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Structural Basis and Genotype-Phenotype Correlations of INSR Mutations

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Structural Basis and Genotype-Phenotype Correlations of INSR Mutations Diabetes Page 2 of 62 Structural Basis and Genotype-phenotype Correlations of INSR Mutations Causing Severe Insulin Resistance Jun Hosoe1*, Hiroko Kadowaki2*, Fuyuki Miya1,3,4,5*, Katsuya Aizu6, Tomoyuki Kawamura7, Ichiro Miyata8, Kenichi Satomura9, Takeru Ito10, Kazuo Hara11, Masaki Tanaka12, Hiroyuki Ishiura12, Shoji Tsuji12, Ken Suzuki1, Minaka Takakura1, Keith A. Boroevich4, Tatsuhiko Tsunoda3,4,5, Toshimasa Yamauchi1, Nobuhiro Shojima1** & Takashi Kadowaki1** * J.H., H.K., and F.M. contributed equally to this work. ** Corresponding authors: Takashi Kadowaki, [email protected], and Nobuhiro Shojima, [email protected]. 1Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2Department of Pediatrics, Sanno Hospital, Tokyo, Japan. 3Department of Medical Science Mathematics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan 4Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Medical 1 Diabetes Publish Ahead of Print, published online August 1, 2017 Page 3 of 62 Diabetes Sciences, Yokohama, Japan 5CREST, JST, Tokyo, Japan 6Division of Endocrinology and Metabolism, Saitama Children's Medical Center, Saitama, Japan 7Department of Pediatrics, Osaka City University Graduate School of Medicine, Osaka, Japan 8Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan 9Department of Pediatric Nephrology and Metabolism, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan 10Department of Pediatrics, Atsugi City Hospital, Kanagawa, Japan 11Department of Endocrinology and Metabolism, Saitama Medical Center, Jichi Medical University, Saitama, Japan 12Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Diabetes Page 4 of 62 Abstract The insulin receptor (INSR) gene was analyzed in four patients with severe insulin resistance, revealing 5 novel mutations and a deletion that removed exon 2. A patient with Donohue syndrome (DS) had a novel p.V657F mutation in the second fibronectin type III domain (FnIII-2), which contains the α-β cleavage site and part of the insulin-binding site. The mutant INSR was expressed in Chinese hamster ovary cells, revealing that it reduced insulin proreceptor processing and impaired activation of downstream signaling cascades. Using online databases, we analyzed 82 INSR missense mutations and demonstrated that mutations causing DS were more frequently located in the FnIII domains than those causing the milder type A insulin resistance (p = 0.016). In silico structural analysis revealed that missense mutations predicted to severely impair hydrophobic core formation and stability of the FnIII domains all caused DS, while those predicted to produce localized destabilization and not affect folding of the FnIII domains all caused the less severe Rabson-Mendenhall syndrome. These results suggest the importance of the FnIII domains, provide insight into the molecular mechanism of severe insulin resistance, and will aid early diagnosis, as well as providing potential novel targets for treating extreme insulin resistance. 3 Page 5 of 62 Diabetes Introduction Mutations of the insulin receptor (INSR) gene result in extreme insulin resistance and dysglycemia (1), leading to several syndromes with various abnormal phenotypes that depend on the severity of INSR dysfunction. Patients with Donohue syndrome (DS, formerly known as leprechaunism) have the most severe insulin resistance (2,3) and patients with type A insulin resistance syndrome (type A-IR) display somewhat less severe manifestations (4,5), while Rabson-Mendenhall syndrome (RMS) represents an intermediate condition (6,7). Patients with type A-IR can live beyond middle age and present with hypertrichosis, acanthosis nigricans, and female hyperandrogenism. Patients with RMS generally survive into childhood or early adulthood and their characteristic symptoms are hypertrichosis, dysplastic dentition, and coarse and dysmorphic facial features. Patients with DS seldom live beyond infancy. They have dysmorphic facial features (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 then processing of the dimer to yield a heterotetramer composed of two α-subunits and two β-subunits (8). Although there are no clear genotype–phenotype correlations for INSR mutations causing severe insulin resistance, it has been suggested that 4 Diabetes Page 6 of 62 homozygous or compound heterozygous mutations of the α-subunit cause more severe syndromes (DS and