Activins are members of the transforming growth factor-β superfamily. They have a wide range of biological effects on cell growth and differentiation. For transmembrane signaling, activins bind directly to activin receptor type 2A (ACVR2A) or 2B (ACVR2B). Transgenic and knock-out mice for the ACVR2B gene display various endocrine pancreas-related abnormalities, including islet hypoplasia and glucose intolerance, demonstrating the crucial role of ACVR2B in the regulation of pancreas development. We have thus examined the contribution of this factor to the development of mature-onset diabetes of the young (MODY) and type 2 diabetes. No evidence of linkage at the ACVR2B locus has been detected in MODY families with unknown etiology for diabetes or found in affected sib pairs from families with type 2 diabetes. Mutation screening of the coding sequence in MODY probands and in a family with severe type 2 diabetes, including a case of pancreatic agenesis, showed single nucleotide polymorphisms that did not cosegregate with MODY and were not associated with type 2 diabetes. Our results indicate that ACVR2B does not represent a common cause of either MODY or type 2 diabetes in the French Caucasian population.

Activins, members of the transforming growth factor-β superfamily, exert a diverse range of biological effects on cell growth and differentiation: they especially govern embryonic axial patterning (1) and may restrict sonic hedgehog (SHH) expression in embryonic chicks, allowing evagination of pancreatic buds from gut endoderm (2). Furthermore, activins, which are expressed in rat islets and rat pancreatic anlage, stimulate insulin secretion even in the absence of glucose by inhibiting the activity of ATP-sensitive K+ channels and modulating voltage-dependent Ca2+ channels (3,4). For transmembrane signaling, activin requires two types of activin receptors: type 1 (ACVR1) and type 2 (ACVR2). Activin binds directly to ACVR2, and this complex associates with and phophorylates ACVR1, which in turn activates the downstream signaling pathways implicating SMAD 2 and 3 (5). Two type 2 receptors (ACVR2A and ACVR2B) that are expressed in the primordia of pancreas and mature pancreatic cells in mice have been identified (6). In a recent study, Kim et al. (7) described the pancreatic phenotype of knockout mice for Acvr2a and/or Acvr2b. They showed that Acvr2b–/– adult mice had hypoplastic islets and higher glycemia 30 min after an oral glucose tolerance test, whereas Acvr2b–/– had no obvious pancreatic abnormalities. Acvr2a+/–b–/– embryos were not viable and showed reduced islet size and number, misexpression of SHH, and reduced expression of insulin and glucagon. Whereas Acvr2a+/–, Acvr2a–/–, and Acvr2b+/– adult mice displayed no detectable malformations of the foregut-derived organs, Acvr2a+/–b+/– mice showed hypoplastic islets and impaired glucose tolerance (resulting from inadequate insulin production or secretion) in a more severe manner than the Acvr2b–/– mice. The endocrine pancreas is thus particularly sensitive to the type and extent of activin receptor defective function. In addition, various abnormalities of the pancreas were reported in two studies of transgenic mice expressing in the pancreas a dominant-negative form of Acvr2b gene under the insulin and the β-actin promoter, respectively (8,9). In both cases, mice had severely hypoplastic islets. In the first study, the mice showed impairment of the insulin response to glucose; in the second one, abnormal endocrine cells were observed outside the islets. The functional studies implicating Acvr2b in the regulation of pancreas development prompted us to examine the contribution of the ACVR2B gene to the development of MODY and type 2 diabetes in the French population. In addition to their pancreatic phenotype, Acvr2b–/– mice have other severe complications: indeed, they also show hypoplastic spleen, abnormal stomach, defects in axial patterning, and lateral asymmetry; 70% of these mice die before weaning, probably because of severe cardiac defects (10). Therefore, we searched for mutations in the ACVR2B gene in a family with a severe form of type 2 diabetes treated with insulin, including one member presenting with cardiac defects and pancreatic agenesis. The latter was not caused by mutations in IPF1, which has been already screened for mutations in this pedigree (data not shown).

