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A Novel Point Mutation in the Insulin Gene Giving Rise to Hyperproinsulinemia¹

Diabetes Research Laboratories (M.G.W.-P., S.E.M., S.M., R.C.T.), Radcliffe Infirmary, Oxford, OX2 6HE England; Section of Endocrinology (D.O., K.P.), Department of Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637; and Scarborough Hospital (P.B.), Scarborough, Yorks, YO12 6QL England

Address all correspondence and requests for reprints to: Prof. Robert C. Turner, Diabetes Research Laboratories, Woodstock Road, Oxford, United Kingdom OX2 6HE.

	Abstract

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A 58-yr-old obese white Caucasian male type 2 diabetic, entered into the UK Prospective Diabetes Study, was found to have raised fasting total proinsulin levels 708 pmol/L^-1 (normal range, 3–16 pmol/L^-1) and normal specific plasma insulin level 29 pmol/L^-1 (normal range, 21–75 pmol/L^-1). Immunoreactive plasma insulin, measured by RIA, was 503 pmol/L^-1. DNA was extracted, the insulin gene amplified by the PCR, and by direct sequencing, a novel point mutation, G1552C, was identified, which resulted in the substitution of proline (CCT) for arginine (CGT) at position 65. This prevented cleavage of the C-peptide A-chain dibasic cleavage site (lys-arg) by the processing protease in the pancreatic ß-cells. The plasma proinsulin and insulin levels were in accord with expression of both the wild-type and the mutant alleles. The G1552C mutation was not linked with diabetes, because it was present in a 37-yr-old nondiabetic daughter and not in a 35-yr-old daughter who had had gestational diabetes.

	Introduction

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INSULIN is produced post translational from its precursor molecule, proinsulin, by site-directed proteolysis in ß-cell granules (1). Conversion involves cleavage at pairs of basic residues that link both the insulin A- and B-chains to C-peptide. Human proinsulin conversion has a preferred sequential route, such that cleavage at the B-chain/C-peptide junction occurs first, producing des-31,32 split proinsulin as the major conversion intermediate (2). Under normal circumstances, proinsulin conversion is largely completed before secretion and low plasma levels of intact proinsulin and conversion intermediates are found. Structural abnormalities in the proinsulin molecule can impair conversion, leading to the accumulation of proinsulin-like material in the circulation. Such defects show an autosomal dominant mode of inheritance and are the main cause of familial hyperproinsulinemia (3, 4, 5, 6, 7, 8). We report here the clinical and laboratory characteristics of a type 2 diabetic patient who had a novel mutation in the insulin gene, giving rise to impaired proinsulin conversion.

	Materials and Methods

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Case report

A 58-yr-old obese white Caucasian man presenting with a fasting plasma glucose of 10.2 mmol/L^-1 was recruited into the United Kingdom Prospective Diabetes Study (9) on diagnosis of noninsulin-dependent diabetes mellitus (NIDDM). Routine laboratory examinations revealed persistent fasting hyperproinsulinemia with a fasting total proinsulin, measured by enzyme-linked immunosorbent assay (ELISA), of 708 pmol/L^-1. Immunoreactive plasma insulin measured by RIA was 503 pmol/L^-1 and specific plasma insulin measured by ELISA was 29 pmol/L^-1. His BW was 87 kg and body mass index 29 kg/m² with triglyceride and HDL cholesterol measurements within the normal range (1.07 and 1.08 mmol/L^-1, respectively). His systolic and diastolic blood pressures were 137 and 80 mmHg, respectively. There was no evidence of any microvascular or macrovascular complications. The patient (GW) has one brother (PW), who is nondiabetic, and two daughters, one of whom (GM) had gestational diabetes during both her pregnancies and the other (JK), normal glucose tolerance .

Assay techniques

Plasma glucose was measured by the hexokinase/G6P dehydrogenase method. Immunoreactive insulin was measured using the Pharmacia Insulin RIA 100 double-antibody RIA (Pharmacia Diagnostics, Milton Keynes, Buckinghamshire, UK). Human proinsulin and all split proinsulins cross-react 100% with this assay. Total proinsulin was measured by means of a sandwich ELISA using murine monoclonal antibodies, HUI-001 binding to insulin A-chain and PEP-001 binding to C-peptide (Novo Nordisk A/S, Bagsværd, Denmark). The proinsulin ELISA cross-reacts with 32,33-split proinsulin (74%) and des 31,32-proinsulin (65%) but not with insulin or C-peptide. Specific insulin was measured by ELISA using murine monoclonal antibodies, HUI-018 reactive to A-loop of insulin and 0XI-005 binding to C-terminal of B-chain (Novo Nordisk). Specific insulin ELISA does not cross-react with C-peptide, intact proinsulin, or its split intermediates.

