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 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 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 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 : 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

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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).

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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 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. We, therefore, K K assessment of the activation of signalling pathways by generated a cell line from 3M-7 (CUL7 / )as GH and IGF1. this patient has a nonsense mutation located within the cullin domain (c.4191delC, p.S1398Pfs*10) with no functional CUL7 protein produced. For Materials and methods OBSL1,approximatelyhalfofthepatientshave the same common nonsense mutation (c.1273dupA, Clinical details p.T425Nfs*40) (Hanson et al. 2009, Huber et al. 2010) and we derived a fibroblast cell line from a patient with We identified a cohort of 13 families with a distinct K K this mutation (OBSL1 / ). We also selected a skin 3-M syndrome phenotype consisting of severe postnatal fibroblast cell line derived from a 3-M syndrome patient growth restriction, prominent fleshy heels, facial with a CCDC8 mutation (c.84insT, p.K29*) in which dysmorphism with prominent forehead, triangular we have previously shown by western blotting no face, full lips and an upturned fleshy tipped nose. Full K K detectable level of CCDC8 protein (CCDC8 / ) clinical details are available for six families (Table 1). (Hanson et al. 2011). Human fibroblasts were main- tained in DMEM media supplemented with 10% FBS, Genomic sequencing 50 U/ml penicillin and 50 U/ml streptomycin (all PAA, Yeovil, Somerset, UK). Experiments were conducted on We obtained informed consent and blood samples from sub-confluent cultures in 75 cm2 flasks. all affected individuals and, when tested, their unaf- fected relatives. Genomic DNA was isolated by standard laboratory procedures. For mutation detection, we Assessment of IGFBP levels in 3-M syndrome amplified all 25 coding exons of CUL7, all 22 exons of OBSL1 and the single exon of CCDC8 using standard Secreted protein was precipitated from fibroblast- PCR methods as described previously (Hanson et al. conditioned cell culture media and resuspended in 2009). PCR products were purified with exonuclease I SDS loading buffer. The protein levels of IGFBP-2, -3 (ExoSAPIT; Amersham Bioscience) according to the and -7 were assessed by immunoblotting and band manufacturer’s instructions and products were density to determine relative expression of IGFBPs sequenced using Applied Biosystems BDv3.1 on an between 3-M cell lines and control fibroblasts. We were ABI3730 automated analyzer (Applied Biosystems) unable to detect secreted IGFBP5, so intracellular levels followed by mutation detection using Sequence analysis were assessed by immunoblotting. software (Applied Biosystems). Signal transduction assays Auxological and biochemical profile We first evaluated the basal protein levels of total IRS1, Height data were collected from our own clinics, MAPK and AKT (antibodies all from Cell Signaling) in including patients in this study, from those we have both patient and control cell lines and then chose to previously reported (Hanson et al. 2009, 2011) and assess STAT5b and MAPK activation in response to GH from the published literature (Akawi et al. 2011, Sasaki and AKT activation in response to IGF1, as robust et al. 2011). Height SDSs were calculated from markers of response to these growth factors. For K K 36 mutation-positive 3-M syndrome patients, 15 with signalling stimulation assays, control, CUL7 / , K K K K CUL7 mutations, 15 with OBSL1 mutations and six with OBSL1 / and CCDC8 / cells were treated with CCDC8 mutations. Peak GH, IGF1 and IGFBP assays either rhGH (Genotropin, Pfizer) (200 ng/ml) or IGF1 www.endocrinology-journals.org Journal of Molecular Endocrinology (2012) 49, 267–275

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Table 2 Mutation screening of 3-M syndrome families. Mutations identified in 13 different 3-M syndrome families investigated: nine have CUL7 mutations (accession numbers NM_014780.4 and NP_055595.2) and three OBSL1 mutations (accession numbers NM_015311.2 and NP_056126.1) and one CCDC8 mutation (accession numbers NM_032040.3 and NP_114429.2)

