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. The product of the second PCR reaction with the oligonucleotide primers used in the first was extracted with chloroform (0.1 ml) and diluted to 2 ml PCR. with water. Single-stranded DNA was separated from the Synthesis of cDNA and Amplification by PCR. cDNA was oligonucleotides and deoxynucleotide triphosphates by fil- synthesized and amplified according to the method of Froh- tration through a Centricon 100 membrane according to the man et al. (29). Total cellular RNA (2.5-10 gg) was heated at manufacturer's instructions (Amicon). The retentate (45,l) 70°C for 10 min, cooled slowly to 42°C, and then reverse- was dried in a Speed Vac concentrator (Savant) and dissolved transcribed at 42°C for 1 hr in a 50-,l reaction mixture in 5 Al of water immediately before use in the DNA sequenc- containing 50 mM Tris HCl (pH 8.8), 100 mM NaCl, 6 mM ing reaction. MgCl2, 10 mM dithiothreitol, deoxyribonucleotide triphos- Labeling of the Primer for the DNA Sequencing Reaction. phates (each at 1 mM), 40,000 units of RNasin (Promega Oligonucleotides 3 and 4 (100 pmol) were phosphorylated for Biotec), 0.5 ,uM oligonucleotide primer, and 10 units ofavian 30 min at 37°C by T4 polynucleotide kinase (13.5 units; myeloblastosis virus reverse transcriptase (Boehringer Mann- Boehringer Mannheim) in a total volume of 42 ,ul of solution heim). The reaction mixture was diluted to 2 ml with water containing MgCI2 (10 mM), dithiothreitol (10 mM), glycine and filtered through a Centricon 100 ultrafiltration device. (50 mM, pH 9.2), and [y-32P]ATP (55 ,Ci; 3000 Ci/mmol; 1 The retentate (45 ,1) containing cDNA was used as template Ci = 37 GBq; New England Nuclear). The 32P-labeled for amplification as described above for the first PCR. oligonucleotide was purified by chromatography through a Sephadex G-25 Quick Spin column (Boehringer Mannheim). The eluate (42 ,1) was dried in the Speed Vac concentrator RESULTS AND DISCUSSION and dissolved in 12 ,ul of water immediately before use in the Identification of a Nonsense Mutation. Levels of insulin sequencing reaction. receptor mRNA in Epstein-Barr virus-transformed lympho- Sequencing of Amplified Single-Stranded DNA. Oligonucle- blasts (16) and cultured skin fibroblasts (21) from the patient otides 3 and 4 were used as primers with the sense and leprechaun/Minn-1 are decreased to <10% of normal. The antisense strands, respectively, as template. Amplified sin- decrease in the level of insulin receptor mRNA leads to a gle-stranded DNA (5 ,ul) was mixed with 3 ,l of 32P-labeled decrease in the rate of receptor biosynthesis and, conse- primer (-25 pmol) and 2 Al of5x polymerase buffer (250 mM quently, a decrease in the number of receptors on the cell Tris HCI, pH 8.3/40 mM MgCl2/150 mM KCI/50 mM di- surface (17, 20). In principle, several different types of thiothreitol) (28). After annealing for 10 min at 95°C, 2.5 ,1 of mutations can cause a decrease in the level ofmRNA (30-34). the mixture was added to each of four tubes, followed by 2 However, the important regulatory regions of the insulin ,l of the appropriate dideoxy terminator/Sequenase mix receptor gene have not yet been mapped completely. Thus, [containing all four deoxyribonucleotide triphosphates (each it is difficult to fully evaluate the possibility that there might at a concentration of 80 ,uM), the appropriate dideoxyribo- be a mutation in the regulatory regions of the gene. Never- Downloaded by guest on September 30, 2021 660 Genetics: Kadowaki et al. Proc. Natl. Acad. Sci. USA 87 (1990) theless, we determined the nucleotide sequence of 876 base pairs (bp) of5' flanking DNA in six independent clones ofthe patient's genomic DNA (data not shown). Because the nucle- Wild Type otide sequence of the 5' flanking DNA is identical to pub- Probe lished sequences (26), we searched elsewhere in the gene for the presence of a mutation. We employed the PCR catalyzed by Taq DNA polymerase A B C D (27) to amplify the individual exons of the insulin receptor gene, a gene composed of 22 exons spanning more than 150 kilobases of DNA (26). Primers were chosen in the interven- Mutant a. ing sequences (26) flanking each exon so that it would be Probe possible to determine the complete sequence of the exons as well as the intron-exon junctions containing consensus se- quences required for RNA splicing. A nonsense mutation was