Endocrine Journal 2015, 62 (4), 353-362
Original
Hyperinsulinemic hypoglycemia syndrome associated with mutations in the human insulin receptor gene: Report of two cases Yohei Kuroda1), Hiromi Iwahashi1), 2), Ikuo Mineo3), Kenji Fukui1), Atsunori Fukuhara1), Ryuya Iwamoto1), Akihisa Imagawa1) and Iichiro Shimomura1) 1)
Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Department of Diabetes Care Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan 3) Diabetes Center, Toyonaka Municipal Hospital, Toyonaka, Osaka, Japan 2)
Abstract. Insulinoma and insulin or insulin receptor (IR) autoantibodies are the main causes of hyperinsulinemic hypoglycemia in adults, but the exact cause in other cases remains obscure. This study is to determine the genetic basis of hyperinsulinemic hypoglycemia in two cases without the above abnormalities. Sequence analysis of IR gene in two patients with adult-onset hyperinsulinemic hypoglycemia and their relatives were performed, and the mutant gene observed in one case was analyzed. Both cases had normal levels of fasting plasma glucose (FPG), fasting hyperinsulinemia, low insulin sensitivity, and hypoglycemia with excessive insulin secretion during oral glucose tolerance test (OGTT). Both reported adult-onset postprandial hypoglycemic symptoms. In one patient, a missense mutation (Arg256Cys) was detected in both alleles of the IR gene, and his parents had the same mutation in only one allele but no hypoglycemia. The other had a novel nonsense mutation (Trp1273X) followed by a mutation (Gln1274Lys) in one allele, and his 9-year old son had the same mutation in one allele, together with hyperinsulinemic hypoglycemia during OGTT. Overexpression experiments of the mutant gene found in Case 1 in mammalian cells showed abnormal processing of the IR protein and demonstrated reduced function of Akt/Erk phosphorylation by insulin in the cells. In two cases of hyperinsulinemic hypoglycemia in adults, we found novel mutations in IR gene considered to be linked to hypoglycemia. We propose a disease entity of adultonset hyperinsulinemic hypoglycemia syndrome associated with mutations in IR gene. Key words: Hypoglycemia, Hyperinsulinemia, Insulin receptor mutation
Hyperinsulinemic hypoglycemia in childhood is commonly caused by genetic beta-cell dysfunction. Mutations in the beta-cell sulfonylurea receptor (SUR1) gene or in the inward-rectifying potassiumchannel (Kir6.2) gene have been identified in some cases and considered to cause the phenotype [1, 2]. In contrast, hyperinsulinemic hypoglycemia in adults is most frequently caused by pancreatic beta-cell tumors, insulinoma [3, 4], and the presence of insulin or insulin receptor autoantibodies [5-7]. In addition, a few case reports were described with hyperinsulinemic hypoglycemia caused by the mutations of glucokinase Submitted Nov. 17, 2014; Accepted Feb. 4, 2015 as EJ14-0547 Released online in J-STAGE as advance publication Mar. 5, 2015
Correspondence to: Hiromi Iwahashi, M.D., Ph.D., Department of Diabetes Care Medicine, Graduate School of Medicine, Osaka University, 2-2 (B5) Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: [email protected] ©The Japan Endocrine Society
[8] and the promoter of monocarboxylate transporter 1 (MCT1) [9]. After ruling out these diseases, however, some sporadic or familial cases of hyperinsulinemic hypoglycemia, especially postprandial hypoglycemia, remain unexplained. Mutations in the IR gene are known to produce some clinical abnormalities identified as leprechaunism (Donohue syndrome), Rabson-Mendenhall, and type A insulin resistance syndrome [10-13]. Patients with these syndromes commonly exhibit insulin resistance, and have some distinct specific manifestations in each syndrome [10]. Type A insulin resistance syndrome was initially characterized as young female patients with acanthosis nigricans, ovarian hyperandrogenism, and hirsutism [14]. Indeed, some patients with leprechaunism or Rabson-Mendenhall syndrome, who seldom survive through childhood, have hyperinsulinemic hypoglycemia from infancy [11], but subjects
Kuroda et al.
