Structural Basis and Genotype–Phenotype Correlations ofINSR Mutations Causing Severe Insulin ResistanceJun Hosoe,1 Hiroko Kadowaki,2 Fuyuki Miya,1,3,4,5 Katsuya Aizu,6 Tomoyuki Kawamura,7 Ichiro Miyata,8

Kenichi Satomura,9 Takeru Ito,10 Kazuo Hara,11 Masaki Tanaka,12 Hiroyuki Ishiura,12 Shoji Tsuji,12

Ken Suzuki,1 Minaka Takakura,1 Keith A. Boroevich,4 Tatsuhiko Tsunoda,3,4,5 Toshimasa Yamauchi,1

Nobuhiro Shojima,1 and Takashi Kadowaki1

Diabetes 2017;66:2713–2723 | https://doi.org/10.2337/db17-0301

The insulin receptor (INSR) gene was analyzed in four pa-tients with severe insulin resistance, revealing five novelmutations and a deletion that removed exon 2. A patient withDonohue syndrome (DS) had a novel p.V657Fmutation in thesecond fibronectin type III domain (FnIII-2), which containsthe a-b cleavage site and part of the insulin-binding site.The mutant INSR was expressed in Chinese hamster ovarycells, revealing that it reduced insulin proreceptor processingand impaired activation of downstream signaling cascades.Using online databases, we analyzed 82 INSRmissensemu-tations and demonstrated that mutations causing DS weremore frequently located in the FnIII domains than those caus-ing the milder type A insulin resistance (P = 0.016). In silicostructural analysis revealed that missense mutations pre-dicted to severely impair hydrophobic core formation andstability of the FnIII domains all caused DS, whereas thosepredicted to produce localized destabilization and to notaffect folding of the FnIII domains all caused the less se-vere Rabson-Mendenhall syndrome. These results sug-gest the importance of the FnIII domains, provide insightinto the molecular mechanism of severe insulin resis-tance, will aid early diagnosis, and will provide potentialnovel targets for treating extreme insulin resistance.

Mutations of the insulin receptor (INSR) gene result in ex-treme insulin resistance and dysglycemia (1), leading to sev-eral syndromes with various abnormal phenotypes thatdepend on the severity of INSR dysfunction. Patients withDonohue syndrome (DS), formerly known as leprechaunism,have the most severe insulin resistance (2,3) and patientswith type A insulin resistance syndrome (type A-IR) displaysomewhat less severe manifestations (4,5), whereas Rabson-Mendenhall syndrome (RMS) represents an intermediate con-dition (6,7). Patients with type A-IR can live beyond middleage and present with hypertrichosis, acanthosis nigricans,and female hyperandrogenism. Patients with RMS generallysurvive into childhood or early adulthood and their charac-teristic symptoms are hypertrichosis, dysplastic dentition,and coarse and dysmorphic facial features. Patients with DSseldom live beyond infancy. They have dysmorphic facial fea-tures (so-called elfin appearance) and little subcutaneous fat.

INSR is a gene consisting of 22 exons and 21 introns.The proreceptor undergoes glycosylation and dimerization,followed by translocation to the Golgi apparatus and thenprocessing of the dimer to yield a heterotetramer composedof two a-subunits and two b-subunits (8). Although there

1Department of Diabetes and Metabolic Diseases, Graduate School of Medicine,University of Tokyo, Tokyo, Japan2Department of Pediatrics, Sanno Hospital, Tokyo, Japan3Department of Medical Science Mathematics, Medical Research Institute, TokyoMedical and Dental University, Tokyo, Japan4Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Med-ical Sciences, Yokohama, Japan5Core Research for Evolutional Science and Technology, Japan Science andTechnology Agency, Tokyo, Japan6Division of Endocrinology and Metabolism, Saitama Children’s Medical Center,Saitama, Japan7Department of Pediatrics, Osaka City University Graduate School of Medicine,Osaka, Japan8Department of Pediatrics, Jikei University School of Medicine, Tokyo, Japan9Department of Pediatric Nephrology and Metabolism, Osaka Women’s andChildren’s Hospital, Osaka, Japan10Department of Pediatrics, Atsugi City Hospital, Kanagawa, Japan

11Department of Endocrinology and Metabolism, Saitama Medical Center, JichiMedical University, Saitama, Japan12Department of Neurology, Graduate School of Medicine, University of Tokyo,Tokyo, Japan

Corresponding authors: Takashi Kadowaki, kadowaki-3im@h.u-tokyo.ac.jp, andNobuhiro Shojima, nshojima-tky@umin.ac.jp.

Received 9 March 2017 and accepted 24 July 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0301/-/DC1.

J.H., H.K., and F.M. contributed equally to this work.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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are no clear genotype–phenotype correlations for INSR mu-tations causing severe insulin resistance, it has been suggestedthat homozygous or compound heterozygous mutations ofthe a-subunit cause more severe syndromes (DS and RMS),whereas heterozygous b-subunit mutations lead to milderinsulin resistance (9,10). Longo et al. (11) reported thatmissense mutations causing the most severe manifestationsaffected the extracellular portion of INSR and markedlyreduced binding of insulin.