ID Country of origin CUL7 OBSL1 CCDC8

3M-1 England c.2TOC, p.M1?; c.1234-2AOG– – 3M-2 Egypt c.1398delC, p.M467* – – 3M-3 India c.2169C1GOA, – – 3M-4 Pakistan c.3379_3380delTG, p.W1127Efs*38 – – 3M-5 England c.3454GOA; p.E1152K, c.3937GOT; p.E1313* – – 3M-6 Egypt c.4108_4111delGGAG, p.G1370Rfs*37 – – 3M-7 Pakistan c.4191delC, p.S1398Pfs*10 – – 3M-8 Morocco c.4450_4451delTG, p.V1484Gfs*68 – – 3M-9 England c.4763TOC, p.L1588P – – 3M-10 Pakistan – c.928COT, p.Q310* – 3M-11 Ireland – c.1273dupA, p.T425Nfs*40 – 3M-12 Egypt – c.1534C2TOC– 3M-13 Pakistan – – c.612dupG, p.K205Efs*58

3M-ID, 3-M syndrome family identification; 3M-1-13, 3-M syndrome family reference number.

(Sigma) (100 ng/ml) for 0, 5, 15 or 60 min. These Results doses were based on previous GH and IGF1 stimulation assays using human skin fibroblast cells (Freeth et al. Genetic analysis 1997, Westwood et al. 2011). Direct sequencing of all coding exons of CUL7 After incubation, cells were washed in PBS and lysates identified pathogenic mutations in nine families: prepared using RIPA buffer and SDS loading buffer. seven patients carried homozygous mutations and Activation of GH signalling pathways was assessed by two patients had compound heterozygous mutations. immunoblotting, probing with antibodies that speci- Two mutations (c.3379_3380delTG, p.W1127Efs*38 fically recognise the phosphorylated isoforms of MAPK and c.4451_4452delTG, p.V1484Gfs*68) have been and STAT5b and antibodies that recognise all isoforms previously described (Huber et al. 2009); however, of MAPK and STAT5b (all from Cell Signalling the remaining nine mutations are novel: three Technologies). b-Actin (Santa Cruz) was used as a frameshift, three missense, two splice-site and one control for protein loading. nonsense mutation. Activation of IGF1 signalling pathways was assessed We also identified pathogenic mutations in OBSL1 by immunoblotting, probing with an antibody that in three families. All mutations were homozygous. One specifically recognises the phosphorylated isoform of mutation has been previously described (c.1273dupA, AKT and an antibody that recognises all isoforms p.T425Nfs*40) (Hanson et al. 2009) and two are novel of AKT (both from Cell Signalling Technologies). mutations – one nonsense and one splice-site mutation GAPDH(SantaCruz)wasusedasacontrolfor (Table 2). None of the novel mutations identified in protein loading. either CUL7 or OBSL1 were present in 210 normal All immunoblots were scanned and band density was , supporting their pathogenicity. A single measured using ImageJ; relative expression of phos- pathogenic mutation in CCDC8 was identified in one phorylated isoforms to total isoforms of MAPK, STAT5b family; the mutation (c.612dupG, p.K205Efs*58) has and AKT was calculated after normalisation to b-actin or been described previously (Hanson et al. 2011). The GAPDH as appropriate. Relative expression of phos- identification of mutations in CUL7, OBSL1 and CCDC8 phorylated to total isoforms in 3-M cells compared with and their absence in control samples confirmed the control cells was calculated. Where appropriate, clinical diagnosis of 3-M syndrome. statistical analysis by one-way (Time) and two-way (Time and Cell) ANOVA (SPSS software) of densito- metry data from three independent experiments (each Growth and GH–IGF axis evaluation repeated in triplicate) was undertaken to determine Height at presentation was measured in 36 mutation- whether there was stimulation of signalling molecules positive 3-M syndrome patients (including those over time and whether there was a difference in above). The mean height SDS of 15 patients with activation between cell types. In view of multiple testing, CUL7 mutations was K5.8, a further 15 patients with a stringent P value !0.005 was used to indicate a OBSL1 mutations had a mean height SDS of K4.7 and significant result. six patients with CCDC8 mutations had a mean height

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0·00 compared with control cells (Fig. 2). IGFBP7 was K K reduced in all 3-M syndrome cell lines with CUL7 / K/K –1·00 and OBSL1 having the lowest levels (Fig. 2).