354
with type A insulin resistance syndrome, except for a few cases [12, 13], rarely have hypoglycemia. In this study, we report two cases of adult-onset hyperinsulinemic hypoglycemia, who were found to have a missense mutation in both alleles or a novel nonsense mutation in one allele of the IR gene. The mutations were considered to be linked to hypoglycemia based on cosegregation of the genotype and phenotype in the family relatives and also on functional analysis of the mutant gene in one case.
materials and methods Measurements Plasma glucose concentrations were measured by the glucose oxidase method, and insulin and C-peptide levels were measured with enzyme immunoassay kits. Hemoglobin A1c (HbA1c) of National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) was calculated by the formula HbA1c (%) = 1.02 × HbA1c (Japan Diabetes Society [JDS], %) + 0.25 (%) [15]. Measurement of serum anti-insulin antibodies was outsourced to SRL, Inc. (Tokyo, Japan), and that of serum anti-insulin receptor antibodies to BML, Inc. (Tokyo, Japan) by radioreceptor assay. The latter is based on the detection of antibodies that inhibit [125I] insulin binding in human IM-9 lymphocytes expressing insulin receptors. The insulin/C-peptide molar ratio was calculated by the formula, 1 μU/mL = 6.0 pmol/L on insulin and 1 ng/mL = 0.331 nmol/L on C-peptide. Three hour 75g OGTT was performed on Case 1 and 5h OGTT was done on Case 2 after overnight fast. Insulin tolerance test (ITT) was performed after overnight fast as described previously [16, 17]. Briefly, blood samples were collected every 3 min for 15 min after intravenous injection of regular insulin (0.1 U/ kg), and insulin sensitivity index with the insulin tolerance test (KITT) was calculated from the linear slope of the plasma glucose concentration curve between 3 and 15 min. Fasting test was performed measuring plasma glucose and immunoreactive insulin levels simultaneously for up to 36 h in Case 1 and 48 h in Case 2 to rule out insulinoma. Polymerase chain reaction (PCR) direct DNA sequencing Genomic DNA was extracted from leukocytes using NucleoSpin Blood XL (Macherey-Nagel, Düren, Germany). Each exon from 1 to 22 of the IR gene
was amplified using primers reported previously [18, 19]. Ex-Taq DNA polymerase (Takara Shuzo Co., Biomedical Group, Shiga, Japan) was used to amplify exons 2–22, while AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA) was employed to amplify exon 1. Amplified PCR products were electrophoresed and the size of each product was confirmed. PCR products were sequenced after purification by QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Sequencing reaction was carried out using ABI Prism dye terminator cycle sequencing kit (Applied Biosystems) and the products were electrophoresed and analyzed on an ABI gene analyzer 1100 system according to the protocol supplied by the manufacturer (Applied Biosystems). Genetic analysis in this study was performed with the approval of the Clinical Genetics Unit in our hospital. In all patients, written informed consent was obtained for analysis of their IR gene after genetic counseling had been performed. In genetic counseling, we explained what we can learn from the genetic test, the merits and demerits of knowing the results, and that each patient has the right to decide whether they wish to know the results or not. After the tests, the results were explained to the patients and families if they wished to know what they were. Restriction Fragment Length Polymorphism (RFLP) study To check whether or not the two mutation sites found in exon 22 in Case 2 (see Results section) were located on the same allele, we performed the following RFLP study. When PCR products of exon 22 were digested with Cac8I, products without mutations ought to be digested to 355, 100, and 55 bps bands and the product with each mutation or both of the mutations should be digested to 410 and 55 bps bands. Thus, the appearance of 355 bps band should denote the presence of a normal allele without mutations, meaning that both mutations should be on the same allele. Plasmid and site directed mutagenesis The pCMV expression vector used was purchased from Clontech (Mountain View, CA). Expression plasmid for Flag-IR was constructed so that the 8 codons of the Flag epitope were fused to the C terminus of IR using PCR primers. IRR256C DNA was generated by PCR-based mutagenesis and the mutant was verified by direct sequencing. The flag epitope was also fused to IRR256C.