Some researchers have performed structural analysis ofmutations of various proteins other than INSR to predictclinical manifestations and establish structure–phenotypecorrelations (12–14), and a structural bioinformatics ap-proach should be useful for predicting the diverse pheno-types caused by monogenic mutations. However, there is noclear evidence of structure–phenotype correlations in pa-tients with severe insulin resistance due to INSR mutations.McKern et al. (15) presented data on the structure of theextracellular portion of INSR, reporting that the extracellu-lar portion of the monomer consists of a leucine-rich repeatdomain (L1), a cysteine-rich region (CR), a second leucine-rich repeat domain (L2), and three fibronectin type III(FnIII) domains (FnIII-1 to FnIII-3). Insulin binds to twosites on INSR, and the FnIII domains contain parts of theprimary and secondary insulin-binding sites (15,16). FnIII-2contains the insert domain within which there is the a-bcleavage site and the carboxy-terminal region of the a-chain(aCT) involved in the primary insulin-binding site.

In this study, we examined four unrelated families withsevere insulin resistance, and we identified five novel muta-tions of INSR and a gross deletion that removed exon 2. Toassess the impact of mutations causing DS on INSR expres-sion, INSR activity, and downstream signaling, we conducteda functional study in Chinese hamster ovary (CHO) cells.Using mutation data from the National Center for Biotech-nology Information ClinVar database, Human Gene Muta-tion Database (HGMD), and UniProt database, we analyzedthe distribution of INSR missense mutations in patientswith severe insulin resistance to investigate the relationshipbetween the mutation location and the severity of insulinresistance. We also performed in silico structural analysis of

pathogenic missense mutations, with the aim of establish-ing structure–phenotype correlations.

RESEARCH DESIGN AND METHODS

SubjectsWe studied four patients with suspected insulin receptorabnormalities who were referred to our hospital (Table 1).Two patients had RMS (RMS-1 and RMS-2), one patienthad DS (DS-1), and one patient had type A-IR (TypeA-IR-1).Detailed clinical information is provided in the SupplementaryData. This research was approved by the ethics committeeof The University of Tokyo (approval numbers G3414 andG10077) and was implemented according to the approvedguidelines. Parents gave written informed consent for ge-netic testing of their children. Genomic DNA was extractedfrom peripheral blood samples.

Sequencing of INSRThe 22 exons of INSR and its intron–exon junctions wereamplified by PCR using the 21 pairs of primers listed inSupplementary Table 1. Then the PCR products were puri-fied and directly sequenced.

Comparative Genomic Hybridization MicroarrayA 60-mer oligonucleotide-based 4 3 44 K comparative geno-mic hybridization (CGH) microarray (INSR array) was custom-designed using the Agilent SureDesign (Agilent Technologies,Santa Clara, CA) web-based application (https://earray.chem.agilent.com/suredesign/). The INSR array contained 40,335probes covering the entire INSR gene. The median probespacing was 193 bp and the array focused on the 14.1 Mbgenomic region encompassing INSR in 19p13.2. Normalmale human reference DNA provided by Agilent in theSureTag Complete DNA Labeling Kit was the control forCGH analysis. After digestion with AluI and RsaI, genomicDNA from the DS-1 patient and his parents was labeledwith Cy5-dUTP, and normal male human reference DNAwas labeled with Cy3-dUTP. Purification of labeled products,array hybridization, washing, and scanning were conductedaccording to the CGH Enzymatic Labeling kit protocol v.7.1(Agilent Technologies). Data analysis was performed using

Table 1—Clinical characteristics of patients with severe insulin resistance

DS-1 RMS-1 RMS-2 TypeA-IR-1

Clinical diagnosis Donohue syndrome Rabson-Mendenhallsyndrome

Rabson-Mendenhallsyndrome

Type A insulin resistance

Age (years) 1 13 5 15

Sex Male Female Female Female

Gestational age 35 weeks, 4 days 37 weeks 40 weeks, 5 days 38 weeks, 5 days

Birth weight (g) 1,470 1,511 2,340 2,090

Length at birth (cm) 41.0 Not assessed 45.0 45.0

Acanthosis nigricans Yes Yes Yes Yes

Hypertrichosis Yes Yes Yes Yes

Other physical findings Elfin appearance of the face,lack of subcutaneous fat

Dental abnormality Dental abnormality Clitoromegaly

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Agilent CytoGenomics 3.0.1.1. Copy number aberration callswere based on a minimum regional absolute average log2 ratioof 0.25 and minimum contiguous probe count of 3. Forbreak point analysis, a pair of primers was used to amplifythe segment across the break point junction (Supplemen-tary Table 1). Amplified junction fragments were directlysequenced.