–2·00 STAT5b and MAPK signalling in response to GH

–3·00 Basal levels of IRS1, MAPK and AKT in 3-M cells were comparable with control cells (data not shown). Control –4·00 cells showed maximal activation of MAPK at 5 min after

Mean height SDS GH exposure, as previously reported (Silva et al. 2002).

–5·00 CCDC8 Maximal STAT5b activation was also seen at 5 min post- OBSL1 mutation, –4·1 stimulation. The levels of activation at 5 min in 3-M cells mutation, –4·7 compared with the normal cells are shown in Fig. 3: –6·00 K/K CUL7 mutation, CUL7 cells showed normal activation, while K K K K –5·8 OBSL1 / and CCDC8 / cells had phospho-MAPK –7·00 77 and 50% of control levels and phospho-STAT5b levels * P<0·05 53 and 42% of control levels respectively (Fig. 3). * P<0·05 Figure 1 Effect of mutation status on height SDS at presentation in 3-M syndrome patients. Height SDSs were calculated from AKT signalling in response to IGF1 36 mutation-positive 3-M syndrome patients; 15 with CUL7 mutations, 15 with OBSL1 mutations and six with CCDC8 The pattern of AKT activation in normal cells showed a mutations. Height data were collected from our own clinics and marked early activation at 5 min, sustained to 60 min. K K K K from the published literature (Akawi et al. 2011, Sasaki et al. 2011). In the 3-M cells at 5 min, CUL7 / and OBSL1 / had reduced levels of phospho-AKT compared with normal K K of K4.1 SDS. Patients with CUL7 mutations are (Figs 3 and 4), while CCDC8 / cells showed normal K K significantly shorter than 3-M syndrome patients with levels. By 60 min, levels of phospho-AKT in CUL7 / either OBSL1 or CCDC8 mutation (Fig. 1). cells had decreased to baseline, while levels in K K K K Peak GH during stimulation testing and basal IGF1 OBSL1 / and CCDC8 / cells were w50% lower levels were measured in 11 and 13 patients respectively than in controls. (Table 3). Ten patients had measurements for both peak GH and IGF1: eight had normal peak GH levels (R7 mg/l) with either normal (one CUL7, one OBSL1 Discussion and one CCDC8) or low IGF1 (SDS !K2, four OBSL1 and 1 CCDC8) and two had low peak GH levels (peak Published series of genetic analyses in 3-M syndrome GH %7 mg/l) with either normal (one OBSL1) or low indicate that the majority of cases are a result of CUL7 IGF1 (one CCDC8). Serum IGFBP3 was not routinely mutations (62/93) but a significant proportion are measured; however, three patients (all OBSL1) were caused by OBSL1 mutations (26/93) (Huber et al. 2005, found to have levels either elevated above or within the 2009, 2010, Maksimova et al. 2007, Hanson et al. 2009, upper normal range. Secreted IGFBP3 was measured by 2011). We recently identified that a smaller subset of western blotting in the three 3-M cell lines, all have patients (5/93) have CCDC8 mutations; two distinct elevated IGFBP3 levels (three- to four-fold) with the mutations have so far been identified but both result in K K highest seen in OBSL1 / cells (Fig. 2). generation of a premature termination codon and Western blotting of secreted IGFBP2 from the same subsequent loss of CCDC8 (Hanson et al. 2011). We now cells revealed that all 3-M cells have reduced IGFBP2 describe the identification of a further nine novel K K levels compared with control cells with CCDC8 / cells mutations in CUL7 and two novel mutations in OBSL1; having the lowest levels (Fig. 2). Intracellular protein this includes the first splice site mutation found in levels of IGFBP5 were either normal or slightly elevated OBSL1. The mutations found in CUL7 are interspersed