identified at codon 897 in exon 14 (26): an arginine codon FIG. 2. Allele-specific oligonucleotide hybridization of amplified (CGA) was converted into the opal stop codon (TGA). The genomic DNA. Genomic DNA including exon 14 of the insulin patient was heterozygous for this mutation as judged by the receptor gene was amplified as described in Fig. 1. One-tenth of the fact that bands corresponding to both C and T could be amplified double-stranded DNA (-50 ng of DNA) was analyzed by detected in the sense strand at nucleotide 2782 in the se- electrophoresis through a 1.8% agarose gel and transferred to nylon membranes (Schleicher & Schuell). The DNA blots were hybridized quencing ladder (Fig. 1). This mutation was confirmed by in buffer containing 5x SSPE (0.9 M NaCl/50 mM sodium phos- amplifying genomic DNA a second time an4 determining the phate/5 mM EDTA, pH 7.7), 5x Denhardt's solution (0.02% poly- sequence of the antisense strand of DNA (data not shown). vinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin), and In contrast, when we determined the nucleotide sequences of 0.1% SDS for6 hr at 37°C with 32P-labeled synthetic oligonucleotides DNA from control subjects (Fig. 1) or from the patient's (1 x 106 cpm/ml). (Upper) Hybridization with the wild-type oligo- mother (data not shown), only C was detected at nucleotide nucleotide (oligonucleotide 5, Table 1). (Lower) Hybridization with 2782. the mutant oligonucleotide (oligonucleotide 6, Table 1). The blots were then washed twice at room temperature in 2x SSPE/0.1% SDS, Moreover, amplified DNA was hybridized with oligonu- followed by a 15-min wash at 63°C in 5x SSPE/0.1% SDS (5, 11). cleotides specific for either the wild-type sequence or the Lane A, patient A-1 (control); lane B, patient RM-1 (control); lane C, mutant sequence (Fig. 2). The oligonucleotide specific for the leprechaun/Minn-1; lane D, mother of leprechaun/Minn-1. mutant sequence hybridized to DNA from the patient but not from the patient's mother or from two unrelated individuals mal levels of mRNA. In fact, we have previously identified (Fig. 2 Lower). In contrast, the oligonucleotide specific for a nonsense mutation at codon 672 in exon 10 of the insulin the wild-type sequence hybridized to DNA from all four receptor gene, which did not decrease the level of insulin samples of amplified DNA (Fig. 2 Upper). These studies receptor mRNA (8). Thus, it was of interest to determine the support the conclusion that the patient is heterozygous for level of mRNA transcribed from each allele of the patient's this nonsense mutation. Furthermore, the mutation was not insulin receptor gene. To address this question, we synthe- inherited from the patient's mother. Most likely the mutation sized single-stranded insulin receptor cDNA by using reverse was inherited from the patient's father. However, because we transcriptase. Thereafter, a 470-bp fragment of cDNA span- could not study the patient's father, we cannot entirely rule ning the region of the nonsense mutation (nucleotides 2741- out the possibility that the mutation arose de novo. 3210) was amplified by using PCR. Hybridization with allele- Expression of Two Alleles of the Insulin Receptor Gene. specific oligonucleotide probes was used to determine the Most nonsense mutations are associated with a decrease in level of transcripts from each allele (Fig. 3). When amplified the level of mRNA (30, 33, 35). Nevertheless, it is clear that cDNA from a control subject was analyzed, only the wild- nonsense mutations can sometimes be associated with nor- type sequence was detected. However, with amplified cDNA Control Lep/Minn-1 Wild Typ-)e Mutant G ATC G AT C Genomic DNA Em

* .. L C L C rC -T/C

la. rn RNA ; ,M__._lw L C L C Val - Arg897 - lie FIG. 3. Allele-specific oligonucleotide hybridization of amplified Normal: 5'- G T G - ,' G A - A T C - 3' cDNA from leprechaun/Minn-1. Genomic DNA including exon 14 of 3!