Whole genome sequencing and alendronate may be of value in diagnosing and managing CED, respectively

Duncan, Emma L

Gregson, Celia L

Hassan, Neelam

Lester, Tracy

Oldridge, Michael

Tobias, Jonathan H

English

Oxford University Research Archive

Letter to the Editor (Case report)Identification of a novel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronateNeelam Hassan 1,2,�, Celia L. Gregson 1, Michael Oldridge3, Tracy Lester3,  Emma L. Duncan 4, Jonathan H. Tobias 1,21Musculoskeletal Research Unit, University of Bristol, Bristol, UK 2Department of Rheumatology, Southmead Hospital, North Bristol NHS Trust, Bristol, UK 3Oxford Regional Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, UK 4Department of Twin Research and Genetic Epidemiology, School of Life Course & Population Sciences, Faculty of Life Sciences and Medicine, King’s College London, London, UK�Correspondence to: Neelam Hassan, Musculoskeletal Research Unit, University of Bristol, Level 1, Learning and Research Building, Southmead Hospital, Bristol BS10 5NB, UK. E-mail: neelam.hassan@bristol.ac.ukDEAR EDITOR, Camurati-Engelmann disease (CED) is a rare autosomal dominant progressive sclerosing dysplasia charac-terized by cortical hyperostosis of the long bones and skull [1]. Affected individuals present with bone pain, proximal muscle weakness and easy fatigability. Headaches, cranial nerve impingement and both conductive and sensorineural hearing loss can occur from skull base involvement. CED type 1 is caused by heterozygous activating mutations in Transforming Growth Factor-Beta-1 (TGFB1). Here, we re-port an individual with a delayed diagnosis of CED and a novel TGFB1 variant.A 34-year-old male presented in 2014 with widespread limb and back pain, arthralgia and muscle weakness since ad-olescence. He had fractured various bones after falls (aged 2, 7, 15 and 21 years). He developed bilateral sensorineural hearing loss in his 20s. He previously had a vitamin D defi-ciency but reported no symptomatic improvement with sup-plementation. There was no relevant family history, and he was not taking any regular medication.Examination showed a slim man (height 173 cm; weight 53.8 kg; BMI 18 kg/m2) wearing hearing aids. He had re-duced proximal (but not distal) muscle volume in his lower limbs and shortened great toes bilaterally. Examination was otherwise unremarkable.Investigations showed mildly elevated ALP (155 U/l [refer-ence range [RR] 20–120 U/l]) but normal adjusted calcium (2.47 mmol/l), phosphate (1.29 mmol/l), parathyroid hor-mone (3.1 pmol/l) and vitamin D (146 nmol/l). Creatinine was low (53 µmol/l) and creatine kinase was normal (37 U/l). Gonadal hormones were unmeasured. Bone turnover markers were markedly elevated (procollagen 1N propeptide [P1NP] 179 µg/l [RR 20–76 µg/l]; serum cross-linked C-telopeptide of type I collagen [CTX] 1.7 µg/l [RR 0.1–0.5 µg/l]). Hand, spine, hip, knee and foot radiographs demonstrated diffuse coarsened sclerotic bones with marked diaphyseal bone expansion (Fig. 1A, B), and Morton’s toes (normal variant affecting 15– 20% of the population). DXA showed increased BMD (Z scores at lumbar spine (L1-4) þ4.1, total hip þ3.0, and femoral neck þ5.3). Muscle biopsy was inconclusive.A diagnosis of juvenile Paget’s disease was made, based on radiographic appearances and hearing loss; however, se-quencing of known monogenic causes did not identify any causative variants. Bisphosphonates were not started due to the elevated BMD.He re-presented in 2020 with worsening bone and joint pain, exacerbated by walking, alongside a deterioration in his hearing. ALP was now elevated at 411 U/l. Audiogram showed progressive severe bilateral sensory hearing loss; cra-nial computed tomography showed diffuse skull thickening with almost complete obliteration of both internal audi-tory canals.His hearing aids were optimized. Alendronate was com-menced, leading to almost complete resolution of musculo-skeletal symptoms within 6 weeks, and partial suppression of CTX (1.03 µg/l) at 9 months (P1NP not tested).Recurrence of bone and joint pains in 2022 prompted a switch to IV zoledronate; however, he reported worsening bone pain and hearing loss within a week of the first dose, persisting for 2 months. CTX was also high (1.2 µg/l) [no pre- dose measurement]. Alendronate was restarted and musculo-keletal symptoms improved within 12 weeks.In 2024, he presented with increased muscle weakness, un-steadiness, and fatigue. Skeletal dysplasia whole genome se-quence (WGS) sequencing panel (R104 version 4.0, https:// nhsgms-panelapp.genomicsengland.co.uk/) was initially reported Rheumatology key message� Whole genome sequencing and alendronate may be of value in diagnosing and managing CED, respectively. Accepted: 27 July 2025 © The Author(s) 2025. Published by Oxford University Press on behalf of the British Society for Rheumatology.  This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Rheumatology, 2025, 64, 6427–6429 https://doi.org/10.1093/rheumatology/keaf441 Advance access publication 14 August 2025 Letter to the Editor Rheumatologyas showing no causative variants. However, upon review of ad-ditional clinical information and variants of uncertain signifi-cance (VUS), a single heterozygous c.31_48 inframe duplication in exon 1 of TGFB1 was identified as a candidate variant. This was predicted to lead to an in-frame duplication of 6 residues p. (Leu11_Leu16dup) in TGFβ1. Protein modelling using AlphaFold2 [2] demonstrated that the variant was located within the N-terminal signal peptide of the TGFβ1 pro-protein (p.Leu11_Leu16dup) (Fig. 1C, D) [2]. Subsequent family testing confirmed the variant to be de novo. His clinical history, biochemistry and imaging were reassessed and considered con-sistent with CED.TGFβ1 