Plasmid ConstructionGFP tagged-pCMV-human INSR cDNA (Origene, Rockville,MD) was used. A mutant INSR expression vector (p.V657F)with the point mutation (NM_000208.2:c.1969G.T) wasconstructed by using the GeneArt Site-Directed Mutagene-sis System (Invitrogen, Carlsbad, CA) according to the man-ufacturer’s instructions. In the same way, mutant INSRexpression vectors with the following mutations of the FnIIIdomains (except for the insert domain) were constructed:c.2504G.T (p.S835I), c.2525C.T (p.A842V), c.2453A.G(p.Y818C), c.1904C.T (p.S635L), c.2465T.C (p.L822P),c.2776C.T (p.R926W), c.2810C.T (p.T937M), c.2621C.T (p.P874L), c.1975T.C (p.W659R), c.2633A.G (p.N878S),and c.2774T.C (p.I925T). Presence of the mutations wasverified by Sanger sequencing.

Transfection of CHO Cells and Stimulation With InsulinCHO cells were maintained at 37°C in Nutrient F-12 Mixture(HAM) medium (Invitrogen) supplemented with 10% FCSin a humidified incubator with 5% CO2/95% air. Transfectionsof either wild-type (WT) constructs or mutant constructs withFnIII mutations was performed with Lipofectamine 3000(Invitrogen). After 72 h, cells were starved of serum for 4 hand stimulated with insulin (0, 10, or 100 nmol/L) (Sigma-Aldrich, St. Louis, MO) for 5 min at 37°C before the phos-phorylation assay. Cells were rinsed with ice-cold PBS andproteins were purified using M-PER Mammalian ProteinExtraction Reagent (Thermo Fisher Scientific, Hudson, NH).

Western Blot AnalysisProtein samples were mixed with NuPAGE sample bufferwith or without NuPAGE sample reducing agent (Invitrogen).The final concentration of DTT in samples with reducingagent was 50 mmol/L. Electrophoresis was performed usingNuPAGE Novex 3–8% Tris-Acetate Protein Gels (Life Tech-nologies, Carlsbad, CA), and proteins were transferred to aHybond P PVDF membrane (GE Healthcare, Milwaukee,WI). After blocking with 5% skim milk in TBS-T, mem-branes were probed overnight at 4°C with primary anti-bodies diluted in TBS-T, followed by secondary antibodiesfor detection using ECL Prime Western Blotting DetectionReagent. An antibody specific for the b-subunit of humanINSR was purchased from Santa Cruz Technologies (sc-711),and antibodies targeting Akt (9272), phospho-INSR (Tyr1150/1151) (3024), and phospho-Akt (Thr308) (9275) were fromCell Signaling Technology Japan. As the secondary antibody,goat anti-rabbit IgG-HRP (sc-2004) was obtained from SantaCruz Technologies. Images were captured with an LAS-3000(Fujifilm, Tokyo, Japan) and were quantified using ImageJsoftware (National Institutes of Health, Bethesda, MD).

Enrichment Analysis of Protein Domains forMissense MutationsWe analyzed the distribution of INSR mutations in the fourpatients. We counted missense mutations within the FnIIIdomains or the other domains of INSR that were identifiedin our study or were registered in the HGMD, ClinVar, andUniProt databases. Each mutation was assigned as a causeof DS, RMS, or type A-IR based on source articles, and the diag-nosis was checked against information from Online MendelianInheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/omim). Next, we used the Fisher exact test to investigatewhether mutations classified as causing DS or RMS weremore frequent in the FnIII domains than mutations classi-fied as causing the milder type A-IR, with reference to Guoet al. (17). We also similarly examined whether mutationscausing type A-IR were more frequent in the tyrosine kinase(TK) domain than mutations causing DS or RMS.

Structural AnalysisThe X-ray crystal structure of INSR was obtained fromProtein Data Bank (PDB) entry 4ZXB (18). Croll et al. (18)presented 4ZXB, which only consists of the relatively well-ordered protein and glycan residues, but they also presentedan extended model of human INSR (model S1) including theinsert domain, which was subjected to energy minimization toobtain a physically reasonable configuration. Model S1 has alsobeen used for in silico structural analysis, but it lacked theatomic coordinates for R759-S763 (including the a-b proteo-lytic processing site). To obtain these missing residues,loop modeling was performed with the SWISS-MODELhomology modeling server (http://swissmodel.expasy.org/)(19). Structural models of mutant INSR were built using Swiss-PdbViewer (20). Each amino acid residue was substituted andenergy minimization was performed to avoid steric hindrance.Then each mutant model and WT structure was comparedusing Waals (Altif Laboratories, Inc., Tokyo, Japan). Calcu-lations for surface structure construction and electrostaticpotential mapping were performed by using eF-surf (http://ef-site.hgc.jp/eF-surf/), and the resulting data were visual-ized with Waals. To identify the folding nucleus of FnIII-2and FnIII-3, nucleation positions were detected by compar-ison with the third FnIII domain of human tenascin (TNfn3),as described previously (21). TNfn3 was the first b-sandwichprotein to be characterized in detail by F-value analysis andwas deposited as PDB entry 1TEN (22).