Table 3 Peak GH and basal IGF1 levels in 3-M syndrome patients. Peak serum GH (mg/ml) were measured after arginine stimulation in 11 3-M syndrome patients and basal serum IGF1 levels were measured and expressed as SDS in 13 patients grouped by mutation status

Gene Peak GH (mg/l) Peak GH range (mg/l) Basal IGF1 SDS Basal IGF1 range SDS

CUL7 10.7(nZ1) – K0.8(nZ3) K0.2toK1.8 OBSL1 19.7(nZ6) 3.7to38.3 K1.9(nZ7) 0.5toK5 CCDC8 10.8(nZ4) 5.4to13.3 K2.0(nZ3) K1.5toK2.4 www.endocrinology-journals.org Journal of Molecular Endocrinology (2012) 49, 267–275

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IGFBP2 protein level in 3-M syndrome IGFBP3 protein level in 3-M syndrome fibroblasts fibroblasts

1·2 CUL7 IGFBP2 6 CUL7 IGFBP3 OBSL1 IGFBP2 OBSL1 IGFBP3 1·0 5 CCDC8IGFBP2 CCDC8 IGFBP3 0·8 4 0·6 3 0·4 2 0·2 1 Relative expression Relative expression 0 0 Control CUL7 –/– OBSL1 –/– Control CUL7 –/– OBSL1 –/–

37 kDa 50 kDa IGFBP-2 IGFBP-3 37 kDa 25 kDa –/– Control CCDC8 Control CCDC8 –/–

37 kDa IGFBP-2 46 kDa IGFBP-3 37 kDa 25 kDa

IGFBP5 protein level in 3-M syndrome IGFBP7 protein level in 3-M syndrome fibroblasts fibroblasts 0·8 CUL7 IGFBP5 2·0 0·7 CUL7 IGFBP7 OBSL1 IGFBP5 0·6 OBSL1 IGFBP7 1·5 CCDC8 IGFBP5 0·5 CCDC8 IGFBP7 0·4 1·0 0·3 0·5 0·2 Relative expression

Relative expression 0·1 00 Control CUL7 –/– OBSL1 –/– Control CUL7 –/– OBSL1 –/–

46 kDa β 37 kDa -Actin IGFBP-7 IGFBP-5 30 kDa 25 kDa

Control CCDC8 –/– Control CCDC8 –/– β 46 kDa -Actin 37 kDa IGFBP-7 IGFBP-5 25 kDa 30 kDa Figure 2 Dysregulation of IGFBPs in 3-M syndrome fibroblast cells. Comparison of the relative levels of IGFBP2, -3, -5 and -7 between 3-M syndrome patient and control fibroblast cells (as secreted proteins for IGFBP2, -3 and -7 and intracellular levels for IGFBP5). Error bars represent S.D.

