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 was normal in CUL7−/− cells but reduced in OBSL1−/− and CCDC8−/− cells compared with controls. Activation of AKT to IGF1 was reduced in CUL7−/− and OBSL1−/− cells at 5 min post-stimulation but normal in CCDC8−/− 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 CUL7−/− cells showed impaired IGF1 signalling, CCDC8−/− cells showed impaired GH signalling and the OBSL1−/− cells showed impairment in both pathways. Dysregulation of the GH–IGF–IGF binding protein axis is a feature of 3-M syndrome.
3-M syndrome is an autosomal recessive disorder characterised by severe pre- and postnatal growth restriction associated with minor dysmorphic facial features, fleshy prominent heels and in some cases skeletal abnormalities including slender bones and relatively tall vertebral bodies (Table 1). We have previously associated the disease to mutations in three separate genes: CUL7 (MIM #273750), OBSL1 (MIM #612921) and CCDC8 (MIM #614205). The majority of genetically confirmed 3-M syndrome patients have been identified with CUL7 mutations (∼70%) with OBSL1 mutations accounting for 25% of cases and CCDC8 mutations identified in the remaining 5% (Huber et al. 2005, 2009, 2010, Maksimova et al. 2007, Hanson et al. 2009, 2011). We have previously shown that 3-M syndrome patients show only a modest response to recombinant human GH (rhGH) treatment, although the molecular mechanism responsible for this remains unclear (Murray et al. 2007, Clayton et al. 2012).
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
|Birth weight (g)||2250||NA||NA||2300||1920||2570||1940||1660|
|Birth weight SDS||−2.8||NA||NA||−3.2||−4.3||−1.5||−4.3||−0.7|
|Age at initial presentation (years)||4.75||10||0.7||24||0.5||0.5||0.4||3.2|
|Height SDS at presentation||−6.1||−6.1||−7.7||−3.7||−5.8||−6.1||−5.8||−3|
|Weight SDS at presentation||NA||−6||−7.1||NA||−5.8||−5||NA||−4.1|
|OFC SDS at presentation||−1.4||NA||NA||NA||NA||−0.4||0.16||NA|
|Slender long bones||−||NA||NA||+||−||−||−||−|
|Tall vertebral bodies||−||NA||NA||−||−||−||−||−|
|Fleshy tipped nose||+||+||+||+||+||+||+||+|
|Full fleshy lips||+||+||−||+||+||+||+||+|
|Other clinical features|
|Transverse chest groove||−||−||−||+||+||+||−||−|
|Hypermobility of joints||−||−||−||+||−||−||−||+|
|Fifth finger clinodactyly||+||+||−||+||−||−||+||+|
|Spina bifida occulta||−||−||−||−||+||+||−||−|
|Developmental dysplasia hip||−||−||−||−||−||−||−||−|
3M-ID, 3-M syndrome family identification; 3M-1-13, 3-M syndrome family reference number; NA, not available; OFC, occipitofrontal head circumference.
CUL7 is a component of an Skp1–Cullin–Fbox (SCF) complex that is responsible for ubiquitin-mediated proteasomal degradation (Dias et al. 2002). The ubiquitin-mediated proteasomal degradation pathway is required for GH receptor endocytosis and regulates downstream signalling molecules including the STAT proteins; however, the involvement of the CUL7–SCF complex in this process has not been investigated (Strous et al. 1997, van Kerkhof et al. 2002). Xu et al. (2008) established that the signalling molecule, insulin receptor substrate 1 (IRS1), is a proteolytic target of the CUL7-SCF ubiquitin ligase. IRS1 is a member of a family of proteins that are adaptor molecules downstream of both the insulin, insulin-like growth factor 1 (IGF1), and GH receptors. Xu et al. (2008) also demonstrated that Cul7−/− mouse embryonic fibroblasts (MEFs) exhibited increased levels of both Akt and Mapk activation which, although pro-mitogenic pathways, when over-stimulated led to poor cell growth and eventually cellular senescence.