We first checked the normal expression of ACVR2B in human embryonic pancreas by reverse transcriptase–polymerase chain reaction (details are available in the online appendix at www.diabetes.org/diabetes/appendix.asp). Then we studied 11 MODY families of French ancestry that were previously found to have no mutations in the five known MODY genes (“MODY X” families) (11). We performed linkage analyses using markers located in the vicinity of the ACVR2B gene. We placed the gene between markers D3S1277 (60 cM) and D3S3521 (62 cM) by radiation hybrid mapping. D3S1277 and D3S3521 were typed in 9 of the 11 French MODY X families (2 of the 11 MODY X families were not suitable for linkage analyses because DNA was available for only 2 patients in those two pedigrees). Cumulative logarithm of odds (LOD) scores showed exclusion of linkage with diabetes at ACVR2B (LOD scores at θ0 for D3S1277 and D3S3521 were –11.74 and –17.05, respectively). The families-individual LOD scores ranged from –4.71 to 0.63 for D3S1277 and from –4.28 to 0.33 for D3S3521, indicating no evidence of linkage with diabetes (details of the results are available in the online appendix at www.diabetes.org/diabetes/appendix.asp).

The 11 exons and flanking introns of ACVR2B were thus screened for mutations in one diabetic proband from each of the 11 MODY X pedigrees. The screening was also performed in seven members of the family mentioned above (called F4854) who presented a severe form of type 2 diabetes treated with insulin, including a member with cardiac defects and pancreas agenesis. We found three nucleotide variations: two silent mutations in exons 3 (E111, GAA/GAG) and 11 (N486N, AAC/AAT) and a T to C variation 13 bp upstream of exon 7 (IVS6nt-13T/C). Table 1 shows the genotypic distribution of the variants (the three variants that we found were in perfect linkage disequilibrium and thus define only two ACVR2B alleles). None of the variants cosegregated with diabetes in the family in which it was found. Searching for the IVS6nt-13T/C variation in 100 additional type 2 diabetic probands and 100 control subjects showed no association between the variation and either diabetes (Table 2) or BMI. Furthermore, no association was found with age at onset of the disease (data not shown). The E111E and N486N silent mutations were already found at high frequency in European and American populations by Kosaki et al. (12) through the screening of the ACVR2B gene in normoglycemic patients with left-right axis malformations. It is noteworthy that we cannot exclude the presence of additional ACVR2B variations located in the promoter and introns in the MODY families tested.

The markers D3S1277 and D3S3521 were also typed in 143 French Caucasian pedigrees with type 2 diabetes (13). We found no evidence of linkage between diabetes and these microsatellites (Table 2) (details are available in the online appendix at www.diabetes.org/diabetes/appendix.asp). The design of our linkage studies in type 2 diabetes pedigrees allowed us to reach the exclusion standard score of –2 for a recurrent sibling risk (λs) of 1.3 (calculated using the Mapmaker/Sib program).

By linkage and/or screening studies, we have found no evidence for a predisposing role of the ACVR2B gene in MODY or type 2 diabetic French Caucasian families. However, studies of patients from other ethnic origins may be of interest to the genetics of type 2 diabetes.

Subjects.

The 11 MODY families are of French ancestry and have been previously described (11). The French family F4854 was recruited at Hospital Robert Debré, Paris, France.

All of the families and patients with type 2 diabetes came from 550 type 2 diabetic French Caucasian pedigrees recruited through a multimedia campaign. All families studied here had no known etiology for diabetes. A total of 143 French Caucasian pedigrees with type 2 diabetes were used for sib-pair analyses and were primarily selected for a genome-wide search for type 2 diabetes–susceptibility genes (13). At least two diabetic subjects in each sibship had to be undergoing treatment for diabetes (to load the families with severe form of type 2 diabetes). The sibships presented no bilineal inheritance of type 2 diabetes, and only one of the subjects was diagnosed before the age of 25 years. Each family contained at least one affected sib pair in which the patients were diagnosed before 65 years of age. The 100 unrelated type 2 diabetic patients and 100 control subjects (spouses of diabetic patients) were randomly selected among the initial group of 550 type 2 diabetic French Caucasian families containing at least one affected sibship.

