Studies on mutant human insulin genes : Identification and sequence analysis of a gene encoding [ SerB 24

Both alleles of the insulin gene of a patient with mild diabetes [maturity-onset-diabetes-of-the-young (MODY)-type syndrome] associated with hyperinsulinemia have been cloned, and the sequences have been determined. One allele contained a mutation (single nucleotide transition) in the coding sequence for the B chain at position 24 (TTC -) TCC), resulting in the loss of a restriction enzyme (Mbo II) cleavage site in the gene. This mutation results in the substitution of serine for phenylalanine in a critically important region of the insulin molecule that is intimately involved in receptor binding. Both insulin alleles were of the a type and, aside from a single nucleotide deletion in the 5' region of the normal allele, their sequences were identical to those previously determined. Diabetes is a heterogeneous group of metabolic disorders characterized by hyperglycemia and a relative or absolute deficiency of insulin. A strong genetic component is particularly evident in the more common noninsulin-dependent or maturity-onset form of the disease (type II diabetes), but the nature of the underlying genetic factor or factors responsible for the disorder remains obscure. Analysis of the inheritance pattern in affected families is complicated by the relatively late onset of the disease in many individuals and the probable existence of secondary contributing factors, which may be either acquired or genetic (1, 2). Despite these complexities, it is important to establish whether defects residing in or near the insulin gene may contribute to the relative insulin deficiency in this group of patients. We have identified three individuals with mild diabetes associated with increased plasma insulin levels and have shown that these patients all have abnormal circulating insulin molecules that can be distinguished from each other and from normal human insulin (3). These abnormal insulins exhibited both impaired biological activity and reduced rates of clearance from the circulation (4-6). In one of these cases, insulin isolated from the patient's pancreas consisted of roughly equal proportions of normal insulin and an insulin of low biological potency, having a leucine-for-phenylalanine substitution at position 25 in the B chain (3, 7). This substitution resulted in the loss of a normal restriction enzyme cleavage site in one allele of the patient's insulin gene in the coding region for the B chain at the phenylalanine-phenylalanine sequence at positions B24 and B25 (8). The codons for these two amino acids contain a pentanucleotide recognition sequence for the enzyme Mbo II. Cloning and nucleotide sequence analysis of the insulin gene of this patient resulted in the identification of a single nucleotide transversion in the codon for phenylalanine-B25, changing it to a codon specifying leucine (9). We have now studied in greater detail another affected individual (J.F.) and her family. This patient had fasting hyperinsulinemia without any resistance to exogenously administered insulin (5). Similar fasting hyperinsulinemia was demonstrated in five additional family members of both sexes over three successive generations, indicating an autosomal dominant pattern of inheritance. Insulin isolated from the patient's serum by immunoaffinity chromatography had markedly reduced biological activity as compared to its immunoreactivity (5). HPLC analysis of the serum insulin revealed a major component that was less hydrophobic than native human insulin and only small amounts of the normal hormone (3). Furthermore, restriction endonuclease cleavage analysis of genomic DNA isolated from the patient's leukocytes revealed the apparent loss in one allele of the insulin gene of the same Mbo II recognition site that had been found to be defective in the first patient (3, 5). However, this abnormal insulin could be distinguished clearly by HPLC from the [Leunm]insulin found in the first case (3). In order to define the nature of this mutation, we cloned and determined the nucleotide sequences of both alleles of the insulin gene of this patient. The results demonstrate that a point mutation has occurred in the codon for position B24 resulting in the substitution of serine for phenylalanine at this site. MATERIALS AND METHODS Preparation of Genomic DNA. Genomic DNA was prepared from the leukocytes from 20-50 ml of blood as described (8). In brief, the leukocytes were digested with proteinase K (100 ,ug/ml) in the presence of 100 mM EDTA, pH 8.0/0.5% NaDodSO4 at 550C overnight and were extracted three times with phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol), and twice with chloroform; the DNA was dialyzed extensively against 10 mM Tris HCl, pH 7.5/1 mM EDTA. DNA was then digested with ribonuclease A (100 ,ug/ml) at 37°C for 2 hr and with proteinase K at 55°C for 2 hr, extracted twice with the phenol solution and once with chloroform, and then dialyzed against 1 mM Tris HCl, pH 7.5/0.1 mM EDTA. DNA concentration was calculated from the absorbancy at 260 nm, and the yields were :"10 ,ug per ml of blood. Restriction Endonuclease Cleavage Analysis. Genomic DNA (30 ,g) was digested with 60 units (unless otherwise specified) of restriction endonuclease at 37°C overnight (== 16 hr) under the conditions suggested by the manufacturers. Digested DNA was separated on an 0.8 or 2% agarose gel and transferred to nitrocellulose filters by the method of Southern (10). The filter was incubated with 14 ml of prehybridizing solution containing 3x NaCl/Cit (1x NaCl/Cit is 0.15 M NaCl/0.015 M sodium citrate, pH 7), 10X Denhardt solution (lx is 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.2% bovine serum albumin), 50 Abbreviations: bp, base pair(s); kb, kilobase(s). 6366 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 6367 mM sodium phosphate, alkali-denatured salmon testis DNA at 200 gg/ml, poly(A) at 10,ug/ml, poly(C) at 10 pg/ml, and 0.1% NaDodSO4 at 650C overnight and then hybridized with 2 x 106 cpm of human insulin cDNA labeled with 32P by nick-translation (11) in 5 ml of the above solution at 650C for 3 days. After hybridization, the filter was washed with 0.1 X NaCl/Cit at 650C, dried, and exposed to x-ray film at -700C for 7 days with an intensifying screen (DuPont Cronex Lightning Plus). Molecular Cloning of the Insulin Genes. Genomic DNA (270 gg) was digested with EcoRI at 370C overnight and was separated on an 0.8% agarose gel. Three 5-mm slices covering the size ranges 16.5-23.5 (fragment A), 12.3-16.5 (fragment B), and 9.8-12.3 kilobases (kb) (fragment C) were cut from gel and isolated by electroelution (12); 1-gg aliquots of each fragment were applied to an 0.8% agarose gel again, along with a marker lane of 30 pg of EcoRI-digested genomic DNA. The autoradiogram of the Southern blot showed that fragment B contained the insulin gene, and this fragment was used for cloning. A gtWES AB phage was grown, and DNA was isolated as described (13). The phage DNA was digested with EcoRI, and the phage arms were prepared by sucrose gradient centrifugation, dialyzed, and precipitated with ethanol (13). The purified EcoRI fragment containing the insulin gene was ligated to the phage arms in equimolar ratios; 1 pg of arms and 0.65 pg of DNA fragment B were hybridized at 420C for 2 hr in 8 ul of 1.25x ligase buffer (12). After cooling slowly, 1 ,ul of 10 mM ATP, 1 /.l of 10 mM dithiothreitol, and 0.5 1. of T4 ligase (2 units) were added, and the mixture was incubated at 9°C overnight. The ligated DNA was then packaged in vitro (14), yielding r5 x 105 recombinant phages per jig of arms. The packaged phage was infected into Escherichia coli LE392, and phage plaques were screened by an in situ plaque hybridization method (15) with 32P-labeled and cloned human insulin cDNA as a probe. Positive plaques were picked and purified by screening at low density through two more cycles. DNA from selected clones were isolated from 400-ml lysates and analyzed by restriction endonuclease mapping. An Xho I fragment containing the insulin gene was then subcloned into the Sal I site of pBR322, and the nucleotide sequence was determined by the method of Maxam and Gilbert (16).