throughout the whole gene. However, the new of mutation status, the associations between mutations in OBSL1 are found in the first eight exons OBSL1/CUL7 and OBSL1/CCDC8 are required to in keeping with our previous findings, further maintain CUL7-SCF E3 ligase activity. Further explora- suggesting that loss of the three main isoforms of tion of this pathway is likely to define new proteins OBSL1 is important in the pathogenicity of controlling mammalian growth and may help to 3-M syndrome. The relative frequency of mutations in identify new genes associated with small for gestational this study (69% CUL7, 23% OBSL1 and 8% CCDC8) age (SGA) and short stature. reflects not only the previous experience but also the We have also hypothesised that the restricted child- date of publication of the mutations – 2005, 2009 and hood growth of 3-M patients could be related to a 2011 respectively. These families have presented over degree of insensitivity to GH and/or IGF1. This has the last 2 years and were screened for CUL7, OBSL1 been supported by two observations. First, the majority and CCDC8 mutations. The different frequencies are, of 3-M children investigated in our unit have normal/- therefore, likely to reflect a true prevalence. high peak GH levels and low or normal IGF1 levels: We hypothesise that the primary molecular normal GH and low IGF1 could be consistent with a mechanism responsible for the pathogenicity of degree of GH resistance, while normal GH and normal 3-M syndrome will include targets of the CUL7-SCF IGF1 in a very short child could be consistent with a E3 ligase or proteins that physically associate with degree of IGF1 resistance. Both interpretations would CUL7, OBSL1 or CCDC8. We have previously shown be concordant with previous descriptions of GH and that OBSL1 interacts with both CUL7 and CCDC8; IGF1 resistance (Rosenfeld & Hwa 2004, Walenkamp & however, CUL7 does not interact with CCDC8, which Wit 2006). Secondly, the growth response to rhGH in may suggest that OBSL1 plays a role as the structural 3-M syndrome, treated on the basis that these children component of the pathway. The importance of these are usually born SGA, was relatively poor. Fifteen 3-M interactions is unclear; however, we postulate that as patients showed no significant change in height SDS the phenotypes of 3-M syndrome is similar regardless over the first year of rhGH treatment (Height SDS K4.4

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(a)STAT5b activation 5 min post- (b) MAPK activation 5 min post- GH stimulation GH stimulation 1·4 1·4 1·2 1·2 1·0 1·0 0·8 0·8 0·6 0·6 0·4 0·4 0·2 0·2 Percentage of control Percentage of control 0 0 Control CUL7–/– OBSL1–/– CCDC8–/– Control CUL7–/– OBSL1–/– CCDC8–/–

(c)AKT activation 5 min post- (d) AKT activation 60 min post- 1·4 IGF1 stimulation 1·4 IGF1 stimulation 1·2 1·2 1·0 1·0 0·8 0·8 0·6 0·6 0·4 0·4 0·2 0·2 Percentage of control Percentage of control 0 0 Control CUL7–/– OBSL1–/– CCDC8–/– Control CUL7–/– OBSL1–/– CCDC8–/– Figure 3 Growth factor signalling in 3-M syndrome fibroblast cells. Cultured normal control fibroblast cells and those derived from 3-M syndrome patients with CUL7, OBSL1 or CCDC8 null mutations were stimulated with GH (200 ng/ml) or IGF1 (100 ng/ml) over an interval of 0, 15, 30 and 60 min. Comparison of activation at 5 min post-GH stimulation of (A) STAT5b and (B) MAPK between control cells and the individual 3-M syndrome cells expressed as a percentage of control cells. Comparison of activation of AKT at (C) 5 min post-IGF1 stimulation and (D) 60 min post-IGF1 stimulation between control cells and the individual 3-M syndrome cells expressed as a percentage of control cells. Error bars represent S.D. at baseline vs K4 at 1 year post-treatment, PZ0.4) IGFBP2 and upregulation of IGFBP5 expression seen in (Murray et al. 2007, Clayton et al. 2012). Over 4 years of fibroblasts from two patients with OBSL1 mutations. rhGH treatment, the increment in height SDS was !C By contrast, upregulation of IGFBP2 is seen in the K K 1 compared with an increment of OC2 in rhGH- Cul7 / mouse (Tsutsumi et al. 2008). We now treated SGA children (Van Pareren et al. 2003). demonstrate that regardless of mutation status, all In addition to possible GH/IGF resistance, dysregula- 3-M cell lines show reduced secreted protein levels of tion of IGFBP2 and IGFBP5 had previously been IGFBP2 and -7. Intracellular IGFBP5 levels were slightly identified (Huber et al. 2010) with downregulation of elevated in 3-M cells. In this study, IGFBP3, the most