High-performance liquid chromatography (HPLC) analysis

Insulin and proinsulin immunoreactivity from serum of the proband and available family members were extracted by immunoaffinity chromatography with a nonspecific guinea pig antiinsulin Ig fraction, coupled to Bio-Rad Affi-Gel 10 agarose beads (Bio-Rad, Richmond, CA), as previously described (10). Circulating insulin, proinsulin, and conversion intermediates were separated by reverse-phase HPLC using a Series 4 liquid chromatography, ISS-100 column oven, a LC-100 recorder/integrator (Fisher, Pittsburgh, PA), and an Ultrasphere C16 column (Beckman, Berkeley, CA), and the fractions were assayed by the proinsulin ELISA (10). The peptides used for HPLC standardization were biosynthetic human insulin, proinsulin, 32–33 split proinsulin, des-31,32 proinsulin, 65–66 split proinsulin, and des-64,65 split proinsulin (Lilly, Indianapolis, IN).

Sequencing of the insulin gene

Genomic DNA was extracted from peripheral leucocytes from the proband and available family members using the Nucleon DNA extraction kit (Scotlab, Coatbridge, Scotland). The PCR was used to amplify exons 2 and 3 of the insulin gene under standard conditions. Oligonucleotide primer pairs used to amplify exon 2 were nucleotides 484 to 502 and complementary to 689 to 708, as described by Bell et al. (11). For exon 3, the primer pair used were nucleotides 1435 to 1454 and complementary to 1682 to 1700 (11). One primer of each pair was biotinylated at the 5' end, and PCR products were separated into single-stranded templates for nucleotide sequencing using the Dynabead biomagnetic separation system (Dynal, Oslo, Norway). Direct DNA sequencing by the dideoxy method was carried out using the Sequenase Version 2.0 kit (USB, Cambridge, UK) and sequence-specific primers.

	Results

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Insulin gene characterization

Sequencing exon 3 of the proband revealed he was heterozygous for a GC point mutation at position 1552. This site corresponds to the lys⁶⁴, arg⁶⁵ dibasic cleavage site at the junction of C-peptide and the A-chain. This substitution results in the amino acid replacement of proline (CCT) for arginine (CGT). Exon 2 showed an entirely normal nucleotide sequence, and thus the normal arg³¹, arg³² dibasic cleavage site was maintained.

The kindred

The proband’s daughter JK showed marked hyperproinsulinemia with immunoreactive insulin measured at 435 pmol/L^-1 with plasma proinsulin 614 pmol/L^-1 and specific insulin 21 pmol/L^-1, despite a normal fasting plasma glucose (4.5 mmol/L^-1). His brother PW and his other daughter GM, who had had gestational diabetes in two pregnancies, both had normal fasting plasma glucose (5.4 and 5.7 mmol/L^-1, respectively) and normal immunoreactive insulin (38 and 47 pmmol/L^-1, respectively).

Genotyping of family members

Daughter JK, with raised proinsulin concentration, was heterozygous for the G1552C mutation in exon 3. The proband’s brother and daughter GM had the normal nucleotide sequence.

Reverse-phase HPLC analysis

Elution profiles of fasting serum insulin and proinsulin immunoreactivities from the proband and his two daughters are shown in Fig. 1. A large abnormal peak was seen in the proband’s sample and that of daughter JK. This peak is assumed to be caused by a structurally abnormal des-31,32-split proinsulin, which eluted at a slightly longer retention time than the normal split intermediate, as a result of the substitution of the more hydrophobic proline residue for arginine. A small peak of immunoreactivity is seen at a position corresponding to normal insulin, suggesting that the normal proinsulin allele is also expressed in these individuals, with conversion in the ß-cell granules to insulin. Daughter GM showed a normal elution profile.

View larger version (20K): [in this window] [in a new window]   Figure 1. HPLC elution profiles of the proband and family members. a, HPLC assay standards; b, IRI from the proband (GW); c, IRI from daughter (GM); d, IRI from daughter (JK).

	Discussion

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The affected individuals with G1552C were heterozygous for the mutation and coexpressed both normal and abnormal alleles, given that both the mutated proinsulin and normal insulin were detected in the plasma. The elevated circulating IRI consisted mainly of the unprocessed mutated proinsulin, which had accumulated because of proinsulin’s relatively low clearance compared with insulin (plasma half-lives approximately 20 and 3.5 min, respectively). Because proinsulin has approximately 3% of normal insulin activity, in the basal state, both the proinsulin and the insulin levels probably contributed to insulin action.

It is unlikely that the G1552C mutation caused the proband’s diabetes, because the daughter with the same mutation had a normal fasting plasma glucose concentration, whereas the daughter who had had gestational diabetes did not have the mutation. Diabetes in this family is likely to be caused by other genetic or environmental factors. The identification of elevated proinsulin levels in the diabetic proband was probably a chance finding from screening large numbers of diabetic patients. Increased proinsulin levels are a feature of NIDDM (12, 13). Raised proinsulin levels have been postulated to be potentially pathogenic, in view of an association in diabetic subjects with cardiovascular risk factors (14). However, the affected members of this family had no evidence of atheroma-related disease, even though they had presumably had raised proinsulin levels since birth.

	Acknowledgments

 
We are grateful to Pauline Sutton, Rachel Morris, and Christopher Groves for technical assistance.

	Footnotes

 
¹ This study was supported by grants from the Wellcome Trust and Alan and Babette Sainsbury Trust and the Diabetes Research and Training Center at the University of Chicago (DK-20595).

Received June 17, 1996.

Revised January 3, 1997.

Accepted January 31, 1997.

	References

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