- C A C - G C T - T A G - 5' the insulin receptor gene was amplified as described in Fig. 1. A 470-bp fragment of cDNA was amplified from total cellular RNA (5 ,ug) by reverse transcription followed by PCR with oligonucleotide primers 7 and 8. Thereafter, amplified genomic DNA (50 ng of DNA) Val - STOP and amplified cDNA (20 ng; labeled mRNA) were analyzed by Mutant: 5' - G T G - .T) G A A T C - 3' Southern blotting as described in the legend to Fig. 2. The blots were 3' - C A C - A C T - T A G - 5' hybridized with the wild-type oligonucleotide (oligonucleotide 5, Table 1) or with the mutant oligonucleotide (oligonucleotide 6, Table FIG. 1. Partial nucleotide sequence of the sense strand of exon 14 1). Amplified genomic DNA and cDNA were derived either from of the insulin receptor gene. The 276-bp fragment of genomic DNA leprechaun/Minn-1 (lanes L) or a control subject (patient A-1; lanes including exon 14 was amplified by PCR using primers 1 and 2. Then C) whose genomic DNA had been sequenced and determined not to the antisense strand of DNA was amplified by using primer 2, and the have a nonsense mutation in exon 14. The autoradiographs were sequencing reaction was carried out by using primer 4. exposed for 8 hr (amplified genomic DNA) or 2 hr (amplified cDNA). Downloaded by guest on September 30, 2021 Genetics: Kadowaki et al. Proc. Natl. Acad. Sci. USA 87 (1990) 661

from leprechaun/Minn-1, both the mutant and the wild-type Table 2. Levels of transcripts of two alleles of the insulin sequences were detected (Fig. 3). Furthermore, the intensi- receptor gene ties of the bands observed with both probes were roughly Ratio equal, just as was observed with amplified genomic DNA Template C831 allele T831 allele (C831/T831) (Fig. 3). Because the two alleles are present in a 1:1 ratio in Genomic DNA we the mRNAs transcribed genomic DNA, conclude that Mother 169 171 0.99 from both alleles must be present in a ratio of -1: 1 as well. Leprechaun 246 152 1.62 Inasmuch as the level of insulin receptor mRNA is reduced Control 436 by 90% in cells from leprechaun/Minn-1 (16, 21), it follows cDNA (mRNA) that the levels of transcripts from both alleles are markedly Mother 2862 298 9.6 decreased. Leprechaun 613 264 2.32 Why is the patient's maternal allele expressed at a level as Control 2982 low as the paternal allele with the nonsense mutation? We have obtained indirect evidence suggesting that there is a The blots presented in Fig. 4 were scanned with a beta scanner cis-acting mutation in this allele even though we have not yet (Ambis, San Diego) to quantitate the radioactivity (cpm) associated succeeded in identifying the mutation directly. This conclu- with each band. sion is based upon studies of the levels of transcripts derived both alleles would be reduced symmetrically. However, from the two alleles of the insulin receptor gene in cells from there is a selective reduction in the level of the transcript the patient's mother. We amplified insulin receptor cDNA derived from the T831 allele. Thus we conclude that there is from cells of leprechaun/Minn-1, the mother, and a control a cis-acting dominant mutation in this T831 allele. Impor- subject (Fig. 4). Both leprechaun/Minn-1 and the mother had tantly, it is this T831 allele that leprechaun/Minn-1 inherited silent polymorphisms at codon 234 [Asp (GAC) vs. Asp from her mother. We have analyzed the entire protein-coding (GAT)] in exon 3 (Fig. 4). In leprechaun/Minn-1, the allele with the nonsense mutation has C at nucleotide 831 (C831 sequence of the maternal T831 allele as well as the sequence allele) (data not shown). Therefore, the presence of T at of the intron-exon boundaries for all 22 exons (data not nucleotide 831 provides a marker for the allele that lepre- shown) but have not yet detected the presumed mutation. chaun/Minn-1 inherited from the mother (T831 allele). As Because the mutation might be located anywhere in the gene, expected, when amplified cDNA from a control subject who which spans in excess of 150 kilobase pairs ofgenomic DNA was homozygous for C at nucleotide 831 was analyzed, only (26), it may be difficult to identify this mutation directly. In the sequence containing C at nucleotide 831 was detected principle, the mutation might either decrease the rate at (Fig. 4). However, with amplified cDNA from leprechaun/ which insulin receptor mRNA is synthesized or, alterna- Minn-1 and the mother, sequences containing both C and T tively, decrease the stability of insulin receptor mRNA. were detected (Fig. 4). In leprechaun/Minn-1, both tran- Allele with Nonsense Mutation Encodes a Truncated Recep- scripts were present in approximately equally low levels as tor Protein. The CGA (arginine) codon has previously been described above (Fig. 3). However, in the patient's mother, identified as a "hot spot" for mutations (36)-presumably' the level of the