No Evidence for Linkage or for Diabetes-Associated Mutations in the Activin Type 2B Receptor Gene (ACVR2B) in French Patients With Mature-Onset Diabetes of the Young or Type 2 Diabetes

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.