Statistical Data AnalysisA Fisher exact test was performed for comparisons and P,0.05 was considered statistically significant. All analyses weredone with R software.

RESULTS

Identification of INSR Mutations in the Patientsand ParentsSanger sequencing of INSR in the patients and their fami-lies revealed the mutations shown in Fig. 1A. Number-ing of the amino acid residues in INSR is based on the

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UniProtKB/Swiss-Prot file P06213, as previously reported(23). Patient RMS-1 was a compound heterozygote fortwo novel mutations, c.2504G.T (p.S835I) and c.2525C.T(p.A842V), both of which were within FnIII. Patient RMS-2was a compound heterozygote for a novel mutation,c.2997T.G (p.Y999*), and c.766C.T (p.R256C), whichwas previously identified in a heterozygous patient withRMS (24). Tyr999 is located near Pro997, which was af-fected by an INSRmissense mutation previously detected inRMS (11). Patient TypeA-IR-1 was a compound heterozy-gote for a novel mutation, c.1465A.G (p.N489D), andc.3160G.A (p.V1054M), which has not previously beenidentified in type A-IR, although the same mutation was

previously reported in a patient with DS (compoundheterozygous with p.Trp659Arg in the a-subunit) (25).With regard to p.N489D, another mutation, c.1466A.G(p.N489S), at the same amino acid position was previ-ously described in a patient with type A-IR (26). The mu-tant alleles were confirmed to have been inherited fromthe parents of each patient. Patient DS-1 was hetero-zygous for a novel mutation, c.1969G.T (p.V657F), inFnIII inherited from his mother. The location of V657 isnear W659, which was affected by a missense mutationpreviously detected in another patient with DS (25). Noother candidate mutations of INSR were detected in thefour patients.

Figure 1—INSR mutations in patients with extreme insulin resistance. A: Sanger sequencing of the identified mutations. B: CGH array data ofpatient DS-1 and his parents. Nucleotide positions are represented on the horizontal axis. Log2 (case/reference signal intensities on CGH array)data are shown on the vertical axis. Dots with log2 (case/reference signal intensity ratio)0 are shown in blue.C: The deletion allele could only be amplified in DS-1 and his father, and the predicted PCR product size was 800 bp. TheWT allele was too largeto be amplified by these primers (25 kb), so no products were found in his mother. M, 100 bp ladder marker. The rightwards blue arrow representsthe forward primer, and the leftwards blue arrow represents the reverse primer. D: Break point junctions of the INSR deletion in patient DS-1.

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Identification of a Deletion Involving Exon 2 of INSRin the DS Patient and his FatherTo investigate the existence of a mutant allele not detected bySanger sequencing, we performed CGH microarray analysis inthe DS-1 patient and his parents. We found that the patientand his father were heterozygous for a deletion mutation ofINSR that removed a sequence containing exon 2 (Fig. 1B),while there was no such deletion in his mother. To determinethe break point, we created primers for the flanking sites andperformed PCR, obtaining a fragment of about 800 bp in thepatient and his father, but not in his mother (Fig. 1C). Wedetermined the break point junction by Sanger sequencing(Fig. 1D), revealing a 24,792 bp deletion (Chr19:7,266,055–7,290,846). The junction sequences were a long interspersedelement and a short interspersed element, with only two basepairs of microhomology at the break point (SupplementaryFig. 1).

Functional Assessment of Mutant INSR ProteinTo assess the impact of the p.V657F mutation in the FnIIIdomains of INSR, CHO cells were transfected with WT ormutant forms of INSR and cell lysates were analyzed byWestern blotting under reducing and nonreducing conditionsusing anti-INSR antibodies. Under reducing conditions, therewas an increase of the proreceptor and a decrease of the maturereceptor in mutant cells (Fig. 2A). To evaluate whether thismutation affected INSR activity and downstream signaling,insulin-induced phosphorylation was assayed in vitro. In cellsexpressing p.V657F, insulin-induced autophosphorylation ofINSR was significantly decreased compared with autophos-phorylation in cells expressing the WT form (Fig. 2B). Post-translational receptor processing involves multiple steps,including dimerization of the precursor form (proreceptor)and proteolytic cleavage of the dimeric form to yield the a2b2tetramer. To investigate which step of posttranslational

Figure 2—Assessment of mutant INSR protein. A: Western blotting of WT and mutant INSR under reducing conditions. CHO cells weretransfected with WT INSR, INSR containing the V657F mutation, or the empty vector (mock). Western blotting was conducted using 5 mg oftotal cellular protein to evaluate levels of the proreceptor and the mature b-subunit of the receptor. B: Analysis of insulin-stimulated autophos-phorylation of the b-subunit of INSR. Transfected CHO cells were stimulated with insulin (0, 10, or 100 nmol/L) for 5 min. Then cell lysates wereanalyzed to detect the autophosphorylated INSR b-subunit by Western blotting using 25 mg of total cellular protein under reducing conditions.C: Western blotting under nonreducing conditions. CHO cells were transfected with WT INSR and INSR containing the V657F mutation. Westernblotting was performed using 5 mg of total cellular protein to assess whether the mutant insulin proreceptor underwent dimerization. D:Phosphorylated and unphosphorylated Akt were detected by Western blotting using 5 mg of total cellular protein under reducing conditions.Conc., concentration; P, phosphorylated.

