An activating variant in CTNNB1 is associated with a sclerosing bone dysplasia and adrenocortical neoplasia
Hui Peng1,2, Zandra A. Jenkins2, Ruby White2, Sam Connors2, Matthew F. Hunter3,4, Anne Ronan5, Andreas Zank|6,7,8, David M. Markie9, Philip B. Daniel2, Stephen P. Robertson2
Affiliations:
1Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital of Central South University, Changsha, Hunan, China;
Otago, Dunedin 9016, New Zealand;
3 Monash Genetics, Monash Medical Centre, Melbourne, VIC, Australia·
4 Department of Paediatrics, Monash University, Melbourne, VIC, Australia.
5 Hunter Genetics, Newcastle, New South Wales, Australia
6 Department of Clinical Genetics, The Children’s Hospital at Westmead, Sydney, New South Wales, Australia.
7Discipline of Genomic Medicine, Sydney Medical School, The University of Sydney, Camperdown, New South Wales, Australia.
8Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.
9 Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand.
* Correspondence:
Professor Stephen Robertson, Department of Women’s and Children’s Health, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand.
E-mail: stephen.robertson@otago.ac.nz.
This work was supported by Curekids NZ and the China Scholarship Council.
Disclosure Summary: The authors have nothing to disclose.
Accepted M-Jetora Ul Sourcesar pt
Abstract
Context. The WNT/B-catenin pathway is central to the pathogenesis of various human diseases including those affecting bone development and tumour progression.
Objective. To evaluate the role of a gain-of-function variant in CTNNB1 in a child with a sclerosing bone dysplasia and an adrenocortical adenoma.
Design. Whole exome sequencing with corroborative biochemical analyses.
Patients. We recruited a child with a sclerosing bone dysplasia and an adrenocortical adenoma together with her unaffected parents.
Intervention. Whole exome sequencing and performance of immunoblotting and luciferase- based assays to assess the cellular consequences of a de novo variant in CTNNB1.
Main Outcome Measure(s)/Result. A de novo variant in CTNNB1 (c.131C>T; p.[Pro44Leu]) was identified in a patient with a sclerosing bone dysplasia and an adrenocortical adenoma. A luciferase-based transcriptional assay of WNT signalling activity verified that the activity of B-catenin was increased in the cells transfected with a CTNNB1P.Pro44Leu construct (p =4.00E-05). The ß-catenin p.Pro44Leu variant was also associated with a decrease in phosphorylation at Ser45 and Ser33/Ser37/Thr41 in comparison to a wild type (WT) CTNNB1 construct (p = 2.16E-03, p = 9.34E-08 respectively).
Conclusion. Increased ß-catenin activity associated with a de novo gain-of-function CTNNB1 variant is associated with osteosclerotic phenotype and adrenocortical neoplasia.
Summary
A de novo likely pathogenic variant in CTNNB1 is associated with osteosclerosis, an adrenal adenoma and activation of WNT signalling through the stabilisation of ß-catenin protein.
Accepted Manuscript
Downloaded from https://academic.oup.com/jcem/advance-article-abstract/doi/10.1210/clinem/dgaa034/5714342 by University of Cambridge user on 04 February 2020
Introduction
The WNT/B-catenin pathway is indispensable for normal bone biology and development1,2. Many studies have demonstrated that pathogenic variants occurring in genes encoding components of the WNT signaling pathway are involved in hereditary syndromes associated with bone defects3-5. The first demonstrated link between abnormal WNT activity and bone phenotypes was the demonstration that pathogenic variants in LRP5 cause osteoporosis pseudoglioma syndrome (OPPG) by a loss-of-function mechanism. Subsequently it was shown that high bone mass diseases result from gain-of-function pathogenic variants in LRP5 6-8. Subsequently, loss of function pathogenic variants in SOST which encodes a WNT antagonist were shown to result in a similar high bone mass phenotype3,9. Additional WNT-sequestering antagonists like SFRP1, SFRP2 and SFRP4, WNT ligands such as WNT1, WNT2 and WNT3, and components of the dual receptor complex comprised of frizzled (FZD) and either hypermorphic LRP5 and LRP6 alleles or hypomorphic LRP4 alleles result in bone dysplasias characterized by abnormal bone density3,10-14. Additionally, our previous work also found that germline pathogenic variants in AMER1, a suppressor of WNT signaling, gives rise to an X-linked sclerosing bone dysplasia, osteopathia striata congenita with cranial sclerosis (OSCS)15,16.
CTNNB1 encodes ß-catenin, which is an important transcriptional co-activator and the final common mediator of the canonical WNT/B-catenin pathway, playing a central role in tissue development and tumorigenesis in some forms of cancer1-3,17. Monoallelic loss-of-function germline pathogenic variants in CTNNB1 have been found to be associated with non- skeletal phenotypes including intellectual disability, autism spectrum disorder and ophthalmological diseases18-21. Notably, mice in which Ctnnb1 has been conditionally inactivated in osteoblasts manifest a low bone mass phenotype through selectively targeting B-catenin degradation22-24, while conditionally active Ctnnb1 alleles expressed within the
osteoblastic lineage result in a high bone mass phenotype25,26. Direct evidence however is lacking that gain-of-function mutations in ß-catenin confers a similar effect in humans.