(a)Control CUL7–/– (b)ControlOBSL1–/– (c) Control CCDC8–/–

0 5 15 60 0 5 15 60 0 5 15 60 0 5 15 60 0 5 15 60 0 5 15 60

pAKT

AKT

GAPDH

0·8 1·0 1·2 0·7 0·9 1·0 0·6 0·8 0·5 0·7 0·8 0·6 0·4 0·5 0·6 0·4 0·3 0·4 0·2 0·3 0·2 0·2 Phospho AKT (AU) Phospho AKT (AU) 0·1 Phospho AKT (AU) 0·1 0 0 0 0 5 15 60 0 5 15 60 0 5 15 60 Time (min) Time (min) Time (min)

One-way ANOVA (time) One–way ANOVA (Ttime) One–way ANOVA (time) Control P<0·005 Control P<0·005 Control P<0·005 CUL7–/– P<0·005 OBSL1–/– P<0·005 CCDC8–/– P<0·005 Two–way ANOVA (time and cell type) Two–way ANOVA (time and cell type) Two–way ANOVA (time and cell type) Control vs CUL7–/– (cell) P<0·005 Control vs OBSL1–/– (cell) P<0·005 Control vs CCDC8–/– (cell) NS (cell* time) P<0·005 Figure 4 Effect of IGF1 on AKT activation in normal control and 3-M syndrome fibroblast cells. Cultured normal control fibroblast cells and those derived from 3-M syndrome patients with CUL7, OBSL1 or CCDC8 null mutations were stimulated with IGF1 (100 ng/ml) over an interval of 0, 15, 30 and 60 min. Expression of phosphorylated AKT (top row), total AKT (middle row) and GAPDH (bottom row) was measured by standard western blot (all antibodies from Cell Signalling Technology). K K Comparison of AKT activation between (a) control (open square) and CUL7 / cells K K (light grey shaded square), (b) control (open square) and OBSL1 / cells (dark grey K K shaded square) and (c) control (open square) and CCDC8 / cells (closed square). Significance levels in one- and two-way ANOVA are shown, error bars represent S.D. www.endocrinology-journals.org Journal of Molecular Endocrinology (2012) 49, 267–275

Downloaded from Bioscientifica.com at 09/30/2021 08:29:34PM via free access 274 D HANSON, P G MURRAY and others . Growth factor signalling in 3-M syndrome