is a multifunctional protein with key roles in bone and muscle physiology; perturbations in the TGFβ1 signalling pathway are associated with multiple skeletal dysplasias (e.g. osteogenesis imperfecta type IV (OMIM 166220), acromicric dysplasia (OMIM 102370) and Loeys-Dietz syndrome types 1–5. Most (>80%) individuals with CED type 1 have activat-ing missense variants in exon 4, affecting the latency- associated peptide of pro-TGFβ1 [1]. Two duplications of 3 Figure 1. Radiographic, structural and genetic characterization of a novel TGFB1 variant in a heterozygous carrier. Diffuse coarse trabecular pattern with cortical bone expansion on pelvic (A) and knee (B) radiographs. (C) Protein model of the wild-type pre-pro-TGFβ1 homodimer (in green) aligned with the mutant pre-pro-TGFβ1 homodimer (in blue), created using AlphaFold2 and PyMOL. The p. Leu11_Leu16 duplication is highlighted in magenta within the signal peptide at the N-terminus of the mutant homodimer. (D) Schematic of wild and mutant pre-pro-TGFβ1 proteins. Please note AlphaFold2 is not validated for predicting the structural effects of variants. This model is shown only to illustrate the position of the variant within the protein. (E) Sanger sequencing traces of the TGFB1 gene in the proband and both parents, shown against a synthesized reference trace using Mutation Surveyor. The proband carries a heterozygous c.31_48dup duplication (highlighted by red arrows), visible as overlapping peaks following nucleotide c.48. This duplication was not present in either parent 6428                                                                                                                                                                                                                  Letter to the Editor and 4 leucine residues within the signal peptide have previ-ously been described [3, 4]. Our case has the largest duplica-tion reported of 6 residues (LLLPLL), extending the hydrophobic region within the signal peptide from 14 to 20 amino acids. This is likely to impact proper cleavage of the sig-nal peptide and reduce pro-TGFβ1 secretion, leading to intra-cellular accumulation. This has been shown to activate TGFβ1 signalling in vitro, possibly via alternative pathways [3].Management of CED is supportive, with inconsistent evi-dence on the symptomatic benefit of corticosteroids, non- steroidal anti-inflammatory drugs, losartan and/or bisphosphonates [5–8]. Unexpectedly, our patient experi-enced discordant effects on his symptoms with alendronate versus zoledronate. This was unexpected, given the greater potency of zoledronate, and has not been observed previ-ously. Speculatively, rapid bone turnover in CED may affect skeletal retention of bisphosphonates, with differential effects from gradual administration versus infusion. Bone turnover marker measurements were limited here; monitoring may help to guide and interpret treatment responses.Our case highlights the importance of WGS analysis in di-agnostically challenging cases. Furthermore, trialling differ-ent bisphosphonates in individuals with CED may aid symptom management.Data availabilityAnonymised data are available upon reasonable request by any qualified researchers who engage in rigorous, indepen-dent scientific research, and will be provided following re-view and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). The data are not publicly available due to privacy or ethical restrictions.FundingN.H. is supported by the Medical Research Council (MR/ V00199X/1).Disclosure statement: The authors have declared no conflicts of interest.Patient consent statement: The patient has provided informed consent for publication.AcknowledgementsWe would like to thank the patient and his family for their help and cooperation with the investigation and publication of this manuscript.References1. Van Hul W, Boudin E, Vanhoenacker FM, Mortier G. Camurati– Engelmann disease. Calcified tissue international. Review 2019; 104:554–60.2. Jumper J, Evans R, Pritzel A et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021;596:583–9.3. Janssens K, Ten Dijke P, Ralston SH, Bergmann C, Van Hul W. Transforming growth factor-β1 mutations in Camurati-Engelmann disease lead to increased signaling by altering either activation or se-cretion of the mutant protein. J Biol Chem 2003;278:7718–24.4. Whyte MP, Totty WG, Novack DV et al. Camurati-engelmann dis-ease: unique variant featuring a novel mutation in TGFβ1 encoding transforming growth factor beta 1 and a missense change in TNFSF11 encoding RANK ligand. J Bone Miner Res 2011;26:920–33.5. Janssens K, Vanhoenacker F, Bonduelle M et al. Camurati- Engelmann disease: review of the clinical, radiological, and molecu-lar data of 24 families and implications for diagnosis and treatment. J Med Genet 2006;43:1–11.6. Combier A, Palazzo E, Forien M et al. Failure of conventional treat-ment and losartan in Camurati-Engelmann disease: a case report. Joint Bone Spine 2018;85:649–50.7. Baroncelli GI, Ferretti E, Pini CM et al. Significant improvement of clinical symptoms, bone lesions, and bone turnover after long-term zoledronic acid treatment in patients with a severe form of Camurati-Engelmann disease. Mol Syndromol 2017;8:294–302.8. Klemm P, Aykara I, Lange U. Camurati-Engelmann disease: a case- based review about an ultrarare bone dysplasia. Eur J Rheumatol 2023;10:34–8.© The Author(s) 2025. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Rheumatology, 2025, 64, 6427–6429https://doi.org/10.1093/rheumatology/keaf441Letter to the EditorLetter to the Editor                                                                                                                                                                                                                 6429

