- Email: [email protected]
P97 The identification of heterozygous CUL7 sequence variants in a cohort of children born with a low birth weight
Poster Presentations / Growth Hormone & IGF Research 20 (2010) S39–S81
content of the liver. Based on preliminary analysis of gene expression, it appears that GH and IGF-1 improve NAFLD via distinct mechanisms. This work was supported by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by NIH grants (DK075436, AG031736 and AG019899), by a grant from the AMVETS organization, by a grants from the Diabetes Research Initiative and the VP for Research at Ohio University. P96 Insulin and insulin-like growth factor ii induce differential endocytic sorting of the insulin receptor isoform A A. Morrione1 . 1 Urology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA The relative abundance of the two isoforms of insulin receptor (IR-A and IR-B) is strictly regulated by developmental and tissue-specific factors and it is important for IR-dependent biological events. IR-A not only binds insulin but also IGF-II and is often over-expressed in cancer cells. There is evidence that IGF-II and insulin stimulation of the IR-A may activate partially different intracellular signaling determining different biological effects. Endocytosis, a major mechanism of signal attenuation, is an integral part of signaling as it provides spatial and temporal regulation of signaling events. We hypothesized that insulin and IGF-II may differently affect IR-A biological effects by inducing differential IR-A trafficking. Using R− /IR cells, which are mouse embryo fibroblasts lacking the IGF-IR and expressing the IR-A, we demonstrated that insulin induced significant IR-A internalization, which was instead modestly affected by IGF-II stimulation. The difference in IR-A internalization was not due to receptor ubiquitination which was detectable at comparable levels in insulin and IGF-II stimulated R− /IR cells. In addition, prolonged stimulation of R− /IR cells with insulin but not IGF-II targeted the IR-A for degradation. Similarly to IR-A, IRS-1 was down-regulated after prolonged insulin stimulation while IGF-II did not significantly affected IRS-1 levels. Using pharmacological and siRNA approaches we then demonstrated that while both clathrin-dependent and independent pathways were required for IR-A internalization but the clathrindependent pathway was critical for insulin-mediated receptor degradation. We also found that inhibition of the clathrin-mediated pathway affected Akt phosphorylation, whereas ERK activation was more sensitive to inhibition of clathrin-independent pathways. In conclusion, our findings support the hypothesis that the lower affinity of IGF-II for the IR-A may on one hand promote lower IR-A phosphorylation and early activation of downstream effectors compared to insulin but may protect IR-A and IRS-1 from feed-back down-regulation mechanism thereby sustaining a more prolonged signaling and more potent mitogenic stimuli. P97 The identification of heterozygous CUL7 sequence variants in a cohort of children born with a low birth weight 1
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2
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E. Saunders , D. Hanson , A. Whatmore . 5th Floor research, Royal Manchester Children’s Hospital, Manchester, United Kingdom; 2 Research, 5th Floor Research, Manchester, United Kingdom Background: There are many causes for a child to be born small for gestational age (SGA) including placental, maternal and fetal factors. A small proportion will be due to genetic syndromes such as Silver Russell syndrome (SRS) and 3M syndrome. The latter is an autosomal recessive primordial growth disorder characterised by pre- and postnatal growth restriction, maxillary hypoplasia, prominent heels and a variable incidence of skeletal abnormalities such as: slender bones and relatively tall vertebral bodies.
