Endocrine-Related (2007) 14 1073–1079

MEN1 mutation analysis in Chinese patients with multiple endocrine neoplasia type 1

Xiao-Hua Jiang1, Jie-Li Lu1, Bin Cui1,2, Yong-Ju Zhao1, Wei-qing Wang1, Jian-Min Liu1, Wen-Qiang Fang 3, Ya-Nan Cao1, Yan Ge1, Chang-xian Zhang4, Huguette Casse4, Xiao-Ying Li 1,5 and Guang Ning1,2

1Shanghai Clinical Center for Endocrine and Metabolic Diseases, Shanghai Institute of and Metabolism and Chinese- French Laboratory of Genomics and Life Sciences, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, China 2Laboratory for Endocrine and Metabolic Diseases, Institute of Health Sciences, SIBS, Chinese Academy of Sciences, China 3Department of Radiological Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 4Laboratoire Genetique et Cancer, Fac. Med. Domaine Rockefeller. Univ-Lyon1, Lyon, France 5Shanghai Key Laboratory for Endocrine Tumours, E institute of Shanghai University, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China (Correspondence should be addressed to X-Y Li; Email: [email protected] or G Ning; Email: [email protected]) X-H Jiang and J-L Lu contributed equally to this work

Abstract Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumour syndrome characterized by the development of tumours of the parathyroid, and pancreatic islets, etc. Heterozygous germ line mutations of MEN1 gene are responsible for the onset of MEN1. We investigated the probands and 31 family members from eight unrelated Chinese families associated with MEN1 and identified four novel mutations, namely 373_374ins18, 822delT, 259delT and 1092delC, as well as three previously reported mutations, such as 357_360delCTGT, 427_428delTA and R108X (CGAOTGA) of MEN1 gene. Furthermore, we detected a loss of heterozygosity (LOH) at chromosome 11q in the removed tumours, including , and parathyroid from two probands of MEN1 families. RT-PCR and direct sequencing showed that mutant MEN1 transcripts remained in the MEN1-associated endocrine tumours, whereas normal menin proteins could not be detected in those tumours by either immunohistochemistry or immunoblotting. In conclusion, MEN1 heterozygous mutations are associated with LOH and menin absence, which are present in MEN1-associated endocrine tumours. Endocrine-Related Cancer (2007) 14 1073–1079

Introduction Hua 2007). More than 400 germ line and somatic Multiple endocrine neoplasia type 1 (MEN1) is an mutations in the MEN1 gene have been identified autosomal dominant inherited disorder characterized (http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/ by frequent occurrence of tumours of parathyroid, 120173.html). However, more mutation detections pancreatic islet and anterior pituitary, sometimes in favour in elucidating the correlation of genotype and combination with other rare tumours. The MEN1 gene phenotype (Kouvaraki et al. 2002, Wautot et al. 2002). was successfully identified in 1997, which is composed The majority of MEN1 patients display heterozygous of ten exons spanning 2.8 kb that codes for a 610 amino germ line mutations. MEN1-associated tumours acid nuclear protein, known as menin (Chandrasekhar- generally have somatic deletions of the MEN1 locus appa et al. 1997, Lemmens et al. 1997). Menin, which and its surrounding regions in chromosome 11, known has no homology to any other known proteins, is as loss of heterozygosity (LOH; Lubensky et al. 1996, considered to play a role in cell growth regulation, cell Debelenko et al. 1997, Hessman et al. 2001). In the cycle, genome stability and synapse plasticity (Yang & present study, we investigated MEN1 gene mutations in