Radiation hybrid mapping.

We localized the ACVR2B gene within the linkage map of chromosome 3 using a Genebridge 3 Radiation Hybrid Panel (Research Genetics, Huntsville, AL) and analyzed the data using the computer program RHMAP3.0 from the statistical package for multipoint radiation hybrid mapping (14).

Linkage studies.

D3S1277 and D3S3521 were typed using automated fluorescent-based procedures. Each genotype was reviewed independently by two members of the research team to confirm the accuracy of allele calling.

The LOD score calculations in MODY families have been computed as previously described (15) with the LINKAGE programs (16).

In the analyses of sib pairs in families with type 2 diabetes, we designed two qualitative traits that took into account the diabetic status: 1) the affection 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 Organization in 1997, and 2) the affection status “strict” only considered as affected those patients who were type 2 diabetic. A maximum number of 677 and 453 affected sib pairs were analyzed with the affection status large and strict, respectively. Nonparametric two-point and multipoint analyses were performed with the programs Mapmaker-Sibs 2.0 (17) (using the unweighted option) and MLBGH 1.0 (18). The maximum-likelihood binomial (MLB) method, based on the binomial distribution of parental marker alleles among affected offspring, overcomes the common problem of multiple sibs by considering the sibship as a whole.

Mutation screening.

Exons and flanking intronic sequences of the human ACVR2B gene were sequenced on both strands using direct DNA sequencing optimized protocols, as previously described (19) (primer sequences and amplification conditions are available in the online appendix at www.diabetes.org/diabetes/appendix.asp). Sequence comparison analyses were scored independently by two readers to ensure maximal accuracy for mutation detection.

TABLE 1

Genotypic distribution of the IVS6nt-13T/C variant found in the unrelated MODY probands, type 2 diabetic subjects, and control subjects studied

nT/TT/CC/C
Unrelated MODY probands 11 
Unrelated T2DM probands 100 28 51 21 
Unrelated control subjects 100 35 40 25 
nT/TT/CC/C
Unrelated MODY probands 11 
Unrelated T2DM probands 100 28 51 21 
Unrelated control subjects 100 35 40 25 

Data are n. T2DM, type 2 diabetic.

TABLE 2

Results of type 2 diabetic sib-pair analyses performed in 143 pedigrees

MarkerPhenotypic group*LOD-MLBPMLSP
D3S1277 Large (T2DM + GI) 0.02 0.37 0.11 0.32 
 Strict (T2DM) 0.00 0.50 0.00 0.50 
D3S3521 Large (T2DM + GI) 0.01 0.42 0.00 0.50 
 Strict (T2DM) 0.00 0.50 0.15 0.27 
MarkerPhenotypic group*LOD-MLBPMLSP
D3S1277 Large (T2DM + GI) 0.02 0.37 0.11 0.32 
 Strict (T2DM) 0.00 0.50 0.00 0.50 
D3S3521 Large (T2DM + GI) 0.01 0.42 0.00 0.50 
 Strict (T2DM) 0.00 0.50 0.15 0.27 
*

The affection status was determined by taking into account the patients’ diabetic status. MLS, maximum LOD score; T2DM, type 2 diabetes.

This research was supported in part by Région Nord-Pas de Calais (S.D.).

We are indebted to all of the families who participated in this study. We thank Christian Dina for help with statistical calculations.

