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Leukemia Research 34 (2010) 258–260

Contents lists available at ScienceDirect

Leukemia Research

journa l homepage: www.e lsev ier .com/ locate / leukres

rief communication

elomerase gene mutation screening in Chinese patients with aplastic anemia

ing Han ∗, Bo Liu, Wei Cui, Xuan Wang, Jie lin, Yongqiang Zhaoepartment of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, No. 1, Shuai Fuyuan, Dongcheng District, Beijing 100730, China

r t i c l e i n f o

rticle history:eceived 2 September 2009

a b s t r a c t

To study the incidence of telomerase gene mutations in Chinese patients with acquired bone marrowfailure (BMF) and explore its relationship with telomere shortening. Blood samples from 66 patients with

eceived in revised form 2 November 2009ccepted 2 November 2009vailable online 25 November 2009

eywords:one marrow failure syndrome (BMFS)plastic anemia (AA)elomerase gene mutations

aplastic anemia (AA) in northern China were collected and TERC mutation analysis was performed. TwoTERC mutations were identified. The incidence of telomerase gene mutations in Chinese people withacquired AA is similar to that of the western people.

© 2009 Elsevier Ltd. All rights reserved.

. Introduction

Acquired bone marrow failure syndromes (BMFS) are a groupf diseases including aplastic anemia (AA), melodysplastic syn-rome (MDS) and paroxysmal nocturnal hemoglobinuria (PNH).ome patients with these diseases have short telomeres in theireripheral nucleated cells [1]. An enzyme, telomerase, can maintainhe length of telomere. The core components of this complex aren RNA template and a reverse transcriptase, called TERC and TERT,espectively [2]. It has been well documented that some autoso-al dominant cases of inherited bone marrow failure—dyskeratosis

ongenita (DKC) have telomerase hTERC or hTERT mutations [3].ecently several studies in the west have disclosed the presence ofelomerase complex gene mutations in a small group of patientsith acquired bone marrow failure [4]. Epidemiologic studies

howed that the incidence rate of AA is about 2–3-fold higher in Asiahan that of western country. Whether the difference of telomeraseene background between different ethnic groups could explain theigher incidence of aplastic anemia in Asian people is still not clear.ecently a Japanese group reported that mutational frequencies

f telomere-synthesizing genes among BMFS patients were lowerhan what had been reported for other ethnic groups [5]. But thereas so far been no study in China about the incidence of telomeraseene mutation in Chinese people with acquired bone marrow fail-re and its relation with telomere length. We therefore collected

∗ Corresponding author. Tel.: +86 10 65295024; fax: +86 10 65295817.E-mail address: hanbing li@sina.com.cn (B. Han).

145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2009.11.001

blood samples from patients with aplastic anemia in northern Chinaand perform mutation analysis.

2. Materials and methods

2.1. Patients and controls

Mononuclear cells (MNC) of peripheral blood from 66 patients with AA diag-nosed between 2003 and 2008 at four hospitals in north part of China wereexamined. Of the 66 patients, 32 were females and 34 were males. Age range wasfrom 9 to 77 years, median age 37 years (Table 1). Patients were diagnosed with AAbased on the criteria of the International Study of agranulocytosis and AA study. MostAA patients received cyclosporine A and some severe cases ATG and cyclosporineA combination therapy. Blood samples from 45 age and sex matched healthy con-trols were analyzed. Our volunteers and patients provided informed consent priorto genetic testing as approved by our institutional review board.

2.2. Mutational analysis

DNA was extracted from the peripheral blood or marrow cells using RelaxGeneBlood DNA system kit (Tiangen, Co Beijing). Polyerase-chain-reaction (PCR) ampli-fication of genes encoding the telomerase RNA component (TERC) was performed.Primers and PCR conditions are listed in Table 2.

Reactions for direct sequencing for the PCR products were performed withBigDye Terminator v3.1 Cyle Sequencing Kit (Applied Biosystems) and analyzed inan ABI 3700 automated genetic-sequencer analyzer (Applied Biosystems). Sequenc-ing results were analyzed using Sequencer 4.5 software (Gene Code, Ann Arbor, MI).All sequences were determined in both directions.

2.3. Statistical analysis

Difference in the frequencies of sequence variations between samples of patientswith bone marrow failure and from controls were evaluated by means of Fisher’sexact test using SPSS 14.0.

B. Han et al. / Leukemia Research 34 (2010) 258–260 259

Fig. 1. Two TERC mutations detected in two chronic AA patients. Left: n37 A

Table 1Patients’ clinical manifestation.

AA (n = 66)

SexMale 34Female 32

Age (range) 9–77

Family history+ 1− 65

Chromosome abnormality+ 1− 65

IS therapya

CR 15PR 41NR 6

3

3

wAttbmwdi

ff

found two TERC mutations in two aplastic anemia patients. The

TP

Blood transfusion only 1Transplantation 1Others 2

a Immunosuppressive therapy.