Endocrine-Related Cancer (2007) 14 1073–1079 Downloaded from Bioscientifica.comDOI:10.1677/ERC-07-0015 at 10/01/2021 02:35:06AM 1351–0088/07/014–001073 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access X-H Jiang, J-L Lu et al.: MEN1 mutation in Chinese MEN1 patients eight Chinese MEN1 families, and analysed LOH on diagnosed and operated at the age of 25, 28, 35, 36, 37 chromosomal 11q and menin expression in MEN1- and 43 years respectively. associated tumours. Mutation analysis DNA was extracted from peripheral blood leukocytes and Subjects and methods from the removed tumour tissues using DNeasy Tissue Subjects Kit (Qiagen). Exons 2–10 of MEN1 gene were amplified The MEN1 probands (one male and seven females, age by PCR in a volume of 50 ml mixture containing 1.5 mM range 18–43 years) from eight unrelated families and 31 MgCl2, 10 mM dNTP, 0.2 mg genomic DNA, 20 mmol of family members, including seven clinically sympto- each primer and 2.5 U Taq DNA polymerase (Sangon, matic (three males and four females, age range 21–66 Shanghai, China). The primer sequences were previously years) and 24 asymptomatic subjects (12 males and 12 reported (Tanaka et al.1998), except for the primer pair amplifying exon 8 (forward 50-agagaccccactgctctcaca-30, females, age range 8–73 years), were investigated in the 0 0 current study. The diagnosis of MEN1 was based on the reverse 5 -ggacacaggctggagctcc-3 ). Amplification was presence of at least two of the three major endocrine performed with preheating at 95 8C for 3 min, followed lesions described in the syndrome, including tumours of by 30 cycles of denaturation at 95 8C for 45 s, annealing at the parathyroid , endocrine and anterior 55–62 8C for 45 s and extension at 72 8C for 1 min. The pituitary. Three representative MEN1 pedigrees were PCR products were purified using a gel extraction kit shown in Supplementary Fig. 1, which can be viewed (Qiagen) and sequenced in both sense and antisense online at http://erc.endocrinology-journals.org/supple- directions on the ABI 3700 Sequencer (Perkin–Elmer mental/. Symptomatic subjects for MEN1 were defined (PE) Applied Biosystems, Foster City, CA, USA). The as any of a variety of clinical manifestations, such as obtained sequences were compared with the reference renal stones, acral growth, amenorrhoea, hypoglycae- sequences of the MEN1 gene (U93237). mia and gastrointestinal bleeding that led them to seek medical attention. The clinical characteristics for all the LOH probands and symptomatic subjects from the eight LOH analysis was performed using DNA from MEN1- families were summarized in Table 1. The protocols for associated endocrine tumour tissues and corresponding the study were approved by the Hospital Ethics peripheral leukocytes. Eighteen short tandem repeat Committee for Human Research, and informed consent (STR) markers were used to determine the scope of was obtained from each individual. LOH in chromosomal arm 11q (Table 2). DNA fragments Seven probands presented with primary hyperpara- were labelled with fluorescent dye using M13 universal thyroidism (87.5%), six with pancreatic lesions (75%), tailed primer method. PCR products were analysed using including five and one gastrinoma, and five Beckman–Coulter CEQ 8800 sequencer (Beckman– with (62.5%), including four Coulter, Fullerton, CA, USA). Data collection and prolactinomas and one mixed prolactinoma, and analysis were performed with Fragment Analysis Module GH-secreting tumour. Interestingly, the thymic carci- (Beckman–Coulter). LOH is defined as positive if the noid, which is considered as an extremely rare allele peak ratio is O1.5. manifestation (prevalence 3.1%) and occurred mostly in male heavy smokers (O95%; Ferolla et al. 2005), was detected in proband 6, a nonsmoking female patient RT-PCR (31 years of age). The patient died from metastasis of RNA was prepared using TRIzol reagent (Invitrogen). thymic in 28 months after surgery, another Total RNA (2 mg) was heated at 70 8C for 5 min and subject in family 1 (father of proband 1) also died of the placed on ice for 5 min. A mixture of 5 ml M-MLV RT thymic in 36 months after surgery, confirming 5! reaction buffer, 10 mM of each of the four dNTPs, that the thymic carcinoid may be an important cause of 200 U M-MLV transcriptase enzyme, 25 pg/ml random death in MEN1. Consequently, prophylactic thymect- primer and 20 U RNase inhibitor (all from Promega) omy should be considered when neck surgery for was added to each sample, followed by incubation at primary hyperparathyroidism in MEN1 patients 40 8C for 60 min and 70 8C for 15 min. cDNA (2 ml) (Brandi et al. 2001, Ferolla et al. 2005) to be undertaken. products were amplified with 1 U Ampli Taq Gold (PE It is also noted that proband 2 had six lesions of Applied Biosystems), together with b-actin as an prolactinoma, gastrinoma, ovary , insulinoma, internal control. The specific primers for MEN1 that and parathyroid , which were spanned exons 2 and 3 (cDNA411 to 699), and the

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Table 1 Clinical manifestations and MEN1 mutations in eight families with MEN1