abundant IGFBP in serum, is expressed at high levels surprising that there are differences in signal transduc- in all three 3-M cell lines, and in the few patients in tion cascades downstream of the IGF1 and GH which serum levels were checked, IGFBP3 was above or receptors between the human and mouse cells. within the upper end of the normal range for age. In our clinical study of response to rhGH, the Elevated levels of IGFBP3 have previously been mutation status of the patients was not known. This identified in Silver–Russell syndrome (SRS) fibroblast study indicates that an analysis of degree of height cells (Montenegro et al. 2012) along with high IGFBP3 restriction, GH–IGF1 axis status, and response to rhGH serum levels in SRS patients (Binder et al. 2008). versus mutation status should be undertaken. We would It, therefore, appears that disruption of IGFBP gene suggest that the signalling abnormalities we have expression and altered levels of pericellular secreted described here could correlate with growth status. We proteins are features of 3-M syndrome. IGFBPs have a have some evidence to support this in that patients with vital role in maintaining the availability of IGFs, and a CUL7 mutation, which is associated with the most these abnormalities are likely to contribute to growth significant IGF1 signalling abnormality, are shorter attenuation and response to rhGH in 3-M syndrome. than those with either OBSL1 or CCDC8 mutations. It is In order to further characterise responses to growth likely that although the 3-M syndrome is primarily a factors, we have taken an in vitro approach, assessing disorder of ubiquitination, the downstream conse- responses to GH and IGF1 in cell lines. Our studies quences can impact on GH–IGF1 signalling and affect have shown differences in activation of signalling response to GH therapy. molecules downstream of both GH and IGF1 receptors both between the three 3-M mutations and between the K K GH and IGF1 pathways. CUL7 / cells show normal Declaration of interest levels of activation of STAT5b and MAPK after rhGH The authors declare no conflict of interest that could be perceived as stimulation, suggesting that there is normal signalling K K prejudicing the impartiality of the research reported. down both pathways. However, both CCDC8 / and K K OBSL1 / cells have moderately reduced levels of pSTAT5b and pMAPK, indicating some impairment of Funding signalling down both pathways. A different pattern emerges for IGF1 signalling. AKT activation in Support from the National Institute of Health Research Manchester K K CUL7 / cells is significantly lower than in control Biomedical Research Centre is acknowledged. During this study, P G M was a Medical Research Council (UK) Clinical Research Fellow. cells and shows a different pattern with early reduced activation that then diminishes to basal levels (Fig. 3). K/K OBSL1 cells show reduced AKT activation, main- Acknowledgements tained at the same level throughout the experiment, K/K while CCDC8 cells show normal early activation of The authors thank all healthcare professionals contributing to the AKT but a later moderate reduction (Fig. 3). Thus, GH care of families described here. K K signalling is normal in CUL7 / cells but IGF1 signalling is disrupted, suggesting possible IGF1 insensitivity. Interestingly, the one patient with a CUL7 References mutation, on whom we have a GH–IGF assessment, had a normal peak GH level and IGF1 SDS within the Akawi NA, Ali BR, Hamamy H, Al-Hadidy A & Al-Gazali L 2011 Is K K normal range. By contrast, in CCDC8 / cells, GH autosomal recessive Silver–Russel syndrome a separate entity or is it part of the 3-M syndrome spectrum? American Journal of Medical signalling is moderately reduced and early activation of Genetics. Part A 155A 1236–1245. (doi:10.1002/ajmg.a.34009) IGF1 signalling is normal, a scenario more consistent Arai T, Kasper JS, Skaar JR, Ali SH, Takahashi C & DeCaprio JA 2003 K K with a degree of GH resistance, and in OBSL1 / cells, Targeted disruption of p185/Cul7 gene results in abnormal both GH and IGF1 signalling are moderately dimin- vascular morphogenesis. PNAS 100 9855–9860. (doi:10.1073/pnas. 1733908100) ished, which may suggest both partial GH and IGF1 Binder G, Seidel AK, Martin DD, Schweizer R, Schwarze CP, resistance. Five OBSL1 patients had normal peak GH Wollmann HA, Eggermann T & Ranke MB 2008 The endocrine levels with three having low IGF1 and one normal IGF1. phenotype in Silver–Russell syndrome is defined by the under- The growth factor signalling data are contradictory to lying epigenetic alteration. Journal of Clinical Endocrinology and K/K Metabolism 93 1402–1407. (doi:10.1210/jc.2007-1897) previous reports in Cul7 MEF cells, which after Clayton PE, Hanson D, Magee L, Murray PG, Saunders E, IGF1 stimulation showed increased AKT and MAPK Abu-Amero SN, Moore GE & Black GC 2012 Exploring the K K activation. In addition, unlike Cul7 / MEFs, we did spectrum of 3-M syndrome, a primordial short stature disorder not see an increase in basal levels of IRS1 (Xu et al. of disrupted ubiquitination. Clinical Endocrinology 77 335–342. K/K (doi:10.1111/j.1365-2265.2012.04428.x) 2008). Cul7 mice, however, die shortly after birth Dias DC, Dolios G, Wang R & Pan ZQ 2002 CUL7: A DOC domain- and have vascular abnormalities (Arai et al. 2003), which containing cullin selectively binds Skp1.Fbx29 to form an SCF-like are inconsistent with 3-M syndrome. Thus, it is not complex. PNAS 99 16601–16606. (doi:10.1073/pnas.252646399)

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Downloaded from Bioscientifica.com at 09/30/2021 08:29:34PM via free access Growth factor signalling in 3-M syndrome . D HANSON, P G MURRAY and others 275

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