Identification of a novel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronate

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Identification of a novel <i>TGFB1 </i>variant in a patient with Camurati-Engelmann disease responsive to alendronate

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                       King’s Research Portal  DOI:10.1093/rheumatology/keaf441Document VersionPeer reviewed versionLink to publication record in King's Research PortalCitation for published version (APA):Hassan, N., Gregson, C. L., Oldridge, M., Lester, T., Duncan, E. L., & Tobias, J. H. (2025). Identification of anovel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronate. Rheumatology,64(12), 6427-6429. https://doi.org/10.1093/rheumatology/keaf441Citing this paperPlease note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this maydiffer from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination,volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you areagain advised to check the publisher's website for any subsequent corrections.General rightsCopyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyrightowners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights.•Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research.•You may not further distribute the material or use it for any profit-making activity or commercial gain•You may freely distribute the URL identifying the publication in the Research PortalTake down policyIf you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access tothe work immediately and investigate your claim.Download date: 27. Dec. 20251Identification of a novel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronateNeelam Hassan1,2, Celia L Gregson1, Michael Oldridge3, Tracy Lester3, Emma L Duncan4, Jonathan H Tobias1,21 Musculoskeletal Research Unit, University of Bristol, Bristol, UK. 2 Department of Rheumatology, Southmead Hospital, North Bristol NHS Trust, Bristol, UK.3 Oxford Regional Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.4Department of Twin Research and Genetic Epidemiology, School of Life Course & Population Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK.Corresponding author details: Full name: Dr Neelam HassanPostal address: Musculoskeletal Research Unit, University of Bristol, Level 1, Learning and Research Building, Southmead Hospital, Bristol, BS10 5NB, UK.Email address: Neelam.hassan@bristol.ac.uk ORCiD:Neelam Hassan: 0000-0001-7052-5708Celia Gregson: 0000-0001-6414-0529Emma Duncan: 0000-0002-8143-4403Jon Tobias: 0000-0002-7475-3932Page 1 of 8 Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960© The Author(s) 2025. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2Key messages: • Whole genome sequencing and alendronate may be of value in diagnosing and managing CED respectivelyPage 2 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603Dear Editor,Camurati-Engelmann disease (CED) is a rare autosomal dominant progressive sclerosing dysplasia characterised by cortical hyperostosis of the long bones and skull(1). Affected individuals present with bone pain, proximal muscle weakness, and easy fatigability. Headaches, cranial nerve impingement and both conductive and sensorineural hearing loss can occur from skull base involvement. CED type 1 is caused by heterozygous activating mutations in Transforming Growth Factor-Beta-1 (TGFB1).  Here we report an individual with delayed diagnosis of CED and a novel TGFB1 variant.A 34-year-old male presented in 2014 with widespread limb and back pain, arthralgia, and muscle weakness since adolescence. He had fractured various bones after falls (aged 2, 7, 15 and 21 years). He developed bilateral sensorineural hearing loss in his 20s. He previously had vitamin D deficiency but reported no symptomatic improvement with supplementation. There was no relevant family history and he was not taking any regular medication. Examination showed a slim man (height 173cm; weight 53.8kg; BMI 18 kg/m2) wearing hearing aids. He had reduced proximal (but not distal) muscle volume in his lower limbs and shortened great toes bilaterally. Examination was otherwise unremarkable.Investigations showed mildly elevated alkaline phosphatase [ALP] (155U/L [reference range [RR] 20-120U/L]) but normal adjusted calcium (2.47mmol/L), phosphate (1.29mmol/L), parathyroid hormone (3.1pmol/L) and vitamin D (146nmol/L). Creatinine was low (53µmol/L); creatine kinase was normal (37U/L).  