Somatic gain-of-function pathogenic variants in CTNNB1 have been identified in a variety of tumors27, and have been observed as recurrent events in adrenocortical adenoma and carcinoma28,29. Most of these missense pathogenic variants substitute residues within the consensus N-terminal phosphorylation Ser33/Ser37/Thr41/Ser45 cluster or at sites immediately adjacent to them. These residues are regarded as “hotspots” for pathogenic variants because they act to stabilise ß-catenin, indicative of the role that phosphorylation plays in the proteosomal inactivation of this protein27,29,30. However, there is no precedent in the literature for germilne genetic alterations in CTNNB1 to confer a predisposition to cancer.
A large number of studies have demonstrated that pathogenic variants in exon 3 of the CTNNB1 leading to the substitution of serine/threonine residues are the most frequent genetic mechanism by which WNT signalling is activated5,9,31,32. In the absence of WNT activation, cytosolic ß-catenin is regulated by APC/Axin destruction complex by a phosphorylation-based mechanism mediated by GSK-3B (glycogen synthase kinase 3ß) and CK1g33,34. CK1a initially phosphorylates Ser45 on ß-catenin, an event which further primes sequential phosphorylation of Thr41/Ser37/Ser33 by GSK-3035,36. This chain reaction of phosphorylation results in the proteosomal degradation of the protein and forms the primary mechanism by which cellular ß-catenin levels are regulated5. Some cancer-associated somatic pathogenic variants occur at Ser45, Thr41, Ser37 and Ser33, and have been shown to disrupt the sequential phosphorylation of B-catenin and thereby lead to the activation of WNT signaling.
In this study, whole exome sequencing analysis of a female with a sclerosing skeletal dysplasia and a congenital adrenocortical adenoma detected a de novo variant in CTNNB1. Biochemical data support the hypothesis that this de novo variant activates WNT/ ß-catenin signaling pathway by inhibiting phosphorylation of ß-catenin. These findings directly support the current understanding that ß-catenin is a central mediator of mineralization of bone tissue during development in humans.
Material and Methods
Whole Exome Sequencing analysis
Genomic DNA was collected and extracted from peripheral whole blood samples according to standard protocols. DNA was enriched with exonic regions with Agilent SureSelect Human All Exon V5+UTR Capture Kit for all samples. Sequencing was performed with 106 bp paired-end reads on the Illumina HiSeq 2000 sequencing platform. Sequence alignment was performed with the Burrows Wheeler Aligner (BWA) using human exome assembly GRCh37 as the reference with variant calling consistent with GATK Best Practice recommendations. Mean coverage ranged from 70X to 85X, and >96% of the exome had a minimum depth of 10X for all samples. Annotation of gene context was carried out with SnpEff and other annotations were added using bcftools. Variants with an allele frequency less than 0.01 in the genome aggregation database (gnomAD) and with a total read depth greater than 10, where at least 20% of the reads comprised the variant allele and a genotype quality (GQ) of greater than 50, were selected. Variants in this subset that were present in the genome of the child but absent in both parents, and had a predicted effect on protein sequence (truncating or missense) were then retained for further analysis.
The region of CTNNB1 containing the variant of interest in this study was amplified by PCR and Sanger sequencing was carried out to confirm the presence of the candidate variant in the proband and its absence in samples obtained from her parents.
Generation of Constructs
HEK293FT cells were purchased from Life Technologies. The full coding sequence of CTNNB1 was amplified from HEK293FT cDNA by RT-PCR and cloned into 3XFLAG vector pCMV7.1 with the TOPO cloning kit (Thermo Fisher). CTNNB1 was subsequently subcloned into EcoRI- and Notl-restriction-digested p3xFLAG-CMV with the Gibson assembly kit (New England Biolabs).
Western Blotting
Hele lek Man been deleted
For quantification of phosphorylated ß-catenin, HEK293FT cells were transfected with 800 ng p3XFLAG ß-catenin (WT, p.Ser45Ala, or p.Pro44Leu) and Lipofectamine2000 in a 24- well plate. After 24 h, cells were lysed and denatured in 1x denaturing loading buffer and followed by sonication. Proteins were resolved by electrophoresis on 5.5% polyacrylamide gels, followed by transfer to nitrocellulose membrane. Membranes were blocked in Odyssey TBS buffer (LI-COR) and probed overnight with anti-FLAG (F1804, Sigma Aldrich; 1:7000), anti-phospho-ß-catenin (Ser45) (1:3000, Cell Signaling Technology, CST9564;), or anti- phospho-ß-catenin (Ser33/37/Thr41) (1:1000, Cell Signaling Technology, CST9561). Detection was carried out with secondary goat anti-mouse IRDye 800CW (1:25,000, LI- COR, 926- 32210) and goat anti-rabbit IRDye 700CW (1:25,000, LI-COR, 926-68071).