355
Hyperinsulinemic hypoglycemia with IR mutation
Transient transfection of HEK293A cells and insulin stimulation HEK293A cells were inoculated at a density of 5×105 cells per well in 6-well plates and incubated for 72 h at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM: 4.5 g/L glucose) (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-G and 1% streptomycin. Then, pCMV-Flag-IR and pCMV-Flag-IRR256C (2 mg/well each) were transfected into HEK293A cells with lipofectamine (Invitrogen, Groningen, The Netherlands). Forty-eight hours later, transfected cells were serum-starved for 12 h, followed by stimulation with 0.1, 1.0 and 10 nM bovine insulin for 5 min. The reaction was stopped immediately by freezing the cells with liquid nitrogen. Western blotting Cells were lysed in RIPA lysis buffer (Millipore, Billerica, MA), containing a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations in the lysates were determined by bicinchoninic acid assay. The lysates were then added with SDS-PAGE sample buffer and 10-18 mg proteins were subjected to 10% SDS-PAGE and western blot analysis using anti-flag antibody (Sigma-Aldrich), anti-insulin receptor antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-Erk antibody, and anti-phosphorylated Erk antibody (Cell Signaling Technology, Beverly, MA). The immune complex was detected using the ECL Advance Western Blot Detection System (GE Healthcare, Buckinghamshire, UK). Signal intensity was quantitated by ImageJ (distributed by NIH). Statistical analysis Data were expressed as mean±SEM. Differences between groups were examined using unpaired Student’s t test. Differences were considered significant when p value was
Original
Hyperinsulinemic hypoglycemia syndrome associated with mutations in the human insulin receptor gene: Report of two cases Yohei Kuroda1), Hiromi Iwahashi1), 2), Ikuo Mineo3), Kenji Fukui1), Atsunori Fukuhara1), Ryuya Iwamoto1), Akihisa Imagawa1) and Iichiro Shimomura1) 1)
Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Department of Diabetes Care Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan 3) Diabetes Center, Toyonaka Municipal Hospital, Toyonaka, Osaka, Japan 2)
Abstract. Insulinoma and insulin or insulin receptor (IR) autoantibodies are the main causes of hyperinsulinemic hypoglycemia in adults, but the exact cause in other cases remains obscure. This study is to determine the genetic basis of hyperinsulinemic hypoglycemia in two cases without the above abnormalities. Sequence analysis of IR gene in two patients with adult-onset hyperinsulinemic hypoglycemia and their relatives were performed, and the mutant gene observed in one case was analyzed. Both cases had normal levels of fasting plasma glucose (FPG), fasting hyperinsulinemia, low insulin sensitivity, and hypoglycemia with excessive insulin secretion during oral glucose tolerance test (OGTT). Both reported adult-onset postprandial hypoglycemic symptoms. In one patient, a missense mutation (Arg256Cys) was detected in both alleles of the IR gene, and his parents had the same mutation in only one allele but no hypoglycemia. The other had a novel nonsense mutation (Trp1273X) followed by a mutation (Gln1274Lys) in one allele, and his 9-year old son had the same mutation in one allele, together with hyperinsulinemic hypoglycemia during OGTT. Overexpression experiments of the mutant gene found in Case 1 in mammalian cells showed abnormal processing of the IR protein and demonstrated reduced function of Akt/Erk phosphorylation by insulin in the cells. In two cases of hyperinsulinemic hypoglycemia in adults, we found novel mutations in IR gene considered to be linked to hypoglycemia. We propose a disease entity of adultonset hyperinsulinemic hypoglycemia syndrome associated with mutations in IR gene. Key words: Hypoglycemia, Hyperinsulinemia, Insulin receptor mutation