RMS), whereas heterozygous β-subunit mutations lead to milder insulin resistance (9,10). Longo et al. reported that missense mutations causing the most severe manifestations affected the extracellular portion of INSR and markedly reduced binding of insulin (11). Some researchers have performed structural analysis of mutations of various proteins other than INSR to predict clinical manifestations and establish structure– phenotype correlations (12-14), and a structural bioinformatics approach should be useful for predicting the diverse phenotypes caused by monogenic mutations. However, there is no clear evidence of structure-phenotype correlations in patients with severe insulin resistance due to INSR mutations. McKern et al. presented data on the structure of the extracellular portion of INSR, reporting that the extracellular portion of the monomer consists of a leucine-rich repeat domain (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) (15). Insulin binds to two sites on INSR, and the FnIII domains contain parts of the primary and secondary insulin-binding sites (15,16). FnIII-2 contains the insert domain (ID) within which there is the α-β cleavage site and the 5 Page 7 of 62 Diabetes carboxy-terminal region of the α-chain (αCT) involved in the primary insulin-binding site. In this study, we examined 4 unrelated families with severe insulin resistance, and we identified 5 novel mutations of INSR and a gross deletion that removed exon 2. To assess the impact of mutations causing DS on INSR expression, INSR activity, and downstream signaling, we conducted a functional study in Chinese hamster ovary (CHO) cells. Using mutation data from the NCBI ClinVar database, Human Gene Mutation Database (HGMD), and UniProt database, we analyzed the distribution of INSR missense mutations in patients with severe insulin resistance to investigate the relationship between the mutation location and the severity of insulin resistance. We also performed in silico structural analysis of pathogenic missense mutations, with the aim of establishing structure–phenotype correlations. Research Design and Methods Subjects We studied 4 patients with suspected insulin receptor abnormalities who were referred to our hospital (Table 1). Two patients had RMS (RMS-1 and RMS-2), one patient had DS (DS-1), and one patient had type A-IR (TypeA-IR-1). Detailed clinical information 6 Diabetes Page 8 of 62 is provided in the Supplementary Data. This research was approved by the ethics committee of The University of Tokyo (approval number: G3414 and G10077) and was implemented according to the approved guidelines. Parents gave written informed consent for genetic testing of their children. Genomic DNA was extracted from peripheral blood samples. Sequencing of INSR The 22 exons of INSR and its intron-exon junctions were amplified by PCR using the 21 pairs of primers listed in Supplementary Table 1. Then the PCR products were purified and directly sequenced. Comparative Genome Hybridization (CGH) Microarray A 60-mer oligonucleotide-based 4×44K CGH microarray (INSR array) was custom-designed using the Agilent SureDesign web-based application (https://earray.chem.agilent.com/suredesign/). The INSR array contained 40,335 probes covering the entire INSR gene. The median probe spacing was 193 bp and the array focused on the 14.1 Mb genomic region encompassing INSR in 19p13.2. Normal male human reference DNA provided by Agilent in the SureTag Complete DNA Labeling Kit 7 Page 9 of 62 Diabetes (Agilent Technologies, Santa Clara, CA) was the control for CGH analysis. After digestion with AluI and RsaI, genomic DNA from the DS-1 patient and his parents was labeled with Cy5-dUTP, while normal male human reference DNA was labeled with Cy3-dUTP. Purification of labeled products, array hybridization, washing, and scanning were conducted according to the CGH Enzymatic Labeling kit protocol v.7.1 (Agilent Technologies). Data analysis was performed using Agilent CytoGenomics 3.0.1.1 (Agilent Technologies). Copy number aberration calls were based on a minimum regional absolute average log2 ratio of 0.25 and minimum contiguous probe count of 3. For breakpoint analysis, a pair of primers was used to amplify the segment across the breakpoint junction (Supplementary Table 1). Amplified junction fragments were directly sequenced. Plasmid construction GFP 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) was constructed by using the GeneArt Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. In the same way, mutant INSR expression vectors with the following
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