Immunohistochemistry
Surgical specimens and autopsy samples were fixed in formalin and embedded in paraffin for routine histology and immunohistochemistry. Immunostaining was performed on an automated platform (Leica Aperio Scanscope USA) using Recombinant anti-ß-catenin antibody (1/500, ab32572) as the primary antibody and 3’3’-diaminobenzadine (DAB) for histochemical detection.
Luciferase assay
Luciferase assays were performed in C2C12 cells, seeded in 24 well plates and transfected with 80ng of reporter plasmid (TOPflash), 10ng of control plasmid (pSV-RL), and 10ng of empty or expression vector per well using Lipofectamine 2000 (0.6 ml per well). Transfected cells were harvested 20hrs later in 100ul passive lysis buffer (Promega) and assayed for Renilla and firefly luciferase activities using a Synergy2 multi-mode reader (BioTek) and injector module, which delivered the respective luciferase assay buffers (Promega). Results were obtained from 5 independent experiments.
Statistical Analysis
Quantitative data from western blots and luciferase assays was imported into Excel and scaled and normalized to appropriate controls. Two-way, unpaired t-tests were performed; critical p-values were Bonferroni corrected and expressed as follows: * p≤0.01, ** p ≤0.001.
Results
A CTNNB1 variant is associated with osteosclerosis and adrenocortical adenoma.
We recruited a family in which a female child had a generalised sclerosing bone dysplasia and a history of childhood adrenocortical tumour. Her parents were not affected and she had no siblings (Figure 1A). She was born after a pregnancy complicated by gestational diabetes and a caesarean section at 38 weeks gestation. At birth she had macrosomia and macrocephaly (weight 4.0kg (94th PC); length 50cm (68th PC); head circumference 38cm (2cm > 95th PC)). Her head circumference has remained well above the 98th PC since birth. Her height has tracked along the 75th PC and weight has tracked on the 90th PC. She developed genu valgum by 3 years of age and walked with a knock-kneed gait. Orthopaedic review at 3 years detected sclerotic bones in the lower limbs, prompting referral to genetics. Her neurodevelopmental milestones were reached within a normal time frame and currently (at age 10 years) she has normal learning and academic ability at school. She was mildly virilised at birth and an abdominal ultrasound detected an adrenal mass. She developed further virilisation by 2 years of age and underwent an adrenalectomy for a congenital adrenal cortical tumour. Her features of virilisation returned to normal by 4 years of age. No Bomthat milk with deltakere germline pathogenic variants in TP53 were detected. At assessment at age 5 years, her phenotype included macrocephaly, wide set eyes, depressed nasal bridge, left-sided choanal atresia, submucous cleft palate with a bifid uvula, low set and externally rotated ears, delayed exfoliation of primary dentition, single palmar crease, and a boot shaped but otherwise normal heart. A full skeletal survey revealed a diffuse dense bone phenotype in the pelvis, long bones and skull, especially around the skull base (Figure 1B). Radiographs of the skulls of both parents did not demonstrate any evidence of bony sclerosis.
Whole exome sequencing was performed on the proband and her parents. After filtering to remove all variants that were either (1) low quality, (2) had an allele frequency greater than
0.01 as represented in the in the 1000 Genomes database and Exome Variant Server, (3) did not affect protein coding sequences to alter encoded amino acids or led to premature truncation of translation, (4) were not represented in in-house databases of variants identified on the same platform or (5) affected canonical splice sites, we tested three segregation models to identify variant(s) that might explain our clinical observations. Under an initial hypothesis that a de novo variant might be causative of this phenotype, we identified a single missense variant, c.131C>T, in CTNNB1 (GenBank: NM_001904.4), which was predicted to result in the substitution to leucine at site Pro44 in the N-terminal domain of ß-catenin (GenBank: NP_001895.1; Figure 2A). No other de novo variants were identified.
Of the variants identified under other models of inheritance that included compound heterozygousity (n = 1; biallelic variants in SYCP2) or homozygousity (n = 0) for potentially pathogenic variants and X-linked recessive variants (n = 0), we identified no potentially pathogenic genotypes that segregated appropriately or represented biologically plausible explanations for the observed phenotype. The filtered data is listed in Supplemental Table 1 which has been uploaded in an online repository37.
The variant in CTNNB1 is not represented in gnomAD. No similar matches to either this genotype or similar phenotypes were obtained through use of the VariantMatcher (https://variantmatcher.org) or GeneMatcher (https://genematcher.org) protocols (as of Jan 14, 2020). Additionally, the case was shared with members of the International Skeletal Dysplasia Society through a listserver but no similar instances were forthcoming.