. Results

.1. Mutations

Two heterozygous TERC mutations, n37 A → G and n66 G → Cere identified in two patients (Fig. 1). The patient carrying n37→ G mutation was a 47-year-old male diagnosed as chronic aplas-

ic anemia. He did not have positive family history and did not reacto the immunosuppressive therapy. This mutation has previouslyeen reported in a DKC patient but not in acquired aplastic ane-ia before. The n66 G → C mutation was detected in a 55-year-oldoman with chronic AA. She has no family history of hematopoietic

isorders. She was treated with cyclosporine A and did not improve

n a 2-year follow-up.Nonsynonymous single-nucleotide polymorphisms were also

ound in the TERC (data not shown). These occurred at similarrequencies in both the patients and the controls.

able 2rimers and PCR conditions for TERC.

Gene Primer PCR

TERC Forward primer: 5′-tcatggccggaaatggaact-3′ 94 ◦

35 cReverse primer: 5′-gtgacggatgcgcacgat-3′

→ G in a 47-year-old male; right: n66 G → C in a 55-year-old woman.

4. Discussion

In our study, two TERC mutations were found among 66 patientswith bone marrow failure. The n37 A → G mutation of TERC genehas previously been reported by Ly et al. in a DKC patient [6]. Then37 A → G mutation is located in the lower strand of a 19-bp bulgedP1 stem that is not highly conserved. Though they found no majordeleterious effect of the 37 A → G mutation in the in vitro assays, thepatient and family members who were heterozygous for this muta-tion had very short telomeres. So it was speculated that the n37A → G did have functionally important effects on the maintenanceof telomere length in vivo, but these were not detected in telom-erase activity assays in vitro. The n66 G → C mutation found in a55-year-old woman with chronic AA has not been reported before.66G is located near the template region of TERC, so its mutationmight directly interfere with telomere elongation. Unfortunatelywe could not obtain enough blood samples to measure the telomerelength of the two patients with TERC mutation.

The incidence of telomerase gene mutation in northern Chi-nese people with acquired bone marrow failure is 3.0%, similarto that of the western people [1,4]. The Asian ethnic groups havea higher incidence rate of bone marrow failure syndrome. But sofar there were only several reports from Japan showing very lowmutational frequencies of telomere-synthesizing genes. Liang etal. had screened 96 Japanese children with AA and found no TERCmutation and two nonsynonymous TERT mutations [7]. Takeuchiet al. found only one patient with a potential pathogenic muta-tion in the hTERC gene and none with pathogenic hTERT mutationsamong a cohort of 220 Japanese men and women [5]. Our resultsdemonstrated a similar mutational frequencies to Japanese group.So the genetic background of telomerase gene was not signifi-cantly different between the west and east and the higher incidenceof AA in the Asians was unlikely to be explained by the sup-posed different background of telomerase gene. In our study, we

n37 A → G mutation, although reported in DKC before, has neverfound in aplastic anemia. The other mutation is first time declared.These results enrich our knowledge of possible gene-telomerasefunction-disease development relationship.

condition PCR kit

C 1 min, 55 ◦C 45 s, 72 ◦C 45 s,ycles; 72 ◦C 5 min

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[6] Ly H, Schertzer M, Jastaniah W, et al. Identification and functional characteriza-

60 B. Han et al. / Leukemia

onflict of interest

The authors report no conflicts of interest. The authors alone areesponsible for the content and writing of the paper.

cknowledgements

We thank the National Important Laboratory of Chinese Basicedicine, Institute Chinese Academy of Medical Science and Cen-

ral Laboratory of Peking Union Medical College Hospital whereost of our study was done, and we also thank Dr. Phil Mason fromashington University, School of Medicine in St. Louis, Drs. Zhi-

an Zhang and Ti Shen from Peking Union Medical College, Chinesecademy of Medical Science for their valuable comments. This workas supported by grant of Clinic and Basic Research program fromhinese Health Department and by research funds of Peking Unionedical College Hospital, Chinese Academy of Medical Science.

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rch 34 (2010) 258–260

References

1] Lee JJ, Kook H, Chung IJ, et al. Telomere length changes in patients with aplasticanaemia. Br J Haematol 2001;112(March (4)):1025–30.

2] Vulliamy T, Marrone A, Goldman F, et al. The RNA component of telomerase ismutated in autosomal dominant dyskeratosis congenita. Nature 2001;413(6854):432–5.

3] Dokal I. Dyskeratosis congenita in all its forms. Br J Haematol2000;110(4):768–79.

4] Vulliamy T, Marrone A, Dokal I, et al. Association between aplastic anemia andmutations in telomerase RNA. Lancet 2002;359:2168–70.

5] Takeuchi J, Ly H, Yamaguchi H, et al. Identification and functional char-acterization of novel telomerase variant alleles in Japanese patients withbone-marrow failure syndromes. Blood Cells Mol Dis 2008;40(March–April (2)):185–91.

tion of 2 variant alleles of the telomerase RNA template gene (TERC) in a patientwith dyskeratosis congenita. Blood 2005;106:1246–52.

7] Liang J, Yagasaki H, Kamachi Y, et al. Mutations in telomerase catalytic pro-tein in Japanese children with aplastic anemia. Haematologica 2006;91(May(5)):656–8.