To date, there is little known about the function of OBSL1, in particular its role in growth and development. OBSL1 is closely related to the giant muscle protein obscurin (Geisler et al. 2007) and has been found to interact physically with the other muscle proteins titin and myomesin (Fukuzawa et al. 2008). However, 3-M syndrome is not recognised as a disease of muscular defects, suggesting that OBSL1 is not primarily a muscle protein. Hanson et al. (2009) postulated that CUL7 and OBSL1 are likely components of a common pathway because pathogenic mutations in both genes cause 3-M syndrome and that loss of OBSL1 by siRNA knockdown also led to reduction in CUL7 expression. Geisler et al. (2007) suggested that OBSL1 is a putative cytoskeletal adaptor protein; therefore, it is possible that OBSL1 acts to facilitate the assembly of the CUL7–SCF complex. CCDC8 is a protein of unknown molecular function; however, we have found that OBSL1 interacts with both CUL7 and CCDC8, suggesting that all three proteins are components of the same molecular pathway (Hanson et al. 2011).
In clinical reports, there are little, if any, data on the status of the GH–IGF axis in 3-M patients, nor are there data on the degree of height restriction by mutation type. We do, however, know that IGFBP gene expression is abnormal in cells derived from 3-M patients (Huber et al. 2010) and in the Cul7−/− mouse (Tsutsumi et al. 2008). This study has addressed the following: i) identification and frequency of mutations in the three genes in those with a clinical diagnosis of 3-M; ii) height at presentation, the status of the GH–IGF axis as measured in serum assays by mutation status and IGFBP generation from cultured cell lines; and iii) in view of the relative clinical resistance to GH treatment and involvement of IRS1, MAPK and AKT, an assessment of the activation of signalling pathways by GH and IGF1.
Materials and methods
We identified a cohort of 13 families with a distinct 3-M syndrome phenotype consisting of severe postnatal growth restriction, prominent fleshy heels, facial dysmorphism with prominent forehead, triangular face, full lips and an upturned fleshy tipped nose. Full clinical details are available for six families (Table 1).
We obtained informed consent and blood samples from all affected individuals and, when tested, their unaffected relatives. Genomic DNA was isolated by standard laboratory procedures. For mutation detection, we amplified all 25 coding exons of CUL7, all 22 exons of OBSL1 and the single exon of CCDC8 using standard PCR methods as described previously (Hanson et al. 2009). PCR products were purified with exonuclease I (ExoSAPIT; Amersham Bioscience) according to the manufacturer's instructions and products were sequenced using Applied Biosystems BDv3.1 on an ABI3730 automated analyzer (Applied Biosystems) followed by mutation detection using Sequence analysis software (Applied Biosystems).
Auxological and biochemical profile
Height data were collected from our own clinics, including patients in this study, from those we have previously reported (Hanson et al. 2009, 2011) and from the published literature (Akawi et al. 2011, Sasaki et al. 2011). Height SDSs were calculated from 36 mutation-positive 3-M syndrome patients, 15 with CUL7 mutations, 15 with OBSL1 mutations and six with CCDC8 mutations. Peak GH, IGF1 and IGFBP assays have not routinely been performed on this category of patients. However, we have been able to collate data on peak GH levels during arginine stimulation testing from 11 3-M syndrome patients, basal serum IGF1 concentrations from 13 patients and serum IGFBP3 from three patients.
We selected representative patients with mutations in either CUL7, OBSL1 or CCDC8 and, with consent and institutional ethical approval, established skin fibroblast cell lines. For comparisons, we also established skin fibroblast cell lines from normal control subjects with consent.
