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Mutations in CUL7, OBSL1 and CCDC8 in 3-M Syndrome Lead to Disordered Growth Factor Signalling

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Mutations in CUL7, OBSL1 and CCDC8 in 3-M Syndrome Lead to Disordered Growth Factor Signalling 267 Mutations in CUL7, OBSL1 and CCDC8 in 3-M syndrome lead to disordered growth factor signalling D Hanson1,2*, P G Murray1,2,3*, T Coulson1, A Sud1,2, A Omokanye1,2, E Stratta1, F Sakhinia1, C Bonshek1,2,3, L C Wilson4, E Wakeling5, S A Temtamy6, M Aglan6, E M Rosser4, S Mansour7, A Carcavilla8, S Nampoothiri9, W I Khan10, I Banerjee3, K E Chandler2, G C M Black2,3 and P E Clayton1,3 1Paediatric Endocrinology and 2Genetic Medicine Research Group, School of Biomedicine, Manchester Academic Health Sciences Centre (MAHSC), University of Manchester, Manchester M13 9WL, UK 3Central Manchester University Hospitals Foundation Trust, Manchester M13 9WL, UK 4Clinical Genetics, Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, UK 5North West Thames Regional Genetic Service, North West London Hospitals NHS Trust, Harrow, Middlesex HA1 3UJ, UK 6Division of Human Genetics and Human Genome Research, Department of Clinical Genetics, National Research Centre, Cairo, Egypt 7South-West Thames Regional Genetics Service, St George’s Healthcare NHS Trust, London SW17 0RE, UK 8Pediatrics Department, Hospital Virgen de la Salud, Toledo, Spain 9Department of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Centre, AIMS Poneakara P O, Cochin 682041, Kerala, India 10Department of Endocrinology, The Children Hospital and The Institute of Child Health, Multan, Pakistan (Correspondence should be addressed to P E Clayton who is now at Manchester Academic Health Sciences Centre (MAHSC), Royal Manchester Children’s Hospital, Oxford Road, Manchester M13 9WL, UK; Email: [email protected]) *(D Hanson and P G Murray contributed equally to this work) Abstract 3-M syndrome is a primordial growth disorder caused by mutations in CUL7, OBSL1 or CCDC8. 3-M patients typically have a modest response to GH treatment, but the mechanism is unknown. Our aim was to screen 13 clinically identified 3-M families for mutations, define the status of the GH–IGF axis in 3-M children and using fibroblast cell lines assess signalling responses to GH or IGF1. Eleven CUL7, three OBSL1 and one CCDC8 mutations in nine, three and one families respectively were identified, those with CUL7 mutations being significantly shorter than those with OBSL1 or CCDC8 mutations. The majority of 3-M patients tested had normal peak serum GH and normal/low IGF1. While the generation of IGF binding proteins by 3-M cells was dysregulated, activation of STAT5b and MAPK in response to GH K K K K K K was normal in CUL7 / cells but reduced in OBSL1 / and CCDC8 / cells compared with controls. Activation of AKT to IGF1 was reduced in CUL7K/K and OBSL1K/K cells at 5 min post-stimulation but normal in CCDC8K/K cells. The prevalence of 3-M mutations was 69% CUL7, 23% OBSL1 and 8% CCDC8. The GH–IGF axis evaluation could reflect a degree of GH resistance and/or IGF1 resistance. This is consistent with the signalling data in which the CUL7K/K cells showed impaired IGF1 signalling, CCDC8K/K cells showed impaired GH signalling and the OBSL1K/K cells showed impairment in both pathways. Dysregulation of the GH–IGF–IGF binding protein axis is a feature of 3-M syndrome. Journal of Molecular Endocrinology (2012) 49, 267–275 Introduction identified with CUL7 mutations (w70%) with OBSL1 mutations accounting for 25% of cases and CCDC8 3-M syndrome is an autosomal recessive disorder mutations identified in the remaining 5% (Huber et al. characterised by severe pre- and postnatal growth 2005, 2009, 2010, Maksimova et al. 2007, Hanson et al. restriction associated with minor dysmorphic facial 2009, 2011). We have previously shown that 3-M features, fleshy prominent heels and in some cases syndrome patients show only a modest response to skeletal abnormalities including slender bones and recombinant human GH (rhGH) treatment, although relatively tall vertebral bodies (Table 1). We have the molecular mechanism responsible for this remains previously associated the disease to mutations in three unclear (Murray et al. 2007, Clayton et al. 2012). separate genes: CUL7 (MIM #273750), OBSL1 (MIM CUL7 is a component of an Skp1–Cullin–Fbox #612921) and CCDC8 (MIM #614205). The majority of (SCF) complex that is responsible for ubiquitin- genetically confirmed 3-M syndrome patients have been mediated proteasomal degradation (Dias et al. 2002). Journal of Molecular Endocrinology (2012) 49, 267–275 DOI: 10.1530/JME-12-0034 0952–5041/12/049–267 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 01:09:33AM via free access 268 D HANSON, P G MURRAY and others . Growth factor signalling in 3-M syndrome Table 1 Clinical information for 3-M syndrome patients. Auxology, radiological and clinical phenotype of eight 3-M syndrome individuals