Diubetes Research and Clinical Practice, 15 (1992) 45-48 0

1992 Elsevier

Science Publishers

B.V. All rights reserved

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0168-8227/92/$05.00

DIABET 00573

Islet amyloid polypeptide (IAPP) gene analysis in a Japanese diabetic with marked islet amyloid deposition Hiroshi Kajio I, Tetsuro Kobayashi 2, Mitsuru Hara3, Koji Nakanishi2, Tadao Sugimoto2, Toshio Murase2, Yasuo Akanuma4, Kinori Kosaka2, Yoshikazu Shibasaki I, Masato Kasuga 5, Nobuhiro Yamada’ and Yoshio Yazaki ’ ’ Third Department of internal Medicine. Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan, ’ Department

of Endocrinology

and Metabolism,

Toranomon Hospital, Toranomon, Mnato-ku,

Tokyo, Japan,

’ Department of

Pathologv, Toranomon Hospital, Toranomon. Minato-ku. Tokyo. Japan, 4 The Institute for Diabetes Care and Research, Asahi Ltfe Foundation, Maruno-Uchi, Chiyoda-ku, Tokyo, Japan and 5 Second Department of Internal Medicine. Faculty of Medicine, Kobe Universitv, Kusunoki-cho,

Chuoh-ku, Kobe, Japan

Summary Islet amyloid polypeptide (IAPP) is a major constituent of pancreatic amyloid deposits in many patients with non-insulin-dependent diabetes mellitus (NIDDM). We analyzed the IAPP gene in a Japanese diabetic with marked islet amyloid deposition. Pancreatic specimens were obtained from an 87-year-old NIDDM patient who had had diabetes for 37 years. All islets (lOO/lOO) in his pancreas had IAPP-positive amyloid deposition, and 70% of the area of the islet was replaced by amyloid. We amplified the coding regions as well as the upstream region of the IAPP gene by polymerase chain reaction (PCR). The products of PCR were sequenced, and the sequences of the coding regions were identical to the Caucasian ones [ 11. However, the nucleotides of two positions of 5’upstream and one position of intron 2 were different from the Caucasian data: the upstream region of the IAPP gene in the patient had cytosine substituted for thymine at - 259, and had two alleles including cytosine and adenine at - 229, respectively. The nucleotide of position 539, that is guanine, was deleted. A possible difference in the IAPP promoting region between the Japanese and Caucasian population was suggested. Key words: Islet amyloid polypeptide (IAPP); Gene; Pathology; Japanese

Introduction Islet amyloid deposition is one of characteristic pathological findings in some patients with nonCorrespondence to: H. Kajio, Third Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo, 113, Japan.

Non-insulin-dependent

diabetes

mellitus (NIDDM);

insulin-dependent diabetes mellitus (NIDDM). Islet amyloid polypeptide (IAPP) was found to be a prominent constituent of the deposition [ 1,2]. Some physiological characteristics [3-51 and amyloidogeneity of this peptide [6] have been investigated. However, a possible variation of the IAPP gene responsible for amyloid deposition over the islet in particular cases remains unclear.

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In this study we performed the IAPP gene sequencing analysis in a Japanese diabetic with marked IAPP-positive amyloid deposition.

Materials and Methods Pancreatic tissue was obtained from an autopsy case, an 87-year-old man with diabetes of 37 years duration. The sections of the formalin-fixed tissues were stained with haematoxylin-eosin and Congo-red, and immunologically stained with anti-IAPP serum (RS 02-l) by the peroxidase antiperoxidase (PAP) method. The frozen pancreatic tissue was used to analyze the IAPP gene. The tissue was incubated at 37°C in 50 mM Tris-HCl (pH S), 0.1 M NaCl, 20 mM EDTA, 1% SDS, 150 pg/ml proteinase K and 1 mg/ml pronase E. Genomic DNA was extracted by following phenol/chloroform extraction, RNase treatment and ethanol precipitation. The IAPP gene was amplified by polymerase chain reaction (PCR). Specific primers, which covered both the upstream region and the coding regions including exon 2 and exon 3, were selected following the already reported sequence of the human IAPP gene [7]. Five pairs were selected; two pairs for 5’-upstream, one pair for exon 2 and two pairs for exon 3. The positions of the primers used were as follows: 5’-upstream -533 b: 17to37 a: -554to -314 to -292 d: - 192 to - 172 c: Exon 2 f: 597 to 616 e: 402 to 420 Exon 3 h: 5499 to 5520 g: 5287 to 5307 j: 5587 to 5609 i: 5339 to 5362 We used the PCR protocol with reference to Gyllenstein’s method [ 81. One hundred microliter of each reaction mixture contained 0.5 pg of genomic DNA, 1 PM of each primer, 2.5 units of Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3) 50 mM KCl, 1.5 mM MgCl,, 10 ,ug/ml gelatin, and 200 PM each of dNTPs. Using a

DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT, U.S.A.), the samples were incubated sequentially for 1 min at 94’ C, 2 min at 58 oC and 2 min at 72’ C, for 25 cycles. The second PCR was performed in a similar manner to the first step procedure except that 10 ~1 of the first PCR product and 1 PM of a single primer were used instead of a pair of primers. The PCR products were extracted with phenol/chloroform, precipitated with ethanol, and purified with a Centricon 100 microconcentrator (Amicon, Danvers, MA, U.S.A.). Sequencing primers were identical to the primers described above. These primers were labeled with 32P using a DNA 5’-labeling kit (Takara, Kyoto, Japan) and 1 pmol of labeled primer was annealed with PCR product in 10 ,ul of 400 mM Tris-HCl (pH 7.5) 20 mM MgCl, and 50 mM NaCl. This mixture was then added to 1 ~1 of 0.1 M dithiothreitol and 2 ~1 of water and 3 1.11of the resulting mixture was incubated with 2.5 ~1 of dideoxynucleotide termination mix and 0.5 ~1 of diluted sequenase at 37°C for 5 min ( SequenaseR version 2.0, 7-deaza-dGTP edition, United States Biochemical Co., Cleveland, OH, U.S.A.). The samples were analyzed with electrophoresis on 6 y0 polyacrylamide and 7 M urea gels and autoradiography.