Family Subject Age Sex Parathyroid Pituitary Pancreas Others Mutation

1 1–1 26 M PTH[ Prolactinoma Insulinoma 357del4 GH-secreting tumour 1–2 45 F PTH[ 1–3 38 M Thymic carcinoid 1–4 21 F Insulinoma 2 2–1 43 F HPT Prolactinoma Insulinoma 427delAT Gastrinoma Ovary teratoma 2–2a 18 F 3 3–1 43 F HPT Insulinoma Adrenal hyperplasia 373ins18b Ovary mucinous 3–2 40 M HPT Pancreas tumour 3–3 66 F HPT 3–4a 7F b

4 4–1 52 F HPT Prolactinoma Thyroid nodule 822delT Cancer Endocrine-Related 4–2a 24 F 5 5–1 37 F HPT Prolactinoma Insulinoma Thyroid nodule R108X (CGA/TGA) Dermatofibroma Lipoma 6 6–1 32 F HPT PRL[ Pancreas tumour Thymic carcinoid 259delTb Ectopic ACTH-secreting tumour 6–2 63 M PTH[ Thyroid nodule Downloaded fromBioscientifica.com at10/01/202102:35:06AM 6–3 30 F HPT Thyroid nodule 7 7–1 32 F HPT Insulinoma R108X (CGA/TGA) 8 8–1 18 F HPT Prolactinoma 1092delCb

PTH[, hyperparathormonemia only; HPT, hyperparathyroidism, hyperparathormonemia plus hypercalcemia; PRL[, hyperprolactinemia only; prolactinoma, hyperprolactinemia plus pituitary adenoma. (2007) aAsymptomatic carrier. bNovel mutation. 14 1073–1079 1075 via freeaccess X-H Jiang, J-L Lu et al.: MEN1 mutation in Chinese MEN1 patients

sequences of the primers for MEN1 amplification are as

K K 0 4463 D11S follows: forward 5 -GAC CTG TCC CTC TAT CCT CG-30 and reverse 50-TGA CCT CAG CTG TCT GCT CC-30. Reactions were carried out in the PTC-225 CC CC 4464 D11S DNA Engine Tetrad (MJ Research Inc., Waltham, MA, USA). Preheating was performed at 95 8C for 8 min, followed by denaturation at 95 8C for 45 s, annealing at KK K K K 1998 D11S 58 8C for 45 s and extension at 72 8C for 1 min for 30 cycles. The PCR products were loaded onto 1.5% agarose gels for electrophoresis and visualized by 1986 D11S ethidium bromide under u.v. light. 2000 D11S Immunohistochemistry CCC CCCCC CCCCC Formalin-fixed and paraffin-embedded sections were 2002 D11S deparaffinized in xylene and rehydrated through descending ethanol concentration to distilled water. K K K CCCC Endogenous peroxidase was quenched by incubating 2371 D11S the slides in 3% H2O2 for 10 min at room temperature. Primary antibody against C19 of menin protein (Santa K C gene is located at 64.3 Mb, between D11S 2006 and D11S1975. Par, 1975 D11S Cruz Biotechnology, Santa Cruz, CA, USA) was added after dilution at 1:200, and then the slides were MEN1 incubated at 4 8C overnight. Next, the sections were K CC CC 2006 D11S covered with biotinylated secondary antibody and incubated for 30 min at room temperature. After the

C sections were finally incubated with ABC com- 1985 D11S plex/HRP for 30 min at room temperature, they were left to develop with 3-30-diaminobenzidine (DAB). As K KK KK CCCCCCCC CCC

2365 soon as the sections were developed, the slides were D11S immersed in distilled water. 1993 D11S Western blot The cell lysates were extracted from parathyroid 1392 D11S adenoma, insulinoma and gastrinoma with lysis buffer (RIPA, 1!PBS, 1% NP40, 5 mM EDTA, 1 mM , LOH positive; –, polymorphism unavailable. The 2361 D11S sodium orthovanadate, 1% phenylmethylsulphonyl C fluoride and complete protease inhibitor cocktail). The protein concentrations were determined with DC KKKK KKKK 1981 D11S Protein Assay Reagent (Bio-Rad Laboratories). Cell lysates (25 mg) were loaded onto SDS/PAGE. The protein bands were stained with Ponceaus solution and 1999 D11S transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences). The membrane was blocked

2362 with a 5% nonfat milk at 4 8C overnight and incubated D11S with anti-menin antibody (1:750) at room temperature for 2 h. Menin expression was visualized with ECL CCCCCCC CCC CCCCCCC CCCCCCC CCC 1984 D11S plus kit (Amersham Biosciences). MEN1C/C and Loss of heterozygosity of chromosomal arm 11q in MEN1-associated tumours MEN1K/K mouse embryonic fibroblasts (MEF) cell lysates were loaded as positive and negative control Par Ins Gas Ins Par ; Ins, insulinoma and Gas, gastrinoma. Mb, the physical distance from the centromere; Family 4 MbFamily 2 1.5 4.9 10.7 17.0 25.9 34.6 43.6 56.0 58.3 59.5 70.0 73.2 79.6 105.1 110.7 117.2 123.1 130.4 Family 3 Table 2 STRs respectively.