For the de novo variant in CTNNB1, genomic evolutionary rate profiling [GERP] gave a score of 5.91 indicating that the p.Pro44Leu substitution occurs at a phylogenetically highly conserved site in both vertebrates and invertebrates (Figure 2B). The predicted effect of this missense variant using different variant effect prediction programmes was deemed either disease-causing (MutationTaster score of 0.99), extremely damaging (scaled CADD score of 28.9) or damaging (MPC score of 2.21).
lot tal , le
Given the primacy of WNT signalling in both bone and adrenal tumour development these data suggested that this de novo variant in CTNNB1 could be responsible for the observed this patient. Due to a lack of prethe development of the adrenocortical adenoma in
de novos functional studies were required to strengthen the case for pathogenicity of this novo variant.
The p.Pro44Leu substitution in @-catenin alters its stabilization.
Previously monoallelic germline loss-of-function alleles in CTNNB1 have been shown to lead to intellectual disability and ophthalmological phenotypes while gain-of-function variants in Ctnnb1 in mice lead to osteosclerosis25,26. This suggests that the CTNNB1 p.Pro44Leu variant being studied here might exert a gain-of-function congruent with the known functions of WNT signalling in bone anabolism. The phosphorylation of residues in the N-terminus of B-catenin plays a pivotal role in the stability of the protein and hence its activity35. Although the substituted residue Pro44 lies within a cluster of serine and threonine residues that are phosphorylated to this end, there is no existing evidence that Pro44 itself is critical for this process. Thus, we questioned whether the Pro44Leu variant could inhibit phosphorylation of target residues in the N-terminus of ß-catenin, stabilising it and thereby activating ß-catenin activity.
We transfected ß-catenin expression constructs specifying the variants p.Ser45Ala (which oblates the priming phosphorylation site in ß-catenin) and p.Pro44Leu into HEK293FT cells and assayed for phosphorylation of the expressed tagged proteins using phospho-specific antibodies. Western analysis revealed that phosphorylation of ß-catenin at Ser45 was ablated by the p.Ser45Ala substitution (p = 2.25E-04, unpaired t test; Figure 3A). In cells transfected with p.Pro44Leu there was less phosphorylation at Ser45 than that observed after transfection with a control WT B-catenin construct (p = 2.16E-03; unpaired t test; Figure 3A) consistent with the suggestion that the Pro44Leu substitution affects phosphorylation at the adjacent Ser45 residue.
We next sought evidence that the inhibition of Ser45 phosphorylation that the Pro44Leu substitution confers on ß-catenin also affects the downstream consequential phosphorylation of residues Ser33/Ser37/Thr41. Using a phospho-specific antibody for these phosphorylation sites we observed a significant reduction in phosphorylation at Ser33/Ser37/Thr41 in ß- catenin proteins harbouring the positive control (Ser45Ala) and variant (Pro44Leu) substitutions (p = 1.11E-07, and p = 9.34E-08 respectively; unpaired t test; Figure 3B). Together, these data are consistent with the proposition that the Pro44Leu substitution significantly inhibits both the priming and sequential phosphorylation of ß-catenin, an action that is predicted to lead to an increase in cellular @-catenin activity.
Considering the data that the p.Pro44Leu substitution reduces the phosphorylation of B- catenin and hence increases its stability, we performed a luciferase-based transcription assay to measure ß-catenin sponsored transcriptional activity in the presence of the Pro44Leu variant. The result showed a significant increase in luciferase activity in cells transfected with Ser45Ala and Pro44Leu variants compared to the WT construct, indicating
an increase in ß-catenin activity (p = 8.25E-06 and p =4.00E-05 respectively; unpaired t test; Figure 3C).
Previous studies have revealed that ß-catenin is located at the cell membrane of cells in the normal adrenal gland, although immunostainable ß-catenin is absent in the cell cytoplasm and nucleus38. In contrast, cytoplasmic and/or nuclear accumulation of ß-catenin correlate with activation of the WNT signalling pathway in adrenal tumours29. We performed immunohistochemistry to ascertain the localization ß-catenin on sections of the adrenal adenoma tissue that was resected from the patient. Adopting the scoring system described by Bonnet et al29, ß-catenin immunostaining in this tumor showed some diffuse nuclear staining in a small fraction of cells (<1% cells) but consistent cytoplasmic staining in the majority (>90%) of cells (Figurcells (Figure 3Di, 3Dii) suggesting ß-catenin activation.
Discussion
We have shown that a de novo missense variant in exon 3 of CTNNB1 is associated with osteosclerosis and an adrenocortical adenoma in a single child. Although the variant was only characterised in blood cells and therefore could conceivably be mosaic in this individual, the widespread nature of the phenotype (generalised skeletal dysplasia, craniofacial anomalies, adrenal tumour) indicates that if present the degree of mosaicism for the de novo CTNNB1 allele must be high. Transfection-based experiments demonstrated results consistent with the conclusion that the Pro44Leu substitution inhibited both the priming and sequential phosphorylation of ß-catenin protein and hence is predicted to confer a stabilizing effect on the protein. Additionally, this variant is also associated with a dramatically increased transcriptional activity sponsored by ß-catenin.