To date, the majority of mutations in CUL7 are located within the cullin domain. We, therefore, generated a cell line from 3M-7 (CUL7−/−) as this patient has a nonsense mutation located within the cullin domain (c.4191delC, p.S1398Pfs*10) with no functional CUL7 protein produced. For OBSL1, approximately half of the patients have the same common nonsense mutation (c.1273dupA, p.T425Nfs*40) (Hanson et al. 2009, Huber et al. 2010) and we derived a fibroblast cell line from a patient with this mutation (OBSL1−/−). We also selected a skin fibroblast cell line derived from a 3-M syndrome patient with a CCDC8 mutation (c.84insT, p.K29*) in which we have previously shown by western blotting no detectable level of CCDC8 protein (CCDC8−/−) (Hanson et al. 2011). Human fibroblasts were maintained in DMEM media supplemented with 10% FBS, 50 U/ml penicillin and 50 U/ml streptomycin (all PAA, Yeovil, Somerset, UK). Experiments were conducted on sub-confluent cultures in 75 cm2 flasks.
Assessment of IGFBP levels in 3-M syndrome
Secreted protein was precipitated from fibroblast-conditioned cell culture media and resuspended in SDS loading buffer. The protein levels of IGFBP-2, -3 and -7 were assessed by immunoblotting and band density to determine relative expression of IGFBPs between 3-M cell lines and control fibroblasts. We were unable to detect secreted IGFBP5, so intracellular levels were assessed by immunoblotting.
Signal transduction assays
We first evaluated the basal protein levels of total IRS1, MAPK and AKT (antibodies all from Cell Signaling) in both patient and control cell lines and then chose to assess STAT5b and MAPK activation in response to GH and AKT activation in response to IGF1, as robust markers of response to these growth factors. For signalling stimulation assays, control, CUL7−/−, OBSL1−/− and CCDC8−/− cells were treated with either rhGH (Genotropin, Pfizer) (200 ng/ml) or IGF1 (Sigma) (100 ng/ml) for 0, 5, 15 or 60 min. These doses were based on previous GH and IGF1 stimulation assays using human skin fibroblast cells (Freeth et al. 1997, Westwood et al. 2011).
After incubation, cells were washed in PBS and lysates prepared using RIPA buffer and SDS loading buffer. Activation of GH signalling pathways was assessed by immunoblotting, probing with antibodies that specifically recognise the phosphorylated isoforms of MAPK and STAT5b and antibodies that recognise all isoforms of MAPK and STAT5b (all from Cell Signalling Technologies). β-Actin (Santa Cruz) was used as a control for protein loading.
Activation of IGF1 signalling pathways was assessed by immunoblotting, probing with an antibody that specifically recognises the phosphorylated isoform of AKT and an antibody that recognises all isoforms of AKT (both from Cell Signalling Technologies). GAPDH (Santa Cruz) was used as a control for protein loading.
All immunoblots were scanned and band density was measured using ImageJ; relative expression of phosphorylated isoforms to total isoforms of MAPK, STAT5b and AKT was calculated after normalisation to β-actin or GAPDH as appropriate. Relative expression of phosphorylated to total isoforms in 3-M cells compared with control cells was calculated. Where appropriate, statistical analysis by one-way (Time) and two-way (Time and Cell) ANOVA (SPSS software) of densitometry data from three independent experiments (each repeated in triplicate) was undertaken to determine whether there was stimulation of signalling molecules over time and whether there was a difference in activation between cell types. In view of multiple testing, a stringent P value <0.005 was used to indicate a significant result.
Direct sequencing of all coding exons of CUL7 identified pathogenic mutations in nine families: seven patients carried homozygous mutations and two patients had compound heterozygous mutations. Two mutations (c.3379_3380delTG, p.W1127Efs*38 and c.4451_4452delTG, p.V1484Gfs*68) have been previously described (Huber et al. 2009); however, the remaining nine mutations are novel: three frameshift, three missense, two splice-site and one nonsense mutation.