Hyperinsulinemic hypoglycemia in childhood is commonly caused by genetic beta-cell dysfunction. Mutations in the beta-cell sulfonylurea receptor (SUR1) gene or in the inward-rectifying potassiumchannel (Kir6.2) gene have been identified in some cases and considered to cause the phenotype [1, 2]. In contrast, hyperinsulinemic hypoglycemia in adults is most frequently caused by pancreatic beta-cell tumors, insulinoma [3, 4], and the presence of insulin or insulin receptor autoantibodies [5-7]. In addition, a few case reports were described with hyperinsulinemic hypoglycemia caused by the mutations of glucokinase Submitted Nov. 17, 2014; Accepted Feb. 4, 2015 as EJ14-0547 Released online in J-STAGE as advance publication Mar. 5, 2015
Correspondence to: Hiromi Iwahashi, M.D., Ph.D., Department of Diabetes Care Medicine, Graduate School of Medicine, Osaka University, 2-2 (B5) Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: [email protected] ©The Japan Endocrine Society
[8] and the promoter of monocarboxylate transporter 1 (MCT1) [9]. After ruling out these diseases, however, some sporadic or familial cases of hyperinsulinemic hypoglycemia, especially postprandial hypoglycemia, remain unexplained. Mutations in the IR gene are known to produce some clinical abnormalities identified as leprechaunism (Donohue syndrome), Rabson-Mendenhall, and type A insulin resistance syndrome [10-13]. Patients with these syndromes commonly exhibit insulin resistance, and have some distinct specific manifestations in each syndrome [10]. Type A insulin resistance syndrome was initially characterized as young female patients with acanthosis nigricans, ovarian hyperandrogenism, and hirsutism [14]. Indeed, some patients with leprechaunism or Rabson-Mendenhall syndrome, who seldom survive through childhood, have hyperinsulinemic hypoglycemia from infancy [11], but subjects
Kuroda et al.
354
with type A insulin resistance syndrome, except for a few cases [12, 13], rarely have hypoglycemia. In this study, we report two cases of adult-onset hyperinsulinemic hypoglycemia, who were found to have a missense mutation in both alleles or a novel nonsense mutation in one allele of the IR gene. The mutations were considered to be linked to hypoglycemia based on cosegregation of the genotype and phenotype in the family relatives and also on functional analysis of the mutant gene in one case.
materials and methods Measurements Plasma glucose concentrations were measured by the glucose oxidase method, and insulin and C-peptide levels were measured with enzyme immunoassay kits. Hemoglobin A1c (HbA1c) of National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) was calculated by the formula HbA1c (%) = 1.02 × HbA1c (Japan Diabetes Society [JDS], %) + 0.25 (%) [15]. Measurement of serum anti-insulin antibodies was outsourced to SRL, Inc. (Tokyo, Japan), and that of serum anti-insulin receptor antibodies to BML, Inc. (Tokyo, Japan) by radioreceptor assay. The latter is based on the detection of antibodies that inhibit [125I] insulin binding in human IM-9 lymphocytes expressing insulin receptors. The insulin/C-peptide molar ratio was calculated by the formula, 1 μU/mL = 6.0 pmol/L on insulin and 1 ng/mL = 0.331 nmol/L on C-peptide. Three hour 75g OGTT was performed on Case 1 and 5h OGTT was done on Case 2 after overnight fast. Insulin tolerance test (ITT) was performed after overnight fast as described previously [16, 17]. Briefly, blood samples were collected every 3 min for 15 min after intravenous injection of regular insulin (0.1 U/ kg), and insulin sensitivity index with the insulin tolerance test (KITT) was calculated from the linear slope of the plasma glucose concentration curve between 3 and 15 min. Fasting test was performed measuring plasma glucose and immunoreactive insulin levels simultaneously for up to 36 h in Case 1 and 48 h in Case 2 to rule out insulinoma. Polymerase chain reaction (PCR) direct DNA sequencing Genomic DNA was extracted from leukocytes using NucleoSpin Blood XL (Macherey-Nagel, Düren, Germany). Each exon from 1 to 22 of the IR gene
