Diabetes Research and Clinical Practice, 18 (1992) 31-34 0

1992 Elsevier Science Publishers

31

B.V. All rights reserved 0168-8227/92/$05.00

DIABET 00669

The islet amyloid polypeptide gene and non-insulin-dependent diabetes mellitus in South Indians M.I. McCarthy”, ,’ Cellular Mechanisms

G.A. Hitman”,

V. Mohanb, A. Ramachandranb, M. Viswanathan b

C. Snehalathab

and

Group, London Hospital Medical College, London, United Kingdom, and b Diabetes Research Centre. Madras, India

(Received 22 July 1991) (Revision accepted 2 May 1992)

Summary Islet amyloid polypeptide (IAPP), otherwise called amylin, is the monomeric component of islet amyloid. Deposition of this amyloid is a characteristic feature of non-insulin-dependent diabetes mellitus in humans and may play a role in the pathogenesis of the disease. As such, abnormalities in the structure or expression of the IAPP gene might contribute to the inheritance of this condition. The IAPP gene was studied in a well-characterised population of 62 unrelated Dravidian subjects with non-insulin-dependent diabetes mellitus and 56 normal Dravidian controls, using a restriction fragment length polymorphism generated by PvuII digestion. Genotype and allele frequencies did not differ between diabetic subjects and controls. Taken together with recent findings in Europid and other racial groups, an abnormality of the IAPP gene is highly unlikely to represent a major gene for the development of non-insulindependent diabetes mellitus. Key words: Islet amyloid polypeptide; Amylin; Population study; South India

Introduction Non-insulin-dependent diabetes mellitus (NIDDM) is a disease with a major inherited component as evidenced by a large number of twin, family and population studies (reviewed in [ 11). Progress in determining the major aetiologCorrespondence to: M.I. McCarthy, The Medical Unit, The Royal London Hospital, London El lBB, UK.

Type 2 (non-insulin-dependent

diabetes

mellitus);

ical gene(s) has however been disappointingly slow, with both association and linkage studies failing to reveal consistent disease-associated genotypes [ 11. This may reflect the likelihood that NIDDM is a heterogeneous disease with a variety of genetic abnormalities producing an apparently singular phenotype. Defects of both insulin action and secretion need to co-exist before appreciable glucose intolerance supervenes, but the molecular basis of

32 these defects and the sequence of events remains uncertain [2]. In some populations at least, the earliest abnormalities detectable in subjects at high risk of NIDDM are related to insulin secretion [3] and it seems likely that a major contribution to the susceptibility to NIDDM results from inherited defect(s) in B-cell function. The rediscovery of islet amyloid as a characteristic feature of NIDDM in humans and other species [4] has led to speculation that it may play a role in the pathogenesis of NIDDM [5]. The monomeric component of islet amyloid has been identified as islet amyloid polypeptide (IAPP, synonyms: diabetes associated peptide, amylin, DAP), a 37 amino-acid peptide, homologous to calcitonin gene-related peptide, synthesised in the b-cell and then co-stored and secreted with insulin [6]. It remains unclear, however, whether amyloid deposition is a primary or secondary event involved in the pathogenesis of NIDDM, or whether it is merely an epiphenomenon [ 71. Since a defect in IAPP gene expression or structure could underlie its deposition as amyloid, the IAPP gene can be considered as a candidate gene for NIDDM: we sought to study the association between an IAPP gene restriction fragment length polymorphism (RFLP) and NIDDM in a wellcharacterised population of South Indian diabetics and controls.

presence of a family history of NIDDM in the majority of cases [9] mean that these criteria effectively exclude a significant risk of future diabetes in the controls. Venous blood was taken into EDTA and stored at -20 “C pending transport to the UK. DNA was extracted by standard methods, and 10 ug digested with PvuII (Northumbria Biologicals Ltd. Cramlington, UK). Following electrophoresis in 0.7:, agarose gels and transfer onto nylon membranes (Gene Screen Plus, New England Nuclear, Boston, USA) by Southern blotting [lo], the membranes were hybridised overnight with a “P-1abelled IAPP cDNA probe (scIAP-8: [ 111). Membranes were washed to a stringency of 1 x SSC and exposed for 1-7 days at -70 “C to Kodak XAR-5 film. Analysis of results was by chi-square analysis.