Gonadal hormones were unmeasured. Bone turnover markers were markedly elevated (procollagen 1 N propeptide [P1NP] 179µg/L [RR 20-76µg/L]; serum cross-lined C-telopeptide of type I collagen [CTX] 1.7µg/L [RR 0.1-0.5µg/L]).  Hand, spine, hip, knee and foot radiographs demonstrated diffuse coarsened sclerotic bones with marked diaphyseal bone expansion (Figures 1A,1B), and Morton’s toes (normal variant affecting 15-20% of the population). Dual-energy X-ray absorptiometry showed increased bone mineral density (BMD) (Z scores at lumbar spine (L1-4) +4.1,  total hip +3.0, and femoral neck +5.3). Muscle biopsy was inconclusive.A diagnosis of juvenile Paget’s disease was made, based on radiographic appearances and hearing loss; however, sequencing of known monogenic causes did not identify any causative variants. Bisphosphonates were not started due to the elevated BMD.He re-presented in 2020 with worsening bone and joint pain, exacerbated by walking, alongside a deterioration in his hearing. ALP was now elevated at 411U/L. Audiogram  showed progressive severe bilateral sensory hearing loss; cranial computed tomography showed diffuse skull thickening with almost complete obliteration of both internal auditory canals.His hearing aids were optimised. Alendronate was commenced, leading to almost complete resolution of musculoskeletal symptoms within six weeks, and partial suppression of CTX (1.03µg/L) at nine months (P1NP not tested).Recurrence of bone and joint pains in 2022 prompted a switch to IV zoledronate; however, he reported worsening bone pain and hearing loss within a week of the first dose, persisting Page 3 of 8 Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604for two months. CTX was also high (1.2µg/L) [no pre-dose measurement]. Alendronate was restarted; and musculokeletal symptoms improved within twelve weeks.In 2024, he presented with increased muscle weakness, unsteadiness, and fatigue. Skeletal dysplasia whole genome sequence (WGS) sequencing panel (R104 version 4.0, https://nhsgms-panelapp.genomicsengland.co.uk/) was initially reported as showing no causative variants. However, upon review of additional clinical information and variants of uncertain significance (VUS), a single heterozygous c.31_48 inframe duplication in exon 1 of TGFB1 was identified as a candidate variant. This was predicted to lead to an in-frame duplication of 6 residues p.(Leu11_Leu16dup) in TGFβ1.  Protein modelling using AlphaFold2(2) demonstrated that the variant was located within the N-terminal signal peptide of the TGFβ1 pro-protein (p.Leu11_Leu16dup) (Figure 1C, 1D)(2). Subsequent family testing confirmed the variant to be de novo. His clinical history, biochemistry, and imaging were reassessed and considered consistent with CED.TGFβ1 is a multifunctional protein with key roles in bone and muscle physiology; perturbations in the TGFβ1 signalling pathway are associated with multiple skeletal dysplasias (e.g. osteogenesis imperfecta type IV (OMIM 166220), acromicric dysplasia (OMIM 102370) and Loeys-Dietz syndrome types 1-5. Most (>80%) individuals with CED type 1 have activating missense variants in exon 4, affecting the latency-associated peptide of pro-TGFβ1(1). Two duplications of 3-4 leucine residues within the signal peptide have previously been described (3,4). Our case has the largest duplication reported of 6 residues (LLLPLL), extending the hydrophobic region within the signal peptide from 14 to 20 amino acids. This is likely to impact proper cleavage of the signal peptide and reduce pro-TGFβ1 secretion, leading to intracellular accumulation. This has been shown to activate TGFβ1 signalling in vitro, possibly via alternative pathways(3).Management of CED is supportive, with inconsistent evidence on the symptomatic benefit of corticosteroids, non-steroidal anti-inflammatory drugs, losartan and/or bisphosphonates(5-8). Unexpectedly, our patient experienced discordant effects on his symptoms with alendronate versus zoledronate. This was unexpected, given the greater potency of zoledronate, and has not been observed previously. Speculatively, rapid bone turnover in CED may affect skeletal retention of bisphosphonates, with differential effects from gradual administration versus infusion.  