Brief Genetics Report No Evidence for Linkage or for Diabetes-Associated Mutations in the Activin Type 2B Receptor (ACVR2B) in French Patients With Mature-Onset Diabetes of the Young or Type 2 Diabetes Sophie Dupont,1 El Habib Hani,1 Corentin Cras-Me´neur,2 Fre´de´rique De Matos,1 Ste´phane Lobbens,1 Ce´cile Lecoeur,1 Martine Vaxillaire,1 Raphae¨l Scharfmann,2 and Philippe Froguel1

Activins are members of the transforming growth fac- pancreatic buds from gut endoderm (2). Furthermore, tor-␤ superfamily. They have a wide range of biological activins, which are expressed in rat islets and rat pancre- effects on cell growth and differentiation. For trans- atic anlage, stimulate insulin secretion even in the absence membrane signaling, activins bind directly to activin of glucose by inhibiting the activity of ATP-sensitive Kϩ receptor type 2A (ACVR2A) or 2B (ACVR2B). Trans- channels and modulating voltage-dependent Ca2ϩ chan- genic and knock-out mice for the ACVR2B gene display nels (3,4). For transmembrane signaling, activin requires various endocrine pancreas-related abnormalities, in- two types of activin receptors: type 1 (ACVR1) and type 2 cluding islet hypoplasia and glucose intolerance, dem- (ACVR2). Activin binds directly to ACVR2, and this com- onstrating the crucial role of ACVR2B in the regulation plex associates with and phophorylates ACVR1, which in of pancreas development. We have thus examined the contribution of this factor to the development of ma- turn activates the downstream signaling pathways impli- ture-onset diabetes of the young (MODY) and type 2 cating SMAD 2 and 3 (5). Two type 2 receptors (ACVR2A diabetes. No evidence of linkage at the ACVR2B and ACVR2B) that are expressed in the primordia of has been detected in MODY families with unknown pancreas and mature pancreatic cells in mice have been etiology for diabetes or found in affected sib pairs from identified (6). In a recent study, Kim et al. (7) described the families with type 2 diabetes. Mutation screening of the pancreatic phenotype of knockout mice for Acvr2a and/or coding sequence in MODY probands and in a family with Acvr2b. They showed that Acvr2b–/– adult mice had hypo- severe type 2 diabetes, including a case of pancreatic plastic islets and higher glycemia 30 min after an oral agenesis, showed single nucleotide polymorphisms that glucose tolerance test, whereas Acvr2b–/– had no obvious did not cosegregate with MODY and were not associated pancreatic abnormalities. Acvr2aϩ/–b–/– embryos were not with type 2 diabetes. Our results indicate that ACVR2B does not represent a common cause of either MODY or viable and showed reduced islet size and number, misex- type 2 diabetes in the French Caucasian population. pression of SHH, and reduced expression of insulin and ϩ/– –/– ϩ/– Diabetes 50:1219–1221, 2001 glucagon. Whereas Acvr2a , Acvr2a , and Acvr2b adult mice displayed no detectable malformations of the foregut-derived organs, Acvr2aϩ/–bϩ/– mice showed hypo- plastic islets and impaired glucose tolerance (resulting ctivins, members of the transforming growth from inadequate insulin production or secretion) in a more factor-␤ superfamily, exert a diverse range of severe manner than the Acvr2b–/– mice. The endocrine biological effects on cell growth and differenti- pancreas is thus particularly sensitive to the type and A ation: they especially govern embryonic axial extent of defective function. In addition, patterning (1) and may restrict sonic hedgehog (SHH) various abnormalities of the pancreas were reported in expression in embryonic chicks, allowing evagination of two studies of transgenic mice expressing in the pancreas a dominant-negative form of Acvr2b gene under the insulin and the ␤-actin promoter, respectively (8,9). In both cases, From the 1Institute of Biology-Centre National de la Recherche Scientifique (CNRS) 8090, Institut Pasteur, Lille, France; and 2 Institut National de la Sante´ mice had severely hypoplastic islets. In the first study, the et de la Recherche Me´dicale (INSERM) U457, Hospital Robert Debre´, Paris, mice showed impairment of the insulin response to glu- France. cose; in the second one, abnormal endocrine cells were Address correspondence and reprint requests to Philippe Froguel, Institut