Germline de novo mutations in CTNNB1 cause intellectual disability, microcephaly and additional individualized clinical presentations19,20,39. In these studies, the mutational spectrum included nonsense, splice site, frameshift mutations, and whole gene deletions indicating that a haploinsufficient mechanism is operative in these instances. The data presented here differentiate these CTNNB1 alleles from the Pro44Leu allele by demonstrating that it confers gain-of-function properties to the ß-catenin protein.
The individual described here presents with a pronounced osteosclerotic disorder. Interestingly, there are some overlapping phenotypic features with individuals with Osteopathia Striata with Cranial Sclerosis (OSCS, OMIM: 300373), which is characterized by macrocephaly, a sclerosing bone dysplasia and cleft palate. OSCS is caused by germline whole gene deletions or truncating variants in the AMER116,40. AMER1 is a negative regulator of the WNT/B-catenin pathway, and loss of AMER1 function therefore results in an inappropriate activation of the WNT/B-catenin pathway, resulting in a sclerosing bone dysplasia. Similarly, gain-of-function pathogenic variants in LRP5, a key regulator in WNT signalling pathway, are associated with high bone mass (HBM)8. Conversely, defective WNT signalling leads to fragile low density bone41. Previously, we identified pathogenic variants in DVL1, a mediator of both canonical and non-canonical Wnt signaling, as the cause of a subtype involving osteosclerosis of Robinow syndrome (RS)42. The RS phenotype overlaps with that observed in the individuals described here, including macrocephaly, wide set eyes, depressed nasal bridge and cleft palate with a bifid uvula42,43. Given that WNT signalling is central to bone homeostasis and activation of WNT signalling pathway can lead to other abnormal hyperostotic phenotypes, we suggest that the variant in CTNNB1 described here might be the cause of the observed osteosclerosis.
Many studies have reported evidence for WNT pathway activation in adrenocortical tumorigenesis29,32,44. Around 27% of adrenocortical adenomas (ACAs), and 31% of adrenocortical carcinomas (ACCs) harbour a somatically-acquired N-terminal activating ß- catenin pathogenic variant as the basis for this increased WNT signalling activity44. Some substitutions at Pro44 are present in the database of Catalogue of Somatic Mutations in Cancer (COSMIC: https://cancer.sanger.ac.uk/cosmic). As presented here, aberrant WNT/ß- catenin stabilisation and activation result from such pathogenic variants45.
Although a single case cannot be considered as a proof of causality, it was well described that variants in CTNNB1 predispose to adrenocortical tumorigenesis. It is unclear if this at other sites such as the colon5,46, and further tumours, either in the adrenal gland or the appropriateness and form of clinical screening for such eventualities remain to be precisely defined.
Acknowledgments
The authors would like to thank the family for their participation in this study. Hui Peng is funded by the China Scholarship Council; The work undertaken in New Zealand was supported by Curekids NZ.
Data availability
All data generated or analyzed during this study are included in this published article or are listed in an online repository37.
References
1. Clevers H. Wnt/B-Catenin signaling in development and disease. Cell. 2006;127(3):469-480.
2. Clevers H, Nusse R. Wnt/ß-catenin signaling and disease. Cell. 2012;149(6):1192- 1205.
3. Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med. 2013;19(2):179-192.
4. Baron R, Rawadi G. Minireview: Targeting the Wnt/B-catenin pathway to regulate bone formation in the adult skeleton. Endocrinology. 2007;148(6):2635-2643.
5.
and diseases. Dev Cell. 2009;17(1):9-26.
6. Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med. 2002;346(20):1513-1521.
7. Bonewald L. The holy grail of high bone mass. Nat Med. 2011;17(6):657-658.
8. Cui Y, Niziolek PJ, MacDonald BT, Zylstra CR, Alenina N, Robinson DR, Zhong Z, Matthes S, Jacobsen CM, Conlon RA, Brommage R, Liu Q, Mseeh F, Powell DR, Yang QM, Zambrowicz B, Gerrits H, Gossen JA, He X, Bader M, Williams BO, Warman ML, Robling AG. Lrp5 functions in bone to regulate bone mass. Nat Med. 2011;17(6):684-691.
9. Gregson CL, Wheeler L, Hardcastle SA, Appleton LH, Addison KA, Brugmans M, Clark GR, Ward KA, Paggiosi M, Stone M, Thomas J, Agarwal R, Poole KE, McCloskey E, Fraser WD, Williams E, Bullock AN, Davey Smith G, Brown MA, Tobias JH, Duncan EL. Mutations in known monogenic high bone mass loci only explain a
small proportion of high bone mass cases. J Bone Miner Res. 2016;31(3):640-649.
10. Nusse R, Clevers H. Wnt/B-Catenin signaling, disease, and emerging therapeutic modalities. Cell. 2017;169(6):985-999.
11. .Moon RT, Kohn AD, Ferrari GV De, Kaykas A. WNT and ß-catenin signalling: diseases and therapies. Nat Rev Genet. 2004;5(9):691-701.