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processing was impaired, Western blot analysis was con-ducted under nonreducing conditions, revealing a predomi-nance of high molecular–weight oligomeric forms with boththe WT and mutant receptors. These results showed thatthe mutant insulin proreceptor also underwent dimeriza-tion (Fig. 2C). Furthermore, phosphorylation of Akt proteindownstream target of the signaling pathway was also re-duced in cells expressing the INSR p.V657F mutant com-pared with cells expressing WT INSR, but unphosphorylatedAkt levels were not different (Fig. 2D). We also investigatedthe other missense mutations in the FnIII domains (Sup-plementary Tables 2 and 3). It was found that mutationscausing both DS and RMS showed a substantially loweramount of the mature insulin receptor (IR) b-subunit ex-pression than in cells with the WT receptor, whereasamount of the mature IR b-subunit was higher with FnIIImutations causing RMS than with mutations causing DS(Supplementary Fig. 2).

Analysis of the Distribution of INSR Mutations CausingSevere Insulin ResistanceAmong our four patients with extreme insulin resistance,the patient with DS (DS-1) and one of the two patients withRMS (RMS-1) had FnIII domain mutations, but the otherRMS patient (RMS-2) and the patient with type A-IR(TypeA-IR-1) did not. Structural analysis of the extracellularportion of INSR (15) has provided insight into its domainstructure, which is shown in Fig. 3A. Because the a-b cleav-age site and some residues of both the primary and secondaryinsulin-binding sites are located within the FnIII domains(Fig. 3A), we suspected that FnIII mutations might be as-sociated with more severe phenotypes. Therefore, we ana-lyzed the distribution of INSR mutations to identify thedomains preferentially affected by mutations causing moresevere insulin resistance. We only assessed missense muta-tions, as previously reported (27), because it is more difficultto determine the impact of mutations and localize impor-tant functional regions by analyzing nonsense mutations aswell as rearrangements and insertions/deletions comparedwith missense mutations (17). We analyzed 82 INSR mis-sense mutations that were detected in our study or regis-tered as pathogenic in databases (Fig. 3B and SupplementaryTable 4), and we found that the frequency of mutationsaffecting the FnIII domains was significantly higher in pa-tients with DS (28.1%) than in patients with type A-IR(3.7%) (P = 0.016, Fisher exact test). The frequency of FnIIImutations was also higher in patients with RMS (17.4%)than in patients with type A-IR (3.7%), but the differencewas not significant (P = 0.17). In addition, mutations of theTK domain showed a significantly higher frequency in pa-tients with type A-IR (59.3%) than in patients with DS(12.5%) (P = 0.00025) or patients with RMS (26.1%) (P =0.024).

Structure–Phenotype CorrelationsMutations causing DS were preferentially associated withthe FnIII domains of INSR, although there were also somemutations causing RMS or type A-IR. Therefore, we performed

structural analysis of missense mutations located in the FnIIIdomains to elucidate the relations with phenotypic severity.

The X-ray crystal structures derived from PDB entry4ZXB and its extended model (model S1) were used forstructural analysis. The structure of the dimeric extracellu-lar portion of INSR (model S1) (18) is shown in Fig. 3C.Because the model lacked residues encoding the a-b cleav-age site, we complemented it by using SWISS-MODEL (Fig.3D). FnIII-2 has the same topology as other proteins in theFnIII family, i.e., seven b-strands composing two b-sheets.The first sheet consists of the A, B, and E b-strands, and thesecond sheet consists of the C9, C, F, and G b-strands. TheB, C, E, and F b-strands form the common hydrophobiccore of the FnIII domains (Fig. 4A). The folding nucleus ofTNfn3 (in layer 3 of the B, C, E, and F strands), anotherprotein belonging to the FnIII family, is essential for form-ing its topology, and the residues in layers 2 and 4 of thestrands that pack onto the folding nucleus contribute sig-nificantly toward stabilizing the transition state for folding(21,28). We compared folding of FnIII-2 in INSR with thatof TNfn3. When the structure of FnIII-2 in INSR was super-imposed on the structure of TNfn3, a root-mean-squaredeviation (RMSD) of only 1.06 Å was observed over thestructurally equivalent positions (72 residues). TNfn3 resi-dues I20, Y36, I59, and V70, which form the folding nu-cleus, overlapped with L640, W659, I809, and I820 of FnIII-2,and the RMSD was only 0.40 Å (Fig. 4B and C). These fourresidues of FnIII-2 form the folding nucleus. That is, resi-dues W659 and I820 in one b-sheet and residues I809 andL640 in the opposite sheet form the folding nucleus ofFnIII-2 through hydrophobic interactions (SupplementaryFig. 3). In the same way, the structure of FnIII-3 was super-imposed on that of TNfn3, and the RMSD was only 1.29 Åover the structurally equivalent positions (62 residues). TheTNfn3 residues forming the folding nucleus overlapped withL869, Y888, L913, and V923 of FnIII-3, and the RMSD wasonly 0.43 Å. These four residues were considered to formthe folding nucleus of FnIII-3 (Supplementary Figs. 3 and4). In FnIII-2 and FnIII-3 of INSR, the hydrophobic core cor-responding to the residues in layers 2–4 of the B, C, E, and Fstrands are also critical for stabilization of the FnIII domains.