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Figure 1 Subclone sequencing of MEN1 gene. Four novel mutations 373ins18, 822delT, 259delT and 1092delC in the probands of families 3, 4, 6 and 8 respectively were shown in A, B, C and D respectively.

Results and discussion Five deletion mutations caused open reading frames shift, one nonsense mutation produced an earlier stop MEN1 mutations codon, and the 373ins18 mutation caused six amino acid Germ line mutations of the MEN1 gene were screened in-frame insertion. In these eight MEN1-associated in 39 individuals from eight families. Seven different families, MEN1 germ line mutations were detected in types of germ line mutations were identified, of 15 symptomatic subjects, as well as in three asympto- which four mutations were novel ones (373ins18, matic subjects (carriers) with an average age of 16.3 years 822delT, 259delT and 1092delC; Fig. 1), and the other (range 7–25 years of age). three (357_360delCTGT, 427_428delAT and R108X Most MEN1 gene mutations have been reported in (CGAOTGA)) had previously been reported. Mutation coding sequences (Kouvaraki et al. 2002, Wautot et al. R108X (CGAOTGA) was identified in families 5 and 7. 2002)). The novel mutation 373ins18, causing six

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confirmed that amino acid substitution in Smad3- binding domains of menin blocked Smad3-mediated transcriptional activation in the TGF-b pathway (Kaji et al. 2001). Blockage of TGF-b signalling may disrupt cell stability, drive cells towards inappropriate growth and ultimately result in tumour formation (Sowa et al. 2004).

LOH analysis in MEN1-associated tumours LOH was performed for the removed MEN1-associated tumours from families 2 and 4. An entire heterozygous deletion of chromosome 11q was identified in gastri- noma, insulinoma and parathyroid adenoma from the probands of families 2 and 4 using 18 STR markers spanning 115.4 Mb (Fig. 2). These findings supported the ‘two hits’ hypothesis in the pathogenesis of MEN1- associated tumours (Knudson & Strong 1971).

Analysis of MEN1 expression in MEN1-associated tumours Using RT-PCR (Fig. 3A) and direct sequencing (data not shown), we had demonstrated that mutant MEN1 transcripts could be identified in gastrinoma, insuli- Figure 2 LOH assay for MEN1-associated insulinoma and noma and parathyroid adenoma from the probands of gastrinoma from the proband of family 2. STR markers families 2 and 4. Moreover, normal menin expression D11S2002 and D11S2006 flanking MEN1 gene were displayed was absent in those tumours determined by immuno- in A and B respectively. PBMC, peripheral blood mononuclear cells. The arrows indicate the allele lost. blotting (Fig. 3B) and immunohistochemistry (Fig. 3C), which is consistent with LOH analysis, amino acid in-frame insertion at position 373 and being although we could not detect the prematurely truncated situated in a putative interacting domain for Smad3 menin by immunoblotting due to the limitation of a binding, possibly affects Smad3 signalling (Kaji et al. C19 antibody against menin used in the present study. 2001), which is a crucial player in transforming growth It indicates that a complete inactivation of the normal factor-b (TGF-b) signalling pathway. In vitro study has MEN1 gene occurs in MEN1-associated tumours.

Figure 3 (A) RT-PCR for MEN1 gene of the MEN1-associated tumours from the probands of families 2 and 4. (B) Immunoblotting for menin of MEN1-associated tumours from the probands of families 2 and 4. Menin expression is absent in those tumours. (C) Immunohistochemistry for menin expression of MEN1-associated tumours from the proband offamily 2. Menin staining is negative in the tumour cells and positive in the surrounding capsule tissues. The amplification is 400!. Ins, insulinoma; Gas, gastrinoma; Par, parathyroid adenoma;Pan, normalpancreas; C/C, MEN1 positive MEF cell lysates;K/K, MEN1 negative (knock-out) MEF cell lysates.