12. Laine CM, Joeng KS, Campeau PM, Kiviranta R, Tarkkonen K, Grover M, Lu JT, Pekkinen M, Wessman M, Heino TJ, Nieminen-Pihala V, Aronen M, Laine T, Kröger H, Cole WG, Lehesjoki A-E, Nevarez L, Krakow D, Curry CJR, Cohn DH, Gibbs RA, Lee BH, Mäkitie O. WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta. N Engl J Med. 2013;368(19):1809-1816.
13. Leupin O, Piters E, Halleux C, Hu S, Kramer I, Morvan F, Bouwmeester T, Schirle M, Bueno-lozano M, Fuentes JR, Itin PH, Boudin E, Freitas F De, Jennes K, Brannetti B, Charara N, Ebersbach H, Geisse S, Lu CX, Bauer A, Hul W Van, Kneissel M. Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function. J Biol Chem. 2011;286(22):19489-19500.
14. Whyte MP, McAlister WH, Zhang F, Bijanki VN, Nenninger A, Gottesman GS, Lin EL, Huskey M, Duan S, Dahir K, Mumm S. New explanation for autosomal dominant high bone mass: Mutation of low-density lipoprotein receptor-related protein 6. Bone 2019;127:228-243.
15. Jenkins ZA, van Kogelenberg M, Morgan T, Jeffs A, Fukuzawa R, Pearl E, Thaller C, Hing A V, Porteous ME, Garcia-Miñaur S, Bohring A, Lacombe D, Stewart F, Fiskerstrand T, Bindoff L, Berland S, Adès LC, Tchan M, David A, Wilson LC, Hennekam RCM, Donnai D, Mansour S, Cormier-Daire V, Robertson SP. Germline mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis. Nat Genet. 2009;41(1):95-100.
16. Herman SB, Holman SK, Robertson SP, Davidson L, Taragin B, Samanich J. Severe osteopathia striata with cranial sclerosis in a female case with whole WTX gene deletion. Am J Med Genet Part A. 2013;161(3):594-599.
17. Gao C, Wang Y, Broaddus R, Sun L, Xue F, Zhang W. Exon 3 mutations of CTNNB1 drive tumorigenesis: a review. Oncotarget. 2018;9(4):5492-5508.
18. Tucci V, Kleefstra T, Hardy A, Heise I, Maggi S, Willemsen MH, Hilton H, Esapa C, Simon M, Buenavista M-T, McGuffin LJ, Vizor L, Dodero L, Tsaftaris S, Romero R, Nillesen WN, Vissers LELM, Kempers MJ, Vulto-van Silfhout AT, Iqbal Z, Orlando M, Maccione A, Lassi G, Farisello P, Contestabile A, Tinarelli F, Nieus T, Raimondi A, Greco B, Cantatore D, Gasparini L, Berdondini L, Bifone A, Gozzi A, Wells S, Nolan PM. Dominant ß-catenin mutations cause intellectual disability with recognizable syndromic features. J Clin Invest. 2014;124(4):1468-1482.
ka esat estar a
19. Sun W, Xiao X, Li S, Jia X, Wang P, Zhang Q. Germline mutations in CTNNB1 associated with syndromic FEVR or Norrie disease. Investig Opthalmology Vis Sci. 2019;60(1):93-97.
20. Kharbanda M, Pilz DT, Tomkins S, Chandler K, Saggar A, Fryer A, Mckay V, Louro P, Smith JC, Burn J, Kini U, De Burca A, FitzPatrick DR, Kinning E. Clinical features associated with CTNNB1 de novo loss of function mutations in ten individuals. Eur J Med Genet. 2017;60(2):130-135.
21. Panagiotou ES, Sanjurjo Soriano C, Poulter JA, Lord EC, Dzulova D, Kondo H, Hiyoshi A, Chung BH-Y, Chu YW-Y, Lai CHY, Tafoya ME, Karjosukarso D, Collin RWJ, Topping J, Downey LM, Ali M, Inglehearn CF, Toomes C. Defects in the cell signaling mediator ß-Catenin cause the retinal vascular condition FEVR. Am J Hum Genet. 2017;100(6):960-968.
22. Grigoryan T, Wend P, Klaus A, Birchmeier W. Deciphering the function of canonical
Wnt signals in development and disease: conditional loss- and gain-of-function mutations of ß-catenin in mice. Genes Dev. 2008;22(17):2308-2341.
23. Song L, Liu M, Ono N, Bringhurst FR, Kronenberg HM, Guo J. Loss of wnt/ß-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes. J Bone Miner Res. 2012;27(11):2344-2358.
24. Bullock WA, Hoggatt A, Horan DJ, Lewis K, Yokota H, Hann S, Warman ML, Sebastian A, Loots GG, Pavalko FM, Robling AG. Expression of a degradation- resistant ß-catenin mutant in osteocytes protects the skeleton from mechanodeprivation-induced bone wasting. J Bone Miner Res. June 2019. doi:10.1002/jbmr.3812.
25. Jia M, Chen S, Zhang B, Liang H, Feng J, Zong Z. Effects of constitutive ß-catenin activation on vertebral bone growth and remodeling at different postnatal stages in mice. PLOS One. 2013;8(9):1-13.