Pasteur de Lille, 1 rue du Pr Calmette, 59000 Lille, France. E-mail: observed outside the islets. The functional studies impli- [email protected]. cating Acvr2b in the regulation of pancreas development Received for publication 19 September 2000 and accepted in revised form 5 February 2001. prompted us to examine the contribution of the ACVR2B Additional information can be found in an online appendix at www. gene to the development of MODY and type 2 diabetes in diabetes.org/diabetes/appendix.asp. the French population. In addition to their pancreatic ACVR1, activin receptor type 1; GI, glucose intolerant; LOD, logarithm of –/– odds; MLB, maximum-likelihood binomial; MODY, mature-onset diabetes of phenotype, Acvr2b mice have other severe complica- the young; SHH, sonic hedgehog. tions: indeed, they also show hypoplastic spleen, abnormal

DIABETES, VOL. 50, MAY 2001 1219 NO T2DM-ASSOCIATED MUTATIONS IN ACVR2B IN FRENCH PATIENTS

TABLE 1 TABLE 2 Genotypic distribution of the IVS6nt-13T/C variant found in the Results of type 2 diabetic sib-pair analyses performed in 143 unrelated MODY probands, type 2 diabetic subjects, and control pedigrees subjects studied Marker Phenotypic group* LOD-MLB P MLS P n T/T T/C C/C D3S1277 Large (T2DM ϩ GI) 0.02 0.37 0.11 0.32 Unrelated MODY probands 11 4 5 2 Strict (T2DM) 0.00 0.50 0.00 0.50 Unrelated T2DM probands 100 28 51 21 D3S3521 Large (T2DM ϩ GI) 0.01 0.42 0.00 0.50 Unrelated control subjects 100 35 40 25 Strict (T2DM) 0.00 0.50 0.15 0.27 Data are n. T2DM, type 2 diabetic. *The affection status was determined by taking into account the patients’ diabetic status. MLS, maximum LOD score; T2DM, type 2 stomach, defects in axial patterning, and lateral asymme- diabetes. try; 70% of these mice die before weaning, probably because of severe cardiac defects (10). Therefore, we pean and American populations by Kosaki et al. (12) searched for mutations in the ACVR2B gene in a family through the screening of the ACVR2B gene in normogly- with a severe form of type 2 diabetes treated with insulin, cemic patients with left-right axis malformations. It is including one member presenting with cardiac defects and noteworthy that we cannot exclude the presence of addi- pancreatic agenesis. The latter was not caused by muta- tional ACVR2B variations located in the promoter and tions in IPF1, which has been already screened for muta- introns in the MODY families tested. tions in this pedigree (data not shown). The markers D3S1277 and D3S3521 were also typed in We first checked the normal expression of ACVR2B in 143 French Caucasian pedigrees with type 2 diabetes (13). human embryonic pancreas by reverse transcriptase– We found no evidence of linkage between diabetes and polymerase chain reaction (details are available in the these microsatellites (Table 2) (details are available in the online appendix at www.diabetes.org/diabetes/appendix. online appendix at www.diabetes.org/diabetes/appendix. asp). Then we studied 11 MODY families of French ances- asp). The design of our linkage studies in type 2 diabetes try that were previously found to have no mutations in the pedigrees allowed us to reach the exclusion standard five known MODY (“MODY X” families) (11). We score of –2 for a recurrent sibling risk (␭s) of 1.3 (calcu- performed linkage analyses using markers located in the lated using the Mapmaker/Sib program). vicinity of the ACVR2B gene. We placed the gene between By linkage and/or screening studies, we have found no markers D3S1277 (60 cM) and D3S3521 (62 cM) by radia- evidence for a predisposing role of the ACVR2B gene in tion hybrid mapping. D3S1277 and D3S3521 were typed in MODY or type 2 diabetic French Caucasian families. 