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In conclusion, we have identified seven MEN1 germ HessmanO,SkogseidB,WestinG&AkerstromG line mutations in eight Chinese families with MEN1, of 2001 Multiple allelic deletions and intratumoral which four mutations have not previously been genetic heterogeneity in men1 pancreatic tumors. reported. The complete inactivation of the MEN1 Journal of Clinical Endocrinology and Metabolism gene and subsequent absence of normal menin 86 1355–1361. Kaji H, Canaff L, Lebrun JJ, Goltzman D & Hendy GN 2001 expression are present in MEN1-associated tumours. Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type b signaling. PNAS Acknowledgements 98 3837–3842. Knudson AG Jr & Strong LC 1971 Mutation and cancer: The present study would not have been possible statistical study of retinoblastoma. PNAS 68 820–823. without the participation of the patients and their Kouvaraki MA, Lee JE, Shapiro SE, Gagel RF, Sherman SI, family members. This study was supported by the Sellin RV, Cote GJ & Evans DB 2002 Genotype– grants from National Nature Science Foundation of phenotype analysis in multiple endocrine neoplasia China (no. 30370666), Shanghai Commission for type 1. Archives of Surgery 137 641–647. Science and Technology (no. 04DZ05907) and the Lemmens I, Van de Ven WJ, Kas K, Zhang CX, Giraud S, E-Institute of Shanghai Universities, Shanghai Munici- Wautot V, Buisson N, De Witte K, Salandre J & Lenoir G 1997 Identification of the multiple endocrine neoplasia pal Education Commission ((No. E03007). The authors type 1 (MEN1) gene. Human Molecular Genetics 6 declare that there is no conflict of interest that would 1177–1183. prejudice the impartiality of this scientific work. Lubensky IA, Debelenko LV, Zhuang Z, Emmert-Buck MR, Dong Q, Chandrasekharappa S, Guru SC, Manickam P, References Olufemi SE & Marx SJ 1996 Allelic deletions on chromosome 11q13 in multiple tumors from individual Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz MEN1 patients. Cancer Research 56 5272–5278. P, Bordi C, Conte-Devolx B, Falchetti A, Gheri RG & Sowa H, Kaji H, Kitazawa R, Kitazawa S, Tsukamoto T, Libroia A 2001 Consensus: guidelines for diagnosis and Yano S, Tsukada T, Canaff L, Hendy GN, Sugimoto T therapy of MEN type 1 and type 2. Journal of Clinical et al. 2004 Menin inactivation leads to loss to Endocrinology and Metabolism 86 5658–5671. transforming growth factor b inhibition of parathyroid cell Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, proliferation and parathyroid secretion. Cancer Collins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Research 64 2223–2228. Lubensky IA & Liotta LA 1997 Positional cloning of the Tanaka C, Yoshimoto K, Yamada S, Nishioka H, Ii S, gene for multiple endocrine neoplasia type 1. Science 276 Moritani M, Yamaoka T & Itakura M 1998 Absence of 404–407. germ-line mutations of the multiple endocrine neoplasia Debelenko LV, Zhuang Z, Emmert-Buck MR, Chandra- type 1 (MEN1) gene in familial pituitary adenoma in sekharappa SC, Manickam P, Guru SC, Marx SJ, contrast to MEN1 in Japanese. Journal of Clinical Skarulis MC, Spiegel AM & Collins FS 1997 Allelic Endocrinology and Metabolism 83 960–965. deletions on chromosome 11q13 in multiple endocrine Wautot V, Vercherat C, Lespinasse J, Chambe B, Lenoir GM, neoplasia type 1-associated and sporadic Zhang CX, Porchet N, Cordier M, Beroud C & Calender A and pancreatic endocrine tumors. Cancer Research 57 2002 Germline mutation profile of MEN1 in multiple 2238–2243. endocrine neoplasia type 1: search for correlation between Ferolla P, Falchetti A, Filosso P, Tomassetti P, Tamburrano G, phenotype and the functional domains of the MEN1 Avenia N, Daddi G, Puma F, Ribacchi R & Santeusanio F protein. Human Mutation 20 35–47. 2005 Thymic neuroendocrine (carcinoid) in Yang Y & Hua X 2007 In search of tumor suppressing functions MEN1 syndrome: the Italian series. Journal of Clinical of menin. Molecular and Cellular Endocrinology 265–266 Endocrinology and Metabolism 90 2603–2609. 34–41.

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