Bone turnover marker measurements were limited here; monitoring may help to guide and interpret treatment responses.Our case highlights the importance of WGS analysis in diagnostically challenging cases. Furthermore, trialling different bisphosphonates in individuals with CED may aid symptom management.Acknowledgements: We would like to thank the patient and his family for their help and cooperation with the investigation and publication of this manuscript. Funding: NH is supported by the Medical Research Council (MR/V00199X/1).Page 4 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859605Conflict of interest statement: The authors have no potential conflicts of interest.Data availability statement: Anonymised data are available upon reasonable requestby any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). The data are not publiclyavailable due to privacy or ethical restrictions.Patient consent statement: The patient has provided informed consent for publication.Page 5 of 8 Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606Figure 1Figure 1. Radiographic, structural and genetic characterisation of a novel TGFB1 variant in a heterozygous carrierDiffuse coarse trabecular pattern with cortical bone expansion on pelvic (A) and knee (B) radiographs. (C) Protein model of the wild type pre-pro-TGFβ1 homodimer (in green) aligned with the mutant pre-pro-TGFβ1 homodimer (in blue), created using AlphaFold2 and PyMOL. The p.Leu11_Leu16 duplication is highlighted in magenta within the signal peptide at the N-terminus of the mutant homodimer. (D) Schematic of wild and mutant pre-pro-TGFB1 proteins. Please note AlphaFold2 is not validated for predicting the structural effects of variants. This model is shown only to illustrate the position of the variant within the protein. (E) Sanger sequencing traces of the TGFB1 gene in the proband and both parents, shown against a synthesised reference trace using Mutation Page 6 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859607Surveyor. The proband carries a heterozygous c.31_48dup duplication, visible as overlapping peaks following nucleotide c.48 (highlighted by red arrows). This duplication was not present in either parent.Alt text: Radiographs, protein model and diagram showing cortical bone expansion and location of novel TGFB1 variant, with subfigures labelled A to D. DNA sequencing traces, labelled E, from the proband and both parents, showing a duplication in the TGFB1 gene which is only present in the proband.References 1. Van Hul W, Boudin E, Vanhoenacker FM, Mortier G. Camurati–Engelmann Disease. Calcified Tissue International. Review 2019;104(5):554-60.2. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. Aug 2021;596(7873):583-9. Epub 20210715.3. Janssens K, Ten Dijke P, Ralston SH, Bergmann C, Van Hul W. Transforming growth factor-β1 mutations in Camurati-Engelmann disease lead to increased signaling by altering either activation or secretion of the mutant protein. Journal of Biological Chemistry. 2003;278(9):7718-24.4. Whyte MP, Totty WG, Novack DV, Zhang X, Wenkert D, Mumm S. Camurati-engelmann disease: Unique variant featuring a novel mutation in TGFβ1 encoding transforming growth factor beta 1 and a missense change in TNFSF11 encoding RANK ligand. Journal of Bone and Mineral Research. 2011;26(5):920-33.5. Janssens K, Vanhoenacker F, Bonduelle M, Verbruggen L, Van Maldergem L, Ralston S, et al. Camurati-Engelmann disease: Review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment. Journal of Medical Genetics. 2006;43(1):1-11.6. Combier A, Palazzo E, Forien M, Gardette A, Dieude P, Ottaviani S. Failure of conventional treatment and losartan in Camurati-Engelmann disease: A case report. Joint Bone Spine. Oct 2018;85(5):649-50. Epub 20180213.7. Baroncelli GI, Ferretti E, Pini CM, Toschi B, Consolini R, Bertelloni S. Significant Improvement of Clinical Symptoms, Bone Lesions, and Bone Turnover after Long-Term Zoledronic Acid Treatment in Patients with a Severe Form of Camurati-Engelmann Disease. Mol Syndromol. Nov 2017;8(6):294-302. Epub 20170909.8. Klemm P, Aykara I, Lange U. Camurati-Engelmann Disease: A Case-Based Review About an Ultrarare Bone Dysplasia. Eur J Rheumatol. Jan 2023;10(1):34-8.Page 7 of 8 Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960 484x521mm (236 x 236 DPI) Page 8 of 8Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