1.
Hoodless PA, Tsukazaki T, Nishimatsu S, Attisano L, Wrana JL, Thomsen GH: Dominant-negative Smad2 mutants inhibit activin/Vg1 signaling and disrupt axis formation in Xenopus.
Dev Biol
207
:
364
–379,
1999
2.
Hebrok M, Kim SK, Melton DA: Notochord repression of endodermal Sonic hedgehog permits pancreas development.
Genes Dev
12
:
1705
–1713,
1998
3.
Furukawa M, Nobusawa R, Shibata H, Eto Y, Kojima I: Initiation of insulin secretion in glucose-free medium by activin A.
Mol Cell Endocrinol
113
:
83
–87,
1995
4.
Mogami H, Kanzaki M, Nobusawa R, Zhang YQ, Furukawa M, Kojima I: Modulation of adenosine triphosphate-sensitive potassium channel and voltage-dependent calcium channel by activin A in HIT-T15 cells.
Endocrinology
136
:
2960
–2966,
1995
5.
Massagué J, Chen Y: Controlling TGF-β signaling.
Genes Dev
14
:
627
–644,
2000
6.
Mathews LS, Vale WW: Molecular and functional characterization of activin receptors.
Receptor
3
:
173
–181,
1993
7.
Kim SK, Hebrok M, Li E, Oh SP, Schrewe H, Harmon EB, Lee JS, Melton DA: Activin receptor patterning of foregut organogenesis.
Genes Dev
14
:
1866
–1871,
2000
8.
Yamaoka T, Idehara C, Yano M, Matsushita T, Yamada T, Ii S, Moritani M, Hata J, Sugino H, Noji S, Itakura M: Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.
J Clin Invest
102
:
294
–301,
1998
9.
Shiozaki S, Tajima T, Zhang YQ, Furukawa M, Nakazato Y, Kojima I: Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor.
Biochim Biophys Acta
1450
:
1
–11,
1999
10.
Oh SP, Li E: The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse.
Genes Dev
11
:
1812
–1826,
1997
11.
Chevre JC, Hani EH, Boutin P, Vaxillaire M, Blanche H, Vionnet N, Pardini 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
:
1017
–1023,
1998
12.
Kosaki R, Gebbia M, Kosaki K, Lewin M, Bowers P, Towbin JA, Casey B: 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
13.
Vionnet N, Hani EH, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P: Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21–q24.
Am J Hum Genet
67
:
1470
–1480,
2000
14.
Lunetta KL, Boehnke M, Lange K, Cox DR: Selected locus and multiple panel models for radiation hybrid mapping.
Am J Hum Genet
59
:
717
–725,
1996
15.
Vaxillaire M, Boccio V, Philippi A, Vigouroux C, Terwilliger J, Passa P, Beckmann JS, Velho G, Lathrop GM, Froguel P: A gene for maturity onset diabetes of the young (MODY) maps to chromosome 12q.
Nat Genet
9
:
418
–423,
1995
16.
Lathrop GM, Lalouel JM: Easy calculations of lod scores and genetic risks on small computers.
Am J Hum Genet
36
:
460
–465,
1984
17.
Abel L, Muller-Myhsok B: Robustness and power of the maximum-likelihood-binomial and maximum-likelihood-score methods, in multipoint linkage analysis of affected-sibship data.
Am J Hum Genet
63
:
638
–647,
1998
18.
Kruglyak L, Lander ES: Complete multipoint sib-pair analysis of qualitative and quantitative traits.
Am J Hum Genet
57
:
439
–454,
1995
19.
Boutin P, Wahl C, Samson C, Vasseur F, Laget F, Froguel P: Big Dye terminator cycle sequencing chemistry: accuracy of the dilution process and application for screening mutations in the TCF1 and GCK genes.
Hum Mutat
15
:
201
–203,
2000

Address correspondence and reprint requests to Philippe Froguel, Institut Pasteur de Lille, 1 rue du Pr Calmette, 59000 Lille, France. E-mail: froguel@mail-good.pasteur-lille.fr.

Received for publication 19 September 2000 and accepted in revised form 5 February 2001.

Additional information can be found in an online appendix at www.diabetes.org/diabetes/appendix.asp.

ACVR1, activin receptor type 1; GI, glucose intolerant; LOD, logarithm of odds; MLB, maximum-likelihood binomial; MODY, mature-onset diabetes of the young; SHH, sonic hedgehog.