WT V657 is structurally close to the folding nucleus ofFnIII-2 and forms the hydrophobic core. This residue is incontact with L640, W659, I809, and I820, which form thefolding nucleus. Using the Swiss-PdbViewer program, wesubstituted V657 with phenylalanine in INSR. Insertion of abulky phenylalanine caused steric clashes with residuesL640, I809, and I820 of the neighboring folding nucleus,which destabilized the folding process and led to defectiveprotein folding (Fig. 4D–G). We found that S835I andA842V in FnIII-2 caused RMS, and S835I created stericclashes with two neighboring residues (P625 and A824),whereas A842V produced a steric clash with the neighbor-ing residue S630 (Supplementary Fig. 5). However, thesemutations are distant from the folding nucleus and struc-tural changes may be small, resulting in only localized struc-tural destabilization.

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We analyzed 12 missense mutations located in FnIII,except for the insert domain, identified in our study orregistered as pathogenic in databases (Fig. 4H and I), andwe divided these mutations into three groups (Supplemen-tary Table 2 and Supplementary Data). Group 1a mutationsdirectly affect the folding nucleus that comprises criticalresidues for folding of the FnIII domains (Fig. 4G and Sup-plementary Fig. 6A–C), resulting in defective folding. Group1b mutations affect the hydrophobic core residues packed

onto the folding nucleus (Supplementary Fig. 6D–F) andalso significantly destabilize the FnIII domains. Group1 (1a and 1b) mutations cause DS. The group 2 mutationcauses loss of the hydrophobic interaction contributing tostabilization of the domain structure (Supplementary Fig.6G) and thus destabilizes the domain to some degree. It hasbeen registered in HGMD as causing DS, and Grasso et al.(29) reported an intermediate phenotype between DS andRMS in the mutation data source article. Group 3 mutations

Figure 3—Structure and missense mutations of INSR. A: Structural map of INSR based on the result of the structural analysis performed byMcKern et al. (15). Interchain disulfide bonds are shown as horizontal lines. ID, insert domain. B: Domains and mutations of INSR. Pins show theloci of the missense mutations identified in our study and the known missense mutations registered as pathogenic in databases as of October2016. Red and purple pins show biallelic defects (homozygous or compound heterozygous mutations) and heterozygous mutations, respec-tively. Missense mutations identified in our study are labeled. Rectangles denote the L1/L2 (InterPro ID: IPR000494), CR (IPR006211), FnIII(IPR003961), and TK (IPR020635) domains.C: Inverted V-shaped arrangement of the domains within model S1, an extended model of PDB entry4ZXB. The model represents the INSR ectodomain homodimer. One monomer is displayed as a tube structure, and the other as a space-fillingmodel. The dashed circle shows missing residues encoding the a-b proteolytic processing site. Each domain is colored as follows: L1, blue; CR,green; L2, orange; FnIII-1, yellow; FnIII-2, magenta; and FnIII-3, red. D: Model S1 does not contain residues encoding the a-b cleavage site andtherefore was complemented using SWISS-MODEL.

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Figure 4—Structural analysis of INSR missense mutations. A: INSR FnIII-2 is formed from seven b-strands. Green triangles show residuesforming the folding nucleus of FnIII-2, and magenta and orange pins represent the loci of the novel FnIII mutations we identified as causing DSand RMS, respectively. B: Simplified view of the structure of INSR FnIII-2. The core of the protein consists of six layers. Four residues form thefolding nucleus, as indicated by the green circles. The hydrophobic core residues are yellow. C: Simplified view of the structure of TNfn3, whichalso belongs to the FnIII family. D: Structure of FnIII-2. WT V657 (green) is in contact with the folding nucleus. The hydrophobic core residues arelabeled red, and residues forming the folding nucleus are orange. Hydrophobic interactions are shown as dashed lines. E: Mutation of V657 withinsertion of a bulky phenylalanine (magenta) causes steric clashes with the neighboring residues (L640, I809, and I820). F: Structure of FnIII-2displayed as sticks and space-filling models. G: The amino acid residues involved in steric clashes with the mutated residue F657 are shown. Hand I: Structures of FnIII-2 (except for the insert domain) and FnIII-3 and location of missense mutations of FnIII identified in this study orregistered as pathogenic in databases. The residues affected by mutations causing DS and RMS are green and light blue, respectively. The Band E b-strands are red, and the C and F b-strands are dark blue. These strands form the common hydrophobic core of the FnIII domains.