                       King’s Research Portal  DOI:10.1093/rheumatology/keaf441Document VersionPeer reviewed versionLink to publication record in King's Research PortalCitation for published version (APA):Hassan, N., Gregson, C. L., Oldridge, M., Lester, T., Duncan, E. L., & Tobias, J. H. (2025). Identification of anovel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronate. Rheumatology.Advance online publication. https://doi.org/10.1093/rheumatology/keaf441Citing this paperPlease note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this maydiffer from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination,volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you areagain advised to check the publisher's website for any subsequent corrections.General rightsCopyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyrightowners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights.•Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research.•You may not further distribute the material or use it for any profit-making activity or commercial gain•You may freely distribute the URL identifying the publication in the Research PortalTake down policyIf you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access tothe work immediately and investigate your claim.Download date: 10. Sep. 20251Identification of a novel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronateNeelam Hassan1,2, Celia L Gregson1, Michael Oldridge3, Tracy Lester3, Emma L Duncan4, Jonathan H Tobias1,21 Musculoskeletal Research Unit, University of Bristol, Bristol, UK. 2 Department of Rheumatology, Southmead Hospital, North Bristol NHS Trust, Bristol, UK.3 Oxford Regional Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.4Department of Twin Research and Genetic Epidemiology, School of Life Course & Population Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK.Corresponding author details: Full name: Dr Neelam HassanPostal address: Musculoskeletal Research Unit, University of Bristol, Level 1, Learning and Research Building, Southmead Hospital, Bristol, BS10 5NB, UK.Email address: Neelam.hassan@bristol.ac.uk ORCiD:Neelam Hassan: 0000-0001-7052-5708Celia Gregson: 0000-0001-6414-0529Emma Duncan: 0000-0002-8143-4403Jon Tobias: 0000-0002-7475-3932Page 1 of 8 Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960© The Author(s) 2025. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2Key messages: • Whole genome sequencing and alendronate may be of value in diagnosing and managing CED respectivelyPage 2 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603Dear Editor,Camurati-Engelmann disease (CED) is a rare autosomal dominant progressive sclerosing dysplasia characterised by cortical hyperostosis of the long bones and skull(1). Affected individuals present with bone pain, proximal muscle weakness, and easy fatigability. Headaches, cranial nerve impingement and both conductive and sensorineural hearing loss can occur from skull base involvement. CED type 1 is caused by heterozygous activating mutations in Transforming Growth Factor-Beta-1 (TGFB1).  Here we report an individual with delayed diagnosis of CED and a novel TGFB1 variant.A 34-year-old male presented in 2014 with widespread limb and back pain, arthralgia, and muscle weakness since adolescence. He had fractured various bones after falls (aged 2, 7, 15 and 21 years). He developed bilateral sensorineural hearing loss in his 20s. He previously had vitamin D deficiency but reported no symptomatic improvement with supplementation. There was no relevant family history and he was not taking any regular medication. Examination showed a slim man (height 173cm; weight 53.8kg; BMI 18 kg/m2) wearing hearing aids. He had reduced proximal (but not distal) muscle volume in his lower limbs and shortened great toes bilaterally. Examination was otherwise unremarkable.Investigations showed mildly elevated alkaline phosphatase [ALP] (155U/L [reference range [RR] 20-120U/L]) but normal adjusted calcium (2.47mmol/L), phosphate (1.29mmol/L), parathyroid hormone (3.1pmol/L) and vitamin D (146nmol/L). Creatinine was low (53µmol/L); creatine kinase was normal (37U/L).  Gonadal hormones were unmeasured. Bone turnover markers were markedly elevated (procollagen 1 N propeptide [P1NP] 179µg/L [RR 20-76µg/L]; serum cross-lined C-telopeptide of type I collagen [CTX] 1.7µg/L [RR 0.1-0.5µg/L]).  