We also identified pathogenic mutations in OBSL1 in three families. All mutations were homozygous. One mutation has been previously described (c.1273dupA, p.T425Nfs*40) (Hanson et al. 2009) and two are novel mutations – one nonsense and one splice-site mutation (Table 2). None of the novel mutations identified in either CUL7 or OBSL1 were present in 210 normal chromosomes, supporting their pathogenicity. A single pathogenic mutation in CCDC8 was identified in one family; the mutation (c.612dupG, p.K205Efs*58) has been described previously (Hanson et al. 2011). The identification of mutations in CUL7, OBSL1 and CCDC8 and their absence in control samples confirmed the clinical diagnosis of 3-M syndrome.
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.2T>C, p.M1?; c.1234-2A>G||–||–|
|3M-5||England||c.3454G>A; p.E1152K, c.3937G>T; p.E1313*||–||–|
3M-ID, 3-M syndrome family identification; 3M-1-13, 3-M syndrome family reference number.
Growth and GH–IGF axis evaluation
Height at presentation was measured in 36 mutation-positive 3-M syndrome patients (including those above). The mean height SDS of 15 patients with CUL7 mutations was −5.8, a further 15 patients with OBSL1 mutations had a mean height SDS of −4.7 and six patients with CCDC8 mutations had a mean height of −4.1 SDS. Patients with CUL7 mutations are significantly shorter than 3-M syndrome patients with either OBSL1 or CCDC8 mutation (Fig. 1).
Peak GH during stimulation testing and basal IGF1 levels were measured in 11 and 13 patients respectively (Table 3). Ten patients had measurements for both peak GH and IGF1: eight had normal peak GH levels (≥7 μg/l) with either normal (one CUL7, one OBSL1 and one CCDC8) or low IGF1 (SDS <−2, four OBSL1 and 1 CCDC8) and two had low peak GH levels (peak GH ≤7 μg/l) with either normal (one OBSL1) or low IGF1 (one CCDC8). Serum IGFBP3 was not routinely measured; however, three patients (all OBSL1) were found to have levels either elevated above or within the upper normal range. Secreted IGFBP3 was measured by western blotting in the three 3-M cell lines, all have elevated IGFBP3 levels (three- to four-fold) with the highest seen in OBSL1−/− cells (Fig. 2).
Peak GH and basal IGF1 levels in 3-M syndrome patients. Peak serum GH (μg/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 (μg/l)||Peak GH range (μg/l)||Basal IGF1 SDS||Basal IGF1 range SDS|
|CUL7||10.7 (n=1)||–||−0.8 (n=3)||−0.2 to −1.8|
|OBSL1||19.7 (n=6)||3.7 to 38.3||−1.9 (n=7)||0.5 to −5|
|CCDC8||10.8 (n=4)||5.4 to 13.3||−2.0 (n=3)||−1.5 to −2.4|
Western blotting of secreted IGFBP2 from the same cells revealed that all 3-M cells have reduced IGFBP2 levels compared with control cells with CCDC8−/− cells having the lowest levels (Fig. 2). Intracellular protein levels of IGFBP5 were either normal or slightly elevated compared with control cells (Fig. 2). IGFBP7 was reduced in all 3-M syndrome cell lines with CUL7−/− and OBSL1−/− having the lowest levels (Fig. 2).
STAT5b and MAPK signalling in response to GH
Basal levels of IRS1, MAPK and AKT in 3-M cells were comparable with control cells (data not shown). Control cells showed maximal activation of MAPK at 5 min after GH exposure, as previously reported (Silva et al. 2002). Maximal STAT5b activation was also seen at 5 min post-stimulation. The levels of activation at 5 min in 3-M cells compared with the normal cells are shown in Fig. 3: CUL7−/− cells showed normal activation, while OBSL1−/− and CCDC8−/− cells had phospho-MAPK 77 and 50% of control levels and phospho-STAT5b levels 53 and 42% of control levels respectively (Fig. 3).