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are located away from the folding nucleus and lead to smallstructural changes (Supplementary Figs. 5 and 6H and I),only producing localized destabilization. These mutationscause RMS. The extent of the structural changes causedby mutations in groups 1–3 is consistent with the clinicalphenotype. On the other hand, two other missense muta-tions of the insert domain contained in FnIII have beenreported (D734A and R762S [30,31]), neither of which in-fluence the domain structure, but they affect the functionalregion or processing site (Supplementary Fig. 6J and K).The influence of these mutations is determined by factorsother than structural defects, leading to a range of phenotypes.

DISCUSSION

We studied four unrelated families with severe insulin re-sistance and identified five novel mutations and a deletionthat removed exon 2 of INSR. The patient with DS and oneof two RMS patients had FnIII mutations. Using onlinedatabases, we demonstrated that missense mutations caus-ing DS were significantly more frequent in the FnIII do-mains than mutations causing type A-IR. This findingsupports the importance of the FnIII domains, which con-tain the a-b cleavage site and part of the insulin-bindingsite of INSR. We also found that missense mutations caus-ing type A-IR were significantly more frequent in the TKdomain than those causing DS or RMS. Patients with DS orRMS (extreme conditions presenting in infancy) have biallelicINSR defects that almost always display recessive inheri-tance, whereas patients with type A-IR (less severe andusually diagnosed around puberty) may have a heterozy-gous mutation of the TK domain that causes insulin resis-tance by a dominant negative effect, unlike mutations ofother INSR domains (32–34). Heterozygosity of the muta-tion should lead to the formation of some fully functionalwt/wt receptors, which would result in a less severe phenotype.

INSR contains three tandem FnIII domains in itsextracellular juxtamembrane region (35). Each FnIII domainis a b-sandwich protein with a Greek key motif and isformed by packing two antiparallel b-sheets to constructa hydrophobic core (36,37). TNfn3 also belongs to the FnIIIfamily, and previous studies have shown that the foldingmechanism is similar throughout this family. For structuralanalysis, we compared folding of FnIII-2 in INSR with fold-ing of TNfn3 and identified the folding nucleus of FnIII-2.We substituted V657 in FnIII-2 of patient DS-1 with phe-nylalanine, which was predicted to cause steric clashes withthe folding nucleus that destabilized the folding process andled to defective folding. Although S835I and A842V (iden-tified in FnIII-2 of patient RMS-1 as causing RMS) alsocaused steric clashes with neighboring residues, they werelocated away from the folding nucleus and unlikely to sig-nificantly destabilize the domain structure.

Review of online databases showed that missense mu-tations of the FnIII domains predicted to result in proteinfolding defects or significant destabilization of the domainstructure all caused DS (Supplementary Table 2). One mu-tation (D734A) causing DS was not predicted to destabilize

the hydrophobic core of the FnIII domains, but it is locatedin aCT (part of the insulin-binding domain) and wouldcause distortion of the insulin-binding site (30). Mutationscausing the less severe RMS probably did not have a largeeffect on FnIII folding or stability. These results indicatethat prediction of the phenotypic expression of INSR mu-tations might be improved by adopting a structural bioinfor-matics approach in addition to biochemical data, particularlyassessment of the reduction of insulin binding that Longoet al. (11) proposed as corresponding to clinical severity.

Transfection of CHO cells with V657F mutation of INSRled to impaired receptor processing and autophosphoryla-tion, as well as reduced phosphorylation of Akt. Under non-reducing conditions, the high molecular–weight form ofINSR (oligomeric form) was predominant when both WTand mutant receptors were analyzed. Therefore, the mutantproreceptor undergoes dimerization before excision of thesubunit processing site, as does the native proreceptor (38).Thus, the mutation probably impairs proreceptor process-ing by disturbing the three-dimensional structure of FnIII-2containing the a-b cleavage site. Several mutations outsidethe cleavage site that disturb a-b cleavage have been re-ported, e.g., p.H236R (39) and p.N42K (40) are not withinthe cleavage site but retard several posttranslational pro-cessing steps, including proteolytic a-b cleavage of INSR. Inour patients, reduced expression of the mature receptorprobably contributed to impaired intramolecular signaltransduction, as reported previously (41,42). Furthermore,we also conducted a functional assessment of INSR proteinswith the other missense mutations in the FnIII domains(except for the insert domain). Patient RMS-1 was com-pound heterozygous for p.S835I and p.A842V. Thoughthe level of the mature IR b-subunit in cells expressingthe p.S835I mutation was much lower than in cells express-ing the WT receptor, proreceptor processing was less im-paired in cells expressing p.A842V, resulting in a less severephenotype in patient RMS-1 (Supplementary Fig. 2). Thelevel of the mature IR b-subunit was substantially lower incells expressing mutations causing DS and RMS than incells expressing the WT receptor, whereas the mature IRb-subunit was not as low in cells expressing the other FnIIImutations causing RMS (p.S635L and p.N878S) as in cellsexpressing mutations causing DS (Supplementary Fig. 2).