9 of the 11 French MODY X families (2 of the 11 MODY X However, studies of patients from other ethnic origins may families were not suitable for linkage analyses because be of interest to the genetics of type 2 diabetes. DNA was available for only 2 patients in those two pedi- grees). Cumulative logarithm of odds (LOD) scores showed RESEARCH DESIGN AND METHODS exclusion of linkage with diabetes at ACVR2B (LOD Subjects. The 11 MODY families are of French ancestry and have been ␪ previously described (11). The French family F4854 was recruited at Hospital scores at 0 for D3S1277 and D3S3521 were –11.74 and –17.05, respectively). The families-individual LOD scores Robert Debre´, Paris, France. All of the families and patients with type 2 diabetes came from 550 type 2 ranged from –4.71 to 0.63 for D3S1277 and from –4.28 to diabetic French Caucasian pedigrees recruited through a multimedia cam- 0.33 for D3S3521, indicating no evidence of linkage with paign. All families studied here had no known etiology for diabetes. A total of diabetes (details of the results are available in the online 143 French Caucasian pedigrees with type 2 diabetes were used for sib-pair appendix at www.diabetes.org/diabetes/appendix.asp). analyses and were primarily selected for a genome-wide search for type 2 diabetes–susceptibility genes (13). At least two diabetic subjects in each The 11 exons and flanking introns of ACVR2B were thus sibship had to be undergoing treatment for diabetes (to load the families with screened for mutations in one diabetic proband from each severe form of type 2 diabetes). The sibships presented no bilineal inheritance of the 11 MODY X pedigrees. The screening was also of type 2 diabetes, and only one of the subjects was diagnosed before the age performed in seven members of the family mentioned of 25 years. Each family contained at least one affected sib pair in which the above (called F4854) who presented a severe form of type patients were diagnosed before 65 years of age. The 100 unrelated type 2 diabetic patients and 100 control subjects (spouses of diabetic patients) were 2 diabetes treated with insulin, including a member with randomly selected among the initial group of 550 type 2 diabetic French cardiac defects and pancreas agenesis. We found three Caucasian families containing at least one affected sibship. nucleotide variations: two silent mutations in exons 3 Radiation hybrid mapping. We localized the ACVR2B gene within the (E111, GAA/GAG) and 11 (N486N, AAC/AAT) andaTtoC linkage map of 3 using a Genebridge 3 Radiation Hybrid Panel (Research Genetics, Huntsville, AL) and analyzed the data using the computer variation 13 bp upstream of exon 7 (IVS6nt-13T/C). Table program RHMAP3.0 from the statistical package for multipoint radiation 1 shows the genotypic distribution of the variants (the hybrid mapping (14). three variants that we found were in perfect linkage Linkage studies. D3S1277 and D3S3521 were typed using automated fluo- disequilibrium and thus define only two ACVR2B alleles). rescent-based procedures. Each genotype was reviewed independently by two None of the variants cosegregated with diabetes in the members of the research team to confirm the accuracy of allele calling. The LOD score calculations in MODY families have been computed as family in which it was found. Searching for the IVS6nt- previously described (15) with the LINKAGE programs (16). 13T/C variation in 100 additional type 2 diabetic probands In the analyses of sib pairs in families with type 2 diabetes, we designed and 100 control subjects showed no association between two qualitative traits that took into account the diabetic status: 1) the affection the variation and either diabetes (Table 2) or BMI. Fur- status “large” considered as affected those subjects who were type 2 diabetic as well as glucose intolerant (GI), as newly defined by the World Health thermore, no association was found with age at onset of Organization in 1997, and 2) the affection status “strict” only considered as the disease (data not shown). The E111E and N486N silent affected those patients who were type 2 diabetic. A maximum number of 677 mutations were already found at high frequency in Euro- and 453 affected sib pairs were analyzed with the affection status large and

