Proc. Nati. Acad. Sci. USA Vol. 87, pp. 658-662, January 1990 Genetics A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction (diabetes mellitus/insulin resistance/leprechaunism) TAKASHI KADOWAKI, HIROKo KADOWAKI, AND SIMEON I. TAYLOR* Biochemistry and Molecular Pathophysiology Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Ora M. Rosen, October 24, 1989 ABSTRACT Mutations in the insulin receptor gene can receptor mRNA and thereby reducing the rate of receptor render the cell resistant to the biological action of insulin. We biosynthesis (15-21). In the present study, using a simplified have studied a patient with leprechaunism (leprechaun/Minn- technique to sequence DNA amplified by the polymerase 1), a genetic syndrome associated with intrauterine growth chain reaction (PCR), we have identified a nonsense mutation retardation and extreme insulin resistance. Genomic DNA in the paternal allele ofthe patient's insulin receptor gene. An from the patient was amplified by the polymerase chain opal chain termination codon (TGA) is substituted for the reaction catalyzed by Thermus aquaticus (Taq) DNA polymer- codon (CGA) encoding Arg-897 in the extracellular domain of ase, and the amplified DNA was directly sequenced. A nonsense the receptor 6 subunit. This nonsense mutation causes a mutation was identified at codon 897 in exon 14 in the paternal reduction in the level of mRNA derived from the paternal allele of the patient's insulin receptor gene. Levels of insulin allele. receptor mRNA are decreased to <10% of normal in Epstein- In addition, we have obtained indirect evidence that there Barr virus-transformed lymphoblasts and cultured skin fibro- is a cis-acting dominant mutation in the maternal allele that blasts from this patient. Thus, this nonsense mutation appears decreases the level of mRNA transcribed from that allele. to cause a decrease in the levels of insulin receptor mRNA. In However, we have not yet identified this mutation directly addition, we have obtained indirect evidence that the patient's despite having determined the nucleotide sequences of all 22 maternal allele ofthe insulin receptor gene contains a cis-acting exons including the intron-exon boundaries. Thus, the pa- dominant mutation that also decreases the level of mRNA, but tient is a compound heterozygote for two mutations in the by a different mechanism. The nucleotide sequence ofthe entire insulin receptor gene, both of which impair receptor biosyn- protein-coding domain and the sequences of the intron-exon thesis by decreasing the levels of insulin receptor mRNA. boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps else- where in the insulin receptor gene. Thus, we conclude that the METHODS patient is a compound heterozygote for two cis-acting dominant Patients. The patient leprechaun/Minn-1 had the syndrome mutations in the insulin receptor gene: (i) a nonsense mutation of leprechaunism, a congenital syndrome associated with in the paternal allele that reduces the level Qf insulin receptor extreme insulin resistance and intrauterine growth retarda- mRNA and (ii) an as yet unidentified mutation in the maternal tion (17). Leprechaun/Minn-1 had a 90% decrease in the allele that either decreases the rate oftranscription or decreases number of insulin receptors on the surface of her Epstein- the stability of the mRNA. Barr virus-transformed lymphoblasts (16, 17). Although the patient's mother is normal from a clinical point of view, there Investigations ofinborn errors ofmetabolism give insight into is a 50% decrease in the number of insulin receptors on the mechanisms of disease and also shed light upon normal surface of her circulating monocytes (3). The patient's father physiology and biochemistry. Studies of mutations in the is not available for study. We have also presented data on two genes encoding the receptors for insulin and low density insulin-resistant patients-patients A-1 (22) and RM-1 (23)- lipoprotein have helped to dissect the pathways of receptor in whom the nucleotide sequence of exon 14 (see Figs. 1 and biosynthesis and intracellular transport (1-3). In addition, 2) is identical to the published normal sequence (24-26). mutational analysis has provided insights into the functional Enzymatic Amplification of Genomic DNA (First PCR). To roles of specific structural domains in the receptors. In some amplify genomic DNA, a PCR (27, 28) was carried out in a patients, mutations have been described that impair post- total volume of 0.1 ml containing the following additions: (i) translational processing and transport of receptors to the genomic DNA template (1 tkg) digested with HindIII, (ii) plasma membrane (4-7) or alter the