AKT signalling in response to IGF1
The pattern of AKT activation in normal cells showed a marked early activation at 5 min, sustained to 60 min. In the 3-M cells at 5 min, CUL7−/− and OBSL1−/− had reduced levels of phospho-AKT compared with normal (Figs 3 and 4), while CCDC8−/− cells showed normal levels. By 60 min, levels of phospho-AKT in CUL7−/− cells had decreased to baseline, while levels in OBSL1−/− and CCDC8−/− cells were ∼50% lower than in controls.
Published series of genetic analyses in 3-M syndrome indicate that the majority of cases are a result of CUL7 mutations (62/93) but a significant proportion are caused by OBSL1 mutations (26/93) (Huber et al. 2005, 2009, 2010, Maksimova et al. 2007, Hanson et al. 2009, 2011). We recently identified that a smaller subset of patients (5/93) have CCDC8 mutations; two distinct mutations have so far been identified but both result in generation of a premature termination codon and subsequent loss of CCDC8 (Hanson et al. 2011). We now describe the identification of a further nine novel mutations in CUL7 and two novel mutations in OBSL1; this includes the first splice site mutation found in OBSL1. The mutations found in CUL7 are interspersed throughout the whole gene. However, the new mutations in OBSL1 are found in the first eight exons in keeping with our previous findings, further suggesting that loss of the three main isoforms of OBSL1 is important in the pathogenicity of 3-M syndrome. The relative frequency of mutations in this study (69% CUL7, 23% OBSL1 and 8% CCDC8) reflects not only the previous experience but also the date of publication of the mutations – 2005, 2009 and 2011 respectively. These families have presented over the last 2 years and were screened for CUL7, OBSL1 and CCDC8 mutations. The different frequencies are, therefore, likely to reflect a true prevalence.
We hypothesise that the primary molecular mechanism responsible for the pathogenicity of 3-M syndrome will include targets of the CUL7-SCF E3 ligase or proteins that physically associate with CUL7, OBSL1 or CCDC8. We have previously shown that OBSL1 interacts with both CUL7 and CCDC8; however, CUL7 does not interact with CCDC8, which may suggest that OBSL1 plays a role as the structural component of the pathway. The importance of these interactions is unclear; however, we postulate that as the phenotypes of 3-M syndrome is similar regardless of mutation status, the associations between OBSL1/CUL7 and OBSL1/CCDC8 are required to maintain CUL7-SCF E3 ligase activity. Further exploration of this pathway is likely to define new proteins controlling mammalian growth and may help to identify new genes associated with small for gestational age (SGA) and short stature.
We have also hypothesised that the restricted childhood growth of 3-M patients could be related to a degree of insensitivity to GH and/or IGF1. This has been supported by two observations. First, the majority of 3-M children investigated in our unit have normal/high peak GH levels and low or normal IGF1 levels: normal GH and low IGF1 could be consistent with a degree of GH resistance, while normal GH and normal IGF1 in a very short child could be consistent with a degree of IGF1 resistance. Both interpretations would be concordant with previous descriptions of GH and IGF1 resistance (Rosenfeld & Hwa 2004, Walenkamp & Wit 2006). Secondly, the growth response to rhGH in 3-M syndrome, treated on the basis that these children are usually born SGA, was relatively poor. Fifteen 3-M patients showed no significant change in height SDS over the first year of rhGH treatment (Height SDS −4.4 at baseline vs −4 at 1 year post-treatment, P=0.4) (Murray et al. 2007, Clayton et al. 2012). Over 4 years of rhGH treatment, the increment in height SDS was <+1 compared with an increment of >+2 in rhGH-treated SGA children (Van Pareren et al. 2003).