from six different families. List of clinical feature taken from Hanson et al. 2009 3M-ID 3M-3 3M-4a 3M-4b 3M-6 3M-7a 3M-7b 3M-9 3M-11 Gender M MMMMMMF Birth weight (g) 2250 NA NA 2300 1920 2570 1940 1660 Birth weight SDS K2.8NANAK3.2 K4.3 K1.5 K4.3 K0.7 Age at initial presentation (years) 4.75 10 0.7240.50.50.43.2 Height SDS at presentation K6.1 K6.1 K7.7 K3.7 K5.8 K6.1 K5.8 K3 Weight SDS at presentation NA K6 K7.1NAK5.8 K5NAK4.1 OFC SDS at presentation K1.4NANANANAK0.40.16 NA Radiological features Slender long bones K NA NA CKKKK Tall vertebral bodies K NA NA KKKKK Facial features Fleshy tipped nose CCCCCCCC Anteverted nares KCKCCCCC Full fleshy lips CCKCCCCC Triangular face CCKCCCCC Dolichocephaly KCCKKKKC Frontal bossing CCCKCCCC Midface hypoplasia KKKCCCCC Long philtrum KCKKCCCC Pointed chin KCKCCCCC Prominent ears KKCKCKCK Other clinical features Short neck CCCKCCCK Winged scapulae KKKKKKKK Square shoulders KKKKCCCC Short thorax KCCKCCCC Transverse chest groove KKKCCCKK Pectus deformity KKKCKKCC Hyperlordosis KCKCKKKK Scoliosis KKKKKKKK Hypermobility of joints KKKCKKKC Fifth finger clinodactyly CCKCKKCC Prominent heels CCCKCCCC Spina bifida occulta KKKKCCKK Developmental dysplasia hip KKKKKKKK 3M-ID, 3-M syndrome family identification; 3M-1-13, 3-M syndrome family reference number; NA, not available; OFC, occipitofrontal head circumference. The ubiquitin-mediated proteasomal degradation development. OBSL1 is closely related to the giant pathway is required for GH receptor endocytosis and muscle protein obscurin (Geisler et al. 2007) and has regulates downstream signalling molecules including been found to interact physically with the other muscle the STAT proteins; however, the involvement of the proteins titin and myomesin (Fukuzawa et al. 2008). CUL7–SCF complex in this process has not been However, 3-M syndrome is not recognised as a disease investigated (Strous et al. 1997, van Kerkhof et al. of muscular defects, suggesting that OBSL1 is not 2002). Xu et al. (2008) established that the signalling primarily a muscle protein. Hanson et al. (2009) molecule, insulin receptor substrate 1 (IRS1), is a postulated that CUL7 and OBSL1 are likely com- proteolytic target of the CUL7-SCF ubiquitin ligase. ponents of a common pathway because pathogenic IRS1 is a member of a family of proteins that are mutations in both genes cause 3-M syndrome and that adaptor molecules downstream of both the insulin, loss of OBSL1 by siRNA knockdown also led to insulin-like growth factor 1 (IGF1), and GH receptors. reduction in CUL7 expression. Geisler et al. (2007) K K Xu et al.(2008)also demonstrated that Cul7 / suggested that OBSL1 is a putative cytoskeletal adaptor mouse embryonic fibroblasts (MEFs) exhibited protein; therefore, it is possible that OBSL1 acts to increased levels of both Akt and Mapk activation facilitate the assembly of the CUL7–SCF complex. which, although pro-mitogenic pathways, when over- CCDC8 is a protein of unknown molecular function; stimulated led to poor cell growth and eventually however, we have found that OBSL1 interacts with both cellular senescence. CUL7 and CCDC8, suggesting that all three proteins To date, there is little known about the function are components of the same molecular pathway of OBSL1, in particular its role in growth and (Hanson et al. 2011). Journal of Molecular Endocrinology (2012) 49, 267–275 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 01:09:33AM via free access Growth factor signalling in 3-M syndrome . D HANSON, P G MURRAY and others 269 In clinical reports, there are little, if any, data on the have not routinely been performed on this category of status of the GH–IGF axis in 3-M patients, nor are there patients. However, we have been able to collate data data on the degree of height restriction by mutation on peak GH levels during arginine stimulation testing type. We do, however, know that IGFBP gene expression from 11 3-M syndrome patients, basal serum IGF1 is abnormal in cells derived from 3-M patients (Huber concentrations from 13 patients and serum IGFBP3 K K et al. 2010) and in the Cul7 / mouse (Tsutsumi et al. from three patients. 2008). This study has addressed the following: We selected representative patients with mutations i) identification and frequency of mutations in the in either CUL7, OBSL1 or CCDC8 and, with consent three genes in those with a clinical diagnosis of 3-M; and institutional ethical approval, established skin ii) height at presentation, the status of the GH–IGF axis fibroblast cell lines. For comparisons, we also estab- as measured in serum assays by mutation status and lished skin fibroblast cell lines from normal control IGFBP generation from cultured cell lines; and iii) in subjects with consent. view of the relative clinical resistance to GH treatment To date, the majority of mutations in CUL7 are and involvement of IRS1, MAPK and AKT, an located within the cullin domain.
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