transcript from the C831 allele was approxi- because the CpG sequence is a substrate for methylation and mately 10-fold higher than the level of the transcript from the the presence of 5-methylcytosine predisposes toward errors T831 allele (Table 2 and Fig. 4). As a methodological control, in DNA replication, which convert the C G base pair to a T-A we confirmed that the alleles containing C and T were present base pair (36, 37). Because the nonsense mutation at codon in a ratio of =1:1 in amplified genomic DNA (Table 2 and Fig. 897 causes the deletion of the C-terminal three-fourths of the 4 Upper). If the low level of expression of the mother's T831 receptor P subunit including the transmembrane anchor and allele were caused by a trans-acting dominant mutation, then the tyrosine kinase domain, it is unlikely that this truncated one would predict that the levels of transcripts derived from receptor would be functional or that it would be located on the cell surface (8, 11, 38). In any case, because the nonsense Wild Type (C) Mutant (T) mutation at codon 897 is associated with a decrease in the level of insulin receptor mRNA, very few molecules of the Genomic DNA _ truncated receptor are synthesized. Consistent with this c M C L M conclusion, we have not reproducibly detected the presence of a truncated form of the receptor in the patient's cultured mRNA - > lymphoblasts (16). However, the patient's cultured lympho- blasts do possess a small number of insulin receptors on their C L M C L M cell surface (17). Furthermore, studies of receptor biosyn- thesis demonstrate that these receptors are derived from a FIG. 4. Allele-specific oligonucleotide hybridization of amplified proreceptor with normal apparent molecular mass of 190 kDa cDNA from the mother of leprechaun/Minn-1. Genomic DNA (16). Presumably, this normal receptor is encoded by the including exon 4 of the insulin receptor gene was amplified by using patient's maternal allele of the insulin receptor gene. oligonucleotides 9 and 10 as primers. A 378-bp fragment of cDNA Direct Sequencing of Single-Stranded Amplified Genomic was amplified from total cellular RNA (2.5 ,ug) by reverse transcrip- tion followed by PCR with oligonucleotide primers 11 and 12. DNA. The use of Taq DNA polymerase to amplify genomic Thereafter, amplified genomic DNA ("20 ng of DNA) and amplified DNA by PCR has enormously increased the ability to identify cDNA (approximately one-tenth of the total product obtained; mutations causing disease. The nucleotide sequences of labeled mRNA) were analyzed by agarose gel electrophoresis fol- DNA fragments amplified by PCR can be determined either lowed by Southern blotting. Blots were hybridized with the oligo- by cloning the fragments into appropriate vectors (39) or by nucleotide specific for either the C831-allele (wild-type probe; oligo- direct sequencing (28, 40). The cloning strategy is labor- nucleotide 13, Table 1) or the T831-allele (mutant probe; oligonucle- intensive and time-consuming. Furthermore, ifthe individual otide 13, Table 1) and washed as described in the legend to Fig. 2 for is heterozygous, as in the present patient, it may be necessary 13 min at 61'C. Amplified genomic DNA and cDNA were derived to sequence multiple clones in order to determine the se- either from leprechaun/Minn-1 (lanes L), her mother (lanes M), or a control subject whose genomic DNA had been sequenced and was quences ofboth alleles. In fact, unless it is possible to identify determined to be homozygous for C at nucleotide 831 (patient RM-1; polymorphic sequences to differentiate the two alleles, it may lane C). The autoradiographs were exposed for 27 hr (amplified not be possible to be certain that the sequences ofboth alleles genomic DNA) or 9 hr (amplified cDNA). have been obtained. The work involved in determining the Downloaded by guest on September 30, 2021 662 Genetics: Kadowaki et al. Proc. Natl. Acad. Sci. USA 87 (1990)