Hand, spine, hip, knee and foot radiographs demonstrated diffuse coarsened sclerotic bones with marked diaphyseal bone expansion (Figures 1A,1B), and Morton’s toes (normal variant affecting 15-20% of the population). Dual-energy X-ray absorptiometry showed increased bone mineral density (BMD) (Z scores at lumbar spine (L1-4) +4.1,  total hip +3.0, and femoral neck +5.3). Muscle biopsy was inconclusive.A diagnosis of juvenile Paget’s disease was made, based on radiographic appearances and hearing loss; however, sequencing of known monogenic causes did not identify any causative variants. Bisphosphonates were not started due to the elevated BMD.He re-presented in 2020 with worsening bone and joint pain, exacerbated by walking, alongside a deterioration in his hearing. ALP was now elevated at 411U/L. Audiogram  showed progressive severe bilateral sensory hearing loss; cranial computed tomography showed diffuse skull thickening with almost complete obliteration of both internal auditory canals.His hearing aids were optimised. Alendronate was commenced, leading to almost complete resolution of musculoskeletal symptoms within six weeks, and partial suppression of CTX (1.03µg/L) at nine months (P1NP not tested).Recurrence of bone and joint pains in 2022 prompted a switch to IV zoledronate; however, he reported worsening bone pain and hearing loss within a week of the first dose, persisting Page 3 of 8 Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604for two months. CTX was also high (1.2µg/L) [no pre-dose measurement]. Alendronate was restarted; and musculokeletal symptoms improved within twelve weeks.In 2024, he presented with increased muscle weakness, unsteadiness, and fatigue. Skeletal dysplasia whole genome sequence (WGS) sequencing panel (R104 version 4.0, https://nhsgms-panelapp.genomicsengland.co.uk/) was initially reported as showing no causative variants. However, upon review of additional clinical information and variants of uncertain significance (VUS), a single heterozygous c.31_48 inframe duplication in exon 1 of TGFB1 was identified as a candidate variant. This was predicted to lead to an in-frame duplication of 6 residues p.(Leu11_Leu16dup) in TGFβ1.  Protein modelling using AlphaFold2(2) demonstrated that the variant was located within the N-terminal signal peptide of the TGFβ1 pro-protein (p.Leu11_Leu16dup) (Figure 1C, 1D)(2). Subsequent family testing confirmed the variant to be de novo. His clinical history, biochemistry, and imaging were reassessed and considered consistent with CED.TGFβ1 is a multifunctional protein with key roles in bone and muscle physiology; perturbations in the TGFβ1 signalling pathway are associated with multiple skeletal dysplasias (e.g. osteogenesis imperfecta type IV (OMIM 166220), acromicric dysplasia (OMIM 102370) and Loeys-Dietz syndrome types 1-5. Most (>80%) individuals with CED type 1 have activating missense variants in exon 4, affecting the latency-associated peptide of pro-TGFβ1(1). Two duplications of 3-4 leucine residues within the signal peptide have previously been described (3,4). Our case has the largest duplication reported of 6 residues (LLLPLL), extending the hydrophobic region within the signal peptide from 14 to 20 amino acids. This is likely to impact proper cleavage of the signal peptide and reduce pro-TGFβ1 secretion, leading to intracellular accumulation. This has been shown to activate TGFβ1 signalling in vitro, possibly via alternative pathways(3).Management of CED is supportive, with inconsistent evidence on the symptomatic benefit of corticosteroids, non-steroidal anti-inflammatory drugs, losartan and/or bisphosphonates(5-8). Unexpectedly, our patient experienced discordant effects on his symptoms with alendronate versus zoledronate. This was unexpected, given the greater potency of zoledronate, and has not been observed previously. Speculatively, rapid bone turnover in CED may affect skeletal retention of bisphosphonates, with differential effects from gradual administration versus infusion.  