26. Glass DA, Bialek P, Ahn JD, Starbuck M, Patel MS, Clevers H, Taketo MM, Long F, McMahon AP, Lang RA, Karsenty G. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 2005;8(5):751-764.
27. Ilyas M. Wnt signalling and the mechanistic basis of tumour development. J Pathol. 2005;205(2):130-144.
28. Barker N, Clevers H. Catenins, Wnt signaling and cancer. BioEssays. 2000;22(11):961-965.
29. Bonnet S, Gaujoux S, Launay P, Baudry C, Chokri I, Ragazzon B, Libé R, René- Corail F, Audebourg A, Vacher-Lavenu M-C, Groussin L, Bertagna X, Dousset B, Bertherat J, Tissier F. Wnt/B-Catenin pathway activation in adrenocortical adenomas is frequently due to somatic CTNNB1-activating mutations, which are associated with larger and nonsecreting tumors: a study in cortisol-secreting and -nonsecreting
tumors. J Clin Endocrinol Metab. 2011;96(2):419-426.
30. Kim S, Jeong S. Mutation hotspots in the ß-Catenin gene: lessons from the human cancer genome databases. Mol Cells. 2019;42(1):8-16.
31. Rebouissou S, Franconi A, Calderaro J, Letouzé E, Imbeaud S, Pilati C, Nault J-C, Couchy G, Laurent A, Balabaud C, Bioulac-Sage P, Zucman-Rossi J. Genotype- phenotype correlation of CTNNB1 mutations reveals different ß-catenin activity associated with liver tumor progression. Hepatology. 2016;64(6):2047-2061.
32. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275(5307):1787-1790.
33.
J. 2012;31(12):2714-2736.
34. Verheyen EM, Gottardi CJ. Regulation of Wnt/ß-catenin signaling by protein kinases. Dev Dyn. 2009;239(1):34-44.
35. Liu C, Li Y, Semenov M, Han C, Baeg G, Tan Y, Zhang Z, Lin X, He X. Control of ß- Catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002;108(6):837-847.
36. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev. 2002;16(9):1066-1076.
37. Peng H, Jenkins ZA, White R, Connors S, Hunter MF, Ronan A, Zankl A, Markie DM , Daniel PB , Robertson SP. Data from: An activating variant in CTNNB1 is associated with a sclerosing bone dysplasia and adrenocortical neoplasia. Figshare 2019. Deposited 24 Sep 2019. 10.6084/m9.figshare.9894425.
38. Tadjine M, Lampron A, Ouadi L, Bourdeau I. Frequent mutations of ß-cateningene in sporadic secreting adrenocortical adenomas *. Clin Endocrinol. 2008;68(2):264-270.
39. Kuechler A, Willemsen MH, Albrecht B, Bacino CA, Bartholomew DW, van Bokhoven H, van den Boogaard MJH, Bramswig N, Büttner C, Cremer K, Czeschik JC, Engels H, van Gassen K, Graf E, van Haelst M, He W, Hogue JS, Kempers M, Koolen D, Monroe G, de Munnik S, Pastore M, Reis A, Reuter MS, Tegay DH, Veltman J, Visser G, van Hasselt P, Smeets EEJ, Vissers L, Wieland T, Wissink W, Yntema H, Zink AM, Strom TM, Lüdecke H-J, Kleefstra T, Wieczorek D. De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum. Hum Genet. 2015;134(1):97-109.
40. Holman S, Morgan T, Baujat G, Cormier-Daire V, Cho T-J, Lees M, Samanich J, Tapon D, Hove H, Hing A, Hennekam R, Robertson S. Osteopathia striata congenita with cranial sclerosis and intellectual disability due to contiguous gene deletions involving the WTX locus. Clin Genet. 2013;83(3):251-256.
41. Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. 2006;116(5):1202-1209.
42. Bunn KJ, Daniel P, Rösken HS, O’Neill AC, Cameron-Christie SR, Morgan T, Brunner HG, Lai A, Kunst HPM, Markie DM, Robertson SP. Mutations in DVL1 cause an osteosclerotic form of robinow syndrome. Am J Hum Genet. 2015;96(4):623-630.
43. White JJ, Mazzeu JF, Coban-Akdemir Z, Bayram Y, Bahrambeigi V, Hoischen A, van Bon BWM, Gezdirici A, Gulec EY, Ramond F, Touraine R, Thevenon J, Shinawi M, Beaver E, Heeley J, Hoover-Fong J, Durmaz CD, Karabulut HG, Marzioglu-Ozdemir E, Cayir A, Duz MB, Seven M, Price S, Ferreira BM, Vianna-Morgante AM, Ellard S, Parrish A, Stals K, Flores-Daboub J, Jhangiani SN, Gibbs RA, Brunner HG, Sutton VR, Lupski JR, Carvalho CMB. WNT signaling perturbations underlie the genetic heterogeneity of Robinow syndrome. Am J Hum Genet. 2018;102(1):27-43.
44. Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagneré A-M, René-Corail F, Jullian E, Gicquel C, Bertagna X, Vacher-Lavenu M-C, Perret C, Bertherat J. Mutations of ß-Catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res. 2005;65(17):7622-7627.
45. Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway. Cancer Sci. 2003;94(3):225-229.
46. Johnson V, Volikos E, Halford SE, Eftekhar Sadat ET, Popat S, Talbot I, Truninger K, Martin J, Jass J, Houlston R, Atkin W, Tomlinson IPM, Silver ARJ. Exon 3 beta- catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome. Gut. 2005;54(2):264-267.
Accepted Maga on oder e
Legends for Figures and Tables
Figure 1. Clinical presentation of the proband with a sclerosing skeletal dysplasia and adrenocortical adenoma.
(A) The pedigree of the family; (B) Radiographs of the proband (age 9 years) demonstrating a thickened, dense and sclerotic calvarium and skull base, sclerosis of pelvis and upper femoral metadiaphyses, undertubulation of the proximal and middle phalanges with diaphyseal sclerosis and cortical thickening of the tibial diaphyses; (C) Sanger sequence chromatograms from the proband and her parents. The c.131C>T transition is indicated by the red arrow.
Figure 2. Location and conservation of the substituted amino acid.
(A) Schematic of the CTNNB1 transcript (NM_001904.4) and ß-catenin protein (NP_001895.1) showing the correspondence of the protein domains with the coding exons. Arrow shows the location of the p.Pro44Leu substitution. (B) Protein homology between selected species for the N-terminus of ß-catenin orthologues. The amino acid residue altered by the variant being studied here is highlighted and adjacent phosphorylation target residues are indicated in blue. ß-catenin is homologous with armadillo in Drosophila melanogaster.
Figure 3 CTNNB 1Pro44Leu alters the WNT signalling activity.
(A) When expressed in HEK293FT cells, CTNNB1Ser45Ala and CTNNB1Pro44Leu have significantly decreased phosphorylation at Ser45 in comparison to CTNNB1WT. (B) When expressed in HEK293FT cells CTNNB1Ser45Ala and CTNNB1Pro44Leu have significantly decreased phosphorylation at Ser33/Ser37/Thr41 in comparison to CTNNB1WT. (C) CTNNB1Ser45Ala and CTNNB1Pro44Leu have significantly increased WNT signaling
transcriptional activity in comparison to CTNNB1WT. EV: empty vector; (D) Immunohistochemistry for anti-ß-catenin antibody in sections of the adrenocortical tumor from the proband (i, ii). Immunostaining at the cell membrane (M), and within the cytoplasm (C) and nucleus (N) is indicated. NC denotes no cytoplasmic staining. Boxed region is in the same place at low (i,) and high (ii) magnification. Scale bars: 50 um on the left side and 20 um on the right side. These experiments were replicated 3 times. Error bars show SD.
Accepted Manuscript89
A
B
C
ACAGCTCCTTCTCTGA
Mother
Father
Patient
1
c. 131C>T
L
L
Accepted Ma
A p.Pro44Leu
amor
B-catenin (protein domain)
N-terminal domain
Central domain: 12-Armadillo repeats
C-terminal domain
| CTNNB1 (mRNA) | 1 | 23 | 4 5 | 6 7 8 9 | 10 11 12 13 14 15 |
|---|---|---|---|---|---|
| 5'UTR | c.131C>T | CDS | 3'UTR |
B ß-catenin
H.sapiens
29. .. SYL-DSGIHSGATTTAPSLSGKGNPEEEDV … 57 NP_001895.1
M.mulatta 29… SYL-DSGIHSGATTTAPSLSGKGNPEEEDV … 57 NP_001244847.1
M.musculus
29… SYL-DSGIHSGATTTAPSLSGKGNPEEEDV … 57 NP_031640.1
C.gallus
29… SYL-DSGIHSGATTTAPSLSGKGNPEEEDV … 57 NP_990412.2
X. tropocalis 29. .. SYL-DSGIHSGATTTAPSLSGKGNPEDEDV … 57 NP_001016958. 1
D.rerio 29… SYL-DSGIHSGATTTAPSLSGKGNPEDDDV … 57 NP_571134.2
D.melanogaster 39. .. SYLGDSGIHSGAVTQVPSLSGKEDEEMEGD. .. 68 NP_476665.2
p.Pro44Leu
Accepted Manuscript
A
Relative Luciferase activity
**
pS45 ß-cat / Total ß-cat
1.6
C
18
**
Total ß-cat
1.2
16
12
pS45 ß-cat
0.8
8
WT
S45A
P44L
0.4
*
4
0.0
**
0
WT S45A P44L
EV
WT
S45A
P44L
B
D
pS33/S33/T41 B-cat /
1.6
i
ii
Total ß-cat
1.2
PS33/37/T41
Total ß-cat
N
ß-cat
0.8
WT S45A P44L
C
M
0.4
NC
0.0
**
**
WT S45A P44L
Accepted Mar