In addition to possible GH/IGF resistance, dysregulation of IGFBP2 and IGFBP5 had previously been identified (Huber et al. 2010) with downregulation of IGFBP2 and upregulation of IGFBP5 expression seen in fibroblasts from two patients with OBSL1 mutations. By contrast, upregulation of IGFBP2 is seen in the Cul7−/− mouse (Tsutsumi et al. 2008). We now demonstrate that regardless of mutation status, all 3-M cell lines show reduced secreted protein levels of IGFBP2 and -7. Intracellular IGFBP5 levels were slightly elevated in 3-M cells. In this study, IGFBP3, the most abundant IGFBP in serum, is expressed at high levels in all three 3-M cell lines, and in the few patients in which serum levels were checked, IGFBP3 was above or within the upper end of the normal range for age. Elevated levels of IGFBP3 have previously been identified in Silver–Russell syndrome (SRS) fibroblast cells (Montenegro et al. 2012) along with high IGFBP3 serum levels in SRS patients (Binder et al. 2008). It, therefore, appears that disruption of IGFBP gene expression and altered levels of pericellular secreted proteins are features of 3-M syndrome. IGFBPs have a vital role in maintaining the availability of IGFs, and these abnormalities are likely to contribute to growth attenuation and response to rhGH in 3-M syndrome.
In order to further characterise responses to growth factors, we have taken an in vitro approach, assessing responses to GH and IGF1 in cell lines. Our studies have shown differences in activation of signalling molecules downstream of both GH and IGF1 receptors both between the three 3-M mutations and between the GH and IGF1 pathways. CUL7−/− cells show normal levels of activation of STAT5b and MAPK after rhGH stimulation, suggesting that there is normal signalling down both pathways. However, both CCDC8−/− and OBSL1−/− cells have moderately reduced levels of pSTAT5b and pMAPK, indicating some impairment of signalling down both pathways. A different pattern emerges for IGF1 signalling. AKT activation in CUL7−/− cells is significantly lower than in control cells and shows a different pattern with early reduced activation that then diminishes to basal levels (Fig. 3). OBSL1−/− cells show reduced AKT activation, maintained at the same level throughout the experiment, while CCDC8−/− cells show normal early activation of AKT but a later moderate reduction (Fig. 3). Thus, GH signalling is normal in CUL7−/− cells but IGF1 signalling is disrupted, suggesting possible IGF1 insensitivity. Interestingly, the one patient with a CUL7 mutation, on whom we have a GH–IGF assessment, had a normal peak GH level and IGF1 SDS within the normal range. By contrast, in CCDC8−/− cells, GH signalling is moderately reduced and early activation of IGF1 signalling is normal, a scenario more consistent with a degree of GH resistance, and in OBSL1−/− cells, both GH and IGF1 signalling are moderately diminished, which may suggest both partial GH and IGF1 resistance. Five OBSL1 patients had normal peak GH levels with three having low IGF1 and one normal IGF1.
The growth factor signalling data are contradictory to previous reports in Cul7−/− MEF cells, which after IGF1 stimulation showed increased AKT and MAPK activation. In addition, unlike Cul7−/− MEFs, we did not see an increase in basal levels of IRS1 (Xu et al. 2008). Cul7−/− mice, however, die shortly after birth and have vascular abnormalities (Arai et al. 2003), which are inconsistent with 3-M syndrome. Thus, it is not surprising that there are differences in signal transduction cascades downstream of the IGF1 and GH receptors between the human and mouse cells.
In our clinical study of response to rhGH, the mutation status of the patients was not known. This study indicates that an analysis of degree of height restriction, GH–IGF1 axis status, and response to rhGH versus mutation status should be undertaken. We would suggest that the signalling abnormalities we have described here could correlate with growth status. We have some evidence to support this in that patients with a CUL7 mutation, which is associated with the most significant IGF1 signalling abnormality, are shorter than those with either OBSL1 or CCDC8 mutations. It is likely that although the 3-M syndrome is primarily a disorder of ubiquitination, the downstream consequences can impact on GH–IGF1 signalling and affect response to GH therapy.
Declaration of interest
The authors declare no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Support from the National Institute of Health Research Manchester Biomedical Research Centre is acknowledged. During this study, P G M was a Medical Research Council (UK) Clinical Research Fellow.
The authors thank all healthcare professionals contributing to the care of families described here.
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(D Hanson and P G Murray contributed equally to this work)