was amplified using primers reported previously [18, 19]. Ex-Taq DNA polymerase (Takara Shuzo Co., Biomedical Group, Shiga, Japan) was used to amplify exons 2–22, while AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA) was employed to amplify exon 1. Amplified PCR products were electrophoresed and the size of each product was confirmed. PCR products were sequenced after purification by QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Sequencing reaction was carried out using ABI Prism dye terminator cycle sequencing kit (Applied Biosystems) and the products were electrophoresed and analyzed on an ABI gene analyzer 1100 system according to the protocol supplied by the manufacturer (Applied Biosystems). Genetic analysis in this study was performed with the approval of the Clinical Genetics Unit in our hospital. In all patients, written informed consent was obtained for analysis of their IR gene after genetic counseling had been performed. In genetic counseling, we explained what we can learn from the genetic test, the merits and demerits of knowing the results, and that each patient has the right to decide whether they wish to know the results or not. After the tests, the results were explained to the patients and families if they wished to know what they were. Restriction Fragment Length Polymorphism (RFLP) study To check whether or not the two mutation sites found in exon 22 in Case 2 (see Results section) were located on the same allele, we performed the following RFLP study. When PCR products of exon 22 were digested with Cac8I, products without mutations ought to be digested to 355, 100, and 55 bps bands and the product with each mutation or both of the mutations should be digested to 410 and 55 bps bands. Thus, the appearance of 355 bps band should denote the presence of a normal allele without mutations, meaning that both mutations should be on the same allele. Plasmid and site directed mutagenesis The pCMV expression vector used was purchased from Clontech (Mountain View, CA). Expression plasmid for Flag-IR was constructed so that the 8 codons of the Flag epitope were fused to the C terminus of IR using PCR primers. IRR256C DNA was generated by PCR-based mutagenesis and the mutant was verified by direct sequencing. The flag epitope was also fused to IRR256C.
355
Hyperinsulinemic hypoglycemia with IR mutation
Transient transfection of HEK293A cells and insulin stimulation HEK293A cells were inoculated at a density of 5×105 cells per well in 6-well plates and incubated for 72 h at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM: 4.5 g/L glucose) (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-G and 1% streptomycin. Then, pCMV-Flag-IR and pCMV-Flag-IRR256C (2 mg/well each) were transfected into HEK293A cells with lipofectamine (Invitrogen, Groningen, The Netherlands). Forty-eight hours later, transfected cells were serum-starved for 12 h, followed by stimulation with 0.1, 1.0 and 10 nM bovine insulin for 5 min. The reaction was stopped immediately by freezing the cells with liquid nitrogen. Western blotting Cells were lysed in RIPA lysis buffer (Millipore, Billerica, MA), containing a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations in the lysates were determined by bicinchoninic acid assay. The lysates were then added with SDS-PAGE sample buffer and 10-18 mg proteins were subjected to 10% SDS-PAGE and western blot analysis using anti-flag antibody (Sigma-Aldrich), anti-insulin receptor antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-Erk antibody, and anti-phosphorylated Erk antibody (Cell Signaling Technology, Beverly, MA). The immune complex was detected using the ECL Advance Western Blot Detection System (GE Healthcare, Buckinghamshire, UK). Signal intensity was quantitated by ImageJ (distributed by NIH). Statistical analysis Data were expressed as mean±SEM. Differences between groups were examined using unpaired Student’s t test. Differences were considered significant when p value was