Results Using PvuII restriction, two bands of 16 kb and 24 kb were identified. Genotype and allele frequencies for subjects with NIDDM and controls are shown in Table 1. There was no association between NIDDM and the IAPP gene RFLP studied.

Discussion Materials and Methods Sixty-two unrelated patients with NIDDM from the clinic population at the M.V. Hospital for Diabetes, Madras, were studied. All were Dravidian (South Indian): other types of diabetes (insulin-dependent diabetes mellitus, fibrocalculous pancreatic diabetes mellitus) were excluded as previously described {S]. Controls were derived from two sources: (a) 24 blood donors from Madras, and (b) 32 members of staff or spouses of patients. Controls had no first degree family history of diabetes, and random blood glucose levels below 6.7 mmol/l. In South Indians, the relatively early age of onset of NIDDM and the

In a well-characterised population of Dravidian diabetic subjects and controls, we failed to detect

‘TABLE IAPP

I

gene:

NIDDM

genotype

I,

Diabetes Controls I’ x’=2.32.

distribution

and

allele distribution

in

and controls

62 56

Allele’

Genotype3 16.16

16,24

24,24

16

24

5 7

18 10

39 39

28 24

96 88

df 2. P=O.31.

’ ~‘=0.03,

df 1. P=O.96

33 an association between NIDDM and an RFLP marker of the IAPP gene. This suggests that amongst South Indians, a population with a moderately high prevalence of NIDDM (age-adjusted urban prevalence of 9-10% [ 121) this RFLP is not in linkage disequilibrium with a diabetespredisposing mutation in the IAPP gene. This locus is relatively non-polymorphic and not amenable to study with other restriction enzymes [ 131. Genetic evidence suggests that Dravidians are Caucasoids, but are distinct from European and Middle Eastern populations [ 141. However, similar findings concerning the IAPP gene and NIDDM have recently been reported in Europid sub.jects [ 151. The Oxford group also reported that linkage with the IAPP gene had effectively been excluded in four pedigrees. The linkage approach is, however, particularly susceptible to disease heterogeneity which may limit its value in NIDDM [ 11. If there are a number of important disease susceptibility genes for NIDDM, then the best prospects for their identification will be offered by large, population-based studies employing suitable controls, though this type of study also has methodological difficulties [ 1,161. An alternative approach is to sequence the IAPP gene for mutations that might contribute to the development of NIDDM. Nishi and colleagues have sequenced the coding region of the IAPP gene in 25 subjects with NIDDM and found it to be the same for all subjects and identical to those previously published [ 171. However, the promoter region was not sequenced in this study: the possibility that abnormalities in this region could lead to increased expression of IAPP and thereby promote amyloid formation was therefore not excluded. In summary, we have failed to detect any association between the IAPP gene and NIDDM in South Indians. When this and previous genetic studies are considered, it seems highly unlikely that the IAPP gene locus is a primary susceptibility gene for NIDDM. This does not imply, however, that IAPP and islet amyloid are irrelevant to the pathogenesis of NIDDM. Amyloid deposition could result from a primary defect in

genes controlling IAPP processing, or as a crucial secondary event exacerbating P-cell dysfunction in susceptible individuals.