It is desirable to consider the properties of mutationscausing DS or RMS in the context of two mutant alleles.The severity of insulin resistance would be related to thefunctional impact of the two mutations. The patientsidentified with 12 missense mutations of the FnIII domains(except for the insert domain) showed either homozygousor compound heterozygous mutations (Supplementary Ta-ble 3). Of the FnIII mutations, those causing DS were eitherhomozygous or compound heterozygous with protein trun-cating mutations (nonsense and frameshift mutations) ordeletions, as well as missense mutations (p.A119V andp.A1055V) situated in functionally important sites. As for themissense mutations, it was reported that p.A119V markedlyimpaired insulin binding when the mutant receptor was

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expressed in vitro (11), whereas p.V1054M is located in theconsensus sequence for ATP binding and a patient withsevere insulin resistance was reported to be heterozygousfor another mutation p.A1055V at the neighboring aminoacid position (43). Taking into account results from func-tional assessment of INSR proteins with FnIII mutations inthis study, we speculated that severe FnIII domains muta-tions that are compound heterozygous with other muta-tions causing severe functional impairment result indeleterious functional impact, leading to DS. However, se-vere mutations, such as FnIII domains mutations, that arecompound heterozygous with other mutations causing lesssevere functional impairment result in less severe form ofRMS. Detailed functional analysis (at the cellular and mo-lecular levels) of mutations causing severe insulin resistancecombined with accumulation of clinical data would furtherdelineate the properties of each mutation.

In patient DS-1, we identified an in-frame deletion in theregion covering exon 2 of INSR. EBV-transformed lympho-cytes from a patient with type A-IR due to an in-frame deletionof exon 2 of INSRmRNA were reported to show impairmentof insulin binding (44). The L1 domain of INSR coded byexon 2 interacts extensively with aCT, which in turn inter-acts directly with insulin (16). Deletion of exon 2 is thoughtto cause destabilization of aCT that binds to insulin inFnIII, leading to impaired insulin binding by INSR mutants.

The syndromes caused by INSR mutations (DS, RMS, andtype A-IR) seem to represent a broad spectrum of diseasedue to considerable variation in the severity of receptor dys-function, rather than each one being a distinct entity. Aseach of the syndromes caused by INSRmutations (DS, RMS,and type A-IR) is rare, diagnosis of the syndrome remainschallenging. Ongoing efforts to apply genomics to healthcare on a larger scale should allow collaboration in identi-fying patients with severe insulin resistance and the causalmutations, leading to refinement of the diagnostic criteriafor these syndromes.

In conclusion, we identified five novel mutations of INSRand a deletion that removed exon 2 in four patients withextreme insulin resistance. Missense mutations causing DSwere significantly more frequently located in the FnIII domainsthan those causing the milder type A-IR. According to struc-tural analysis, DS was caused by all of the missense muta-tions that were predicted to severely impair formation of thehydrophobic core and stability of the FnIII domains, whereasRMS was caused by all of the mutations predicted to producelocalized destabilization and not affect folding of the FnIIIdomains. Thus, the genotype–phenotype and structure–phenotype correlations of INSR mutations identified in thisstudy provide insights into the molecular mechanisms of se-vere insulin resistance, would assist with early diagnosis ofthese syndromes, and could lead to new treatment approaches.

Acknowledgments. The authors thank K. Ishinohachi (Department ofDiabetes and Metabolic Diseases, Graduate School of Medicine, The University ofTokyo) for providing excellent technical support during this study.

Funding. This study was supported by a grant-in-aid for scientific research inpriority areas (C) from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT) (grant 15K09409 to N.S.). This work was also partlysupported by a grant-in-aid for scientific research from MEXT (grant 16K07211 toF.M.) and by grants from Core Research for Evolutional Science and Technology,Japan Science and Technology Agency (to F.M. and T.T.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.H., H.K., F.M., N.S., and T.Kad. designed the studyand wrote the manuscript. J.H., F.M., K.H., M.Tan., K.Su., M.Tak., and N.S.conducted the experimental research and analyzed the data. H.K., K.A., T.Kaw., I.M.,K.Sa., T.I., K.A.B., T.T., and T.Y. contributed to data analysis and preparation of themanuscript. H.I., S.T., and all coauthors read the manuscript and contributed to thefinal version of the manuscript. T.Kad. is the guarantor of this work and, as such, hadfull access to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Data Availability. The INSR mutations identified in this study have beendeposited in National Center for Biotechnology Information ClinVar with accessionnumbers SCV000503034, SCV000503035, SCV000503036, and SCV000503037.Prior Presentation. Parts of this study were presented at the 77th ScientificSessions of the American Diabetes Association, San Diego, CA, 9–13 June 2017.

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