pre-B T Early Pre-B Early Pre-B Early Pre-B NA T T Early Pre-B Pro-B Early Pre-B Early Pre-B Early Pre-B Early Pre-B Early Pre-B B Early Pre-B Pro-B Early Pre-B NA Mixed lineage Pre-B Early Pre-B Early Pre-B Early Pre-B Early Pre-B Early Pre-B Pro-B Pre-B Pre-B NA T Early Pre-B T T Pre-B Pre-B Early Pre-B Pre-B Early Pre-B Pro-B T

++ ++ ++ − − − − ++ − − + − ++ − − − + − + − − − − − + − + − − + − + +++ − +++ − − ++ + ++ ++ +++ ++ ++ +++

+ + + + − + + + + + + + + + − − + − + − − + + − + + + + + + + + + + + − + + + + + + + + +

+ + + + − + + + − − − + + + − − + − + − − + + − + + + + + + + + + + + − + + + + + + + + +

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Patient code

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No.

All patient’s samples were obtained from BM except those with * which were taken from BM and peripheral blood. +++ means similar bands in Fig. 1 Lanes c2 and c7; ++ means similar band in Fig. 1 Lane c6; + means similar bands in Fig. 1 Lanes c4 and c5.

+ + + + + + + − − + + ++ +++ + Early Pre-B Pre-B Pre-B B Pre-B T Early Pre-B 93 >90 35 NA NA >95 65 23.2 2.3 27.9 2.5 10.1 46.1 10.1 ND69* ND70 ND71 ND73 ND74 ND76 ND77 46. 47. 48. 49. 50. 51. 52.

6 40 88 60 10 NA 92

3.8 3.1 5.5 2.9 3 5.3 1.9

11.2 8.3 14.7 7.8 9.6 15.3 5.1

39 36 41 39 43 104 15

NA NA Hypo. NA Hypo. Normal Hyper.

L1 NA L1 L3 L1 NA L1

Nested PCR TSGA10 expresstion first PCR Immunophenotype (BM) FAB Blast (%) (BM) Cellularity (BM) PLT × 103 HB (g/dl) RBC × 106 cell/ml Lym (%) WBC × 103 Patient code No.

Table 1 (Continued )

+ + + + + + +

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false positive results caused by DNA contamination. The sequence of the internal primers used were: Forward-GSP9: 5 -CAACGGCACATGCTATTCTC-3 ); reverese-R10: 5 GATGATGCGAAGTAGGTC-3 to amplify a 269 bp fragment of TSGA10. Primers were positioned in different exons of the gene. The TSGA10 semi-nested amplification reactions consisted of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 30 s for 30 cycles and a 7 min final extension at 72 ◦ C. PCR product was subjected to electrophoresis in a 1.8% agarose gel containing ethidium bromide (EB), and then photographed under ultraviolet light. 2.4. PCR-RFLP and polyacrylamide gel electrophoresis For each sample, the amplified PCR product was digested with the restriction enzyme XbaI and BglII (Fermentas, Russia). The enzyme digestion mixtures contained 10 ␮l PCR product, 6 units of enzyme, 2 ␮l the enzyme 10Xbuffer and H2 O to total volume of 20 ␮l. The reactions were allowed to proceed for 12 h at 37 ◦ C. The resulting fragments were separated by 8% polyacrylamide gel electrophoresis and visualized by standard silver staining. 2.5. Computer analysis Bioinformatics and computer analysis was carried out using SPSS software and programs at the following sites: http://ca.expasy.org/, http://www.ensembl.org/ and http://www.webcutter.com.

3. Results and discussion We used RT-PCR and semi-nested PCR to analyze expression of human TSGA10 in 52 bone marrow and 14 peripheral blood samples of patients with ALL and 10 peripheral blood samples from healthy donors as controls. TSGA10 expression pattern is summarized in Table 2. All RT-PCR products were amplified using internal primers, if no expression was detected from running RT-PCR samples on agarose gel. We detected TSGA10 expression in 44 out of 52 (84.6%) of bone marrow samples from ALL patients. In bone marrow samples, 27 (52%) cases with high level of expression were identified by first round of RT-PCR and 16 (30.7%) cases by semi-nested PCR or second amplification which represented low level of TSGA10 expression. Representative results are shown in Fig. 1. Negative results from nested PCR showed presence of no TSGA10 transcript in those ALL samples (Table 2). Gene expression analysis of TSGA10 was examined in 33 adult patients and 19 children with ALL but no association was detected between age and the gene expression. We also found no correlation between type of ALL and TSGA10 expression. RT-PCR amplification of cDNA made from patient samples using GSP9 and R11 primers showed differing levels

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887

Table 2 TSGA10 gene expression analysis of all samples after two rounds amplification (first PCR and semi-nested PCR) TSGA10 expression

Positive in the first PCR (high expression) Positive in the semi-nested PCR out of negatives in first PCR (low expression) Negative in the semi-nested PCR out of negatives in first PCR (no expression) Total-positive expression Total samples

of expression with both alternative splice products present in almost all of the actively dividing bone marrow samples (Figs. 1 and 2). Semi-quantitative expression analysis was carried out by nested PCR. Fourteen peripheral blood samples from ALL patients were examined and expression of TSGA10 was present in all of them. Eight patients had high and others showed low expression of TSGA10 in their peripheral blood samples. However, the transcript was not found in the peripheral blood sample from healthy donors using RT-PCR (Table 1 and Fig. 2). This result may suggest that TSGA10 is an important prognostic factor and a tool for monitoring MRD in ALL. Expression of this gene in blood cells may predict survival or help early diagnosis. To date, 44 cancer testis gene families have been identified and their expression studied in numerous cancer types [5,6,11]. TSGA10 could be regarded as a new member of cancer-testis genes selectively expressed in various types of human neoplasms [8,9,12] and ALL but not in normal tissues other than testis [7]. This characteristic feature of these specific genes makes them promising antigens for cancerspecific immunotherapy. In order to confirm sequencing results and to check isoform of the TSGA10 gene, PCR-RFLP analysis was performed. We found the isoform of the TSGA10 gene which is the result of its mRNA alternative splicing in three patient