S75
Missense and nonsense mutations in CUL7 account for the majority of 3M cases. The CUL7 protein forms an SCF (SKP-CUL7-FBX)-like ubiquitin ligase complex with FBXW8 and ROC1. Mutations in CUL7 are thought to disrupt the ubiquitination pathway and thus lead to disordered growth. It has been proposed that obligate 3M heterozygotes may also display growth restriction. We therefore hypothesized that the frequency of heterozygous CUL7 mutations could be greater in the SGA population compared to the background population. Methods: Genomic DNA from a cohort of SGA+/-IUGR (n = 59) and SRS (n = 33) children was analysed for the presence of CUL7 sequence variants using the denaturing high-performance liquid chromatography transgenomic WAVE® system which relies on heteroduplex formation to identify anomalous dsDNA. These were subsequently confirmed using genomic DNA sequencing and compared to a cohort of 345 controls obtained from the Genetic archive, National Genetics Reference Laboratories, Manchester UK. Results: 2 heterozygous in cis sequence variants in CUL7 were found in the cohort. The first was a 3 base pair (bp) deletion in exon 4 (c.1171_1173delGAG p.E390del). This would lead to a loss of a glutamate residue at position 390 within the CUL7-PARC-HERC2 (CPH) domain of CUL7. The CPH domain is vital for p53 interaction and the correct cytoplasmic localisation of CUL7. This deletion was detected in 2 patients in the cohort (n = 92) and in one of 345 controls (p = 0.117). The second was a missense variant in exon 24 (c.4463 T>C p.L1488P). This amino acid is located within the cullin domain of CUL7, essential for the interaction between ROC1 and CUL7 in the SCF complex. This variant was found in 2 patients in the cohort (n = 92) and was not detected in 96 controls (p = 0.243). This variant has since been described as a single nucleotide polymorphism (SNP ID: rs41274912) although currently no population frequency data are available. The 2 patients carrying the 3 bp deletion also carried the missense variant. This combination was not detected in 345 controls (p = 0.045). The first patient was a female born IUGR with a birth weight SDS of −2.4, the second was a female SRS patient with a birth weight SDS of −1.86. Conclusion: The prevalence of these 2 CUL7 variants in combination appears to be higher in this cohort than in normal controls, albeit at a low frequency (1.7% in the SGA/IUGR group and 3.0% in the SRS group). The functional significance of each variant alone and in combination needs investigation to assess their impact on ubiquitination. P98 Extreme body growth and protein accretion under normal serum IGF-I concentrations in male but not in female mice – new models and new challenges for the somatomedin hypothesis M. Sawitzky1 , U. Renne1 , M. Langhammer1 , J. Brenmoehl1 , A. Zeissler1 , M. Bielohuby2 , M. Bidlingmaier2 , A. Hoeflich1 . 1 Genetics and Biometry, FBN Dummerstorf, Dummerstorf, Germany; 2 Department of Medicine Inner City, LMU Munich, Munich, Germany According to the original somatomedin hypothesis somatic growth is mediated by IGF-I produced in the liver under control of growth hormone. Later on, by the development of single-gene modified animal models characterized by decreased serum IGF-I concentrations it was demonstrated that different tissues may be involved in somatic growth control or even IGF-binding protein through control of free versus bound IGF-I may have an impact on IGF activity. Here we introduce a novel type of mouse model which has been established by long term selection of male mice for high protein amount of the carcass at an age of 42 days after birth. The allelic composition of our mouse model was developed from an outbred background originally, characterized by high genetic variability. Since the selection period spans more than 30 years
content of the liver. Based on preliminary analysis of gene expression, it appears that GH and IGF-1 improve NAFLD via distinct mechanisms. This work was supported by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by NIH grants (DK075436, AG031736 and AG019899), by a grant from the AMVETS organization, by a grants from the Diabetes Research Initiative and the VP for Research at Ohio University. P96 Insulin and insulin-like growth factor ii induce differential endocytic sorting of the insulin receptor isoform A A. Morrione1 . 1 Urology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA The relative abundance of the two isoforms of insulin receptor (IR-A and IR-B) is strictly regulated by developmental and tissue-specific factors and it is important for IR-dependent biological events. IR-A not only binds insulin but also IGF-II and is often over-expressed in cancer cells. There is evidence that IGF-II and insulin stimulation of the IR-A may activate partially different intracellular signaling determining different biological effects. Endocytosis, a major mechanism of signal attenuation, is an integral part of signaling as it provides spatial and temporal regulation of signaling events. We hypothesized that insulin and IGF-II may differently affect IR-A biological effects by inducing differential IR-A trafficking. Using R− /IR cells, which are mouse embryo fibroblasts lacking the IGF-IR and expressing the IR-A, we demonstrated that insulin induced significant IR-A internalization, which was instead modestly affected by IGF-II stimulation. The difference in IR-A internalization was not due to receptor ubiquitination which was detectable at comparable levels in insulin and IGF-II stimulated R− /IR cells. In addition, prolonged stimulation of R− /IR cells with insulin but not IGF-II targeted the IR-A for degradation. Similarly to IR-A, IRS-1 was down-regulated after prolonged insulin stimulation while IGF-II did not significantly affected IRS-1 levels. Using pharmacological and siRNA approaches we then demonstrated that while both clathrin-dependent and independent pathways were required for IR-A internalization but the clathrindependent pathway was critical for insulin-mediated receptor degradation. We also found that inhibition of the clathrin-mediated pathway affected Akt phosphorylation, whereas ERK activation was more sensitive to inhibition of clathrin-independent pathways. In conclusion, our findings support the hypothesis that the lower affinity of IGF-II for the IR-A may on one hand promote lower IR-A phosphorylation and early activation of downstream effectors compared to insulin but may protect IR-A and IRS-1 from feed-back down-regulation mechanism thereby sustaining a more prolonged signaling and more potent mitogenic stimuli. P97 The identification of heterozygous CUL7 sequence variants in a cohort of children born with a low birth weight 1
1
2
1
E. Saunders , D. Hanson , A. Whatmore . 5th Floor research, Royal Manchester Children’s Hospital, Manchester, United Kingdom; 2 Research, 5th Floor Research, Manchester, United Kingdom Background: There are many causes for a child to be born small for gestational age (SGA) including placental, maternal and fetal factors. A small proportion will be due to genetic syndromes such as Silver Russell syndrome (SRS) and 3M syndrome. The latter is an autosomal recessive primordial growth disorder characterised by pre- and postnatal growth restriction, maxillary hypoplasia, prominent heels and a variable incidence of skeletal abnormalities such as: slender bones and relatively tall vertebral bodies.