Results Pathological fidings

Congo-red staining showed that amyloid deposition was observed in and around all the pancreatic islets. Immunohistochemical staining demonstrated that the pancreatic amyloid was positive for IAPP. IAPP-positive amyloid was observed in all (lOO/lOO) islets. Mean islet area replaced by IAPP-positive amyloid was 70%. Genetic &dings

The results of gel electrophoresis of the first PCR products indicated that there was little non-specific amplification and the primers were highly specific (data not shown). The sequences of the

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2 I

ACGT

3 I

ACGT

\AC

GT

539

G 4 deleted

Fig. 1. The demonstration

N-

of the sequences different from those reported by Nishi et al. [ 11. Two positions at - 259 and - 229 belong to the upstream region and one position at 539 belongs to intron 2.

PCR products corresponding to the coding region were the same as the data already reported [ 71. However, the nucleotides at positions - 259 and - 229 were different (Fig. 1). At position - 259, cytosine was substituted for thymine, while we found two alleles, including cytosine and adenine at - 229, respectively. Moreover, the nucleotide at 539 in exon 2, that is guanine in a previous study [7] was deleted.

Discussion We performed IAPP gene analysis in a Japanese diabetic, who had marked IAPP-positive amyloid deposition over an autopsied pancreas. Nishi et al. sequenced IAPP coding regions in American NIDDM using leukocyte specimens [ 91. They suggested that a primary structural abnormality of IAPP or its precursor is unlikely to play a significant role in the formation of islet amyloid. We autopsied pancreatic tissues which were confirmed pathologically to have IAPPpositive amyloid. As a consequence, concordant results were obtained as to the IAPP gene with a previous study [7]. Mosselman et al. [lo] demonstrated that the upstream region of IAPP is

indispensable for the regulation of IAPP gene expression. We also carried out sequence analysis on this region. We found two different positions from those by Nishi et al. [7]. However, the nucleotide at - 259, cytosine, is identical to the data in non-diabetic cases reported by van Mansfeld et al. [ 111. Therefore, this difference is not likely to be important for amyloid formation. On the other hand, the fact that there are two alleles including cytosine and adenine at - 229, respectively, is a new finding. The significance of this position is unknown. One possibility may be related to racial differences in the IAPP promoting region between Japanese and Caucasian populations. Over-secretion of IAPP could be a cause of islet amyloid deposition. In this respect, mutations in the upstream region could induce over-expression of IAPP, leading to subsequent islet amyloid deposition.

Acknowledgements We wish to thank Dr. Tatsuhiko Kodama for his generous help in the laboratory. We also thank Mitsue Shibata and Fumie Takano for their assistance in the preparation of the manuscript.

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References 1 Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. U.S.A. 84, 8628-8632. 2 Westermark, P., Wernstedt, C., O’Brien, T.D., Hayden, D.W. and Johnson, K.H. (1987) Islet amyloid in Type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. Am. J. Pathol. 127, 414-417. 3 Ohsawa, H., Kanatsuka, A., Yamaguchi, T., Makino, H. and Yoshida, S. (1989) Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion for isolated rat pancreatic islets. Biochem. Biophys. Res. Commun. 160, 961-967. 4 Leighton, B. and Cooper, G.J.S. (1988) Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335, 632-635. 5 Cooper, G.J.S., Leighton, B., Dimitriadis, G.D. et al. (1988) Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 85, 7763-7766. 6 Westermark, P., Wernstedt, C., Wilander, E., Hayden,

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D.W., O’Brien, T.D. and Johnson, K.H. (1987) Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. U.S.A. 84, 3881-3885. Nishi, M., Sanke, T., Seino, S. et al. (1989) Human islet amyloid polypeptide gene: complete nucleotide sequence, chromosomal localization, and evolutionary history. Mol. Endocrinol. 3, 1775-1781. Gyllenstein, V.B. and Erlich, H.A. (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. U.S.A. 85, 7652-7656. 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. Mosselman, S., Hoppener, J.W.M., de Wit, L., Soeller, W., Lips, C.J.M. and Jansz, H.S. (1990) IAPP/amylin gene transcriptional control region: evidence for negative regulation. FEBS Lett. 271, 33-36. Mansfeld, A.D.M., Mosselman, S., Hoppener, J.W.M. et al. (1990) Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man. Biochim. Biophys. Acta 1087, 235-240.

Islet amyloid polypeptide (IAPP) gene analysis in a Japanese diabetic with marked islet amyloid deposition.

Islet amyloid polypeptide (IAPP) is a major constituent of pancreatic amyloid deposits in many patients with non-insulin-dependent diabetes mellitus (...
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