nucleotide sequence has been greatly reduced by the ability 12. Grunberger, G., Zick, Y. & Gorden, P. (1984) Science 223, to directly sequence amplified DNA. We have made two 932-934. simplifications in the method developed by Gibbs et al. (28) 13. Taira, M., Taira, M., Hashimoto, N., Shimada, F., Suzuki, Y., to sequence amplified DNA: (i) elimination of the need to Kanatsuka, A., Nakamura, F., Ebina, Y., Tatibana, M., the double-stranded DNA in the first Makino, H. & Yoshida, S. (1989) Science 245, 63-66. purify amplified poly- 14. Moller, D. E. & Flier, J. S. (1988) N. Engl. J. Med. 319, merase chain reaction and (ii) use of ultrafiltration to remove 1526-1529. the oligonucleotides used as primers for the PCR, thereby 15. Hedo, J. A., Moncada, V. Y. & Taylor, S. I. (1985) J. Clin. enabling the use of the same oligonucleotides to prime the Invest. 76, 2355-2361. sequencing reaction and eliminating the need to synthesize 16. Ojamaa, K., Hedo, J. A., Roberts, C. T., Jr., Moncada, V. Y., additional primers. Gorden, P., Ullrich, A. & Taylor, S. 1. (1988) Mol. Endocrinol. In light of the central role of insulin resistance in predis- 2, 242-247. posing to development of non-insulin-dependent diabetes 17. Taylor, S. I., Samuels, B., Roth, J., Kasuga, M., Hedo, J. A., mellitus (NIDDM), it is reasonable to inquire whether pa- Gorden, P., Brasel, D. E., Pokora, T. & Engel, R. R. (1982) J. tients with NIDDM may have mutations in the insulin re- Clin. Endocrinol. Metab. 54, 919-930. the effort 18. Podskalny, J. M. & Kahn, C. R. (1982) J. Clin. Endocrinol. ceptor gene. Because of large required previously, Metab. 54, 261-268. it has not been feasible to study enough patients to determine 19. Endo, F., Nagata, N., Priest, J. H., Longo, N. & Elsas, L. J., the prevalence of mutations in the insulin receptor gene in 11 (1987) Am. J. Hum. Genet. 41, 402-417. patients with NIDDM. However, as a consequence of recent 20. Reddy, S. S-K., Lauris, V. & Kahn, C. R. (1988) J. Clin. technological advances, it is now feasible to undertake a Invest. 82, 1359-1365. study to evaluate this possibility. 21. Muller-Wieland, D., Taub, R., Tewari, D. S., Kriauciunas, K. M., Reddy, S. S-K. & Kahn, C. R. (1989) Diabetes 38, Note Added in Proof. We have identified nonsense mutations in the 31-38. insulin receptor genes of two additional patients, a patient with type 22. Kahn,C. R., Flier,J. S., Bar, R. S., Archer,J. A.,Gorden, P., A extreme insulin resistance (A-1) and a patient with the Rabson- Martin, M. M. & Roth, J. (1976) N. Engl. J. Med. 294,739-745. Mendenhall syndrome (RM-1). Both of these nonsense mutations 23. Moncada,V. Y.,Hedo,J. A., Serrano-Rios, M. & Taylor, S. 1. have cis-acting dominant effects to reduce mRNA levels. (1986) Diabetes 35, 802-807. 24. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, We are grateful to Drs. S. Seino and G. I. Bell (Howard Hughes L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Medical Institute, University of Chicago) forgenerously providing us Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., with partial nucleotide sequences for the introns of the insulin Rosen, 0. M. & Ramachandran, J. (1985) Nature (London) 313, receptor gene and guidance in designing primers for amplification of 756-761. the DNA. We also thank Dr. Richard A. Gibbs (Baylor College of 25. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, Medicine) and Dr. Hiroyuki Aburatani (Massachusetts Institute of E., Ou, J., Masiarz, F., Kan, Y. M., Goldfine, I. D., Roth, Technology) for helpful discussions in developing our modified R. A. & Rutter, W. J. (1985) Cell 40, 747-758. technique for direct sequencing. We gratefully acknowledge many 26. Seino, S., Seino, M., Nishi, S. & Bell, G. I. (1989) Proc. Natl. colleagues at the National Institutes of Health for helpful discussions Acad. Sci. USA 86, 114-118. and assistance in carrying out this work: Domenico Accili, Alessan- 27. Saiki, R. K., Gelfand, D. H., Stoffel, S., Sharf, S. J., Higuchi, dro Cama, Phillip Gorden, Maxine Lesniak, Bernice Marcus- R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science Samuels, Catherine McKeon, Victoria Moncada, Maria Rojeski, 239, 487-491. Jesse Roth, and Alan Shuldiner. Finally, H.K. was supported by a 28. Gibbs, R. A., Nguyen, P.-N., McBride, L. J., Koepf, S. M. & Mentor-Based Fellowship provided by the American Diabetes As- Caskey, C. T. (1989) Proc. Natl. Acad. Sci. USA 86, 1919- sociation. 1923. 29. Frohman, M. A., Dush, M. K. & Martin, G. R. (1988) Proc. 1. Brown, M. S. & Goldstein, J. L. (1986) Science 232, 34-47. Natl. Acad. Sci. USA 85, 8998-9002. 2. Taylor, S. I. (1987) Clin. Res. 35, 459-467. 30. Urlaub, G., Mitchell, P. J., Ciudad, C. J. & Chasinm L. A. 3. Taylor, S. I. (1985) Diabetes/Metab. Rev. 1, 171-202. (1989) Mol. Cell. Biol. 9, 2868-2880. 4. 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