affinity with which the upstream and downstream oligonucleotide primers (100 pmol receptor binds its ligand (6-9). Furthermore, mutations have of each) [primers 1 and 2 were used for exon 14, and primers been described that interfere with other functions of the 9 and 10 were used for exon 3 (Table 1)], (iii) 10 ,ul of 10x receptor such as receptor-mediated endocytosis and delivery buffer [500 mM KCI/100 mM Tris-HCI, pH 8.3/15 mM of low density lipoprotein for utilization within the cell (10) or MgCI2/0.1% (wt/vol) gelatin], (iv) 16 ,ul of a solution con- transmembrane signaling by inactivating the insulin receptor taining dATP, dCTP, dGTP, and dTTP (each at a concen- tyrosine kinase activity (11-14). tration of 1.25 mM), (v) Thermus aquaticus (Taq) DNA Previously, we and others have studied a patient (lepre- polymerase (2.5 units) (Cetus/Perkin-Elmer), and (vi) water chaun/Minn-1) who is the prototype of a type of defect that to make a total volume of 0.1 ml. Amplification was carried causes insulin resistance by decreasing the level of insulin out for 30 cycles; each cycle consisted of incubations for 60 sec at 94°C for denaturation, 90 sec at 55°C for annealing, and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: National Insti- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tutes of Health, Building 10, Room 8N-250, Bethesda, MD 20892. 658 Downloaded by guest on September 30, 2021 Genetics: Kadowaki et al. Proc. Natl. Acad. Sci. USA 87 (1990) 659 Table 1. Sequences of synthetic oligonucleotides Oligonucleotide Sequence Location 1 5'-TGGACACTCCCAGATGTGCA-3' nt -60 -41; intron 13 2 5'-ACCATGCTCAGTGCTAAGCA-3' nt +56 - +37; intron 14 3 5'-GTCTGTCACGTAGAAATAG-3' nt 2850 2832; exon 14 4 5'-AAGCTCAGCCACCCTCCTTCTC-3' nt -40 -19; intron 13 5 5'-TACAGCGTGCGAATCCGGG-3' nt 2773 - 2791; exon 14; WT 6 5'-TACAGCGTGTGAATCCGGG-3' nt 2773 2791; exon 14; mutant 7 5'-GCTGCAGGCTGCGTGGGCTGTC-3' nt 2741 - 2762; exon 14 8 5'-TCGGAGACTGGCTGACTCGT-3' nt 3210 - 3191; exon 17 9 5'-ACAGGAATTGGACAAAGCCAT-3' nt -89 -69; intron 2 10 5'-AGAGCAGAGACCTCACTCATAGCCAA-3' nt +129 - +104; intron 3 11 5'-CTATCGACTGGTCCCGTAT-3' nt 518 536; exon 2 12 5'-AGTTCACACAGCGCCAGTCCTG-3' nt 895 - 874; exon 3 13 5'-CTACCTGGACGGCAGGTGT-3' nt 822 - 840; exon 3, C831 allele 14 5'-CTACCTGGATGGCAGGTGT-3' nt 822 840; exon 3, T831 allele The oligonucleotides were synthesized on a DuPont Coder 300 by using phosphoramidite chemistry and were purified by reverse-phase chromatography on NEN-Sorb Prep columns (DuPont/NEN). Nucleotides (nt) in exons are numbered according to the system of Ullrich et al. (24). Nucleotides in introns are numbered with respect to the distance from the junction with nearest exon. For example, nucleotide -40 in intron 13 is 40 nucleotides upstream from the 5' end of exon 14; nucleotide +40 in intron 14 is 40 nucleotides downstream from the 3' end of exon 14. Oligonucleotides 1, 4, 5, 6, 7, 9, 11, 13, and 14 correspond to the nucleotide sequence of the sense strand of DNA; oligonucleotides 2, 3, 8, 10, and 12 correspond to the nucleotide sequence of the antisense strand of DNA. Oligonucleotide 5 has the wild-type (WT) sequence, whereas oligonucleotide 6 has the mutant sequence with the C -* T transition indicated by the boldface T. Oligonucleotides 13 and 14 have the sequences of two polymorphic alleles: the C831 and T831 alleles (indicated in boldface type), respectively. 90 sec at 72°C for primer extension. However, at the begin- nucleotide triphosphate (8 ,uM), plus Sequenase (1.2 units)]. ning of the first cycle, DNA was denatured for 5 min; in the After incubation at 50°C for 40 min, the reaction was termi- last cycle, the 72°C incubation lasted 5 min. nated by adding 3 pu of stop solution [95% (vol/vol) form- Synthesis of Single-Stranded DNA (Second PCR). Single- amide/20 mM EDTA/0.05% bromphenol blue/0.05% xylene stranded DNA was synthesized according to the protocol cyanol FF]. The samples were analyzed by electrophoresis described above for the first PCR (27, 28), except for three through a 6% polyacrylamide/8 M urea gel. In this case, we changes. First, to amplify only one strand of DNA selec- have used sequencing primers that are different from the tively, only one ofthe two oligonucleotide primers (either the oligonucleotides used as primers in the PCR. However, upstream or the downstream primer) was included. Second, because filtration through the Centricon 100 membrane effi- amplified DNA (1-5 ng) synthesized in the first PCR was used ciently removes the oligonucleotide primers used in the PCR as template. Finally, the second PCR reaction was carried out reaction, we have found it possible to prime the sequencing for 25 rather than 30 cycles.