Fig. 1. RT-PCR results from some of bone marrow samples. All samples were controlled for presence of cDNA using a housekeeping gene, PGM (a). RT-PCRs were carried out as described in Section 2 and produced two bands (b) from TSGA10 cDNA (Lanes 2, 7 and 10). All PCR products were subjected to electrophoresis in a 1.8% agarose gel containing EB. Lane 1 is a DNA marker and Lanes 2–8 are cDNAs from bone marrow. Lane 9 is the negative control (water) and Lane 10 is cDNA from an adult human testis. Nested PCR was performed to analyze low expression of TSGA10 gene in ALL samples (c).

Samples from Healthy donors

Patients with ALL

Peripheral blood

Bone marrow

Peripheral blood

0 (0%) 0 (0%) 10 (100%) 0 (0%) 10

27 (52%) 16 (30.7%) 8 (15.4%) 44 (84.6%) 52

8 (57.1%) 6 (42.9%) 0 (0%) 14 (100%) 14

samples. Two patients had large spliced isoforms of the gene and the other patient had the small spliced isoform of the TSGA10 transcript (Fig. 3). It has been reported that an alternative splicing at nucleotide 511 leads to an mRNA product in testis which includes an additional 44 bp in the 5 UTR [7]. The site of alternative splicing was located between GSP9 and R11 primers. Both RT-PCR product bands were analysed using specific restriction enzymes to confirm that those bands derive from alternatively spliced TSGA10 mRNAs (Fig. 3). The mouse homologue of TSGA10 mRNAs is corresponding to the large isoform of TSGA10 transcript and this alternative splicing was not seen in mouse [9]. There were no

Fig. 2. Expression analysis in peripheral blood samples from ALL patients and healthy individuals. Presence of cDNA was checked in all samples using a housekeeping gene, PGM (1a and 2a). Lane 1 is a DNA marker. (1) Lanes 2–5 are results of RT-PCR from some of peripheral blood samples obtained from ALL patients. RT-PCR products (1b) from TSGA10 transcripts were seen in Lanes 2 and 3. Nested PCR was performed (1c). It showed lower expression of TSGA10 gene in ALL blood samples (1c: Lanes 4 and 5). Lanes 6 and 7 are RT-PCR products from large spliced form of TSGA10 cDNAs from bone marrow. Lane 8 was a negative control (water) and Lane 9 was RT-PCR from an adult human testis as a positive control. (1) Lanes 2–4 are results of RT-PCR from some of normal peripheral blood samples (2b). Nested PCR was carried out (2c) and showed no expression of TSGA10 gene in normal blood samples (2c: Lanes 2–4). Lane 5 was a negative control (water) and Lane 6 was RT-PCR from an adult human testis as a positive control.

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(ERM) family domain (pfam00769) and also a high degree of similarity to MAD (pfam05557). Mitotic arrest deficient (MAD) is a mitotic checkpoint protein. The mitotic spindle checkpoint monitors proper attachment of the bipolar spindle to the kinetochores of aligned sister chromatids [17]. TSGA10 is expressed during spermatogenesis and there is evidence for the possibility of TSGA10 involvement in cell proliferation (14). Presence of TSGA10 expression in ALL may open a window to functional study of mitotic checkpoint proteins in leukemia.

Acknowledgements We thank the patients who took part in this study. This work was supported by grants from Tehran University of Medical Sciences Research Fund. Fig. 3. Restriction fragment analysis of PCR products in PAGE. PCR products from bone marrow (BM) and human testis were digested (D) using XbaI endonuclease which has a restriction site in large spliced form of TSGA10 transcript. Undigested PCR products (U) were run side by side with the digested products (D). Alternative splicing creates a restriction site for XbaI endonuclease. Digestion of the 679 bp PCR product with the enzyme resulted in two restriction fragments of 371 bp and 308 bp where the small band, 635 bp, remains undigested.

significant associations between clinical or haematological presentations of the patients and the expressed isoforms of TSGA10 (Table 1). Increasing the length of a 5 UTR appears to be especially common in mRNAs encoding proto-oncogenes, transcription factors, growth factors, and their receptors, which suggests that their translation is tightly controlled. The 5 UTR of the TSGA10 gene is 626 nucleotides with (or 582 nucleotides without) the alternative spliced fragment (Fig. 2) which is relatively long for a 5 UTR. The question arises whether the mechanism of alternative splicing is a result of 5 UTR controlling mechanism as is used in other actively dividing tissues such as cancers [13]. However, the mechanisms responsible for generating most alternative transcripts in spermatogenic cells and actively dividing tissues are still not known. We previously showed that the gene for human TSGA10 maps to 2q11.2 and Mtsga10, was mapped to the B region of the mouse chromosome one that is syntenic to the human 2q11.2 chromosome region [9]. A significant number of neighboring genes including MGAT4, LAF4 and REV1L are involved in cancer. For example, the highest expression of MGAT4 is in the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line [14]. Another example is LAF4, which has been suggested to function as nuclear transcription factor in lymphoid development and oncogenesis [15,16]. Bioinformatics analysis of the 65 kDa protein of TSGA10 gene revealed the presence of regions with significant similarity to the myosin tail (pfam 01576), the ezrin/radixin/moesin

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