Bone turnover marker measurements were limited here; monitoring may help to guide and interpret treatment responses.Our case highlights the importance of WGS analysis in diagnostically challenging cases. Furthermore, trialling different bisphosphonates in individuals with CED may aid symptom management.Acknowledgements: We would like to thank the patient and his family for their help and cooperation with the investigation and publication of this manuscript. Funding: NH is supported by the Medical Research Council (MR/V00199X/1).Page 4 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859605Conflict of interest statement: The authors have no potential conflicts of interest.Data availability statement: Anonymised data are available upon reasonable requestby any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). The data are not publiclyavailable due to privacy or ethical restrictions.Patient consent statement: The patient has provided informed consent for publication.Page 5 of 8 Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606Figure 1Figure 1. Radiographic, structural and genetic characterisation of a novel TGFB1 variant in a heterozygous carrierDiffuse coarse trabecular pattern with cortical bone expansion on pelvic (A) and knee (B) radiographs. (C) Protein model of the wild type pre-pro-TGFβ1 homodimer (in green) aligned with the mutant pre-pro-TGFβ1 homodimer (in blue), created using AlphaFold2 and PyMOL. The p.Leu11_Leu16 duplication is highlighted in magenta within the signal peptide at the N-terminus of the mutant homodimer. (D) Schematic of wild and mutant pre-pro-TGFB1 proteins. Please note AlphaFold2 is not validated for predicting the structural effects of variants. This model is shown only to illustrate the position of the variant within the protein. (E) Sanger sequencing traces of the TGFB1 gene in the proband and both parents, shown against a synthesised reference trace using Mutation Page 6 of 8Rheumatology1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859607Surveyor. The proband carries a heterozygous c.31_48dup duplication, visible as overlapping peaks following nucleotide c.48 (highlighted by red arrows). This duplication was not present in either parent.Alt text: Radiographs, protein model and diagram showing cortical bone expansion and location of novel TGFB1 variant, with subfigures labelled A to D. DNA sequencing traces, labelled E, from the proband and both parents, showing a duplication in the TGFB1 gene which is only present in the proband.References 1. Van Hul W, Boudin E, Vanhoenacker FM, Mortier G. Camurati–Engelmann Disease. Calcified Tissue International. Review 2019;104(5):554-60.2. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. Aug 2021;596(7873):583-9. Epub 20210715.3. Janssens K, Ten Dijke P, Ralston SH, Bergmann C, Van Hul W. Transforming growth factor-β1 mutations in Camurati-Engelmann disease lead to increased signaling by altering either activation or secretion of the mutant protein. Journal of Biological Chemistry. 2003;278(9):7718-24.4. Whyte MP, Totty WG, Novack DV, Zhang X, Wenkert D, Mumm S. Camurati-engelmann disease: Unique variant featuring a novel mutation in TGFβ1 encoding transforming growth factor beta 1 and a missense change in TNFSF11 encoding RANK ligand. Journal of Bone and Mineral Research. 2011;26(5):920-33.5. Janssens K, Vanhoenacker F, Bonduelle M, Verbruggen L, Van Maldergem L, Ralston S, et al. Camurati-Engelmann disease: Review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment. Journal of Medical Genetics. 2006;43(1):1-11.6. Combier A, Palazzo E, Forien M, Gardette A, Dieude P, Ottaviani S. Failure of conventional treatment and losartan in Camurati-Engelmann disease: A case report. Joint Bone Spine. Oct 2018;85(5):649-50. Epub 20180213.7. Baroncelli GI, Ferretti E, Pini CM, Toschi B, Consolini R, Bertelloni S. Significant Improvement of Clinical Symptoms, Bone Lesions, and Bone Turnover after Long-Term Zoledronic Acid Treatment in Patients with a Severe Form of Camurati-Engelmann Disease. Mol Syndromol. Nov 2017;8(6):294-302. Epub 20170909.8. Klemm P, Aykara I, Lange U. Camurati-Engelmann Disease: A Case-Based Review About an Ultrarare Bone Dysplasia. Eur J Rheumatol. Jan 2023;10(1):34-8.Page 7 of 8 Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960 484x521mm (236 x 236 DPI) Page 8 of 8Rheumatology123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

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Identification of a novel TGFB1 variant in a patient with Camurati-Engelmann disease responsive to alendronate

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