Acknowledgement The probe ScIAP-8 was kindly donated by Dr. J.W.M. Hdppener (University of Utrecht, Netherlands). References 1 Hitman,

2

3

4 5

6

7

8

G.A. and McCarthy, MI. (1991) Genetics of non-insulin dependent diabetes mellitus. Bailhere’s Clin. Endocrinol. Metab. S(3), 455-476. Turner, R.C., O’Rahilly, S., Levy, J.. Rudenski, A. and Clark, A. (1989) Does type II diabetes arise from a major gene defect producing insulin resistance or beta cell dysfunction? In: J. Nerup, T. Mandrup-Poulsen and B. Hofeldt (Eds.), Genes and Gene Products in the Development of Diabetes Mellitus. Elsevier, Amsterdam, pp. 171-183. O’Rahilly, S., Turner. R.C. and Matthews, D.R. (1988) Impaired pulsatile secretion of insulin in relatives of patients with non-insulin-dependent diabetes. N. Engi. J. Med. 318. 1225-1230. Clark. A. (1989) Islet amyloid and type 2 diabetes. Diabetic Med. 6. 561-567. Porte, D. and Kahn, S.E. (1989) Hyperproinsulinaemia and amyloid in NIDDM: clues to etiology of islet B-cell dysfunction? Diabetes 38, 1333-1336. Kahn, S.E., D’Alessio, D.A., Schwartz. M.W. et al. (I 990) Evidence of cosecretion of islet amyloid polypeptide and insulin by p-cells. Diabetes 39. 634-638. Steiner, D.F., Ohagi, S., Nagamatsu, S., Bell, G.I. and Nishi. M. (1991) Is islet amyloid polypeptide a significant factor in pathogenesis or pathophysiology of diabetes? Diabetes 40, 305-309. Kambo. P.K., Hitman, G.A., Mohan. V. et al. (1989) The genetic predisposition to fibrocalculous pancreatic diabetes. Diabetologia 32. 45-51. Hitman, G.A., Viswanathan, M.. McCarthy, M.I. et al. (1991) Genetic markers for non-insulin dependent diabetes mellitus and obesity. In: H. Rifkin. J.A. Colweli and S.I. Taylor (Eds.). Diabetes 1991. Elsevier. Amsterdam (in press). Hitman, G.A., Niven, M.J.. Festenstein. H. et al. (1987) HLA Class II alpha chain gene polymorphisms in patients with insulin dependent diabetes mellitus, dermatitis herpetiformis and coeliac disease. J. Clin. Invest. 79, 609615.

34 11 Mosselman, S., Hbppener, J.W.M., Lips, C.J.M. and Jansz, H.S. (1989) The complete islet amyloid polypeptide precursor is encoded by two exons. FEBS Lett. 247, 154158. 12 Ramachandran. A., Jali, M.V., Mohan, V., Snehalatha. C. and Viswanathan, M. (1988) High prevalence of diabetes in an urban population in south India. Br. Med. J. 297, 587-590. 13 Patel, P.P., Mosselman. S., Hbppener, J.W.M. et al. (1989) An RFLP associated with insulinoma amyloid polypeptide locus. Nucleic Acids Res. 17, 6758. 14 Cavalli-Sforza, L., Piazza, A., Menozzi, P. and Mountain. J. (1988) Reconstruction of human evolution: bringing to-

gether genetic, archeological and linguistic data. Proc. Natl. Acad. Sci. USA 85, 6002-6006. 15 Cook, J.T.E., Patel, P.P., Clark, A. et al. (1991) Nonlinkage of the islet amyloid polypeptide gene with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 34, 103-108. 16 O’Rahilly, S.. Wainscoat, J.S. and Turner, R.C. (1988) Type 2 (non-insulin-dependent) diabetes mellitus. New genetics for old nightmares. Diabetologia 31, 407-414. 17 Nishi, M., Bell, G.I. and Steiner, D.F. (1990) Islet amyloid polypeptide (amylin): no evidence of an abnormal precursor sequence in 25 type 2 (non-insulin-dependent) diabetic patients. Diabetologia 33, 628-630.

The islet amyloid polypeptide gene and non-insulin-dependent diabetes mellitus in south Indians.

Islet amyloid polypeptide (IAPP), otherwise called amylin, is the monomeric component of islet amyloid. Deposition of this amyloid is a characteristic...
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