S75
Missense and nonsense mutations in CUL7 account for the majority of 3M cases. The CUL7 protein forms an SCF (SKP-CUL7-FBX)-like ubiquitin ligase complex with FBXW8 and ROC1. Mutations in CUL7 are thought to disrupt the ubiquitination pathway and thus lead to disordered growth. It has been proposed that obligate 3M heterozygotes may also display growth restriction. We therefore hypothesized that the frequency of heterozygous CUL7 mutations could be greater in the SGA population compared to the background population. Methods: Genomic DNA from a cohort of SGA+/-IUGR (n = 59) and SRS (n = 33) children was analysed for the presence of CUL7 sequence variants using the denaturing high-performance liquid chromatography transgenomic WAVE® system which relies on heteroduplex formation to identify anomalous dsDNA. These were subsequently confirmed using genomic DNA sequencing and compared to a cohort of 345 controls obtained from the Genetic archive, National Genetics Reference Laboratories, Manchester UK. Results: 2 heterozygous in cis sequence variants in CUL7 were found in the cohort. The first was a 3 base pair (bp) deletion in exon 4 (c.1171_1173delGAG p.E390del). This would lead to a loss of a glutamate residue at position 390 within the CUL7-PARC-HERC2 (CPH) domain of CUL7. The CPH domain is vital for p53 interaction and the correct cytoplasmic localisation of CUL7. This deletion was detected in 2 patients in the cohort (n = 92) and in one of 345 controls (p = 0.117). The second was a missense variant in exon 24 (c.4463 T>C p.L1488P). This amino acid is located within the cullin domain of CUL7, essential for the interaction between ROC1 and CUL7 in the SCF complex. This variant was found in 2 patients in the cohort (n = 92) and was not detected in 96 controls (p = 0.243). This variant has since been described as a single nucleotide polymorphism (SNP ID: rs41274912) although currently no population frequency data are available. The 2 patients carrying the 3 bp deletion also carried the missense variant. This combination was not detected in 345 controls (p = 0.045). The first patient was a female born IUGR with a birth weight SDS of −2.4, the second was a female SRS patient with a birth weight SDS of −1.86. Conclusion: The prevalence of these 2 CUL7 variants in combination appears to be higher in this cohort than in normal controls, albeit at a low frequency (1.7% in the SGA/IUGR group and 3.0% in the SRS group). The functional significance of each variant alone and in combination needs investigation to assess their impact on ubiquitination. P98 Extreme body growth and protein accretion under normal serum IGF-I concentrations in male but not in female mice – new models and new challenges for the somatomedin hypothesis M. Sawitzky1 , U. Renne1 , M. Langhammer1 , J. Brenmoehl1 , A. Zeissler1 , M. Bielohuby2 , M. Bidlingmaier2 , A. Hoeflich1 . 1 Genetics and Biometry, FBN Dummerstorf, Dummerstorf, Germany; 2 Department of Medicine Inner City, LMU Munich, Munich, Germany According to the original somatomedin hypothesis somatic growth is mediated by IGF-I produced in the liver under control of growth hormone. Later on, by the development of single-gene modified animal models characterized by decreased serum IGF-I concentrations it was demonstrated that different tissues may be involved in somatic growth control or even IGF-binding protein through control of free versus bound IGF-I may have an impact on IGF activity. Here we introduce a novel type of mouse model which has been established by long term selection of male mice for high protein amount of the carcass at an age of 42 days after birth. The allelic composition of our mouse model was developed from an outbred background originally, characterized by high genetic variability. Since the selection period spans more than 30 years