1220 DIABETES, VOL. 50, MAY 2001 S. DUPON AND ASSOCIATES strict, respectively. Nonparametric two-point and multipoint analyses were Hata J, Sugino H, Noji S, Itakura M: Hypoplasia of pancreatic islets in performed with the programs Mapmaker-Sibs 2.0 (17) (using the unweighted transgenic mice expressing activin receptor mutants. J Clin Invest 102: option) and MLBGH 1.0 (18). The maximum-likelihood binomial (MLB) 294–301, 1998 method, based on the binomial distribution of parental marker alleles among 9. Shiozaki S, Tajima T, Zhang YQ, Furukawa M, Nakazato Y, Kojima I: affected offspring, overcomes the common problem of multiple sibs by Impaired differentiation of endocrine and exocrine cells of the pancreas in considering the sibship as a whole. transgenic mouse expressing the truncated type II activin receptor. Mutation screening. Exons and flanking intronic sequences of the human Biochim Biophys Acta 1450:1–11, 1999 ACVR2B gene were sequenced on both strands using direct DNA sequencing 10. Oh SP, Li E: The signaling pathway mediated by the type IIB activin optimized protocols, as previously described (19) (primer sequences and receptor controls axial patterning and lateral asymmetry in the mouse. amplification conditions are available in the online appendix at www. Genes Dev 11:1812–1826, 1997 diabetes.org/diabetes/appendix.asp). Sequence comparison analyses were 11. Chevre JC, Hani EH, Boutin P, Vaxillaire M, Blanche H, Vionnet N, Pardini scored independently by two readers to ensure maximal accuracy for muta- tion detection. VC, Timsit J, Larger E, Charpentier G, Beckers D, Maes M, Bellanne- Chantelot C, Velho G, Froguel P: Mutation screening in 18 Caucasian families suggest the existence of other MODY genes. Diabetologia 41: ACKNOWLEDGEMENTS 1017–1023, 1998 This research was supported in part by Re´gion Nord-Pas 12. Kosaki R, Gebbia M, Kosaki K, Lewin M, Bowers P, Towbin JA, Casey B: de Calais (S.D.). Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB. Am J Med Genet 82:70–76, 1999 We are indebted to all of the families who participated in 13. Vionnet N, Hani EH, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, this study. We thank Christian Dina for help with statistical Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P: calculations. Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early-onset diabetes on REFERENCES chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21–q24. Am J Hum Genet 67:1470–1480, 2000 1. Hoodless PA, Tsukazaki T, Nishimatsu S, Attisano L, Wrana JL, Thomsen 14. Lunetta KL, Boehnke M, Lange K, Cox DR: Selected locus and multiple GH: Dominant-negative Smad2 mutants inhibit activin/Vg1 signaling and panel models for radiation hybrid mapping. Am J Hum Genet 59:717–725, disrupt axis formation in Xenopus. Dev Biol 207:364–379, 1999 1996 2. Hebrok M, Kim SK, Melton DA: Notochord repression of endodermal Sonic 15. Vaxillaire M, Boccio V, Philippi A, Vigouroux C, Terwilliger J, Passa P, hedgehog permits pancreas development. Genes Dev 12:1705–1713, 1998 3. Furukawa M, Nobusawa R, Shibata H, Eto Y, Kojima I: Initiation of insulin Beckmann JS, Velho G, Lathrop GM, Froguel P: A gene for maturity onset secretion in glucose-free medium by activin A. Mol Cell Endocrinol diabetes of the young (MODY) maps to chromosome 12q. Nat Genet 113:83–87, 1995 9:418–423, 1995 4. Mogami H, Kanzaki M, Nobusawa R, Zhang YQ, Furukawa M, Kojima I: 16. Lathrop GM, Lalouel JM: Easy calculations of lod scores and genetic risks Modulation of adenosine triphosphate-sensitive potassium channel and on small computers. Am J Hum Genet 36:460–465, 1984 voltage-dependent calcium channel by activin A in HIT-T15 cells. Endo- 17. Abel L, Muller-Myhsok B: Robustness and power of the maximum- crinology 136:2960–2966, 1995 likelihood-binomial and maximum-likelihood-score methods, in multipoint 5. Massague´ J, Chen Y: Controlling TGF-␤ signaling. Genes Dev 14:627–644, linkage analysis of affected-sibship data. Am J Hum Genet 63:638–647, 2000 1998 6. Mathews LS, Vale WW: Molecular and functional characterization of 18. Kruglyak L, Lander ES: Complete multipoint sib-pair analysis of qualitative activin receptors. Receptor 3:173–181, 1993 and quantitative traits. Am J Hum Genet 57:439–454, 1995 7. Kim SK, Hebrok M, Li E, Oh SP, Schrewe H, Harmon EB, Lee JS, Melton 19. Boutin P, Wahl C, Samson C, Vasseur F, Laget F, Froguel P: Big Dye DA: Activin receptor patterning of foregut organogenesis. Genes Dev terminator cycle sequencing chemistry: accuracy of the dilution process 14:1866–1871, 2000 and application for screening mutations in the TCF1 and GCK genes. Hum 8. Yamaoka T, Idehara C, Yano M, Matsushita T, Yamada T, Ii S, Moritani M, Mutat 15:201–203, 2000

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